Researcher Profile and Settings

Affiliation

• Institute for Genetic Medicine　Molecular Pathogenesis

Job Title

• Professor

J-Global ID

• 201301087274865066

Research Interests

• ゲートウェイ反射   IL-6アンプ   Endothelial cells   Helper T cells   Neural Singlaing pathway   Chronic Inflammation   Cytokines   

Research Areas

• Life sciences / Immunology
• Life sciences / Experimental pathology

Educational Organization

•  Master's degree program　Graduate School of Medicine
•  Doctoral (PhD) degree program　Graduate School of Medicine

Academic & Professional Experience

• 2014 - Today Institute for Genomic Medicine and Graduate School of Medicine Division of Molecular Neuroimmunology Professor

Education

• 1989/04 - 1993/03  Osaka University  Graduate School of Medicine
• 1984/04 - 1989/03  Hokkaido University  School of Veterinary Medicine

Association Memberships

• ISNI - International Society of Neuroimmunology   AAI - THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS   日本神経免疫学会   北海道病理談話会   量子生命科学会   日本サイトカイン学会   日本生化学会   日本リウマチ学会北海道・東北支部   日本リウマチ学会   日本免疫学会   

Research Activities

Published Papers

• DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation

  Seiichiro Naito, Hiroki Tanaka, Jing-Jing Jiang, Masato Tarumi, Ari Hashimoto, Yuki Tanaka, Kaoru Murakami, Shimpei I. Kubota, Shintaro Hojyo, Shigeru Hashimoto, Masaaki Murakami

  Biochemical and Biophysical Research Communications 703 149666 - 149666 0006-291X 2024/04
• Gateway reflexes describe novel neuro-immune communications that establish immune cell gateways at specific vessels.

  Hiroki Tanaka, Rie Hasebe, Kaoru Murakami, Toshiki Sugawara, Takeshi Yamasaki, Masaaki Murakami

  Bioelectronic medicine 9 (1) 24 - 24 2023/11/08 

  Neuroinflammation is an important biological process induced by complex interactions between immune cells and neuronal cells in the central nervous system (CNS). Recent research on the bidirectional communication between neuronal and immunological systems has provided evidence for how immune and inflammatory processes are regulated by nerve activation. One example is the gateway reflex, in which immune cells bypass the blood brain barrier and infiltrate the CNS to cause neuroinflammation. We have found several modes of the gateway reflex in mouse models, in which gateways for immune cells are established at specific blood vessels in the spinal cords and brain in experimental autoimmune encephalomyelitis and systemic lupus erythematosus models, at retinal blood vessels in an experimental autoimmune uveitis model, and the ankle joints in an inflammatory arthritis model. Several environmental stimulations, including physical and psychological stresses, activate neurological pathways that alter immunological responses via the gateway reflex, thus contributing to the development/suppression of autoimmune diseases. In the manuscript, we describe the discovery of the gateway reflex and recent insights on how they regulate disease development. We hypothesize that artificial manipulation of specific neural pathways can establish and/or close the gateways to control the development of autoimmune diseases.
• Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

  Takeshi Kurosu, Daisuke Okuzaki, Yusuke Sakai, Mohamad Al Kadi, Supranee Phanthanawiboon, Yasusi Ami, Masayuki Shimojima, Tomoki Yoshikawa, Shuetsu Fukushi, Noriyo Nagata, Tadaki Suzuki, Daisuke Kamimura, Masaaki Murakami, Hideki Ebihara, Masayuki Saijo

  PLoS neglected tropical diseases 17 (11) e0011743  2023/11/08 

  Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/β and γ receptor knockout mice (IFN-α/β/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/β/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1β, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.
• High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore.

  Kaoru Murakami, Shimpei I Kubota, Kumiko Tanaka, Hiroki Tanaka, Keiichiroh Akabane, Rigel Suzuki, Yuta Shinohara, Hiroyasu Takei, Shigeru Hashimoto, Yuki Tanaka, Shintaro Hojyo, Osamu Sakamoto, Norihiko Naono, Takayui Takaai, Kazuki Sato, Yuichi Kojima, Toshiyuki Harada, Takeshi Hattori, Satoshi Fuke, Isao Yokota, Satoshi Konno, Takashi Washio, Takasuke Fukuhara, Takanori Teshima, Masateru Taniguchi, Masaaki Murakami

  Lab on a chip 23 (22) 4909 - 4918 2023/11/07 

  A digital platform that can rapidly and accurately diagnose pathogenic viral variants, including SARS-CoV-2, will minimize pandemics, public anxiety, and economic losses. We recently reported an artificial intelligence (AI)-nanopore platform that enables testing for Wuhan SARS-CoV-2 with high sensitivity and specificity within five minutes. However, which parts of the virus are recognized by the platform are unknown. Similarly, whether the platform can detect SARS-CoV-2 variants or the presence of the virus in clinical samples needs further study. Here, we demonstrated the platform can distinguish SARS-CoV-2 variants. Further, it identified mutated Wuhan SARS-CoV-2 expressing spike proteins of the delta and omicron variants, indicating it discriminates spike proteins. Finally, we used the platform to identify omicron variants with a sensitivity and specificity of 100% and 94%, respectively, in saliva specimens from COVID-19 patients. Thus, our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants.
• An inflammatory bowel disease-associated SNP increases local thyroglobulin expression to develop inflammation in miniature dachshunds

  Yong Bin Teoh, Jing-Jing Jiang, Takeshi Yamasaki, Noriyuki Nagata, Toshiki Sugawara, Rie Hasebe, Hiroshi Ohta, Noboru Sasaki, Nozomu Yokoyama, Kensuke Nakamura, Yumiko Kagawa, Mitsuyoshi Takiguchi, Masaaki Murakami

  Frontiers in Veterinary Science 10 2023/07/14 

  Inflammatory colorectal polyp (ICRP) in miniature dachshunds (MDs) is a chronic inflammatory bowel disease (IBD) characterized by granulomatous inflammation that consists of neutrophil infiltration and goblet cell hyperplasia in the colon. Recently, we identified five MD-associated single-nucleotide polymorphisms (SNPs), namely PLG, TCOF1, TG, COL9A2, and COL4A4, by whole-exome sequencing. Here, we investigated whether TG c.4567C>T (p.R1523W) is associated with the ICRP pathology. We found that the frequency of the T/T SNP risk allele was significantly increased in MDs with ICRP. In vitro experiments showed that TG expression in non-immune cells was increased by inducing the IL-6 amplifier with IL-6 and TNF-α. On the other hand, a deficiency of TG suppressed the IL-6 amplifier. Moreover, recombinant TG treatment enhanced the activation of the IL-6 amplifier, suggesting that TG is both a positive regulator and a target of the IL-6 amplifier. We also found that TG expression together with two NF-κB targets, IL6 and CCL2, was increased in colon samples isolated from MDs with the T/T risk allele compared to those with the C/C non-risk allele, but serum TG was not increased. Cumulatively, these results suggest that the T/T SNP is an expression quantitative trait locus (eQTL) of TG mRNA in the colon, and local TG expression triggered by this SNP increases the risk of ICRP in MDs via the IL-6 amplifier. Therefore, TG c.4567C>T is a diagnostic target for ICRP in MDs, and TG-mediated IL-6 amplifier activation in the colon is a possible therapeutic target for ICRP.
• GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation.

  Shiina Matsuyama, Reiji Yamamoto, Kaoru Murakami, Nobuhiko Takahashi, Rieko Nishi, Asuka Ishii, Junko Nio-Kobayashi, Nobuya Abe, Kumiko Tanaka, Jing-Jing Jiang, Tadafumi Kawamoto, Toshihiko Iwanaga, Yuta Shinohara, Takeshi Yamasaki, Izuru Ohki, Shintaro Hojyo, Rie Hasebe, Shimpei I Kubota, Noriyuki Hirata, Daisuke Kamimura, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami

  Journal of immunology (Baltimore, Md. : 1950) 211 (1) 34 - 42 2023/07/01 

  We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common β chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.
• Computer model of IL-6-dependent rheumatoid arthritis in F759 mice

  Reiji Yamamoto, Satoshi Yamada, Toru Atsumi, Kaoru Murakami, Ari Hashimoto, Seiichiro Naito, Yuki Tanaka, Izuru Ohki, Yuta Shinohara, Norimasa Iwasaki, Akihiko Yoshimura, Jing-Jing Jiang, Daisuke Kamimura, Shintaro Hojyo, Shimpei I Kubota, Shigeru Hashimoto, Masaaki Murakami

  International Immunology 2023/05/25 

  Abstract The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.
• Calcineurin inhibitor inhibits tolerance induction by suppressing terminal exhaustion of donor T cells after allo-HCT.

  Hajime Senjo, Shinpei Harada, Shimpei I Kubota, Yuki Tanaka, Takahiro Tateno, Zixuan Zhang, Satomi Okada, Xuanzhong Chen, Ryo Kikuchi, Naoki Miyashita, Masahiro Onozawa, Hideki Goto, Tomoyuki Endo, Yuta Hasegawa, Hiroyuki Ohigashi, Takahide Ara, Yoshinori Hasegawa, Masaaki Murakami, Takanori Teshima, Daigo Hashimoto

  Blood 2023/05/22 

  Calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis is standard in allogeneic hematopoietic stem cell transplantation (HCT) but fails to induce long-term tolerance without chronic GVHD in a considerable number of patients. In this study, we addressed this long-standing question in mouse models of HCT. After HCT, alloreactive donor T cells rapidly differentiated into PD-1+ TIGIT+ terminally exhausted T cells (terminal-Tex). GVHD prophylaxis with cyclosporine (CSP) suppressed donor T-cell expression of TOX, a master regulator to promote differentiation of transitory exhausted T cells (transitory-Tex), expressing both inhibitory receptors and effector molecules, into terminal-Tex, and inhibited tolerance induction. Adoptive transfer of transitory-Tex, but not terminal-Tex, into secondary recipients developed chronic GVHD. Transitory-Tex maintained alloreactivity and thus PD-1 blockade restored graft-versus-leukemia (GVL) activity of transitory-Tex, not terminal-Tex. In conclusion, CSP inhibits tolerance induction by suppressing the terminal exhaustion of donor T cells, while maintaining GVL effects to suppress leukemia relapse.
• In situ Microinflammation Detection Using Gold Nanoclusters and a Tissue-clearing Method.

  Fayrouz Naim, Rie Hasebe, Shintaro Hojyo, Yukatsu Shichibu, Asuka Ishii, Yuki Tanaka, Kazuki Tainaka, Shimpei I Kubota, Katsuaki Konishi, Masaaki Murakami

  Bio-protocol 13 (7) e4644  2023/04/05 

  Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4+ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au13 nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.
• Combination therapy with bevacizumab and a CCR2 inhibitor for human ovarian cancer: An in vivo validation study.

  Tianyue Zhai, Takashi Mitamura, Lei Wang, Shimpei I Kubota, Masaaki Murakami, Shinya Tanaka, Hidemichi Watari

  Cancer medicine 12 (8) 9697 - 9708 2023/04 

  BACKGROUND: Anti-angiogenic therapy with bevacizumab (BEV), an anti-VEGF antibody, plays a critical role in the treatment of ovarian cancer. However, despite an encouraging initial response, most tumors become resistant to BEV over time, and a new strategy that enables sustainable treatment using BEV is therefore needed. METHODS: To overcome the resistance to BEV in patients with ovarian cancer, we performed a validation study of combination therapy with BEV (10 mg/kg) and the CCR2 inhibitor BMS CCR2 22 (20 mg/kg) (BEV/CCR2i) using 3 consecutive patient-derived xenografts (PDXs) of immunodeficient mice. RESULTS: BEV/CCR2i demonstrated a significant effect of growth suppression in the BEV-resistant serous PDX and BEV-sensitive serous PDX compared with BEV (30.4% after the second cycle and 15.5% after the first cycle, respectively), and treatment cessation did not attenuate this effect. Tissue clearing and immunohistochemistry with an anti-α-SMA antibody suggested that BEV/CCR2i suppressed angiogenesis from the host mice more than BEV. In addition, human CD31 immunohistochemistry revealed that BEV/CCR2i decreased microvessels originating from the patients to a significantly greater degree than BEV. Regarding the BEV-resistant clear cell PDX, the effect of BEV/CCR2i was unclear during the first five cycles, but the following two cycles of increased-dose BEV/CCR2i (CCR2i 40 mg/kg) significantly suppressed tumor growth compared with BEV (28.3%) by inhibiting the CCR2B-MAPK pathway. CONCLUSIONS: BEV/CCR2i showed a sustained anticancer immunity-independent effect in human ovarian cancer that was more significant in serous carcinoma than in clear cell carcinoma.
• Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients.

  Takeshi Yamasaki, Noriyuki Nagata, Toru Atsumi, Rie Hasebe, Yuki Tanaka, Izuru Ohki, Shimpei Kubota, Yuta Shinohara, Yong Bin Teoh, Nozomu Yokoyama, Noboru Sasaki, Kensuke Nakamura, Hiroshi Ohta, Takehiko Katsurada, Yoshihiro Matsuno, Shintaro Hojyo, Shigeru Hashimoto, Mitsuyoshi Takiguchi, Masaaki Murakami

  International immunology 35 (7) 313 - 326 2023/03/18 

  Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD), and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2, and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G>T and c.478A>T, and found that ICRPs with the T/T SNP risk-alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk-alleles but no differences in serum. Moreover, we show that MMP9, which is a NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were colocalized with epithelial cells expressing MMP9 in normal colons with the risk-alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn's disease also colocalized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.
• Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees

  Takeshi Inoue, Ryo Shinnakasu, Chie Kawai, Hiromi Yamamoto, Shuhei Sakakibara, Chikako Ono, Yumi Itoh, Tommy Terooatea, Kazuo Yamashita, Toru Okamoto, Noritaka Hashii, Akiko Ishii-Watabe, Noah S. Butler, Yoshiharu Matsuura, Hisatake Matsumoto, Shinya Otsuka, Kei Hiraoka, Takanori Teshima, Masaaki Murakami, Tomohiro Kurosaki

  Journal of Experimental Medicine 220 (2) 0022-1007 2023/02/06 

  In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)–specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.
• Dupuytren's contracture-associated SNPs increase SFRP4 expression in nonimmune cells including fibroblasts to enhance inflammation development.

  Hiroaki Kida, Jing-Jing Jiang, Yuichiro Matsui, Ikuko Takahashi, Rie Hasebe, Daisuke Kawamura, Takeshi Endo, Hiroki Shibayama, Makoto Kondoh, Yasuhiko Nishio, Kinya Nishida, Yoshihiro Matsuno, Tsukasa Oikawa, Shimpei Kubota, Shintaro Hojyo, Norimasa Iwasaki, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami

  International immunology 35 (7) 303 - 312 2023/01/31 

  Dupuytren's contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two SNPs associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors, and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and βTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.
• Regulation of NETosis and Inflammation by Cyclophilin D in Myeloperoxidase-Positive Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

  Takashi Kudo, Daigo Nakazawa, Kanako Watanabe-Kusunoki, Masatoshi Kanda, Satoka Shiratori-Aso, Nobuya Abe, Saori Nishio, Jun-Ichiro Koga, Sari Iwasaki, Takahiro Tsuji, Yuichiro Fukasawa, Miwako Yamasaki, Masahiko Watanabe, Sakiko Masuda, Utano Tomaru, Masaaki Murakami, Yasuaki Aratani, Akihiro Ishizu, Tatsuya Atsumi

  Arthritis & rheumatology (Hoboken, N.J.) 75 (1) 71 - 83 2023/01 

  OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.
• SARS-CoV-2 Omicron detection by antigen tests using saliva.

  Kaoru Murakami, Sumio Iwasaki, Satoshi Oguri, Kumiko Tanaka, Rigel Suzuki, Kasumi Hayasaka, Shinichi Fujisawa, Chiaki Watanabe, Satoshi Konno, Isao Yokota, Takasuke Fukuhara, Masaaki Murakami, Takanori Teshima

  Journal of clinical virology plus 2 (4) 100109 - 100109 2022/11 

  The Omicron emerged in November 2021 and became the predominant SARS-CoV-2 variant globally. It spreads more rapidly than ancestral lineages and its rapid detection is critical for the prevention of disease outbreaks. Antigen tests such as immunochromatographic assay (ICA) and chemiluminescent enzyme immunoassay (CLEIA) yield results more quickly than standard polymerase chain reaction (PCR). However, their utility for the detection of the Omicron variant remains unclear. We herein evaluated the performance of ICA and CLEIA in saliva from 51 patients with Omicron and 60 PCR negative individuals. The sensitivity and specificity of CLEIA were 98.0% (95%CI: 89.6-100.0%) and 100.0% (95%CI: 94.0-100.0%), respectively, with fine correlation with cycle threshold (Ct) values. The sensitivity and specificity of ICA were 58.8% (95%CI: 44.2-72.4%) and 100.0% (95%CI: 94.0-100.0%), respectively. The sensitivity of ICA was 100.0% (95%CI: 80.5-100.0%) when PCR Ct was less than 25. The Omicron can be efficiently detected in saliva by CLEIA. ICA also detects high viral load Omicron using saliva.
• Interleukin-17A released from intestinal γδT cells induces cytokine storm in mice with severe dengue

  Takeshi Kurosu, Daisuke Okuzaki, Yusuke Sakai, Mohamad Al Kadi, Supranee Phanthanawiboon, Yasusi Ami, Masayuki Shimojima, Tomoki Yoshikawa, Shuetsu Fukushi, Noriyo Nagata, Tadaki Suzuki, Daisuke Kamimura, Masaaki Murakami, Hideki Ebihara, Masayuki Saijo

  2022/09/16
• 【自己免疫疾患 層別化する新時代へ 臨床検体のマルチオミクス解析、腸内細菌によって見えてきた免疫経路の全容】(第2章)自己免疫疾患の基盤メカニズムの最新知見 ゲートウェイ反射による自己免疫疾患の制御

  村上 薫, 西 李依子, 北條 慎太郎, 田中 勇希, 村上 正晃

  実験医学 (株)羊土社 40 (15) 2456 - 2466 0288-5514 2022/09 

  計測技術、解析技術の進歩に伴い神経免疫学は日進月歩の分野となった。そのなかでもわれわれは自己免疫疾患を含む炎症性疾患の発症について、非免疫細胞においてNF-κBとSTAT3が同時に活性化されることでNF-κB経路の過剰な活性化が惹起され、サイトカイン、ケモカイン、および成長因子などの炎症性メディエーターの産生が相乗的かつ局所的に増強する「IL-6アンプ」と、特定の血管領域においてIL-6アンプを誘導するために必要な特異的な神経回路の活性化機構である「ゲートウェイ反射」を発見した。本稿では、最近我々が発表した、RAに新規の診断・治療標的をもたらす遠隔炎症ゲートウェイ反射を含む各ゲートウェイについて概説する。(著者抄録)
• Pathogenic neuropsychiatric effect of stress-induced microglial interleukin 12/23 axis in systemic lupus erythematosus.

  Nobuya Abe, Masato Tarumi, Yuichiro Fujieda, Nobuhiko Takahashi, Kohei Karino, Mona Uchida, Michihito Kono, Yuki Tanaka, Rie Hasebe, Masaru Kato, Olga Amengual, Yoshiyuki Arinuma, Kenji Oku, Wakiro Sato, Khin Khin Tha, Miwako Yamasaki, Masahiko Watanabe, Tatsuya Atsumi, Masaaki Murakami

  Annals of the rheumatic diseases 81 (11) 1564 - 1575 2022/07/11 

  OBJECTIVES: The central nervous system disorder in systemic lupus erythematosus (SLE), called neuropsychiatric lupus (NPSLE), is one of the most severe phenotypes with various clinical symptoms, including mood disorder, psychosis and delirium as diffuse neuropsychological manifestations (dNPSLE). Although stress is one of the aggravating factors for neuropsychiatric symptoms, its role in the pathogenesis of dNPSLE remains to be elucidated. We aimed to investigate stress effects on the neuropsychiatric pathophysiology in SLE using lupus-prone mice and patients' data. METHODS: Sleep disturbance stress (SDS) for 2 weeks was placed on 6-8-week-old female MRL/lpr and control mice. Behavioural phenotyping, histopathological analyses and gene and protein expression analyses were performed to assess SDS-induced neuroimmunological alterations. We also evaluated cytokines of the cerebrospinal fluid and brain regional volumes in patients with dNPSLE and patients with non-dNPSLE. RESULTS: SDS-subjected MRL/lpr mice exhibited less anxiety-like behaviour, whereas stressed control mice showed increased anxiety. Furthermore, stress strongly activated the medial prefrontal cortex (mPFC) in SDS-subjected MRL/lpr. A transcriptome analysis of the PFC revealed the upregulation of microglial activation-related genes, including Il12b. We confirmed that stress-induced microglial activation and the upregulation of interleukin (IL) 12/23p40 proteins and increased dendritic spines in the mPFC of stressed MRL/lpr mice. IL-12/23p40 neutralisation and tyrosine kinase 2 inhibition mitigated the stress-induced neuropsychiatric phenotypes of MRL/lpr mice. We also found a higher level of cerebrospinal fluid IL-12/23p40 and more atrophy in the mPFC of patients with dNPSLE than those with non-dNPSLE. CONCLUSIONS: The microglial IL-12/23 axis in the mPFC might be associated with the pathogenesis and a promising therapeutic target for dNPSLE.
• 【基盤病態としての慢性炎症】IL-6アンプおよびゲートウェイ反射による関節リウマチ発症メカニズム

  田中 勇希, 長谷部 理絵, 村上 正晃

  医学のあゆみ 医歯薬出版(株) 282 (1) 92 - 99 0039-2359 2022/07 

  関節リウマチ(RA)は関節滑膜増殖および軟骨破壊を伴う慢性炎症性疾患であり、わが国での有病率は0.5〜1%と報告されている。関節滑膜には自己反応性を含むと思われる活性化CD4+T細胞をはじめとした免疫細胞の浸潤が認められるだけでなく、滑膜線維芽細胞(FLS)を含め組織の非免疫細胞からサイトカイン、ケモカイン、増殖因子(GF)などが産生され炎症を誘導し、骨の破壊まで生じる。また、当該疾患ではその症状が両側対称性に現れることも診断基準とされており、神経系が関与していることも示唆されているが、その詳細なメカニズムはいまだ明らかとなっていない。本稿では、これまで筆者らが世界に先がけて報告してきた炎症誘導機構である"IL-6アンプ"および固有神経の活性化によりIL-6アンプが特異血管部で活性化し、血中免疫細胞の組織侵入口を形成するメカニズムである"ゲートウェイ反射"を基盤としたRA発症メカニズムについて議論したい。(著者抄録)
• ATP spreads inflammation to other limbs through crosstalk between sensory neurons and interneurons.

  Rie Hasebe, Kaoru Murakami, Masaya Harada, Nada Halaka, Hiroshi Nakagawa, Fuminori Kawano, Yoshinobu Ohira, Tadafumi Kawamoto, Fiona E Yull, Timothy S Blackwell, Junko Nio-Kobayashi, Toshihiko Iwanaga, Masahiko Watanabe, Nobuhiro Watanabe, Harumi Hotta, Toshihide Yamashita, Daisuke Kamimura, Yuki Tanaka, Masaaki Murakami

  The Journal of experimental medicine 219 (6) 2022/06/06 

  Neural circuits between lesions are one mechanism through which local inflammation spreads to remote positions. Here, we show the inflammatory signal on one side of the joint is spread to the other side via sensory neuron-interneuron crosstalk, with ATP at the core. Surgical ablation or pharmacological inhibition of this neural pathway prevented inflammation development on the other side. Mechanistic analysis showed that ATP serves as both a neurotransmitter and an inflammation enhancer, thus acting as an intermediary between the local inflammation and neural pathway that induces inflammation on the other side. These results suggest blockade of this neural pathway, which is named the remote inflammation gateway reflex, may have therapeutic value for inflammatory diseases, particularly those, such as rheumatoid arthritis, in which inflammation spreads to remote positions.
• 多発性硬化症モデル動物におけるMaresin-1の保護効果

  菅原 季起, 田中 勇希, 北條 慎太郎, 村上 正晃, 南 雅文

  日本薬学会年会要旨集 (公社)日本薬学会 142年会 27F - pm10S 0918-9823 2022/03
• Glycogen synthase kinase 3β/CCR6-positive bone marrow cells correlate with disease activity in multicentric Castleman disease-TAFRO.

  Nobuya Abe, Michihito Kono, Michihiro Kono, Naoki Ohnishi, Tomoya Sato, Masato Tarumi, Masaru Yoshimura, Taiki Sato, Kohei Karino, Yuka Shimizu, Yuichiro Fujieda, Masaru Kato, Rie Hasebe, Kenji Oku, Masaaki Murakami, Tatsuya Atsumi

  British journal of haematology 196 (5) 1194 - 1204 2022/03 

  Multicentric Castleman disease-thrombocytopenia, anasarca, reticulin fibrosis of bone marrow, renal dysfunction and organomegaly (MCD-TAFRO)-is an emergent phenotype characterized by lymphoproliferation, fluid collection, hemocytopenia and multiple organopathy. Although studies have demonstrated an aberrant blood cytokine/chemokine profile referred to as "chemokine storm", the pathogenesis remains unclear. We aimed to identify pathogenic key molecules, potential diagnostic targets and therapeutic markers in MCD-TAFRO using serum cytokine/chemokine profiles. We performed the targeted cytokine/chemokine multiplex analysis in six cases of MCD-TAFRO with remission or non-remission status. We observed significant changes in serum concentrations of CCL2, CCL5, and Chitinase-3-like-1 in the MCD-TAFRO patients with active state compared to inactive state. Ingenuity pathway analysis revealed that glycogen synthase kinase 3 (GSK3) and CCR6, which is expressed in megakaryocytes, were detected as upstream positive regulators for activating MCD-TAFRO status. More GSK3β+ CCR6+ cells like megakaryocytes were detected in the bone marrow of patients with MCD-TAFRO than in those with systemic lupus erythematosus, MCD-not otherwise specified or autoimmune haemophagocytic lymphohistiocytosis. The cellularity of GSK3β+ CCR6+ cells was correlated with disease activity, including thrombocytopenia and anaemia. In conclusion, GSK3β and CCR6 of bone marrow cells were potentially involved in the pathogenesis of MCD-TAFRO and may act as diagnostic targets and therapeutic markers.
• Cytokine and chemokine multiplex analysis-based exploration for potential treatment and prognostic prediction in large-vessel vasculitis: A preliminary observational study.

  Nobuya Abe, Michihiro Kono, Michihito Kono, Takayuki Katsuyama, Kazumasa Ohmura, Taiki Sato, Kohei Karino, Yuichiro Fujieda, Masaru Kato, Rie Hasebe, Masaaki Murakami, Tatsuya Atsumi

  Frontiers in immunology 13 1066916 - 1066916 2022 

  Large-vessel vasculitis (LVV) is subclassified into two phenotypes; Takayasu arteritis and giant cell arteritis. Although the pathogenesis of LVV is not fully established, IL-6-IL-17 axis and IL-12-IFN-γ axis play critical roles in the disease development. We aimed to clarify the association between the disease state and cytokine/chemokine levels, to assess disease course as prognosis and to predict regulators in patients with LVV using the blood profiles of multiple cytokines/chemokines. This retrospective analysis comprised 35 LVV patients whose blood were collected, and multiplex cytokine/chemokine analysis with 28 analytes was performed. The differences of cytokines/chemokines corresponding disease status, upstream regulator analysis, pathway analysis and cluster analysis were conducted using the cytokines/chemokines profile. Relapse-free survival rate was calculated with Kaplan-Meier analysis in the classified clusters. In the robust analysis, IL-4, CCL2/MCP-1, TNFSF13/APRIL, TNFSF13B/BAFF, CHI3L1 and VEGF-A levels were significantly changed after treatment. Untreated LVV patients demonstrated activation of NFκB-related molecules and these patients are potentially treated with JAK/STAT inhibitors, anti-TNF-α inhibitors and IL-6 inhibitors. Cluster analysis in active LVV patients revealed two clusters including one with high blood levels of IL-1β, IL-6, IL-17, IL-23 and CCL20/MIP-3. A subgroup of the LVV patients showed activated IL-17 signature with high relapse frequency, and JAK/TyK2 inhibitors and IFN-γ inhibitors were detected as potentially upstream inhibitors. Blood cytokine/chemokine profiles would be useful for prediction of relapse and potentially contributes to establish therapeutic strategy as precision medicine in LVV patients.
• Cyclophilin D regulates NETosis and inflammation in myeloperoxidase‐antineutrophil cytoplasmic antibody‐associated vasculitis

  Takashi Kudo, Daigo Nakazawa, Kanako Watanabe‐Kusunoki, Masatoshi Kanda, Satoka Shiratori‐Aso, Nobuya Abe, Saori Nishio, Jun‐Ichiro Koga, Sari Iwasaki, Takahiro Tsuji, Yuichiro Fukasawa, Miwako Yamasaki, Masahiko Watanabe, Sakiko Masuda, Utano Tomaru, Masaaki Murakami, Yasuaki Aratani, Akihiro Ishizu, Tatsuya Atsumi

  Arthritis & Rheumatology in press (1) 71 - 83 2326-5191 2022 [Refereed]
   

  OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. METHODS: We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. RESULTS: In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CONCLUSION: CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.
• Depletion of Retinal Dopaminergic Activity in a Mouse Model of Rod Dysfunction Exacerbates Experimental Autoimmune Uveoretinitis: A Role for the Gateway Reflex.

  Andrea Stofkova, Miloslav Zloh, Dominika Andreanska, Ivana Fiserova, Jan Kubovciak, Jan Hejda, Patrik Kutilek, Masaaki Murakami

  International journal of molecular sciences 23 (1) 2021/12/31 

  The gateway reflex is a mechanism by which neural inputs regulate chemokine expression at endothelial cell barriers, thereby establishing gateways for the invasion of autoreactive T cells into barrier-protected tissues. In this study, we hypothesized that rod photoreceptor dysfunction causes remodeling of retinal neural activity, which influences the blood-retinal barrier and the development of retinal inflammation. We evaluated this hypothesis using Gnat1rd17 mice, a model of night blindness with late-onset rod-cone dystrophy, and experimental autoimmune uveoretinitis (EAU). Retinal remodeling and its effect on EAU development were investigated by transcriptome profiling, target identification, and functional validation. We showed that Gnat1rd17 mice primarily underwent alterations in their retinal dopaminergic system, triggering the development of an exacerbated EAU, which was counteracted by dopamine replacement with L-DOPA administered either systemically or locally. Remarkably, dopamine acted on retinal endothelial cells to inhibit NF-κB and STAT3 activity and the expression of downstream target genes such as chemokines involved in T cell recruitment. These results suggest that rod-mediated dopamine release functions in a gateway reflex manner in the homeostatic control of immune cell entry into the retina, and the loss of retinal dopaminergic activity in conditions associated with rod dysfunction increases the susceptibility to autoimmune uveitis.
• 【コロナウイルス感染の免疫学】サイトカインストームとIL-6アンプ

  村上 薫, 北條 慎太郎, 田中 くみ子, 村上 正晃

  炎症と免疫 (株)先端医学社 30 (1) 18 - 27 0918-8371 2021/12 

  昨今、新型コロナウイルス(Severe acute respiratory syndrome coronavirus 2:SARS-CoV-2)が世界中で猛威を振るっている。11月1日現在、累計の患者数は2億5,000万人、死者は500万人となっており、ワクチンが各国の国民に接種されてなお、変異型がつぎつぎ登場するこのウイルスによるパンデミックは終わる様相をみせない。SARS-CoV-2の最も恐ろしい点は、高齢者や基礎疾患のある人に対する重症化からの多臓器不全による死亡だ。これには免疫系の暴走とも評されるサイトカインストームが深くかかわっている。サイトカインストームを引き起こす原因の一つに免疫系細胞の過剰活性化とあわせて非免疫細胞における核内因子κB(nuclear factor-kappa B:NF-κB)の過剰活性化機構であるインターロイキン-6(IL-6)アンプが知られており、サイトカインストーム、ひいてはSARS-CoV-2の病態の理解のためには、まずIL-6アンプの理解も大切だろう。(著者抄録)
• The gateway reflex: breaking through the blood barriers.

  Kaoru Murakami, Yuki Tanaka, Masaaki Murakami

  International immunology 33 (12) 743 - 748 2021/11/25 

  We have been studying inflammatory diseases, with a special focus on IL-6, and discovered two concepts related to inflammation development. One is the gateway reflex, which is induced by the activation of specific neural circuits followed by establishing gateways for autoreactive CD4+ T cells to pass through blood barriers toward the central nervous system (CNS) and retina during tissue-specific inflammatory diseases. We found that the formation of these gateways is dependent on the IL-6 amplifier, which is machinery for enhanced NF-κB activation in endothelial cells at specific sites. We have found five gateway reflexes in total. Here, we introduce the gateway reflex and the IL-6 amplifier.
• 関節リウマチにおける軟骨細胞の新たな役割 IL-6アンプとTMEM147

  太田 光俊, 田中 勇希, 岩崎 倫政, 村上 正晃

  リウマチ科 (有)科学評論社 66 (4) 393 - 403 0915-227X 2021/10
• 【脳とからだ】腸,免疫系,脳の相互作用 ゲートウェイ反射による血液脳関門への免疫細胞ゲート形成制御と病態の誘導

  田中 勇希, 村上 正晃

  生体の科学 (公財)金原一郎記念医学医療振興財団 72 (5) 405 - 408 0370-9531 2021/10 

  ＜文献概要＞これまでに筆者らは,局所神経活性化が固有の血管に免疫細胞の中枢への侵入口を形成するゲートウェイ反射を報告してきた。本稿では,これまでに報告してきたゲートウェイ反射を介した血液脳関門の制御と,組織特異的な病態誘導との関係について論じる。
• Sjögren's syndrome-associated SNPs increase GTF2I expression in salivary gland cells to enhance inflammation development.

  Shuhei Shimoyama, Ikuma Nakagawa, Jing-Jing Jiang, Isao Matsumoto, John A Chiorini, Yoshinori Hasegawa, Osamu Ohara, Rie Hasebe, Mitsutoshi Ota, Mona Uchida, Daisuke Kamimura, Shintaro Hojyo, Yuki Tanaka, Tatsuya Atsumi, Masaaki Murakami

  International immunology 33 (8) 423 - 434 2021/07/23 

  Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammation with lymphoid infiltration and destruction of the salivary glands. Although many genome-wide association studies have revealed disease-associated risk alleles, the functions of the majority of these alleles are unclear. Here, we show previously unrecognized roles of GTF2I molecules by using two SS-associated single nucleotide polymorphisms (SNPs), rs73366469 and rs117026326 (GTF2I SNPs). We found that the risk alleles of GTF2I SNPs increased GTF2I expression and enhanced nuclear factor-kappa B (NF-κB) activation in human salivary gland cells via the NF-κB p65 subunit. Indeed, the knockdown of GTF2I suppressed inflammatory responses in mouse endothelial cells and in vivo. Conversely, the over-expression of GTF2I enhanced NF-κB reporter activity depending on its p65-binding N-terminal leucine zipper domain. GTF2I is highly expressed in the human salivary gland cells of SS patients expressing the risk alleles. Consistently, the risk alleles of GTF2I SNPs were strongly associated with activation of the IL-6 amplifier, which is hyperactivation machinery of the NF-κB pathway, and lymphoid infiltration in the salivary glands of SS patients. These results demonstrated that GTF2I expression in salivary glands is increased in the presence of the risk alleles of GTF2I SNPs, resulting in activation of the NF-κB pathway in salivary gland cells. They also suggest that GTF2I could be a new therapeutic target for SS.
• Gateway reflexes, neuronal circuits that regulate the gateways for autoreactive T cells in organs that have blood barriers.

  Mona Uchida, Reiji Yamamoto, Shiina Matsuyama, Kaoru Murakami, Rie Hasebe, Shintaro Hojyo, Yuki Tanaka, Masaaki Murakami

  International immunology 2021/05/12 

  Gateway reflexes are neural circuits that maintain homeostasis of the immune system. They form gateways for autoreactive T cells to infiltrate the central nervous system in a noradrenaline-dependent manner despite the blood-brain barrier. This mechanism is critical not only for maintaining organ homeostasis but also for inflammatory disease development. Gateway reflexes can be regulated by environmental or artificial stimuli including electrical stimulation, suggesting that the infiltration of immune cells can be controlled by bioelectronic medicine. In this review, we describe the discovery of gateway reflexes and their future directions with special focus on bioelectronic medicine.
• Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4+CD25+Foxp3+ T cells and ameliorates allograft rejection in an antigen-specific manner.

  Shin Emoto, Susumu Shibasaki, Akihisa Nagatsu, Ryoichi Goto, Hitoshi Ono, Yasutomo Fukasaku, Rumi Igarashi, Takuji Ota, Moto Fukai, Tsuyoshi Shimamura, Kan Saiga, Akinobu Taketomi, Masaaki Murakami, Satoru Todo, Kenichiro Yamashita

  Transplant immunology 65 101338 - 101338 2021/04 

  We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST). BALB/c (H-2d) heart graft were transplanted into C57BL/6 (H-2b) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2k). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8+ T cells in the spleen and inhibited infiltration of CD8+ T cells into the graft. Depletion of CD25+ cells inhibited the graft prolonging effect of the NK026680 plus DST treatment. NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.
• 【免疫系の暴走 サイトカインストーム 多様な疾患で生じる全身性の炎症反応その共通機構から病態を理解する】IL-6アンプとCOVID-19におけるサイトカインストーム誘導機構

  内田 萌菜, 田中 くみ子, 北條 慎太郎, 田中 勇希, 長谷部 理絵, 村上 正晃

  実験医学 (株)羊土社 39 (4) 499 - 504 0288-5514 2021/03 

  2021年1月8日、イギリス政府は、IL-6受容体に対する2種類の中和抗体であるトシリズマブとサリルマブが、新型コロナウイルス感染症(COVID-19)の重症患者の治療に有効であると発表した。本発表は重篤化したCOVID-19の病態形成にIL-6シグナルが関与していることを示すものであり、重篤化の本態であるサイトカインストームとの関連性を示唆するものである。COVID-19の重症化には、サイトカインストームの引き金である活性化T細胞に加えて、肺胞上皮細胞や血管内皮細胞などの非免疫細胞におけるIL-6アンプが関連していると考えられる。本稿では、今後さまざまな病態でのサイトカインストームの治療標的として注目されるであろうIL-6アンプについて、その誘導機構を解説する。(著者抄録)
• COVID-19 サイトカインストームとリンパ球機能

  北條 慎太郎, 内田 萌菜, 田中 くみ子, 長谷部 理絵, 村上 正晃

  臨床免疫・アレルギー科 (有)科学評論社 75 (1) 94 - 100 1881-1930 2021/01
• 新型コロナウイルス感染症(COVID-19)におけるサイトカインストーム

  高橋 郁子, 木田 博朗, 田中 くみ子, 北條 慎太郎, 長谷部 理絵, 村上 正晃

  老年内科 (有)科学評論社 3 (1) 74 - 82 2435-1881 2021/01
• Rhodobacter azotoformans LPS (RAP99-LPS) Is a TLR4 Agonist That Inhibits Lung Metastasis and Enhances TLR3-Mediated Chemokine Expression.

  Kaoru Murakami, Daisuke Kamimura, Rie Hasebe, Mona Uchida, Nobuya Abe, Reiji Yamamoto, Jing-Jing Jiang, Yasuhiro Hidaka, Yuko Nakanishi, Shuzo Fujita, Yuki Toda, Nobuhiro Toda, Hiroki Tanaka, Shizuo Akira, Yuki Tanaka, Masaaki Murakami

  Frontiers in immunology 12 675909 - 675909 2021 

  The lipopolysaccharides (LPSs) of Rhodobacter are reported to be TLR4 antagonists. Accordingly, the extract of Rhodobacter azotoformans (RAP99) is used as a health supplement for humans and animals in Japan to regulate immune responses in vivo. We previously analyzed the LPS structure of RAP99 (RAP99-LPS) and found it is different from that of E. coli-LPS but similar to lipid A from Rhodobacter sphaeroides (RSLA), a known antagonist of TLR4, with both having three C14 fatty acyl groups, two C10 fatty acyl groups, and two phosphates. Here we show that RAP99-LPS has an immune stimulatory activity and acts as a TLR4 agonist. Pretreatment of RAP99-LPS suppressed E. coli-LPS-mediated weight loss, suggesting it is an antagonist against E. coli-LPS like other LPS isolated from Rhodobacter. However, injections of RAP99-LPS caused splenomegaly and increased immune cell numbers in C57BL/6 mice but not in C3H/HeJ mice, suggesting that RAP99-LPS stimulates immune cells via TLR4. Consistently, RAP99-LPS suppressed the lung metastasis of B16F1 tumor cells and enhanced the expression of TLR3-mediated chemokines. These results suggest that RAP99-LPS is a TLR4 agonist that enhances the activation status of the immune system to promote anti-viral and anti-tumor activity in vivo.
• 腎移植後慢性拒絶反応診断のためのLiquid Biopsy 炎症回路に関わる新規尿中バイオマーカーの研究

  高田 祐輔, 上村 大輔, 樋口 はるか, 岩見 大基, 堀田 記世彦, 岩原 直也, 篠原 信雄, 村上 正晃

  日本泌尿器科学会総会 (一社)日本泌尿器科学会総会事務局 108回 872 - 872 2020/12
• 【新型コロナウイルス感染症(COVID-19)-私たちが今知っておくべきこと】新型コロナウイルス感染症(COVID-19)重症化のメカニズム

  長谷部 理絵, 北條 慎太郎, 田中 勇希, 内田 萌菜, 村上 正晃

  カレントテラピー (株)ライフメディコム 38 (12) 1145 - 1150 0287-8445 2020/12 

  新型コロナウイルス感染症(COVID-19)では、患者の多くは無症候または軽度の症状を示した後に回復するが、一部の患者は急性呼吸窮迫症候群(acute respiratory distress syndrome:ARDS)、多臓器不全などの重度の症状を示し、死亡する。重症化患者では血中サイトカインレベルが上昇しており、特に血中IL-6レベルは死亡率と相関があることから、致死性のCOVID-19は、IL-6を基軸とするサイトカインストームが引き起こすcytokine releasing syndromeであると考えられる。新型コロナウイルス感染を起点とするサイトカインストームの誘導には、肺胞上皮細胞や血管内皮細胞などの非免疫細胞でSTAT3/NF-κBシグナルの同時活性化により、NF-κBの過剰活性化が起こるIL-6アンプが関連しているものと考えられ、IL-6シグナリングの阻害は、重症COVID-19の治療標的の有力な候補のひとつとして挙げられる。(著者抄録)
• Bidirectional communication between neural and immune systems.

  Daisuke Kamimura, Yuki Tanaka, Rie Hasebe, Masaaki Murakami

  International immunology 32 (11) 693 - 701 2020/10/20 

  The immune and nervous systems share many features, including receptor and ligand expression, enabling efficient communication between the two. Accumulating evidence suggests that the communication is bidirectional, with the neural system regulating immune cell functions and vice versa. Steroid hormones from the hypothalamus-pituitary-adrenal gland axis are examples of systemic regulators for this communication. Neural reflexes describe regional regulation mechanisms that are a historically new concept that helps to explain how the neural and body systems including immune system communicate. Several recently identified neural reflexes, including the inflammatory reflex and gateway reflex, significantly impact the activation status of the immune system and are associated with inflammatory diseases and disorders. Either pro-inflammatory or anti-inflammatory effects can be elicited by these neural reflexes. On the other hand, the activities of immune cells during inflammation, for example the secretion of inflammatory mediators, can affect the functions of neuronal systems via neural reflexes and modulate biological outputs via specific neural pathways. In this review article, we discuss recent advances in the understanding of bidirectional neuro-immune interactions, with a particular focus on neural reflexes.
• 腎移植後慢性拒絶反応のためのLiquid Biopsy 尿中エクソソームSYT17(Synaptotagmin 17)の有用性

  高田 祐輔, 上村 大輔, 樋口 はるか, 岩見 大基, 堀田 記世彦, 岩原 直也, 篠原 信雄, 村上 正晃

  移植 (一社)日本移植学会 55 (総会臨時) 339 - 339 0578-7947 2020/10
• Increased urinary exosomal SYT17 levels in chronic active antibody-mediated rejection after kidney transplantation via the IL-6 amplifier.

  Yusuke Takada, Daisuke Kamimura, Jing-Jing Jiang, Haruka Higuchi, Daiki Iwami, Kiyohiko Hotta, Yuki Tanaka, Mitsutoshi Ota, Madoka Higuchi, Saori Nishio, Tatsuya Atsumi, Nobuo Shinohara, Yoshihiro Matsuno, Takahiro Tsuji, Tatsu Tanabe, Hajime Sasaki, Naoya Iwahara, Masaaki Murakami

  International immunology 32 (10) 653 - 662 2020/09/30 

  Chronic active antibody-mediated rejection (CAAMR) is a particular problem in kidney transplantation (KTx), and ~25% of grafts are lost by CAAMR. Further, the pathogenesis remains unclear, and there is no effective cure or marker. We previously found that a hyper NFκB-activating mechanism in non-immune cells, called the IL-6 amplifier, is induced by the co-activation of NFκB and STAT3, and that this activation can develop various chronic inflammatory diseases. Here, we show that synaptotagmin-17 (SYT17) is increased in an exosomal fraction of the urine from CAAMR patients, and that this increase is associated with activation of the IL-6 amplifier. Immunohistochemistry showed that SYT17 protein expression was increased in renal tubule cells of the CAAMR group. While SYT17 protein was not detectable in whole-urine samples by western blotting, urinary exosomal SYT17 levels were significantly elevated in the CAAMR group compared to three other histology groups (normal, interstitial fibrosis and tubular atrophy, and calcineurin inhibitors toxicity) after KTx. On the other hand, current clinical laboratory data could not differentiate the CAAMR group from these groups. These data suggest that urinary exosomal SYT17 is a potential diagnostic marker for CAAMR.
• ケロイド組織で炎症が遷延するメカニズム 炎症性サイトカインIL-6とCD4陽性T細胞のケロイド線維芽細胞に対する作用

  村尾 尚規, 藤田 宗純, 林 利彦, 清野 研一郎, 村上 正晃, 山本 有平

  瘢痕・ケロイド治療ジャーナル (株)全日本病院出版会 (14) 4 - 6 2185-7776 2020/09
• 【線維化 慢性疾患のキープロセス 多彩な間質細胞が織りなす組織リモデリング"fibrosis"の理解】(第2章)線維化を制御する細胞間ネットワーク 炎症の誘導機構 IL-6アンプの病気への関与

  北條 慎太郎, 内田 萌菜, 村上 薫, 松山 詩菜, 田中 くみ子, 村上 正晃

  実験医学 (株)羊土社 38 (12) 2032 - 2039 0288-5514 2020/08 

  線維化には炎症の誘導が伴う。われわれはIL-6サイトカインと関節リウマチモデルの研究を通して炎症誘導の基盤的機構であるIL-6アンプを発見し、その分子機序について研究を続けている。その過程で、特異的な血管部のIL-6アンプが血液脳関門に免疫細胞の侵入口を形成することを発見し、ゲートウェイ反射と名付けた。本稿では、これら一連の研究を紹介する。(著者抄録)
• Corrigendum: Orosomucoid 1 is involved in the development of chronic allograft rejection after kidney transplantation.

  Haruka Higuchi, Daisuke Kamimura, Jing-Jing Jiang, Toru Atsumi, Daiki Iwami, Kiyohiko Hotta, Hiroshi Harada, Yusuke Takada, Hiromi Kanno-Okada, Kanako C Hatanaka, Yuki Tanaka, Nobuo Shinohara, Masaaki Murakami

  International immunology 32 (7) 493 - 493 2020/06/26
• 【あたらしい臓器連関】ゲートウェイ反射によるあたらしい臓器機能連関

  田中 勇希, 田中 くみ子, 村上 正晃

  細胞 (株)ニュー・サイエンス社 52 (7) 352 - 356 1346-7557 2020/06 

  中枢神経系(CNS)には特殊な血管構造である血液脳関門が存在し、免疫細胞や高分子の侵入を制限してCNSの恒常性を維持する。しかし、免疫細胞は、CNSにも少数であるが存在して発癌、感染を防いでいる。これら免疫細胞が特異的な侵入口(ゲート)を介してCNSに侵入するのか否かは最近まで明らかではなかった。筆者らは、多発性硬化症のマウスモデルを用いて、血液脳関門に特異的ゲートがあることを証明し、特異的な神経回路によるその形成機構を「ゲートウェイ反射」として発表した。また、ゲートウェイ反射の分子基盤として血管内皮細胞におけるケモカイン大量発現機構「IL-6アンプ」を明らかにした。本稿では、主にゲートウェイ反射の観点から臓器機能連関に関して議論する。(著者抄録)
• Role of Chondrocytes in the Development of Rheumatoid Arthritis Via Transmembrane Protein 147-Mediated NF-κB Activation.

  Mitsutoshi Ota, Yuki Tanaka, Ikuma Nakagawa, Jing-Jing Jiang, Yasunobu Arima, Daisuke Kamimura, Tomohiro Onodera, Norimasa Iwasaki, Masaaki Murakami

  Arthritis & rheumatology (Hoboken, N.J.) 72 (6) 931 - 942 2020/06 

  OBJECTIVE: We have previously reported that the coactivation of NF-κB and STAT3 in nonimmune cells, including synovial fibroblasts, enhances the expression of NF-κB target genes and plays a role in chronic inflammation and rheumatoid arthritis (RA). This study was undertaken to examine the role of NF-κB activation in chondrocytes and better understand the pathogenesis of RA. Furthermore, transmembrane protein 147 (TMEM147) was investigated as a representative NF-κB activator in chondrocytes. METHODS: Clinical samples from RA patients were analyzed by immunohistochemistry. Specimens obtained from patients with polydactyly were used as control samples. The functional contribution of chondrocytes and TMEM147 to arthritis was examined in several murine models of RA. In vitro experiments (quantitative polymerase chain reaction, RNA interference, immunoprecipitation, and confocal microscopy) were performed to investigate the mechanism of action of TMEM147 in chondrocytes. RESULTS: Samples obtained from RA patients and mouse models of RA showed coactivation of NF-κB and STAT3 in chondrocytes (P < 0.001). This coactivation induced a synergistic expression of NF-κB targets in vitro (P < 0.01). Chondrocyte-specific deletion of STAT3 significantly suppressed the development of cytokine-induced RA (P < 0.01). TMEM147 was highly expressed in chondrocytes from RA patient samples and the mouse models of RA. Gene silencing of TMEM147 or anti-TMEM147 antibody treatment inhibited the cytokine-mediated activation of NF-κB in vitro (P < 0.01) and suppressed cytokine-induced RA in vivo (P < 0.01). Mechanistically, TMEM147 molecules acted as scaffold proteins for the NF-κB complex, which included breakpoint cluster region and casein kinase 2, and enhanced NF-κB activity. CONCLUSION: These results suggest that chondrocytes play a role in the development of RA via TMEM147-mediated NF-κB activation and indicate a novel therapeutic strategy for RA.
• COVID-19: A New Virus, but a Familiar Receptor and Cytokine Release Syndrome.

  Toshio Hirano, Masaaki Murakami

  Immunity 52 (5) 731 - 733 2020/05/19 

  Zhou et al. (Nature) and Hoffmann et al. (Cell) identify ACE2 as a SARS-CoV-2 receptor, and the latter show its entry mechanism depends on cellular serine protease TMPRSS2. These results may explain proinflammatory cytokine release via the associated angiotestin II pathway and a possible therapeutic target via the IL-6-STAT3 axis.
• How COVID-19 induces cytokine storm with high mortality.

  Shintaro Hojyo, Mona Uchida, Kumiko Tanaka, Rie Hasebe, Yuki Tanaka, Masaaki Murakami, Toshio Hirano

  Inflammation and regeneration 40 37 - 37 2020 

  The newly emerging coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, but has rapidly spread all over the world. Some COVID-19 patients encounter a severe symptom of acute respiratory distress syndrome (ARDS) with high mortality. This high severity is dependent on a cytokine storm, most likely induced by the interleukin-6 (IL-6) amplifier, which is hyper-activation machinery that regulates the nuclear factor kappa B (NF-κB) pathway and stimulated by the simultaneous activation of IL-6-signal transducer and activator of transcription 3 (STAT3) and NF-κB signaling in non-immune cells including alveolar epithelial cells and endothelial cells. We hypothesize that IL-6-STAT3 signaling is a promising therapeutic target for the cytokine storm in COVID-19, because IL-6 is a major STAT3 stimulator, particularly during inflammation. We herein review the pathogenic mechanism and potential therapeutic targets of ARDS in COVID-19 patients.
• Gateway reflex: Local neuroimmune interactions that regulate blood vessels.

  Daisuke Kamimura, Takuto Ohki, Yasunobu Arima, Mitsutoshi Ota, Masaaki Murakami

  Neurochemistry international 130 104303 - 104303 0197-0186 2019/11 [Refereed][Not invited]
   

  Neuroimmunology is a research field that intersects neuroscience and immunology, with the larger aim of gaining significant insights into the pathophysiology of chronic inflammatory diseases such as multiple sclerosis. Conventional studies in this field have so far mainly dealt with immune responses in the nervous system (i.e. neuroinflammation) or systemic immune regulation by the release of glucocorticoids. On the other hand, recently accumulating evidence has indicated bidirectional interactions between specific neural activations and local immune responses. Here we discuss one such local neuroimmune interaction, the gateway reflex. The gateway reflex represents a mechanism that translates specific neural stimulations into local inflammatory outcomes by changing the state of specific blood vessels to allow immune cells to extravasate, thus forming the gateway. Several types of gateway reflex have been identified, and each regulates distinct blood vessels to create gateways for immune cells that induce local inflammation. The gateway reflex represents a novel therapeutic strategy for neuroinflammation and is potentially applicable to other inflammatory diseases in peripheral organs.
• 尿中エキソソーム性KIAMタンパク質は腎移植後の活性型の抗体を介した慢性拒絶反応に関する新規の非侵襲的診断バイオマーカーである(Urinary exosomal KIAM protein is a novel non-invasive diagnostic biomarker for chronic active antibody-mediated rejection after kidney transplantation)

  高田 祐輔, 上村 大輔, 樋口 はるか, 岩見 大基, 堀田 記世彦, 篠原 信雄, 村上 正晃

  西日本泌尿器科 81 (増刊) 134 - 134 0029-0726 2019/10
• 膵癌ドライバー変異はmRNA翻訳と蛋白質プレニル化を介しARF6が駆動する癌免疫回避を促進する(Pancreatic KRAS/TP53 mutations promote ARF6-based immune evasion via activating mRNA translation and protein prenylation)

  橋本 あり, 橋本 茂, 古川 聖太郎, 蔦保 暁生, 小野寺 康人, 半田 悠, 及川 司, 水上 裕輔, 西川 義浩, 児玉 裕三, 村上 正晃, 平野 聡, 佐邊 壽孝

  日本癌学会総会記事 78回 P - 3033 0546-0476 2019/09
• ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer.

  Shigeru Hashimoto, Shotaro Furukawa, Ari Hashimoto, Akio Tsutaho, Akira Fukao, Yurika Sakamura, Gyanu Parajuli, Yasuhito Onodera, Yutaro Otsuka, Haruka Handa, Tsukasa Oikawa, Soichiro Hata, Yoshihiro Nishikawa, Yusuke Mizukami, Yuzo Kodama, Masaaki Murakami, Toshinobu Fujiwara, Satoshi Hirano, Hisataka Sabe

  Proceedings of the National Academy of Sciences of the United States of America 116 (35) 17450 - 17459 0027-8424 2019/08/27 [Refereed][Not invited]
   

  Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.
• Mast cells play role in wound healing through the ZnT2/GPR39/IL-6 axis.

  Keigo Nishida, Aiko Hasegawa, Satoru Yamasaki, Ryota Uchida, Wakana Ohashi, Yosuke Kurashima, Jun Kunisawa, Shunsuke Kimura, Toshihiko Iwanaga, Hiroshi Watarai, Koji Hase, Hideki Ogura, Manabu Nakayama, Jun-Ichi Kashiwakura, Yoshimichi Okayama, Masato Kubo, Osamu Ohara, Hiroshi Kiyono, Haruhiko Koseki, Masaaki Murakami, Toshio Hirano

  Scientific reports 9 (1) 10842 - 10842 2019/07/25 [Refereed][Not invited]
   

  Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.
• 【"適応&修復"のサイエンスと臨床応用の最前線】ゲートウェイ反射による血管・臓器調節機構

  田中 勇希, 村上 正晃

  別冊Bio Clinica: 慢性炎症と疾患 (株)北隆館 8 (1) 33 - 37 2019/07 

  末梢と中枢神経系(CNS)を隔てる血管には血液脳関門が存在し、高分子や免疫細胞の侵入を防ぐことでCNSの恒常性を維持している。これまでに我々は神経活性化が免疫細胞のCNSへの侵入を可能にする「ゲートウェイ反射」を報告してきた。当研究から、神経-免疫の相互作用により生体内の血管や臓器の機能が制御されることが明らかとなった。また痛みによる炎症の再発誘導機構が存在することもわかった。本総説では、ゲートウェイ反射がどのように血管や末梢臓器の組織適応を破綻させ疾患を制御するのか議論する。(著者抄録)
• Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response.

  Hiroki Tanaka, Yasunobu Arima, Daisuke Kamimura, Yuki Tanaka, Noriyuki Takahashi, Takuya Uehata, Kazuhiko Maeda, Takashi Satoh, Masaaki Murakami, Shizuo Akira

  The Journal of experimental medicine 216 (6) 1431 - 1449 2019/06/03 [Refereed][Not invited]
   

  Regnase-1 (also known as Zc3h12a or MCPIP-1) is an endoribonuclease involved in mRNA degradation of inflammation-associated genes. Regnase-1 is inactivated in response to external stimuli through post-translational modifications including phosphorylation, yet the precise role of phosphorylation remains unknown. Here, we demonstrate that interleukin (IL)-17 induces phosphorylation of Regnase-1 in an Act1-TBK1/IKKi-dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released from the endoplasmic reticulum (ER) into the cytosol, thereby losing its mRNA degradation function, which leads to expression of IL-17 target genes. By using CRISPR/Cas-9 technology, we generated Regnase-1 mutant mice, in which IL-17-induced Regnase-1 phosphorylation is completely blocked. Mutant mice (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD ) were resistant to the IL-17-mediated inflammation caused by T helper 17 (Th17) cells in vivo. Thus, Regnase-1 plays a critical role in the development of IL-17-mediated inflammatory diseases via the Act1-TBK1-IKKi axis, and blockade of Regnase-1 phosphorylation sites may be promising for treatment of Th17-associated diseases.
• Pleiotropy and Specificity: Insights from the Interleukin 6 Family of Cytokines.

  Masaaki Murakami, Daisuke Kamimura, Toshio Hirano

  Immunity 50 (4) 812 - 831 2019/04/16 

  Since the molecular cloning of interleukin-6 (IL-6) in 1986, many other cytokines have been found to share the same signal transducer, gp130, in their receptor complexes. Thus, the IL-6 family of cytokines now consists of ten members. Although some of the family members' functions are redundant as a result of the expression of gp130, there are also functional distinctions between members. The mechanisms that determine functional redundancies and distinctions are not completely understood. Yet, research has clarified the role of IL-6 family cytokines in autoimmune diseases and has led to effective therapies that target them. Here, we review the IL-6 family of cytokines in autoimmune diseases, with a particular focus on the prototypical member IL-6, from the viewpoints of their structure, signaling, and biological features and discuss possible mechanisms of their functional pleiotropy.
• Photopic light-mediated down-regulation of local α1A-adrenergic signaling protects blood-retina barrier in experimental autoimmune uveoretinitis.

  Andrea Stofkova, Daisuke Kamimura, Takuto Ohki, Mitsutoshi Ota, Yasunobu Arima, Masaaki Murakami

  Scientific reports 9 (1) 2353 - 2353 2019/02/20 

  We have reported the gateway reflex, which describes specific neural activations that regulate immune cell gateways at specific blood vessels in the central nervous system (CNS). Four types of gateway reflexes exist, all of which induce alterations in endothelial cells at specific vessels of the blood-brain barrier followed by inflammation in the CNS in the presence of CNS-autoreactive T cells. Here we report a new gateway reflex that suppresses the development of retinal inflammation by using an autoreactive T cell-mediated ocular inflammation model. Exposure to photopic light down-regulated the adrenoceptor pathway to attenuate ocular inflammation by suppressing breaching of the blood-retina barrier. Mechanistic analysis showed that exposure to photopic light down-regulates the expression of α1A-adrenoceptor (α1AAR) due to high levels of norepinephrine and epinephrine, subsequently suppressing inflammation. Surgical ablation of the superior cervical ganglion (SCG) did not negate the protective effect of photopic light, suggesting the involvement of retinal noradrenergic neurons rather than sympathetic neurons from the SCG. Blockade of α1AAR signaling under mesopic light recapitulated the protective effect of photopic light. Thus, targeting regional adrenoceptor signaling might represent a novel therapeutic strategy for autoimmune diseases including those that affect organs separated by barriers such as the CNS and eyes.
• NEDD4 Is Involved in Inflammation Development during Keloid Formation.

  Munezumi Fujita, Yuhei Yamamoto, Jing-Jing Jiang, Toru Atsumi, Yuki Tanaka, Takuto Ohki, Naoki Murao, Emi Funayama, Toshihiko Hayashi, Masayuki Osawa, Taku Maeda, Daisuke Kamimura, Masaaki Murakami

  The Journal of investigative dermatology 139 (2) 333 - 341 0022-202X 2019/02 [Refereed][Not invited]
   

  Keloids mark a chronic inflammatory disease characterized by a fibroproliferative disorder of the skin. A genome-wide association study showed that single-nucleotide polymorphism rs8032158 in the neural precursor cell-expressed NEDD4 gene, which has six protein-coding transcript variants (TVs), is genetically linked to keloids. Here, we show that the high frequency of risk allele C in rs8032158 in keloid patients is associated with a selectively higher expression of TV3 of NEDD4 to activate the NF-κB pathway. Comparisons of keloid scars with normal skin samples that do not have the single-nucleotide polymorphism allele and were derived from different anatomical sites showed stronger expressions of NEDD4 TV3 and activated forms of NF-κB and STAT3 in keloid scars. Forced expression or selective knockdown of NEDD4 TV3 increased or decreased NF-κB activation in vitro. Furthermore, NEDD4 knockdown suppressed NF-κB-dependent inflammation development in vivo. Mechanistic analysis showed that NEDD4 TV3 is involved in NF-κB activation through its association with the adaptor protein RIP. These results suggest that NEDD4 TV3 is a potential diagnostic marker and therapeutic target for chronic skin diseases, including keloid.
• Neural activity regulates autoimmune diseases through the gateway reflex.

  Andrea Stofkova, Masaaki Murakami

  Bioelectronic medicine 5 14 - 14 2019 

  The brain, spinal cord and retina are protected from blood-borne compounds by the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB) and blood-retina barrier (BRB) respectively, which create a physical interface that tightly controls molecular and cellular transport. The mechanical and functional integrity of these unique structures between blood vessels and nervous tissues is critical for maintaining organ homeostasis. To preserve the stability of these barriers, interplay between constituent barrier cells, such as vascular endothelial cells, pericytes, glial cells and neurons, is required. When any of these cells are defective, the barrier can fail, allowing blood-borne compounds to encroach neural tissues and cause neuropathologies. Autoimmune diseases of the central nervous system (CNS) and retina are characterized by barrier disruption and the infiltration of activated immune cells. Here we review our recent findings on the role of neural activity in the regulation of these barriers at the vascular endothelial cell level in the promotion of or protection against the development of autoimmune diseases. We suggest nervous system reflexes, which we named gateway reflexes, are fundamentally involved in these diseases. Although their reflex arcs are not completely understood, we identified the activation of specific sensory neurons or receptor cells to which barrier endothelial cells respond as effectors that regulate gateways for immune cells to enter the nervous tissue. We explain this novel mechanism and describe its role in neuroinflammatory conditions, including models of multiple sclerosis and posterior autoimmune uveitis.
• Short-Term Administration of Single-Agent Toceranib in Six Cases of Inoperable Massive Canine Hepatocellular Carcinoma.

  Heishima K, Iwasaki R, Kawabe M, Murakami M, Sakai H, Maruo K, Mori T

  Journal of the American Animal Hospital Association 0587-2871 2018/11 [Refereed][Not invited]
   

  Six dogs with massive hepatocellular carcinoma that was not amenable to surgery were treated by oral administration of single-agent toceranib at a dose of 2.0-3.0 mg/kg every other day for a minimum of 60 days. Partial response was achieved in three dogs, stable disease was achieved in one dog, and progressive disease occurred in two dogs, according to the canine Response Evaluation Criteria in Solid Tumors v1.0. Observed adverse events were mild to moderate in severity and reported in accordance with the Veterinary Cooperative Oncology Group's common terminology criteria for adverse events v1.1. Activities of alanine aminotransferase and alkaline phosphatase decreased in the cases that were sensitive to treatment with toceranib, whereas the activities remained high in resistant cases. Additionally, the level of phospho-vascular endothelial growth factor receptor 2 was found to be increased in a resistant case. Single-agent toceranib might prove to be an effective treatment for canine hepatocellular carcinoma pending further validation.
• Presenilin 1 Regulates NF-κB Activation via Association with Breakpoint Cluster Region and Casein Kinase II.

  Tanaka Y, Sabharwal L, Ota M, Nakagawa I, Jiang JJ, Arima Y, Ogura H, Okochi M, Ishii M, Kamimura D, Murakami M

  Journal of immunology (Baltimore, Md. : 1950) 201 (8) 2256 - 2263 0022-1767 2018/10 [Refereed][Not invited]
   

  We recently reported that NF-κB-mediated inflammation caused by breakpoint cluster region (BCR) is dependent on the α subunit of casein kinase II (CK2α) complex. In the current study, we demonstrate that presenilin 1 (Psen1), which is a catalytic component of the γ-secretase complex and the mutations of which are known to cause familial Alzheimer disease, acts as a scaffold of the BCR-CK2α-p65 complex to induce NF-κB activation. Indeed, Psen1 deficiency in mouse endothelial cells showed a significant reduction of NF-κB p65 recruitment to target gene promoters. Conversely, Psen1 overexpression enhanced reporter activation under NF-κB responsive elements and IL-6 promoter. Furthermore, the transcription of NF-κB target genes was not inhibited by a γ-secretase inhibitor, suggesting that Psen1 regulates NF-κB activation in a manner independent of γ-secretase activity. Mechanistically, Psen1 associated with the BCR-CK2α complex, which is required for phosphorylation of p65 at serine 529. Consistently, TNF-α-induced phosphorylation of p65 at serine 529 was significantly decreased in Psen1-deficient cells. The association of the BCR-CK2α-p65 complex was perturbed in the absence of Psen1. These results suggest that Psen1 functions as a scaffold of the BCR-CK2α-p65 complex and that this signaling cascade could be a novel therapeutic target for various chronic inflammation conditions, including those in Alzheimer disease.
• Bmi1 Regulates IκBα Degradation via Association with the SCF Complex.

  Okuyama Y, Tanaka Y, Jiang JJ, Kamimura D, Nakamura A, Ota M, Ohki T, Higo D, Ogura H, Ishii N, Atsumi T, Murakami M

  Journal of immunology (Baltimore, Md. : 1950) 201 (8) 2264 - 2272 0022-1767 2018/10 [Refereed][Not invited]
   

  Bmi1 is a polycomb group protein and regulator that stabilizes the ubiquitination complex PRC1 in the nucleus with no evidently direct link to the NF-κB pathway. In this study, we report a novel function of Bmi1: its regulation of IκBα ubiquitination in the cytoplasm. A deficiency of Bmi1 inhibited NF-κB-mediated gene expression in vitro and a NF-κB-mediated mouse model of arthritis in vivo. Mechanistic analysis showed that Bmi1 associated with the SCF ubiquitination complex via its N terminus and with phosphorylation by an IKKα/β-dependent pathway, leading to the ubiquitination of IκBα. These effects on NF-κB-related inflammation suggest Bmi1 in the SCF complex is a potential therapeutic target for various diseases and disorders, including autoimmune diseases.
• 神経系のバリアー機能と病態 ゲートウェイ反射を起点とした神経-免疫のクロストーク

  田中 勇希, 村上 正晃

  神経免疫学 (一社)日本神経免疫学会 23 (1) 50 - 50 0918-936X 2018/09
• 腎移植の慢性抗体関連型拒絶反応における早期診断バイオマーカー、治療ターゲットOrosomucoid1(ORM1)の可能性

  樋口 はるか, 堀田 記世彦, 岩見 大基, 村上 正晃, 篠原 信雄

  移植 (一社)日本移植学会 53 (総会臨時) 483 - 483 0578-7947 2018/09
• Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines

  Sakai K, Maeda S, Saeki K, Nakagawa T, Murakami M, Endo Y, Yonezawa T, Kadosawa T, Mori T, Nishimura R, Matsuki N

  Vet Comp Oncol. 16 (4) 642 - 649 2018/09 [Refereed][Not invited]
  
• Cell- and stage-specific localization of galectin-3, a β-galactoside-binding lectin, in a mouse model of experimental autoimmune encephalomyelitis

  Tetsuya Itabashi, Yasunobu Arima, Daisuke Kamimura, Kotaro Higuchi, Yoshio Bando, Hiromi Takahashi-Iwanaga, Masaaki Murakami, Masahiko Watanabe, Toshihiko Iwanaga, Junko Nio-Kobayashi

  Neurochemistry International 118 176 - 184 1872-9754 2018/09/01 [Refereed][Not invited]
   

  Multiple sclerosis (MS) is an autoimmune disease in which pathogenic T cells play an important role, and an experimental autoimmune encephalomyelitis (EAE) is used as an animal model of MS. Galectins are β-galactoside-binding lectins and involved in various physiological and pathological events. Among fifteen members of galectins, galectin-1, -8, and -9 play immunosuppressive roles in MS and EAE however, the role of galectin-3 (gal-3) is complex and controversial. We examined expression of gal-3 in the spinal cord and nerve roots of EAE mice. No immunohistochemical signals were detected in naïve mice, whereas gal-3 appeared at lower lumbar levels of the spinal cord and nerve roots in EAE mice. In the spinal cord, gal-3-positive cells were activated microglia and/or infiltrating macrophages, which were round in shape and intensified for the lysosomal enzyme, cathepsin D, indicating elevated phagocytic activity. Gal-3-positive cells in the spinal cord were most abundant during the peak symptomatic period. In the recovery period, they disappeared from the spinal parenchyma but remained at moderate levels in the pia mater. Interestingly, gal-3-positive cells selectively appeared in ventral, but not dorsal, nerve roots running through the spinal canal, with expression peaking during the recovery period. In ventral nerve roots, the major cell type expressing gal-3 was a specific population of Schwann cells that surround unmyelinated axons and express the biosynthetic enzyme for L-serine, a potent neurotrophic amino acid. Gal-3 was also induced in Iba1/F4/80-positive macrophages, which engulf damaged myelin and axon debris. Thus, gal-3 is induced in distinct cell types that are engaged in removal of damaged axons and cell debris and axon regeneration and remyelination, suggesting a potential neuroprotective role of gal-3 in EAE mice.
• Chemical Landscape for Tissue Clearing based on Hydrophilic Reagents

  Kazuki Tainaka, Tatsuya C. Murakam, Etsuo A. Susaki, Chika Shimizu, Rie Saito, Kei Takahashi, Akiko Hayashi-Takagi, Hiroshi Sekiya, Yasunobu Arima, Satoshi Nojima, Masako Ikemura, Tetsuo Ushiku, Yoshihiro Shimizu, Masaaki Murakami, Kenji F. Tanaka, Masamitsu Iino, Haruo Kasai, Toshikuni Sasaoka, Kazuto Kobayashi, Kohei Miyazono, Eiichi Morii, Tadashi Isa, Masashi Fukayama, Akiyoshi Kakita, Hiroki R. Ueda

  Cell Reports Cell Press 24 (8) 2196 - 2210 2018/08/24 [Refereed][Not invited]
   

  We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.
• Differences in activated clotting time and initial heparin dosage during atrial fibrillation ablation for patients with edoxaban compared with warfarin

  Hirosuke Yamaji, Takashi Murakami, Kazuyoshi Hina, Shunichi Higashiya, Hiroshi Kawamura, Masaaki Murakami, Shigeshi Kamikawa, Satoshi Hirohata, Shozo Kusachi

  Journal of Cardiovascular Electrophysiology 29 (6) 835 - 843 1540-8167 2018/06/01 [Refereed][Not invited]
   

  Background: Different target activated clotting times (ACTs) during atrial fibrillation (AF) ablation have been proposed. Moreover, relationships between initial bolus dose of heparin at the start of AF ablation in patients receiving edoxaban anticoagulation therapy and ACT are unclear. Methods: Patients who received anticoagulation with uninterrupted warfarin (control n = 120) or interrupted edoxaban (n = 120) on the morning of day of ablation were studied. An initial dose of 100 U/kg heparin was administered as a reliable control for warfarin. Initial heparin doses of 120, 130, 140, or 150 U/kg were randomly administered to the edoxaban group. Results: Edoxaban group showed shorter baseline ACT before the procedure (130 ± 16 seconds) than the warfarin group (152 ± 26 seconds, P <  0.0001). In the warfarin group, 100 U/kg heparin showed 361 ± 48 seconds 15-minute ACT. In the edoxaban group, an increase in initial dose induced prolongation of 15-minute ACT (i.e., 15-minute ACTs of 293 ± 56, 306 ± 39, 311 ± 45, and 319 ± 45 seconds for 120, 130, 140, and 150 U/kg initial doses, respectively). The total heparin required during the procedure was higher in the edoxaban group than in the warfarin group (109 ± 37 vs. 77 ± 21 U/kg/h, P <  0.0001). The 120–150 U/kg dose of heparin in edoxaban group did not cause thromboembolic or major bleeding complications. Conclusion: Edoxaban interrupted on the day of ablation showed a shorter baseline ACT than uninterrupted warfarin. Edoxaban required a higher initial heparin dose to achieve a similar 15-minute ACT to warfarin. These results are useful for determining the initial heparin dose required to achieve variable target ACTs.
• Gateway reflex: neural activation-mediated immune cell gateways in the central nervous system.

  Kamimura D, Ohki T, Arima Y, Murakami M

  International immunology 30 (7) 281 - 289 0953-8178 2018/06 [Refereed][Not invited]
   

  The neural regulation of organs can be categorized as systemic or local. Whereas systemic regulation by the hypothalamus-pituitary-adrenal gland-mediated release of steroid hormones has been well studied, the mechanisms for local regulation have only recently emerged. Two types of local neural regulation are known, the gateway reflex and the inflammatory reflex. The gateway reflex describes a mechanism that converts regional neural stimulations into inflammatory outputs by changing the state of specific blood vessels. Molecularly, the enhancement of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity in endothelial cells by neurotransmitters, such as noradrenaline and ATP, induces an enhanced production of pro-inflammatory mediators, including chemokines, which form immune cell gateways at specific vessels. Several types of gateway reflex have been identified, and each regulates distinct organs by creating gateways for autoreactive T cells that induce local inflammation. On the other hand, the inflammatory reflex elicits an anti-inflammatory response through vagal nerves. Here, we summarize recent works on these two local neuro-immune interactions, giving special focus to the gateway reflex.
• Existence of NEU1 sialidase on mouse thymocytes whose natural substrate is CD5

  Shigeko Kijimoto-Ochiai, Tokuko Matsumoto-Mizuno, Daisuke Kamimura, Masaaki Murakami, Miwako Kobayashi, Ichiro Matsuoka, Hiroshi Ochiai, Hideharu Ishida, Makoto Kiso, Keiko Kamimura, Toshiaki Koda

  Glycobiology 28 (5) 306 - 317 1460-2423 2018/05/01 [Refereed][Not invited]
   

  Membrane-bound sialidases in themouse thymus are unique and mysterious because their activity at pH 6.5 is equal to or higher than that in the acidic region. The pH curve like this has never been reported in membrane-bound form. To clarify this enzyme, we studied the sialidase activities of crude membrane fractions from immature-T, mature-T and non-T cells from C57BL/6 mice and from SM/J mice, a strain with a defect in NEU1 activity. Non-T cells fromC57BL/6 mice had high activity at pH 6.5, but those from SM/J mice did not. Neu1 and Neu3 mRNA was shown by real-time PCR to be expressed in T cells and also in non-T cells, whereas Neu2 was expressed mainly in non-T cells and Neu4 was scarcely expressed. However, the in situ hybridization study on the localization of four sialidases in the thymus showed that Neu4 was clearly expressed. We then focused on a sialidase on the thymocyte surface because the possibility of the existence of a sialidase on thymocytes was suggested by peanut agglutinin (PNA) staining after incubation of the cells alone in PBS. This activity was inhibited by NEU1-selective sialidase inhibitor C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The natural substrate for the cell surface sialidase was identified as clustered differentiation 5 (CD5) by PNA-blot analysis of anti-CD5 immunoprecipitate. We conclude that NEU1 exists on the cell surface of mouse thymocytes and CD5 is a natural substrate for it. Although this is not themain reaction of themembrane-bound thymus-sialidases, itmust be important for the thymus.
• Verification of radiodynamic therapy by medical linear accelerator using a mouse melanoma tumor model.

  Junko Takahashi, Mami Murakami, Takashi Mori, Hitoshi Iwahashi

  Scientific reports 8 (1) 2728 - 2728 2018/02/09 [Refereed][Not invited]
   

  Combined treatment with 5-aminolevulinic acid (5-ALA) and X-rays improves tumor suppression in vivo. This is because the accumulated protoporphyrin IX from 5-ALA enhances the generation of ROS by the X-ray irradiation. In the present study, a high-energy medical linear accelerator was used instead of a non-medical low energy X-ray irradiator, which had been previously used. Tumor-bearing mice implanted with B16-BL6 melanoma cells were treated with fractionated doses of irradiation (in total, 20 or 30 Gy), using two types of X-ray irradiator after 5-ALA administration. Suppression of tumor growth was enhanced with X-ray irradiation in combination with 5-ALA treatment compared with X-ray treatment alone, using both medical and non-medical X-ray irradiators. 5-ALA has been used clinically for photodynamic therapy. Thus, "radiodynamic therapy", using radiation from medical linacs as a physical driving force, rather than the light used in photodynamic therapy, may have potential clinical applications.
• Rbm10 regulates inflammation development via alternative splicing of Dnmt3b

  Toru Atsumi, Hironao Suzuki, Jing-Jing Jiang, Yuko Okuyama, Ikuma Nakagawa, Mitsutoshi Ota, Yuki Tanaka, Takuto Ohki, Kokichi Katsunuma, Koichi Nakajima, Yoshinori Hasegawa, Osamu Ohara, Hideki Ogura, Yasunobu Arima, Daisuke Kamimura, Masaaki Murakami

  International Immunology 29 (12) 581 - 591 1460-2377 2017/12/01 [Refereed][Not invited]
   

  RNA-binding motif 10 (Rbm10) is an RNA-binding protein that regulates alternative splicing, but its role in inflammation is not well defined. Here, we show that Rbm10 controls appropriate splicing of DNA (cytosine-5)-methyltransferase 3b (Dnmt3b), a DNA methyltransferase, to regulate the activity of NF-κB-responsive promoters and consequently inflammation development. Rbm10 deficiency suppressed NF-κB-mediated responses in vivo and in vitro. Mechanistic analysis showed that Rbm10 deficiency decreased promoter recruitment of NF-κB, with increased DNA methylation of the promoter regions in NF-κB-responsive genes. Consistently, Rbm10 deficiency increased the expression level of Dnmt3b2, which has enzyme activity, while it decreased the splicing isoform Dnmt3b3, which does not. These two isoforms associated with NF-κB efficiently, and overexpression of enzymatically active Dnmt3b2 suppressed the expression of NF-κB targets, indicating that Rbm10-mediated Dnmt3b2 regulation is important for the induction of NF-κB-mediated transcription. Therefore, Rbm10-dependent Dnmt3b regulation is a possible therapeutic target for various inflammatory diseases.
• Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells

  Kayo Ikeda, Makoto Kinoshita, Hisako Kayama, Shushi Nagamori, Pornparn Kongpracha, Eiji Umemoto, Ryu Okumura, Takashi Kurakawa, Mari Murakami, Norihisa Mikami, Yasunori Shintani, Satoko Ueno, Ayatoshi Andou, Morihiro Ito, Hideki Tsumura, Koji Yasutomo, Keiichi Ozono, Seiji Takashima, Shimon Sakaguchi, Yoshikatsu Kanai, Kiyoshi Takeda

  CELL REPORTS 21 (7) 1824 - 1838 2211-1247 2017/11 [Refereed][Not invited]
   

  Foxp3(+) regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp(3+) Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp(3+) Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation.
• The Gateway Reflex, a Novel Neuro-immune interaction for the Regulation of Regional vessels

  Yuki Tanaka, Yasunobu Arima, Daisuke Kamimura, Masaaki Murakami

  FRONTIERS IN IMMUNOLOGY 8 1321  1664-3224 2017/10 [Refereed][Not invited]
   

  The gateway reflex is a new phenomenon that explains how immune cells bypass the blood-brain barrier to infiltrate the central nervous system (CNS) and trigger neuroinflammation. To date, four examples of gateway reflexes have been discovered, each described by the stimulus that evokes the reflex. Gravity, electricity, pain, and stress have all been found to create gateways at specific regions of the CNS. The gateway reflex, the most recently discovered of the four, has also been shown to upset the homeostasis of organs in the periphery through its action on the CNS. These reflexes provide novel therapeutic targets for the control of local neuroinflammation and organ function. Each gateway reflex is activated by different neural activations and induces inflmammation at different regions in the CNS. Therefore, it is theoretically possible to manipulate each independently, providing a novel therapeutic strategy to control local neuroinflammation and peripheral organ homeostasis.
• Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit

  Yasunobu Arima, Takuto Ohki, Naoki Nishikawa, Kotaro Higuchi, Mitsutoshi Ota, Yuki Tanaka, Junko Nio-Kobayashi, Mohamed Elfeky, Ryota Sakai, Yuki Mori, Tadafumi Kawamoto, Andrea Stofkova, Yukihiro Sakashita, Yuji Morimoto, Masaki Kuwatani, Toshihihiko Iwanaga, Yoshichika Yoshioka, Naoya Sakamoto, Akihiko Yoshimura, Mitsuyoshi Takiguchi, Saburo Sakoda, Marco Prinz, Daisuke Kamimura, Masaaki Murakami

  ELIFE 6 2050-084X 2017/08 [Refereed][Not invited]
   

  Impact of stress on diseases including gastrointestinal failure is well-known, but molecular mechanism is not understood. Here we show underlying molecular mechanism using EAE mice. Under stress conditions, EAE caused severe gastrointestinal failure with high-mortality. Mechanistically, autoreactive-pathogenic CD4+ T cells accumulated at specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus to establish brain micro-inflammation via stress gateway reflex. Importantly, induction of brain micro-inflammation at specific vessels by cytokine injection was sufficient to establish fatal gastrointestinal failure. Resulting micro-inflammation activated new neural pathway including neurons in paraventricular-nucleus, dorsomedial-nucleus-ofhypothalamus, and also vagal neurons to cause fatal gastrointestinal failure. Suppression of the brain micro-inflammation or blockage of these neural pathways inhibited the gastrointestinal failure. These results demonstrate direct link between brain micro-inflammation and fatal gastrointestinal disease via establishment of a new neural pathway under stress. They further suggest that brain micro-inflammation around specific vessels could be switch to activate new neural pathway(s) to regulate organ homeostasis.
• BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Trypanosoma cruzi infection

  Shoko Kitada, Hisako Kayama, Daisuke Okuzaki, Ritsuko Koga, Masao Kobayashi, Yasunobu Arima, Atsushi Kumanogoh, Masaaki Murakami, Masahito Ikawa, Kiyoshi Takeda

  JOURNAL OF EXPERIMENTAL MEDICINE 214 (5) 1313 - 1331 0022-1007 2017/05 [Refereed][Not invited]
   

  Inappropriate IL-17 responses are implicated in chronic tissue inflammation. IL-23 contributes to Trypanosoma cruzi-specific IL-17 production, but the molecular mechanisms underlying regulation of the IL-23-IL-17 axis during T. cruzi infection are poorly understood. Here, we demonstrate a novel function of BATF2 as a negative regulator of Il23a in innate immune cells. IL-17, but not IFN-gamma, was more highly produced by CD4(+) T cells from spleens and livers of T. cruzi-infected Batf2(-/-) mice than by those of wild-type mice. In this context, Batf2(-/-) mice showed severe multiorgan pathology despite reduced parasite burden. T. cruzi-induced IL-23 production was increased in Batf2(-/-) innate immune cells. The T. cruzi-induced enhanced Th17 response was abrogated in Batf2(-/-) Il23a(-/-) mice. The interaction of BATF2 with c-JUN prevented c-JUN-ATF-2 complex formation, inhibiting Il23a expression. These results demonstrate that IFN-gamma-inducible BATF2 in innate immune cells controls Th17-mediated immunopathology by suppressing IL-23 production during T. cruzi infection.
• Recent advances in neuroimmune interactions

  Masaaki Murakami

  Clinical and Experimental Neuroimmunology 8 (1) 3 - 4 1759-1961 2017/02/01 [Refereed][Not invited]
  
• Gateway Reflexes are Stimulated by Neural Activations and Promote the Pathogenesis of Multiple Sclerosis Models

  K. Higuchi, D. Kamimura, A. Stofkova, N. Nishikawa, T. Ohki, Y. Arima, M. Murakami

  Nutrition and Lifestyle in Neurological Autoimmune Diseases: Multiple Sclerosis 39 - 45 2017/01/18 [Refereed][Not invited]
   

  The central nervous system (CNS) is tightly regulated by the blood-brain barrier. This regulation is compromised in patients with neuroinflammatory diseases such as multiple sclerosis (MS), which allows immune cells to infiltrate the CNS. We identified a gateway, dorsal vessels of the fifth lumbar (L5) cord, for this infiltration using MS mouse models. The gateway is regulated by local neural activation. Sensory neurons activated by gravity induce local norepinephrine production via sympathetic nerve activations, which increases the expression of chemokines to attract autoreactive CD4+ T cells at L5 dorsal vessels. Neural activation caused by other stimulations, such as electric pulses and pain sensation, also results in gateways. This chapter focuses on these gateway reflexes, which are critical for immune reactions in the CNS.
• Breakpoint cluster region-mediated inflammation is dependent on casein kinase II (vol 197, pg 3111, 2016)

  J. Meng, J. -J. Jiang, T. Atsumi, H. Bando, Y. Okuyama, L. Sabharwal, I. Nakagawa, H. Higuchi, M. Ota, M. Okawara, R. Ishitani, O. Nureki, D. Higo, Y. Arima, H. Ogura, D. Kamimura, M. Murakami

  JOURNAL OF IMMUNOLOGY 198 (2) 971 - 971 0022-1767 2017/01 [Refereed][Not invited]
  
• Inflammation amplifier and gateway reflex: The regulation of inflammation by neuroimmune interaction

  Ikuma Nakagawa, Masaaki Murakami

  Japanese Journal of Clinical Immunology 40 (3) 160 - 168 1349-7413 2017 [Refereed][Not invited]
   

  Central nervous system (CNS), which is made up of brain and spinal cord, is protected from the invasion of harmful agents, such as various pathogens, chemical products or immune cells by a special structure “Blood Brain Barrier (BBB)”. BBB highly preserves the homeostasis of CNS environment. On the other hand, there are many diseases in CNS regions which is associated with infection or autoimmunity, that means there may exist the “gateway” for pathogens or immune cells to attack CNS. Until recently, the molecular mechanism of the gateway formation has not been elucidated. Through studies in the multiple sclerosis model experimental autoimmune encephalomyelitis, we have clarified the mechanism of the gateway formation, and also the locations of gateways which depend on the regional neural activation. Further more, we have also discovered a massive chemokine-inducing mechanism “inflammation amplifier” via co-activation of NF-κB pathway and STAT3 pathway. It is essential for the development of inflammation in various diseases and is a molecular basis of BBB breakdown.
• Lactobacillus helveticus SBT2171 Attenuates Experimental Autoimmune Encephalomyelitis in Mice.

  Maya Yamashita, Ken Ukibe, Yumi Matsubara, Tomohiro Hosoya, Fumihiko Sakai, Shigeyuki Kon, Yasunobu Arima, Masaaki Murakami, Hisako Nakagawa, Tadaaki Miyazaki

  Frontiers in microbiology 8 2596 - 2596 2017 [Refereed][Not invited]
   

  We recently reported that Lactobacillus helveticus SBT2171 (LH2171) inhibited the proliferation and inflammatory cytokine production of primary immune cells in vitro, and alleviated collagen-induced arthritis (CIA) in mice, a model of human rheumatoid arthritis (RA). In this study, we newly investigated whether LH2171 could relieve the severity of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), which is an autoimmune disease, but develop the symptoms by different mechanisms from RA. In MS and EAE, main cause of the disease is the abnormality in CD4+ T cell immunity, whereas in RA and CIA, is that in antibody-mediated immunity. The intraperitoneal administration of LH2171 significantly decreased the incidence and clinical score of EAE in mice. LH2171 also reduced the numbers of pathogenic immune cells, especially Th17 cells, in the spinal cord at the peak stage of disease severity. Interestingly, before the onset of EAE, LH2171 administration remarkably decreased the ratio of Th17 cells to CD4+ T cells in the inguinal lymph nodes (LNs), where pathogenic immune cells are activated to infiltrate the central nervous system, including the spinal cord. Furthermore, the expression of interleukin (IL)-6, an inflammatory cytokine essential for Th17 differentiation, decreased in the LNs of LH2171-administered mice. Moreover, LH2171 significantly inhibited IL-6 production in vitro from both DC2.4 and RAW264.7 cells, model cell lines of antigen-presenting cells. These findings suggest that LH2171 might down-regulate IL-6 production and the subsequent Th17 differentiation and spinal cord infiltration, consequently alleviating EAE symptoms.
• Bacteremic kidney cyst infection caused by Helicobacter cinaedi.

  Ito K, Yamamoto T, Nishio H, Sawaya A, Murakami M, Kitagawa A, Matsuo Y, Matsuo K, Tanaka S, Mori N

  CEN case reports 5 (2) 121 - 124 2016/11 [Refereed][Not invited]
  
• Breakpoint Cluster Region-Mediated Inflammation Is Dependent on Casein Kinase II

  Jie Meng, Jing-Jing Jiang, Toru Atsumi, Hidenori Bando, Yuko Okuyama, Lavannya Sabharwal, Ikuma Nakagawa, Haruka Higuchi, Mitsutoshi Ota, Momoko Okawara, Ryuichiro Ishitani, Osamu Nureki, Daisuke Higo, Yasunobu Arima, Hideki Ogura, Daisuke Kamimura, Masaaki Murakami

  JOURNAL OF IMMUNOLOGY 197 (8) 3111 - 3119 0022-1767 2016/10 [Refereed][Not invited]
   

  The breakpoint cluster region (BCR) is known as a kinase and cause of leukemia upon fusing to Abl kinase. In this study, we demonstrate that BCR associated with the alpha subunit of casein kinase II (CK2 alpha), rather than BCR itself, is required for inflammation development. We found that BCR knockdown inhibited NF-kappa B activation in vitro and in vivo. Computer simulation, however, suggested that the putative BCR kinase domain has an unstable structure with minimal enzymatic activity. Liquid chromatography-tandem mass spectrometry analysis showed that CK2 alpha associated with BCR. We found the BCR functions are mediated by CK2 alpha. Indeed, CK2 alpha associated with adaptor molecules of TNF-alpha R and phosphorylated BCR at Y177 to establish a p65 binding site after TNF-alpha stimulation. Notably, p65 S529 phosphorylation by CK2 alpha creates a p300 binding site and increased p65-mediated transcription followed by inflammation development in vivo. These results suggest that BCR-mediated inflammation is dependent on CK2 alpha, and the BCR-CK2 alpha complex could be a novel therapeutic target for various inflammatory diseases.
• 移植腎における炎症アンプの活性化

  樋口 はるか, 岩見 大基, 堀田 記世彦, 佐々木 元, 広瀬 貴行, 篠原 信雄, 村上 正晃

  移植 (一社)日本移植学会 51 (総会臨時) 381 - 381 0578-7947 2016/09
• Strong TCR-mediated signals suppress integrated stress responses induced by KDELR1 deficiency in naive T cells

  Daisuke Kamimura, Yasunobu Arima, Mineko Tsuruoka, Jing-jing Jiang, Hidenori Bando, Jie Meng, Lavannya Sabharwal, Andrea Stofkova, Naoki Nishikawa, Kotaro Higuchi, Hideki Ogura, Toru Atsumi, Masaaki Murakami

  INTERNATIONAL IMMUNOLOGY 28 (3) 117 - 126 0953-8178 2016/03 [Refereed][Not invited]
   

  KDEL receptor 1 (KDELR1) regulates integrated stress responses (ISR) to promote naive T-cell survival in vivo. In a mouse line having nonfunctional KDELR1, T-Red (naive T-cell reduced) mice, polyclonal naive T cells show excessive ISR and eventually undergo apoptosis. However, breeding T-Red mice with TCR-transgenic mice bearing relatively high TCR affinity rescued the T-Red phenotype, implying a link between ISR-induced apoptosis and TCR-mediated signaling. Here, we showed that strong TCR stimulation reduces ISR in naive T cells. In mice lacking functional KDELR1, surviving naive T cells expressed significantly higher levels of CD5, a surrogate marker of TCR self-reactivity. In addition, higher TCR affinity/avidity was confirmed using a tetramer dissociation assay on the surviving naive T cells, suggesting that among the naive T-cell repertoire, those that receive relatively stronger TCR-mediated signals via self-antigens survive enhanced ISR. Consistent with this observation, weak TCR stimulation with altered peptide ligands decreased the survival and proliferation of naive T cells, whereas stimulation with ligands having higher affinity had no such effect. These results suggest a novel role of TCR-mediated signals in the attenuation of ISR in vivo.
• Adenovirus-mediated ICOSIg gene therapy in a presensitized murine model of allergic rhinitis

  Jun Sato, Nobuhiro Konno, Masaaki Murakami, Toshimitsu Uede, Tetsuo Himi

  Advances in Oto-Rhino-Laryngology 77 59 - 66 0065-3071 2016 [Refereed][Not invited]
   

  Allergic rhinitis is a chronic inflammatory disease of the upper airways caused by Th2 cell-type cytokines in response to allergen exposure. The inducible costimulator (ICOS), the third member of the CD28/CTLA4 family, plays an important role in immune response. In this study, adenovirus vectors containing ICOSIg (Adex1CAICOSIg) were administered to effectively inhibit the ICOS/ICOSL interaction, and the effects of Adex1CAICOSIg on allergic rhinitis were examined. Intranasal administration of Adex1CAICOSIg attenuated airway inflammation, as demonstrated by a decrease in nasal symptoms and infiltration of eosinophils into the nasal mucosa, as well as by a decrease in local IL-5 expression. Therefore, the ICOS/ICOSL pathway significantly contributes to the progression of allergic rhinitis.
• Epidemiological study of canine neoplasia based on tumor registration data of domestic dogs in Gifu prefecture between April 2013 and March 2014.

  Komazawa S, Shibata S, Sakai S, Ito Y, Kawabe M, Murakami M, Mori M, Maruo K

  J Jpn Vet Med Assoc 日本獣医師会 69 (7) 395 - 400 0446-6454 2016 [Refereed][Not invited]
  
• Naive T Cell Homeostasis Regulated by Stress Responses and TCR Signaling

  Daisuke Kamimura, Toru Atsumi, Andrea Stofkova, Naoki Nishikawa, Takuto Ohki, Hironao Suzuki, Kokichi Katsunuma, Jing-jing Jiang, Hidenori Bando, Jie Meng, Lavannya Sabharwal, Hideki Ogura, Toshio Hirano, Yasunobu Arima, Masaaki Murakami

  FRONTIERS IN IMMUNOLOGY 6 638  1664-3224 2015/12 [Refereed][Not invited]
   

  The survival of naive T cells is believed to require signals from TCR-pMHC interactions and cytokines such as IL-7. In contrast, signals that negatively impact naive T cell survival are less understood. We conducted a forward genetic screening of mice and found a mutant mouse line with reduced number of naive T cells (T-Red mice). T-Red mice have a point mutation in the Kdelr1 gene, and their naive T cells show enhanced integrated stress response (ISR), which eventually induces their apoptosis. Therefore, naive T cells require a KDEL receptor-mediated mechanism that efficiently relieves cellular stress for their survival in vivo. Interestingly, naive T cells expressing TCR with higher affinity/avidity to self-antigens survive in T-Red mice, suggesting the possible link between TCR-mediated survival and ISR-induced apoptosis. In this article, we discuss the regulation of naive T cell homeostasis, keeping special attention on the ISR and TCR signal.
• Klotho-related Molecules Upregulated by Smoking Habit in Apparently Healthy Men: A Cross-sectional Study

  Kaori Nakanishi, Makoto Nishida, Masaya Harada, Tohru Ohama, Noritaka Kawada, Masaaki Murakami, Toshiki Moriyama, Keiko Yamauchi-Takihara

  SCIENTIFIC REPORTS 5 14230  2045-2322 2015/09 [Refereed][Not invited]
   

  While aging is unavoidable, the aging mechanism is still unclear because of its complexity. Smoking causes premature death and is considered as an environmental aging accelerator. In the present study, we focused on the influence of smoking to the serum concentration of anti-aging protein alpha-klotho (alpha Kl) and the beta-klotho-associated protein fibroblast growth factor (FGF)-21 in men. Subjects consisted of apparently healthy men over 40 years of age who underwent health examination. Physical and biochemical parameters, including the levels of several cytokines and growth factors, were obtained from the subjects. Among middle-aged men (46.1 +/- 5.1 years), serum levels of FGF-21, soluble alpha Kl (s alpha Kl), and inflammation-related cytokine interleukin (IL)-6 were significantly higher in smokers than in never-smokers. Serum levels of FGF-21 increased and correlated with alanine transaminase, gamma guanosine-5'-triphosphate, and total cholesterol only in smokers, suggesting FGF-21 as a metabolic disorder-related factor in smokers. In aged men (60.3 +/- 1.7 years), although the serum levels of s alpha Kl in never-smokers were low, smokers showed highly increased serum levels of s alpha Kl. Serum levels of s alpha Kl was correlated with IL-6 in middle-aged never-smokers, suggesting s alpha Kl regulates IL-6. However, this correlation was disrupted in smokers and aged men.
• Role of T cell—glial cell interactions in creating and amplifying central nervous system inflammation and multiple sclerosis disease symptoms

  Eric S. Huseby, Daisuke Kamimura, Yasunobu Arima, Caitlin S. Parello, Katsuhiro Sasaki, Masaaki Murakami

  Frontiers in Cellular Neuroscience 9 (AUGUST) 295  1662-5102 2015/08/05 [Refereed][Not invited]
   

  Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System (CNS) that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons and axons. Historically, MS has been thought of as a T cell-mediated autoimmune disease of CNS white matter. However, recent studies have identified gray matter lesions in MS patients, suggesting that CNS antigens other than myelin proteins may be involved during the MS disease process. We have recently found that T cells targeting astrocyte-specific antigens can drive unique aspects of inflammatory CNS autoimmunity, including the targeting of gray matter and white matter of the brain and inducing heterogeneous clinical disease courses. In addition to being a target of T cells, astrocytes play a critical role in propagating the inflammatory response within the CNS induced NF-κB signaling. Here, we will discuss the pathophysiology of CNS inflammation mediated by T cell—glial cell interactions and its contributions to CNS autoimmunity.
• CD147/Basigin Limits Lupus Nephritis and Th17 Cell Differentiation in Mice by Inhibiting the Interleukin-6/STAT-3 Pathway

  Kayaho Maeda, Tomoki Kosugi, Waichi Sato, Hiroshi Kojima, Yuka Sato, Daisuke Kamimura, Noritoshi Kato, Naotake Tsuboi, Yukio Yuzawa, Seiichi Matsuo, Masaaki Murakami, Shoichi Maruyama, Kenji Kadomatsu

  ARTHRITIS & RHEUMATOLOGY 67 (8) 2185 - 2195 2326-5191 2015/08 [Refereed][Not invited]
   

  Objective. Interleukin-17 (IL-17)-producing T cells (Th17 cells) play critical roles in the pathogenesis of immune-related diseases, including systemic lupus erythematosus. However, the fundamental mechanism regulating Th17 cell differentiation is not fully understood. Recently, we demonstrated that plasma levels of CD147/basigin (Bsg) in patients with lupus nephritis (LN) were closely associated with disease activity. but the molecular mechanism involving Bsg has been elusive. Here, we addressed the role of Bsg in the pathogenesis of LN. Methods. Injections of pristane (2,6,10,14-tetramethylpentadecane [TMPD]) were administered to Bsg(-/-) or Bsg(+/+) mice to induce LN. The mice were killed 6 months after being injected, for histologic and biochemical analyses of the kidneys and spleens. Results. Pristane induced LN more strikingly in Bsg(-/-) mice than in Bsg(+/+) mice, even though humoral autoimmunity was similarly increased in both genotypes. The increased number of Th17, but not Th1, Treg cells, was augmented in Bsg(-/-) mice. The expression of IL-17 was also increased in the kidneys of Bsg(-/-) mice, in proportion to LN disease activity. Furthermore, treatment with anti-IL-17 antibody reduced LN disease activity in Bsg(-/-) mice. Complementary to these phenotypes of Bsg(-/-) mice, Bsg expression was enhanced in activated CD4+ T cells in vivo and in vitro. Bsg deficiency selectively augmented in vitro differentiation of naive CD4+ T cells to Th17 cells and STAT-3 phosphorylation during this differentiation. Moreover, STAT-3 phosphorylation was suppressed by crosslinking of Bsg with its antibody. Conclusion. Bsg plays an indispensable role in Th17 cell differentiation as a negative regulator by suppressing the IL-6/STAT-3 pathway.
• mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

  Daisuke Ito, Satoshi Nojima, Masayuki Nishide, Tatsusada Okuno, Hyota Takamatsu, Sujin Kang, Tetsuya Kimura, Yuji Yoshida, Keiko Morimoto, Yohei Maeda, Takashi Hosokawa, Toshihiko Toyofuku, Jun Ohshima, Daisuke Kamimura, Masahiro Yamamoto, Masaaki Murakami, Eiichi Morii, Hiromi Rakugi, Yoshitaka Isaka, Atsushi Kumanogoh

  Journal of immunology (Baltimore, Md. : 1950) 195 (3) 934 - 43 0022-1767 2015/08/01 [Refereed][Not invited]
   

  Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling.
• A pain-mediated neural signal induces relapse in murine autoimmune encephalomyelitis, a multiple sclerosis model

  Yasunobu Arima, Daisuke Kamimura, Toru Atsumi, Masaya Harada, Tadafumi Kawamoto, Naoki Nishikawa, Andrea Stofkova, Takuto Ohki, Kotaro Higuchi, Yuji Morimoto, Peter Wieghofer, Yuka Okada, Yuki Mori, Saburo Sakoda, Shizuya Saika, Yoshichika Yoshioka, Issei Komuro, Toshihide Yamashita, Toshio Hirano, Marco Prinz, Masaaki Murakami

  ELIFE 4 2050-084X 2015/08 [Refereed][Not invited]
   

  Although pain is a common symptom of various diseases and disorders, its contribution to disease pathogenesis is not well understood. Here we show using murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), that pain induces EAE relapse. Mechanistic analysis showed that pain induction activates a sensory-sympathetic signal followed by a chemokine-mediated accumulation of MHC class II+CD11b+ cells that showed antigen-presentation activity at specific ventral vessels in the fifth lumbar cord of EAE-recovered mice. Following this accumulation, various immune cells including pathogenic CD4+ T cells recruited in the spinal cord in a manner dependent on a local chemokine inducer in endothelial cells, resulting in EAE relapse. Our results demonstrate that a pain-mediated neural signal can be transformed into an inflammation reaction at specific vessels to induce disease relapse, thus making this signal a potential therapeutic target. DOI: 10.7554/eLife.08733.001
• KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR

  Daisuke Kamimura, Kokichi Katsunuma, Yasunobu Arima, Toru Atsumi, Jing-jing Jiang, Hidenori Bando, Jie Meng, Lavannya Sabharwal, Andrea Stofkova, Naoki Nishikawa, Hironao Suzuki, Hideki Ogura, Naoko Ueda, Mineko Tsuruoka, Masaya Harada, Junya Kobayashi, Takanori Hasegawa, Hisahiro Yoshida, Haruhiko Koseki, Ikuo Miura, Shigeharu Wakana, Keigo Nishida, Hidemitsu Kitamura, Toshiyuki Fukada, Toshio Hirano, Masaaki Murakami

  NATURE COMMUNICATIONS 6 7474  2041-1723 2015/06 [Refereed][Not invited]
   

  KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naive T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naive T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naive T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2 alpha and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naive T-cell homeostasis by controlling ISR.
• Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension

  Takahiro Hashimoto-Kataoka, Naoki Hosen, Takashi Sonobe, Yoh Arita, Taku Yasui, Takeshi Masaki, Masato Minami, Tadakatsu Inagaki, Shigeru Miyagawa, Yoshiki Sawa, Masaaki Murakami, Atsushi Kumanogoh, Keiko Yamauchi-Takihara, Meinoshin Okumura, Tadamitsu Kishimoto, Issei Komuro, Mikiyasu Shirai, Yasushi Sakata, Yoshikazu Nakaoka

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 112 (20) E2677 - E2686 0027-8424 2015/05 [Refereed][Not invited]
   

  IL-6 is a multifunctional proinflammatory cytokine that is elevated in the serum of patients with pulmonary arterial hypertension (PAH) and can predict the survival of patients with idiopathic PAH (IPAH). Previous animal experiments and clinical human studies indicate that IL-6 is important in PAH; however, the molecular mechanisms of IL-6-mediated pathogenesis of PAH have been elusive. Here we identified IL-21 as a downstream target of IL-6 signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly up-regulated after hypoxia exposure in the lungs of mice treated with control antibody but not in the lungs of mice treated with MR16-1. Although IL-17 blockade with an anti-IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. In accordance with these findings, IL-21 promoted the polarization of primary alveolar macrophages toward the M2 phenotype. Of note, significantly enhanced expressions of IL-21 and M2 macrophage markers were detected in the lungs of IPAH patients who underwent lung transplantation. Collectively, these findings suggest that IL-21 promotes PAH in association with M2 macrophage polarization, downstream of IL-6-signaling. The IL-6/IL-21-signaling axis may be a potential target for treating PAH.
• [Gateway Reflex, a regulator of the inflammation feedback loop by regional neural activation].

  Arima Y, Kamimura D, Atsumi T, Murakami M

  Nihon rinsho. Japanese journal of clinical medicine 日本臨床社 73 (4) 693 - 700 0047-1852 2015/04 [Refereed][Not invited]
  
• Temporal Expression of Growth Factors Triggered by Epiregulin Regulates Inflammation Development

  Masaya Harada, Daisuke Kamimura, Yasunobu Arima, Hitoshi Kohsaka, Yuji Nakatsuji, Makoto Nishida, Toru Atsumi, Jie Meng, Hidenori Bando, Rajeev Singh, Lavannya Sabharwal, Jing-Jing Jiang, Noriko Kumai, Nobuyuki Miyasaka, Saburo Sakoda, Keiko Yamauchi-Takihara, Hideki Ogura, Toshio Hirano, Masaaki Murakami

  JOURNAL OF IMMUNOLOGY 194 (3) 1039 - 1046 0022-1767 2015/02 [Refereed][Not invited]
   

  In this study, we investigated the relationship between several growth factors and inflammation development. Serum concentrations of epiregulin, amphiregulin, betacellulin, TGF-alpha, fibroblast growth factor 2, placental growth factor (PLGF), and tenascin C were increased in rheumatoid arthritis patients. Furthermore, local blockades of these growth factors suppressed the development of cytokine-induced arthritis in mice by inhibiting chemokine and IL-6 expressions. We found that epiregulin expression was early and followed by the induction of other growth factors at different sites of the joints. The same growth factors then regulated the expression of epiregulin at later time points of the arthritis. These growth factors were increased in patients suffering from multiple sclerosis (MS) and also played a role in the development of an MS model, experimental autoimmune encephalomyelitis. The results suggest that the temporal expression of growth factors is involved in the inflammation development seen in several diseases, including rheumatoid arthritis and MS. Therefore, various growth factor pathways might be good therapeutic targets for various inflammatory diseases.
• Regulation of Inflammation Responses by Neural Activation

  Murakami Masaaki

  Shinzo 公益財団法人 日本心臓財団 47 (2) 261 - 268 0586-4488 2015
• Role of cytokine-mediated crosstalk between T cells and nonimmune cells in the pathophysiology of multiple sclerosis

  Daisuke Kamimura, Yasunobu Arima, Toru Atsumi, Jie Meng, Lavannya Sabharwal, Hidenori Bando, Hideki Ogura, Jing-Jing Jiang, Eric S. Huseby, Masaaki Murakami

  Multiple Sclerosis: A Mechanistic View 101 - 125 2015/01/01 [Refereed][Not invited]
   

  Multiple sclerosis (MS) is a progressive demyelinating disease associated with chronic inflammation in the central nervous system (CNS). Genetic linkage is found in genes related to T cell function, and T cell infiltration is evident in MS lesions, suggesting a pathophysiological role for these cells. In the MS animal model experimental autoimmune encephalomyelitis (EAE), activated CD4+ and CD8+ T cells that recognize CNS antigens can induce MS-like symptoms. Various cytokines from these T cells trigger inflammation, critically contributing to the pathogenesis of the CNS disease. Evidence suggests an important role for cytokine-mediated crosstalk between T cells and nonimmune cells, including endothelial cells, glial cells, and fibroblasts in enhancing CNS inflammation. Counterbalancing T cell-induced inflammation, certain regulatory CD4+ and CD8+ T cell subsets can suppress CNS disease progression. Here, we discuss the pathophysiological role of T cells during MS and EAE, including neuro-immune interactions that allow T cells to invade the CNS.
• Role of Inflammation Amplifier-Induced Growth Factor Expression in the Development of Inflammatory Diseases

  Ikuma Nakagawa, Daisuke Kamimura, Toru Atsumi, Yasunobu Arima, Masaaki Murakami

  CRITICAL REVIEWS IN IMMUNOLOGY 35 (5) 365 - 378 1040-8401 2015 [Refereed][Not invited]
   

  Inflammation is a fundamental response induced by the immune system to protect the body against pathogens, tissue damage, and stress. At the same time, recent studies have suggested that chronically induced inflammation is involved in various human diseases and disorders. Thus, understanding the molecular mechanisms of chronic inflammation could provide therapeutic value. Many mediators such as cytokines or chemokines regulate inflammatory responses. Among them, interleukin(IL)-6 is a prominent cytokine that induces and maintains inflammatory reactions. It is expressed by activated CD4+ T cells and also non-immune cells such as fibroblasts and epithelial cells. We discovered an inflammation-induction machinery, the inflammation amplifier, which is activated by the simultaneous stimulation of nuclear factor-kappa B (NF-kappa B) and signal transducers and activator of transcription 3 (STAT3) via various cytokines like IL-17 and IL-6 in non-immune cells. Activation of the inflammation amplifier induces a synergistic increase of IL-6, inflammatory chemokines, and growth factors. Using genome-wide screening, we identified several growth factors as mediators of the inflammation amplifier. In this review, we highlight the role of growth factors in the inflammation mechanism with special attention on the inflammation amplifier.
• The Gateway Reflex, which is mediated by the inflammation amplifier, directs pathogenic immune cells into the CNS

  Lavannya Sabharwal, Daisuke Kamimura, Jie Meng, Hidenori Bando, Hideki Ogura, Chiemi Nakayama, Jing-Jing Jiang, Noriko Kumai, Hironao Suzuki, Toru Atsumi, Yasunobu Arima, Masaaki Murakami

  JOURNAL OF BIOCHEMISTRY 156 (6) 299 - 304 0021-924X 2014/12 [Refereed][Not invited]
   

  The brain-blood barrier (BBB) tightly limits immune cell migration into the central nervous system (CNS), avoiding unwanted inflammation under the normal state. However, immune cells can traverse the BBB when inflammation occurs within the CNS, suggesting a certain signal that creates a gateway that bypasses the BBB might exist. We revealed the inflammation amplifier as a mechanism of this signal, and identified dorsal vessels of the fifth lumber (L5) spinal cord as the gateway. The inflammation amplifier is driven by a simultaneous activation of NF-kappa B and STATs in non-immune cells, causing the production of a large amount of inflammatory chemokines to open the gateway at L5 vessels. It was found that the activation of the amplifier can be modulated by neural activation and artificially operated by electric pulses followed by establishment of new gateways, Gateway Reflex, at least in mice. Furthermore, genes required for the inflammation amplifier have been identified and are highly associated with various inflammatory diseases and disorders in the CNS. Thus, physical and/or pharmacological manipulation of the inflammation amplifier holds therapeutic value to control neuro-inflammation.
• Zinc transporter SLC39A10/ZIP10 controls humoral immunity by modulating B-cell receptor signal strength

  Shintaro Hojyo, Tomohiro Miyai, Hitomi Fujishiro, Masami Kawamura, Takuwa Yasuda, Atsushi Hijikata, Bum-Ho Bin, Tarou Irie, Junichi Tanaka, Toru Atsumi, Masaaki Murakami, Manabu Nakayama, Osamu Ohara, Seiichiro Himeno, Hisahiro Yoshida, Haruhiko Koseki, Tomokatsu Ikawa, Kenji Mishima, Toshiyuki Fukada

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 (32) 11786 - 11791 0027-8424 2014/08 [Refereed][Not invited]
   

  The humoral immune response, also called the antibody-mediated immune response, is one of the main adaptive immune systems. The essential micronutrient zinc (Zn) is known to modulate adaptive immune responses, and dysregulated Zn homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this Zn-mediated modulation are largely unknown. Here, we show that the Zn transporter SLC39A10/ZIP10 plays an important role in B-cell antigen receptor (BCR) signal transduction. Zip10-deficiency in mature B cells attenuated both T-cell-dependent and -independent immune responses in vivo. The Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. The perturbed signaling was found to be triggered by a reduction in CD45R phosphatase activity and consequent hyperactivation of LYN, an essential protein kinase in BCR signaling. Our data suggest that ZIP10 functions as a positive regulator of CD45R to modulate the BCR signal strength, thereby setting a threshold for BCR signaling in humoral immune responses.
• Temporal Changes in Lower-Lumbar Spinal Cord in EAE Mouse

  Mori Y, Murakami M, Arima Y, Zhu D, Yoshioka Y

  Japanese J Magn Reson Med 34 (3) 96 - 99 2014/08 [Refereed][Not invited]
  
• CD4⁺ T cell cytokine gene and protein expression in duodenal mucosa of dogs with inflammatory bowel disease.

  Hiroshi Ohta, Kanae Takada, Yuji Sunden, Yu Tamura, Tatsuyuki Osuga, Sue Yee Lim, Masahiro Murakami, Noboru Sasaki, Bandula Kumara Wickramasekara Rajapakshage, Kensuke Nakamura, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  The Journal of veterinary medical science 76 (3) 409 - 14 0916-7250 2014/03 [Refereed][Not invited]
   

  Inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal signs in dogs. In humans, T helper cells have important roles in the pathogenesis of IBD. In contrast, no specific involvement of a distinct T cell subset has been described in canine IBD. The present study evaluated the gene and protein expression of cytokines of T cell subsets in duodenal mucosa from dogs with IBD. Relative quantification of interleukin (IL)-17A, interferon (IFN)-γ, IL-4 and IL-10 mRNA transcription was performed using duodenal mucosa from 27 IBD dogs and 8 controls. Duodenal mucosal IL-17A, IFN-γ and IL-10 protein levels were determined by ELISA in 15 IBD dogs and 8 controls. There was no significant difference in each cytokines mRNA transcription level between groups. There was no significant difference in IL-17A, IFN-γ and IL-10 protein expression levels between groups. Thus, there is no clear evidence for the involvement of distinct Th cytokine in the pathogenesis of canine IBD.
• The Reverse-Direction Method Links Mass Experimental Data to Human Diseases

  Hideki Ogura, Toru Atsumi, Hidenori Bando, Lavannya Sabharwal, Moe Yamada, Jing-Jing Jiang, Akihiro Nakamura, Yasunobu Arima, Daisuke Kamimura, Masaaki Murakami

  ARCHIVUM IMMUNOLOGIAE ET THERAPIAE EXPERIMENTALIS 62 (1) 41 - 45 0004-069X 2014/02 [Refereed][Not invited]
   

  Genome-wide analyses such as DNA microarray, RNA sequencing and RNA interference-based high-throughput screening are prevalent to decipher a biological process of interest, and provide a large quantity of data to be processed. An ultimate goal for researchers must be extrapolation of their data to human diseases. We have conducted functional genome-wide screenings to elucidate molecular mechanisms of the inflammation amplifier, a NF kappa B/STAT3-dependent machinery that potently drives recruitment of immune cells to promote inflammation. Using a public database of genome-wide association studies (GWAS), we recently reported the reverse-direction method by which our mass screening data were successfully linked to many human diseases. As an example, the epiregulin-epidermal growth factor receptor pathway was identified as a regulator of the inflammation amplifier, and associated with human diseases by GWAS. In fact, serum epiregulin levels were higher in patients with chronic inflammatory disorders. The reverse-direction method can be a useful tool to narrow mass data down to focus on human disease-related genes.
• Early pathological alterations of lower lumbar cords detected by ultrahigh-field MRI in a mouse multiple sclerosis model

  Yuki Mori, Masaaki Murakami, Yasunobu Arima, Dasong Zhu, Yasuo Terayama, Yutaka Komai, Yuji Nakatsuji, Daisuke Kamimura, Yoshichika Yoshioka

  INTERNATIONAL IMMUNOLOGY 26 (2) 93 - 101 0953-8178 2014/02 [Refereed][Not invited]
   

  Changes in the lower lumbar region during EAE are detectable by sensitive MRI.Magnetic resonance imaging (MRI) is widely employed for the diagnosis of multiple sclerosis (MS). However, sometimes, the lesions found by MRI do not correlate with the neurological impairments observed in MS patients. We recently showed autoreactive T cells accumulate in the fifth lumbar cord (L5) to pass the bloodbrain barrier and cause inflammation in the central nervous system of experimental autoimmune encephalomyelitis (EAE) mice, an MS model. We here investigated this early event using ultrahigh-field MRI. T2-weighted image signals, which conform to the water content, increased in L4 and L5 during the development of EAE. At the same time, the sizes of L4 and L5 changed. Moreover, angiographic images of MRI showed branch positions of the blood vessels in the lower lumbar cords were significantly altered. Interestingly, EAE mice showed occluded and thickened vessels, particularly during the peak phase, followed by reperfusion in the remission phase. Additionally, demyelination regions of some MS patients had increased lactic acid content, suggesting the presence of ischemic events. These results suggest that inflammation-mediated alterations in the lower lumbar cord change the homeostasis of the spinal cord and demonstrate that ultrahigh-field MRI enables the detection of previously invisible pathological alterations in EAE.
• Inflammation Amplifier, a New Paradigm in Cancer Biology

  Toru Atsumi, Rajeev Singh, Lavannya Sabharwal, Hidenori Bando, Jie Meng, Yasunobu Arima, Moe Yamada, Masaya Harada, Jing-Jing Jiang, Daisuke Kamimura, Hideki Ogura, Toshio Hirano, Masaaki Murakami

  CANCER RESEARCH 74 (1) 8 - 14 0008-5472 2014/01 [Refereed][Not invited]
   

  Tumor-associated inflammation can induce various molecules expressed from the tumors themselves or surrounding cells to create a micro environment that potentially promotes cancer development. Inflammation, particularly chronic inflammation, is often linked to cancer development, even though its evolutionary role should impair nonself objects including tumors. The inflammation amplifier, a hyperinducer of chemokines in nonimmune cells, is the principal machinery for inflammation and is activated by the simultaneous stimulation of NF-kappa B and STAT3. We have redefined inflammation as local activation of the inflammation amplifier, which causes an accumulation of various immune cells followed by dysregulation of local homeostasis. Genes related to the inflammation amplifier have been genetically associated with various human inflammatory diseases. Here, we describe how cancer-associated genes, including interleukin (IL)-6, Ptgs2, ErbB1, Gas1, Serpine1, cMyc, and Vegf-alpha, are strongly enriched in genes related to the amplifier. The inflammation amplifier is activated by the stimulation of cytokines, such as TNF-alpha, IL-17, and IL-6, resulting in the subsequent expression of various target genes for chemokines and tumor-related genes like BCL2L11, CPNE7, FAS, HIF1-alpha, IL-1RAP, and SOD2. Thus, we conclude that inflammation does indeed associate with the development of cancer. The identified genes associated with the inflammation amplifier may thus make potential therapeutic targets of cancers. (C)2013 AACR.
• IL-6 and inflammatory diseases

  Daisuke Kamimura, Yasunobu Arima, Toshio Hirano, Hideki Ogura, Masaaki Murakami

  Cytokine Frontiers: Regulation of Immune Responses in Health and Disease 53 - 78 2014/01/01 [Refereed][Not invited]
   

  Interleukin-6 (IL-6) is a multifunctional cytokine that plays key roles not only in the immune system but also in a variety of biological processes. It is a primary regulator of both acute and chronic inflammations. Moreover, it has proven an excellent target for clinical treatment, as the anti-IL-6 receptor antibody has been successfully used against autoimmune disorders such as rheumatoid arthritis, juvenile idiopathic arthritis, and Castleman’s disease. In fact, it could be argued that IL-6 is the best example of basic cytokine research extending into clinical application. Here, we summarize IL-6 and its biological functions, with particular emphasis on inflammation and chronic inflammatory diseases, and a recently discovered inflammation control mechanism, the inflammation amplifier (formerly known as the IL-6 amplifier). We also describe a recent finding that indicates neural stimulations can modulate the activation of the inflammation amplifier at local blood vessels, creating a gate for the influx of immune cells into the central nervous system, which suggests the entry of immune cells into target organs can be artificially manipulated by local neural stimulation.
• Qualitative and Quantitative Contrast Enhanced Ultrasonography of the Pancreas Using Bolus Injection and Continuous Infusion Methods in Normal Dogs

  Sue Yee Lim, Kensuke Nakamura, Keitaro Morishita, Noboru Sasaki, Masahiro Murakami, Tatsuyuki Osuga, Hiroshi Ohta, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (12) 1601 - 1607 0916-7250 2013/12 [Refereed][Not invited]
   

  Quantitative contrast enhanced ultrasound is a major breakthrough for ultrasound imaging in recent years. However, contrast enhancement of the pancreas is brief with bolus injection. To assess if continuous infusion of Sonazoid (R) can prolong the duration of pancreatic enhancement over bolus injections, eight adult dogs received bolus injection and continuous infusion of Sonazoid (R) on separate days. Contrast enhanced ultrasound of the pancreatic parenchyma and proximal descending duodenum was performed, and time intensity curves reflecting tissue perfusions were generated. Perfusion parameters- time to initial upslope, peak time, time to wash-out and peak intensity were calculated and evaluated. Fast wash-in to intense peak, followed by rapid wash-out was observed for time intensity curves of bolus injection. With continuous infusion, contrast wash-in to peak intensity was gradual, followed by long plateau and slow wash-out. Median contrast enhancement durations of the pancreas and duodenum were significantly prolonged by continuous infusion from 11 sec (range, 10 to 23 sec) and 16 sec (range, 3 to 43 sec) at bolus injection to 205 sec (range, 170 to 264 sec, P<0.01) and 193 sec (range, 169 to 216 sec, P<0.05), respectively. Median peak intensity of the pancreas was 100.9 MPV (range, 80.2 to 124.3 MPV) at bolus injection and 77.6 MPV (range, 58.2 to 99.5 MPV, P<0.05) at continuous infusion. Prolonged continuous imaging is afforded by continuous infusion of contrast agent. Peak intensity of the pancreas was slightly diminished in continuous infusion, but offered adequate imaging subjectively.
• Markedly increased expression of interleukin-8 in the colorectal mucosa of inflammatory colorectal polyps in miniature dachshunds

  Yu Tamura, Hiroshi Ohta, Shidow Torisu, Masashi Yuki, Nozomu Yokoyama, Masahiro Murakami, Sue Yee Lim, Tatsuyuki Osuga, Keitaro Morishita, Kensuke Nakamura, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 156 (1-2) 32 - 42 0165-2427 2013/11 [Refereed][Not invited]
   

  Inflammatory colorectal polyps (ICRPs) in miniature dachshunds were recently recognized as a major cause of large bowel diarrhea in this dog breed in Japan. ICRPs are characterized by the formation of multiple small polyps and/or space-occupying large polyps in the colorectal area and are thought to be a novel form of inflammatory bowel disease (IBD). To explore key mediators in the pathogenesis of ICRPs, we analyzed several pro-inflammatory cytokine (IL-1 beta, IL-6, TNF-alpha, IL-8, IL-12p35, IL-12/23p40, and IL-23p19) mRNA expressions in colorectal polyps in ICRP dogs by quantitative PCR. Among these cytokines, IL-8 mRNA expression was markedly up-regulated in large polyps. To examine IL-8 protein expression, we analyzed IL-8 protein level and its location in colorectal mucosal specimens of ICRP dogs by ELISA and immunofluorescence microscopy. IL-8 protein was significantly increased in large polyps and serum in dogs with ICRPs compared to controls. By immunofluorescence microscopy, IL-8 was only localized in macrophages, but not in mucosal epithelial cells or neutrophils. IL-8-positive macrophages were significantly increased in large polyps compared to controls. These results suggest that IL-8 is produced mainly by macrophages and may induce neutrophil infiltration in the colorectal area of ICRP dogs. (C) 2013 Elsevier B.V. All rights reserved.
• Short-term Outcome of Hypofractionated Three-dimensional Conformal Radiation Therapy for Pituitary Tumors in Doge

  MORI Takashi, ITO Yusuke, YAMAZAKI Mifumi, MURAKAMI Mami, NOGUCHI Shunsuke, YAMADA Nami, MARUO Kohji

  Journal of the Japan Veterinary Medical Association 日本獸医師会 66 (10) 709 - 712 0446-6454 2013/10/20 [Not refereed][Not invited]
   

  Fifteen dogs with pituitary tumors were treated with a weekly schedule of hypofractionated three-dimensional conformal radiation therapy (3D-CRT). The median total dose was 48 Gy (range: 35-52 Gy), with a median irradiation dose/fraction of 7 Gy (5-8 Gy). The median follow-up time was 298 days. Twelve dogs were alive at the time of analysis. One dog died before completing the course of radiation therapy. Two died at 372 and 479 days after 3D-CRT. Although six of the 13 dogs with pituitary-dependent hyperadrenocorticism had improvement of clinical signs, none were able to stop receiving trilostane. Of the 13 dogs with neurologic signs, 10 had complete resolution and three had partial resolution. None of the dogs developed acute or late adverse effects due to 3D-CRT. Our preliminary results suggest that hypofractionated 3D-CRT was feasible in dogs with pituitary tumors.
• Expression of CD4(+) T cell cytokine genes in the colorectal mucosa of inflammatory colorectal polyps in miniature dachshunds

  Hiroshi Ohta, Kanae Takada, Shidow Torisu, Masashi Yuki, Yu Tamura, Nozomu Yokoyama, Tatsuyuki Osuga, Sue Yee Lim, Masahiro Murakami, Noboru Sasaki, Kensuke Nakamura, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 155 (4) 259 - 263 0165-2427 2013/10 [Refereed][Not invited]
   

  Inflammatory colorectal polyps (ICRPs) in miniature dachshunds are recently recognized as a major cause of large bowel diarrhea in this dog breed in Japan. ICRPs are characterized by the formation of multiple small polyps and a space-occupying large polyp in the colorectal area, and are thought to be a novel form of inflammatory bowel disease (IBD). In humans, specific cytokine patterns attributed to T helper (Th)1, Th17 and regulatory T cells have important roles in the pathogenesis of IBD. Thus, the aim of the present study was to assess the gene expression of cytokines of T cell subsets in the colorectal mucosa from dogs with ICRPs. Colorectal mucosal specimens from 10 dogs with ICRPs and 14 control dogs were used in this study. Interferon (IFN)-gamma, interleukin (IL)-4, IL-17A and IL-10 mRNA expression was assessed using quantitative real-time PCR. IL-17A mRNA expression was significantly increased in large polyps compared to small polyps and controls. IFN-gamma and IL-10 mRNA expression in large polyps were significantly higher than in controls. There was no significant difference in IL-4 mRNA expression among the three groups. IL-17A is thought to play important roles in the pathogenesis of ICRPs. IL-10 up-regulation could oppose the proinflammatory function of IL-17A. (C) 2013 Elsevier B.V. All rights reserved.
• Adaptive-servo ventilation combined with deep sedation is an effective strategy during pulmonary vein isolation

  Takashi Murakami, Hirosuke Yamaji, Kenji Numa, Hiroshi Kawamura, Masaaki Murakami, Shunichi Higashiya, Shigeshi Kamikawa, Kazuyoshi Hina, Satoshi Hirohata, Shozo Kusachi

  Europace 15 (7) 951 - 956 1099-5129 2013/07 [Refereed][Not invited]
   

  AimsPulmonary vein isolation (PVI) by catheter ablation for atrial fibrillation (AF) requires suppression of patient restlessness by sufficient sedation in addition to maintaining stable respiration. We applied adaptive-servo ventilation (ASV) and examined the effects of ASV combined with deep propofol sedation on PVI using a NavX.Methods and resultsWe analysed 75 paroxysmal AF (PAF) patients (62 ± 11 years 53 men and 22 women) who underwent PVI for treatment of PAF using an ASV system combined with deep sedation (ASV group). Control patients included 75 consecutive PAF patients (62 ± 11 years 51 men and 24 women) who underwent PVI just before introduction of the ASV system. Deep sedation was defined as a Ramsay sedation score of 6. The ASV group had a lower frequency of restless body movements compared with the control group during PVI (1.5 ± 0.7 vs. 7.8 ± 1.4 times, P < 0.01). The frequency of respiratory compensation and EnGuide alignment of catheter position by the NavX was lower in the ASV (4.2 ± 3.3 and 8.8 ± 7.1 times) than control group (7.1 ± 5.1 and 15.2 ± 10.0 times, P < 0.05 and < 0.01, respectively). Consequently, significantly lower total electrical energy supply (48.7 ± 6.0 KJ) was required in the ASV than control group (64.5 ± 24.9 KJ, P < 0.01). Further, significantly shorter fluoroscopy and procedural times were observed in the ASV (28 ± 5 and 109 ± 25 min) than the control group (33 ± 6 and 141 ± 38 min, respectively, P < 0.01) and the AF recurrence rate was significantly lower in the ASV than the control group (12 vs. 25%, P < 0.01).ConclusionASV combined with deep sedation is an effective strategy during PVI using the NavX in patients with PAF. © 2013 The Author.
• Repeatability and reproducibility of measurements obtained via two-dimensional speckle tracking echocardiography of the left atrium and time-left atrial area curve analysis in healthy dogs

  Tatsuyuki Osuga, Kensuke Nakamura, Sue Yee Lim, Yu Tamura, Wickramasekara Rajapakshage Bandula Kumara, Masahiro Murakami, Noboru Sasaki, Keitaro Morishita, Hiroshi Ohta, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  AMERICAN JOURNAL OF VETERINARY RESEARCH 74 (6) 864 - 869 0002-9645 2013/06 [Refereed][Not invited]
   

  Objective-To evaluate left atrial phasic function in healthy dogs by means of 2-D speckle tracking echocardiography with time-left atrial area curve analysis and to assess repeatability and reproducibility of obtained measurements. Animals-6 healthy Beagles. Procedures-Each dog underwent echocardiography twice on different days (3 nonconsecutive examinations/d). Images were analyzed with offline software; area of the left atrium was automatically calculated in each frame throughout the cardiac cycle to derive time-left atrial area curves. Variables used to assess left atrial phasic function (total, passive, and active emptying area and emptying fractions and mean active and total emptying rates) were calculated. Agreement between variables measured via speckle tracking echocardiography and a manual tracing method was assessed with modified Bland-Altman analysis. Withinday and between-day coefficients of variation were determined. Results-Mean +/- SD total, passive, and active emptying fractions of the left atrium were 49.8 +/- 3.5%, 27.7 +/- 4.0%, and 30.5 +/- 4.3%, respectively. Mean +/- SD total and active emptying rates were 16.0 +/- 2.5 cm(2)/s and 25.1 +/- 4.9 cm(2)/s, respectively. Within-day and between-day coefficients of variation were < 20% (range, 0.41% to 16.4%) for all variables except mean active emptying rate (between-day coefficient of variation, 29.2%). Agreement between variables measured via speckle tracking echocardiography and the manual tracing method was good, and differences between methods were nonsignificant. Conclusions and Clinical Relevance-Evaluation of left atrial phasic function via speckle tracking echocardiography was feasible; repeatability and reproducibility of measurements were adequate in healthy dogs. Studies are needed to determine clinical applicability in canine patients. (Am J Vet Res 2013;74:864-869)
• Usefulness of dabigatran etexilate as periprocedural anticoagulation therapy for atrial fibrillation ablation

  Hirosuke Yamaji, Takashi Murakami, Kazuyoshi Hina, Shunichi Higashiya, Hiroshi Kawamura, Masaaki Murakami, Shigeshi Kamikawa, Satoshi Hirohata, Shozo Kusachi

  Clinical Drug Investigation 33 (6) 409 - 418 1173-2563 2013/06 [Refereed][Not invited]
   

  Background: The usefulness of dabigatran etexilate for the prevention of stroke in patients with atrial fibrillation (AF) has been reported. Objectives: In this study the efficacy and safety of dabigatran etexilate for anticoagulation for AF ablation were examined. Method: Patients were divided into three groups: Group 1, interrupted warfarin bridged by heparin between pre- and post-ablation Group 2, continuous warfarin therapy and Group 3, dabigatran etexilate therapy. Anticoagulation therapy with warfarin or dabigatran etexilate was performed from 30 days before to at least 90 days after AF ablation. Dabigatran etexilate was administered at 110 or 150 mg twice daily, depending on renal function and age. Results: Patients' clinical characteristics, associated disorders, echocardiographic parameters and arrhythmia status were not different among the three groups. Procedural parameters such as procedural time and radiofrequency energy supply were also not different among the three groups. The dabigatran etexilate group and the warfarin groups had no embolic complications (stroke, cerebral transient ischaemic attack, deep venous thrombosis or pulmonary embolism). No pericardial tamponade was observed in the dabigatran etexilate group, while two patients in each of Group 1 (2/194, 1.0 %) and Group 2 (2/203, 0.98 %) developed cardiac tamponade, though the differences were not significant. Pericardial effusion and groin haematoma were observed in one patient each (1/105, 0.9 %) in the dabigatran etexilate group, and the incidences were not different from the warfarin group (Group 1: 4/194, 2.1 % and 2/194, 1.0 % Group 2: 3/203, 1.5 % and 2/203, 1.0 %, respectively). As a whole, the safety outcomes did not differ among the three groups. Conclusion: Dabigatran etexilate is an effective and safe anticoagulation therapy for AF ablation. Thus, dabigatran etexilate appears to be useful as an alternative anticoagulant therapy to warfarin for AF ablation. © 2013 Springer International Publishing Switzerland.
• Commensal microbe-derived butyrate induces colonic regulatory T cells.

  Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, Nakanishi Y, Uetake C, Kato K, Kato T, Takahashi M, Fukuda NR, Murakami M, Miyauchi E, Hino S, Atarashi K, Onawa S, Fujimura Y, Lockett T, Clarke JM, Topping DL, Tomita M, Hori S, Ohara O, Morita T, Koseki H, Kikuchi J, Honda K, Hase K, Ohno H

  Nature 500 232-236  2013/06 [Refereed][Not invited]
  
• Erratum to Disease-Association Analysis of an Inflammation-Related Feedback Loop [Cell Reports 3, (2013) 946-959]

  Masaaki Murakami, Masaya Harada, Daisuke Kamimura, Hideki Ogura, Yuko Okuyama, Noriko Kumai, Azusa Okuyama, Rajeev Singh, Jing-Jing Jiang, Toru Atsumi, Sayaka Shiraya, Yuji Nakatsuji, Makoto Kinoshita, Hitoshi Kohsaka, Makoto Nishida, Saburo Sakoda, Nobuyuki Miyasaka, Keiko Yamauchi-Takihara, Toshio Hirano

  Cell Reports 3 (5) 1754  2211-1247 2013/05/30 [Refereed][Not invited]
  
• IL-6 amplifier activation in epithelial regions of bronchi after allogeneic lung transplantation

  Jihye Lee, Tomoyuki Nakagiri, Daisuke Kamimura, Masaya Harada, Takahiro Oto, Yoshiyuki Susaki, Yasushi Shintani, Masayoshi Inoue, Shinichiro Miyoshi, Eiichi Morii, Toshio Hirano, Masaaki Murakami, Meinoshin Okumura

  INTERNATIONAL IMMUNOLOGY 25 (5) 319 - 332 0953-8178 2013/05 [Refereed][Not invited]
   

  An IL-6-triggered NF-B loop: the IL-6 amplifier occurs in epithelia of a transplanted human lung.The IL-6 amplifier, a positive feedback loop for NFB signaling, which was originally found to be activated by IL-17A and IL-6 stimulation in non-immune cells, is molecularly a simultaneous activator of NFB and signal transducer and activator of transcription 3 (STAT3), functionally a local chemokine inducer and pathologically a machinery for inflammation development. It has been shown that IL-6 amplifier activation in epithelial cells contributes to rejection responses in a mouse chronic rejection model that develops a bronchiolitis obliterans (BO)-like disease. We investigated whether the IL-6 amplifier is activated in BO regions of a human lung graft after allogeneic transplantation. NFB and STAT3 molecules were phosphorylated in the epithelial regions of bronchi that localized in the BO regions. Additionally, chemokine ligand 2 (CCL2), and CD4 T cells and macrophages increased in these regions. Furthermore, human lung epithelial cells expressed CCL2 after stimulation by IFN in the presence of IL-6 and epidermal growth factor via enhanced STAT3 signaling, which parallels behavior seen in the mouse model. Thus, our results suggest that the IL-6 amplifier in the epithelial cells of grafts is involved in chronic rejection after lung transplantation, suggesting that the amplifier may be a valuable therapeutic target to prevent chronic rejection after lung transplantation.
• Disease-association analysis of an inflammation-related feedback loop.

  Masaaki Murakami, Masaya Harada, Daisuke Kamimura, Hideki Ogura, Yuko Okuyama, Noriko Kumai, Azusa Okuyama, Rajeev Singh, Jing-Jing Jiang, Toru Atsumi, Sayaka Shiraya, Yuji Nakatsuji, Makoto Kinoshita, Hitoshi Kohsaka, Makoto Nishida, Saburo Sakoda, Nobuyuki Miyasaka, Keiko Yamauchi-Takihara, Toshio Hirano

  Cell reports 3 (3) 946 - 59 2013/03/28 [Refereed][Not invited]
   

  The IL-6-triggered positive feedback loop for NFκB signaling (or the IL-6 amplifier/Inflammation amplifier) was originally discovered as a synergistic-activation signal that follows IL-17/IL-6 stimulation in nonimmune cells. Subsequent results from animal models have shown that the amplifier is activated by stimulation of NFκB and STAT3 and induces chemokines and inflammation via an NFκB loop. However, its role in human diseases is unclear. Here, we combined two genome-wide mouse screens with SNP-based disease association studies, revealing 1,700 genes related to the IL-6 amplifier, 202 of which showed 492 indications of association with ailments beyond autoimmune diseases. We followed up on ErbB1 from our list. Blocking ErbB1 signaling suppressed the IL-6 amplifier, whereas the expression of epiregulin, an ErbB1 ligand, was higher in patients with inflammatory diseases. These results indicate that the IL-6 amplifier is indeed associated with human diseases and disorders and that the identified genes may make for potential therapeutic targets.
• Autoimmune disorder phenotypes in Hvcn1-deficient mice

  Mari Sasaki, Akihiro Tojo, Yoshifumi Okochi, Nana Miyawaki, Daisuke Kamimura, Akihito Yamaguchi, Masaaki Murakami, Yasushi Okamura

  BIOCHEMICAL JOURNAL 450 295 - 301 0264-6021 2013/03 [Refereed][Not invited]
   

  H-v channels (voltage-gated proton channels) are expressed in blood cells, microglia and some types of epithelial cells. In neutrophils H-v channels regulate the production of reactive oxygen species through regulation of membrane potential and intracellular pH. H-v channels have also been suggested to play a role in sperm physiology in the human. However, the functions of the H-v channel at the whole-body level are not fully understood. In the present paper we show that Hvcn1 (voltage-gated hydrogen channel 1)-knockout mice show splenomegaly, autoantibodies and nephritis, that are reminiscent of human autoimmune diseases phenotypes. The number of activated T-cells was larger in Hvcn1-deficient mice than in the wild-type mice., Upon viral infection this was remarkably enhanced in Hvcn1-deficient mice. The production of superoxide anion in T-cells upon stimulation with PMA was significantly attenuated in the Hvcn1-deficient mice. These results suggest that H-v channels regulate T-cell homoeostasis in vivo.
• Ecto-Nucleoside Triphosphate Diphosphohydrolase 7 Controls Th17 Cell Responses through Regulation of Luminal ATP in the Small Intestine

  Takashi Kusu, Hisako Kayama, Makoto Kinoshita, Seong Gyu Jeon, Yoshiyasu Ueda, Yoshiyuki Goto, Ryu Okumura, Hiroyuki Saiga, Takashi Kurakawa, Kayo Ikeda, Yuichi Maeda, Jun-ichi Nishimura, Yasunobu Arima, Koji Atarashi, Kenya Honda, Masaaki Murakami, Jun Kunisawa, Hiroshi Kiyono, Meinoshin Okumura, Masahiro Yamamoto, Kiyoshi Takeda

  JOURNAL OF IMMUNOLOGY 190 (2) 774 - 783 0022-1767 2013/01 [Refereed][Not invited]
   

  Extracellular ATP is released from live cells in controlled conditions, as well as dying cells in inflammatory conditions, and, thereby, regulates T cell responses, including Th17 cell induction. The level of extracellular ATP is closely regulated by ATP hydrolyzing enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases). ENTPDase1/CD39, which is expressed in immune cells, was shown to regulate immune responses by downregulating the ATP level. In this study, we analyzed the immunomodulatory function of ENTPDase7, which is preferentially expressed in epithelial cells in the small intestine. The targeted deletion of Entpd7 encoding ENTPDase7 in mice resulted in increased ATP levels in the small intestinal lumen. The number of Th17 cells was selectively increased in the small intestinal lamina propria in Entpd7(-/-) mice. Th17 cells were decreased by oral administration of antibiotics or the ATP antagonist in Entpd7(-/-) mice, indicating that commensal microbiota-dependent ATP release mediates the enhanced Th17 cell development in the small intestinal lamina propria of Entpd7(-/-) mice. In accordance with the increased number of small intestinal Th17 cells, Entpd7(-/-) mice were resistant to oral infection with Citrobacter rodentium. Entpd7(-/-) mice suffered from severe experimental autoimmune encephalomyelitis, which was associated with increased numbers of CD4(+) T cells producing both IL-17 and IFN-gamma. Taken together, these findings demonstrate that ENTPDase7 controls the luminal ATP level and, thereby, regulates Th17 cell development in the small intestine. The Journal of Immunology, 2013, 190: 774-783.
• Regulation of immune cell infiltration into the CNS by regional neural inputs explained by the gate theory

  Yasunobu Arima, Daisuke Kamimura, Lavannya Sabharwal, Moe Yamada, Hidenori Bando, Hideki Ogura, Toru Atsumi, Masaaki Murakami

  Mediators of Inflammation 2013 898165  0962-9351 2013 [Refereed][Not invited]
   

  The central nervous system (CNS) is an immune-privileged environment protected by the blood-brain barrier (BBB), which consists of specific endothelial cells that are brought together by tight junctions and tight liner sheets formed by pericytes and astrocytic end-feet. Despite the BBB, various immune and tumor cells can infiltrate the CNS parenchyma, as seen in several autoimmune diseases like multiple sclerosis (MS), cancer metastasis, and virus infections. Aside from a mechanical disruption of the BBB like trauma, how and where these cells enter and accumulate in the CNS from the blood is a matter of debate. Recently, using experimental autoimmune encephalomyelitis (EAE), an animal model of MS, we found a "gateway" at the fifth lumber cord where pathogenic autoreactive CD4+ T cells can cross the BBB. Interestingly, this gateway is regulated by regional neural stimulations that can be mechanistically explained by the gate theory. In this review, we also discuss this theory and its potential for treating human diseases. © 2013 Yasunobu Arima et al.
• The gateway theory: Bridging neural and immune interactions in the CNS

  Daisuke Kamimura, Moe Yamada, Masaya Harada, Lavannya Sabharwal, Jie Meng, Hidenori Bando, Hideki Ogura, Toru Atsumi, Yasunobu Arima, Masaaki Murakami

  Frontiers in Neuroscience 7 (7) 204  1662-4548 2013 [Refereed][Not invited]
   

  The central nervous system (CNS) is considered an immune-privileged tissue protected by a specific vessel structure, the blood-brain barrier (BBB). Upon infection or traumatic injury in the CNS, the BBB is breached, and various immune cells are recruited to the affected area. In the case of autoimmune diseases in the CNS like multiple sclerosis (MS), autoreactive T cells against some CNS-specific antigens can theoretically attack neurons throughout the CNS. The affected CNS regions in MS patients can be detected as multiple focal plaques in the cerebrum, thoracic cord, and other regions. Vision problems are often associated with the initial phase of MS, suggesting a disturbance in the optic nerves. These observations raise the possibility that there exist specific signals that direct autoreactive T cells past the BBB and into particular sites of the CNS. Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we recently defined the mechanism of the pathogenesis in which regional neural stimulations modulate the status of the blood vessel endothelium to allow the invasion of autoreactive T cells into specific sites of the CNS via the fifth lumbar cord. This gate for autoreactive T cells can be artificially manipulated by removing gravity forces on the hind legs or by electric pulses to the soleus muscles, quadriceps, and triceps of mice, resulting in an accumulation of autoreactive T cells in the intended regions via the activation of regional neurons. Gating blood vessels by regional neural stimulations, a phenomenon we call the gateway theory, has potential therapeutic value not only in preventing autoimmunity, but also in augmenting the effects of cancer immunotherapies. © 2013 Kamimura, Yamada, Harada, Sabharwal, Meng, Bando, Ogura, Atsumi, Arima and Murakami.
• The gateway theory: How regional neural activation creates a gateway for immune cells via an inflammation amplifier

  Hideki Ogura, Yasunobu Arima, Daisuke Kamimura, Masaaki Murakami

  Biomedical Journal 36 (6) 269 - 273 2319-4170 2013 [Refereed][Not invited]
   

  The inflammation amplifier, a nuclear factor-kappa B (NF-kB)feedback loop in non-immune cells including fibroblasts and endothelial cells, describes how NF-kB-mediated transcriptions are enhanced to induce the inflammation in the presence of signal Tranducer and Activator of Transcription 3 (STAT3) activation. It was originally discovered in rheumatoid arthritis and multiple sclerosis mouse models and has since been shown to be associated with various human diseases and disorders including autoimmune diseases, metabolic syndromes, neurodegenerative diseases, and other inflammatory diseases. The amplifier begins with IL-17, which acts as the main signal to express NF-kB-mediated transcriptions, and IL-6, an NF-kB target, which functions as a fuel for the inflammation amplifier. Indeed, other NF-kB targets including various chemokines also act as effector molecules that cause local accumulation of various immune cells and subsequent inflammation. Through extensive studies in the multiple sclerosis model experimental autoimmune encephalomyelitis, we recently demonstrated that regional neural activation induces excess activation of the inflammation amplifier at specific blood vessels in the fifth lumbar cord, creating a gateway for immune cells to enter the central nervous system (CNS). We thus propose the gateway theory to describe how regional neural activation enables immune cells to enter the CNS from the blood and argue that this theory might provide novel therapeutic targets for inflammatory diseases and disorders.
• Outcome of chemoradiotherapy using orthovoltage X-ray for canine intranasal tumors.

  Yanase S, Mori T, Hoshino Y, Ito Y, Murakami M, Nishitani Y, Noguchi S, Sakai H, Yanai T, Maruo K

  Jpn J Vet Anesth Surg Japanese Society of Veterinary Anesthesia & Surgery 43 (3&4) 41 - 45 0916-5908 2013 [Not refereed][Not invited]
   

  We retrospectively reviewed 11 canine cases of malignant intranasal tumors treated with chemoradiotherapy. All the dogs received orthovoltage radiation in weekly 6.3 Gy fractions and multi-agent chemotherapy containing doxorubicin and carboplatin. The dogs were irradiated 4-11 times; the total dose of irradiation was 25.2-69.3 Gy. The median irradiation dose was 37.8 Gy. Clinical symptoms improved in 9 cases. Computed tomography (CT) images taken before and after treatment showed tumor regression in 8 cases; complete remission was achieved in 2 dogs and partial remission in 6. The median survival time was 478 days (99-969 days). Although adverse effects of radiation were observed in 7 cases, the symptoms were mild and improved with symptomatic treatment. In conclusion, our retrospective study suggests that chemoradiotherapy using orthovoltage might be effective for managing canine malignant intranasal tumors and for prolonging the survival period without affecting the quality of life.
• [IL-6 signal transduction and its physiological roles].

  Murakami M, Arima Y, Hirano T

  Nihon rinsho. Japanese journal of clinical medicine 70 Suppl 8 192 - 206 0047-1852 2012/11 [Refereed][Not invited]
  
• サイトカインとケモカイン-1 MTF-1の新たな標的遺伝子であるmolecular-XはNF-κB活性化を介してIL-6アンプ活性化を制御する(Molecular-X, which is a novel MTF-1 target gene regulates the IL-6 amplifier activation via NF κB activation)

  Otsubo Ryota, Okuyama Yuko, Andrews Glen K., Hirano Toshio, Murakami Masaaki

  日本免疫学会総会・学術集会記録 41 65 - 65 0919-1984 2012/11
• Involvement of Mitochondrial Genes of Babesia gibsoni in Resistance to Diminazene Aceturate

  Bandula Kumara Wickramasekara Rajapakshage, Masahiro Yamasaki, Shiang-Jyi Hwang, Noboru Sasaki, Masahiro Murakami, Yu Tamura, Sue Yee Lim, Kensuke Nakamura, Hiroshi Ohta, Mitsuyoshi Takiguchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (9) 1139 - 1148 0916-7250 2012/09 [Refereed][Not invited]
   

  The stability of the characteristics of the diminazene aceturate (DA)-resistant B. gibsoni isolate was initially determined in vitro. Part of the DA-resistant B. gibsoni isolate was cultured without DA for 4 weeks, and then newly exposed to 200 ng/m/ DA. As a result, this isolate could proliferate the same as the DA-resistant isolate, indicating that the characteristic of DA resistance was stable in the DA-resistant isolate. Additionally, the level of parasitemia in the DA-resistant isolate was comparatively lower than in the wild-type, suggesting that the proliferation potential of the DA-resistant isolate would be lower than that of the wild-type. Subsequently, to investigate the involvement of mitochondrial DNA (mtDNA) in DA resistance in B. gibsoni, the nucleotide sequences and deduced amino acid sequences of mitochondrial genes such as COXI, COXIII, and CYTb genes of the DA-resistant isolate, were compared with those of the wild-type. As a result, these three genes were not altered in the DA-resistant B. gibsoni isolate. Moreover, the transcription levels of COXI,COXIII, and CYTb genes were observed by semi-quantitative RT-PCR. As a result, the gene transcription of those genes in the DA-resistant isolate was not significantly altered. These results indicated that DA did not affect mtDNA directly in DA-resistant B. gibsoni. Thus, it is suggested that mtDNA should not be deeply involved in DA resistance in B. gibsoni.
• IL-6 Amplifier, NF-kappa B-Triggered Positive Feedback for IL-6 Signaling, in Grafts Is Involved in Allogeneic Rejection Responses

  Jihye Lee, Tomoyuki Nakagiri, Takahiro Oto, Masaya Harada, Eiichi Morii, Yasushi Shintani, Masayoshi Inoue, Yoichiro Iwakura, Shinichiro Miyoshi, Meinoshin Okumura, Toshio Hirano, Masaaki Murakami

  JOURNAL OF IMMUNOLOGY 4 189 (4) 1928 - 1936 0022-1767 2012/08 [Refereed][Not invited]
   

  The IL-6-amplifier first was discovered as a synergistic activation mechanism for NF-kappa B/STAT3 in type 1 collagen(+) cells. This process is marked by the hyperinduction of chemokines and subsequent local inflammation that leads to autoimmune diseases. In this study, we show that IL-6 amplifier activation in grafts plays important roles in allogeneic graft rejection by using a tracheal heterotopic transplantation model that includes bronchiolitis obliterans, a pathological marker for chronic rejection. IL-6, epidermal growth factor, and IFN-gamma all stimulate IL-6 amplifier activation, whereas CCL2, a chemotactic factor for Th1 cells, was one of the amplifier's main targets. Interestingly, IFN-gamma hyperinduced CCL2 in type 1 collagen(+) cells via the IL-6 amplifier at least in vitro. In addition, we detected IL-6, CCL2, phospho-STAT3, and phospho-NF-kappa B in epithelial type 1 collagen(+) cells of allogeneic tracheal grafts. These results show that IL-6 amplifier activation in grafts plays a critical role for graft rejection responses after allogeneic transplantation, including chronic rejection. From these results, we consider whether the IL-6 amplifier in grafts might be a valuable therapeutic target for the prevention of transplant rejection, including chronic rejection. The Journal of Immunology, 2012, 189: 1928-1936.
• Analysis of energy generation and glycolysis pathway in diminazene aceturate-resistant Babesia gibsoni isolate in vitro

  Bandula Kumara Wickramasekara Rajapakshage, Masahiro Yamasaki, Masahiro Murakami, Yu Tamura, Sue Yee Lim, Tatsuyuki Osuga, Noboru Sasaki, Kensuke Nakamura, Hiroshi Ohta, Mitsuyoshi Takiguchi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (2-3) 51 - 61 0047-1917 2012/08 [Refereed][Not invited]
   

  In our previous study, the level of parasitemia of the diminazene aceturate (DA)-resistant B. gibsoni isolate was continuously lower than that of the wild-type, indicating the possible alteration of energy metabolism in that isolate. Therefore, in the present study, the concentrations of ATP, glucose, lactate, and pyruvate, and the activities of lactate dehydrogenase and pyruvate kinase in the wild-type and DA-resistant isolate of B. gibsoni were measured and compared to investigate the amount of energy generation and the activity of the glycolysis pathway. As a result, the intracellular ATP and glucose concentrations in the DA-resistant B. gibsoni isolate were significantly higher than those in the wild-type. Meanwhile, the concentrations of lactate and pyruvate and the activities of lactate dehydrogenase and pyruvate kinase in the DA-resistant B. gibsoni isolate were not different from those in the wild-type. These results indicated that the DA-resistant B. gibsoni isolate contained a higher ATP concentration than the wild-type, but the activity of the glycolysis pathway was not altered in the DA-resistant B. gibsoni isolate. However, we could not determine the mechanism of the high energy production of the DA-resistant B. gibsoni isolate. Further studies on the energy metabolism of B. gibsoni are necessary to clarify the mechanism of the high energy production in the DA-resistant B. gibsoni isolate.
• Intestinal CX3C chemokine receptor 1(high) (CX(3)CR1(high)) myeloid cells prevent T-cell-dependent colitis

  Hisako Kayama, Yoshiyasu Ueda, Yukihisa Sawa, Seong Gyu Jeon, Ji Su Ma, Ryu Okumura, Atsuko Kubo, Masaru Ishii, Taku Okazaki, Masaaki Murakami, Masahiro Yamamoto, Hideo Yagita, Kiyoshi Takeda

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (13) 5010 - 5015 0027-8424 2012/03 [Refereed][Not invited]
   

  Adequate activation of CD4(+) T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4(+) T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX3C chemokine receptor 1(high) (CX(3)CR1(high)) CD11b(+) CD11c(+) cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4(+) T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX(3)CR1(high) CD11b(+) CD11c(+) cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX(3)CR1(high) CD11b(+) CD11c(+) cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid
• Regional Neural Activation Defines a Gateway for Autoreactive T Cells to Cross the Blood-Brain Barrier

  Yasunobu Arima, Masaya Harada, Daisuke Kamimura, Jin-Haeng Park, Fuminori Kawano, Fiona E. Yull, Tadafumi Kawamoto, Yoichiro Iwakura, Ulrich A. K. Betz, Gabriel Marquez, Timothy S. Blackwell, Yoshinobu Ohira, Toshio Hirano, Masaaki Murakami

  CELL 148 (3) 447 - 457 0092-8674 2012/02 [Refereed][Not invited]
   

  Although it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.
• The Pathological and Physiological Roles of IL-6 Amplifier Activation

  Masaaki Murakami, Toshio Hirano

  INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES 8 (9) 1267 - 1280 1449-2288 2012 [Refereed][Not invited]
   

  The NF kappa B-triggered positive feedback loop for IL-6 signaling in type 1 collagen+ non-immune cells (IL-6 amplifier) was first discovered to be a synergistic signal that is activated following IL-17A and IL-6 stimulation in type 1 collagen+ non-immune cells. Subsequent disease models have shown that it can also be stimulated by the simultaneous activation of NF kappa B and STAT3, functions as a local chemokine inducer, and acts as a mechanism for local inflammation, particularly chronic ones like rheumatoid arthritis and a multiple sclerosis. Moreover, we have recently shown that hyper activation of the IL-6 amplifier via regional neural activation establishes a gateway for immune cells including autoreactive T cells to pass the blood-brain barrier at dorsal vessels in 5th lumbar cord. Here we review how the IL-6 amplifier is activated by neural activation and the physiological relevance of the gateway to the central nervous system. Accumulating evidences continues to suggest that the IL-6 amplifier offers a potential molecular mechanism for the relationship between neural activation and the development of inflammatory diseases, which could establish a new interdisciplinary field that fuses neurology and immunology.
• The molecular mechanisms of chronic inflammation development

  Masaaki Murakami, Toshio Hirano

  FRONTIERS IN IMMUNOLOGY 3 323  1664-3224 2012 [Refereed][Not invited]
  
• ACTIVATION OF MICROBUBBLES BY SHORT-PULSED ULTRASOUND ENHANCES THE CYTOTOXIC EFFECT OF CIS-DIAMMINEDICHLOROPLATINUM (II) IN A CANINE THYROID ADENOCARCINOMA CELL LINE IN VITRO

  Noboru Sasaki, Nobuki Kudo, Kensuke Nakamura, Sue Yee Lim, Masahiro Murakami, W. R. Bandula Kumara, Yu Tamura, Hiroshi Ohta, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  ULTRASOUND IN MEDICINE AND BIOLOGY 38 (1) 109 - 118 0301-5629 2012/01 [Refereed][Not invited]
   

  Ultrasound targeted microbubble destruction has succeeded in delivering drugs and genes. This study was designed to explore characteristics of ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. Canine thyroid adenocarcinoma cells were exposed to short-pulsed diagnostic ultrasound in the presence of cis-diamminedichloroplatinum (II) (cisplatin) and ultrasound contrast agent Sonazoid (R) microbubbles. The cytotoxic effect of cisplatin was enhanced by short-pulsed diagnostic ultrasound and microbubbles. Incubation time with microbubbles influenced the cytotoxic effect of cisplatin. However, exposure duration did not affect the cytotoxic effect of cisplatin. Therefore, short-pulsed diagnostic ultrasound may activate microbubbles near cells and deliver cisplatin into cells. In addition, activation of microbubbles may be concluded in a short time. Our results suggest that short exposure duration could be potentially sufficient to induce efficient drug delivery by ultrasound targeted microbubble destruction using short-pulsed diagnostic ultrasound. (E-mail: mtaki@vetmed.hokudai.ac.jp) (C) 2012 World Federation for Ultrasound in Medicine & Biology.
• Sufficient pulmonary vein image quality of non-enhanced multi-detector row computed tomography for pulmonary vein isolation by catheter ablation

  Hirosuke Yamaji, Kazuyoshi Hina, Hiroshi Kawamura, Takashi Murakami, Masaaki Murakami, Satoshi Hirohata, Natsuki Ohmaru, Shozo Kusachi

  Europace 14 (1) 52 - 59 1099-5129 2012/01 [Refereed][Not invited]
   

  Aims: We evaluated the quality of non-enhanced multi-detector row computed tomography (MDCT) images of the pulmonary vein (PV) and the clinical results of catheter ablation to isolate the PV for treatment of atrial fibrillation (AF) without the use of contrast medium in patients with chronic kidney disease (CKD). Methods and Results: We compared PV images quantitatively and qualitatively between non-enhanced and enhanced images (n 50). Procedural parameters and clinical outcomes were compared between catheter ablation for AF referring solely to non-enhanced MDCT in CKD patients (n 20) and using enhanced MDCT images integrated with electroanatomic mapping in non-CKD patients (n 30). In gross anatomy, complete agreement was obtained between non-enhanced and enhanced MDCT images. BlandAltman plots and cumulative coefficient variation showed good agreement in PV diameter determination between non-enhanced and enhanced MDCT images. There were no statistically significant differences in procedural or fluoroscopic times between PV isolation only referring to non-enhanced MDCT images and that using enhanced MDCT images integrated with electroanatomic mapping. Similarly, the ablation success rate and AF-free status at 3 months after PV isolation did not differ between PV isolation referring only to non-enhanced MDCT images and that using an electroanatomic integration system. No complications occurred in PV isolation with or without enhanced MDCT. Conclusion: sNon-enhanced MDCT provides adequate PV image quality both quantitatively and qualitatively. The present study suggests that catheter ablation referring solely to non-enhanced MDCT images for AF could be performed with clinically acceptable results. These findings warrant further studies involving a much larger number of patients to confirm the present results. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2011. For permissions please.
• Inguinal skin window for chronic two-photon In vivo imaging of mouse lymph node

  Yutaka Komai, Punniyakoti Thanikachalam Veeraveedu, Daisuke Kamimura, Masaaki Murakami

  2012 WORLD AUTOMATION CONGRESS (WAC) 2012 [Refereed][Not invited]
   

  We developed an animal model for chronic observation with using two-photon microscopy. To demonstrate potency of the model, T-cell migration and proliferation in inguinal lymph node were observed for two weeks on singlemice. The image quality was maintained enough high during a observation period to count and trace cell population and migration.
• Effects and Mechanisms of Action of Polyene Macrolide Antibiotic Nystatin on Babesia gibsoni In Vitro

  Masahiro Yamasaki, Norihisa Tamura, Kensuke Nakamura, Noboru Sasaki, Masahiro Murakami, Wickramasekara Rajapakshage, Bandula Kumara, Yu Tamura, Sue Yee Lim, Hiroshi Ohta, Mitsuyoshi Takiguchi

  JOURNAL OF PARASITOLOGY 97 (6) 1190 - 1192 0022-3395 2011/12 [Refereed][Not invited]
   

  Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 mu g/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.
• サイトカイン・ケモカイン 新しい生理機能 MTF-1標的分子がIL-6アンプの活性化を制御する(A MTF-1 target, which regulates the IL-6 amplifier activation)

  大坪 亮太, 奥山 祐子, Andrews Glen K., 平野 俊夫, 村上 正晃

  日本免疫学会総会・学術集会記録 40 112 - 112 0919-1984 2011/11
• Zinc homeostasis and signaling in health and diseases

  Toshiyuki Fukada, Satoru Yamasaki, Keigo Nishida, Masaaki Murakami, Toshio Hirano

  JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY 16 (7) 1123 - 1134 0949-8257 2011/10 [Refereed][Not invited]
   

  The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems that include growth retardation, immunodeficiency, hypogonadism, and neuronal and sensory dysfunctions. Zn homeostasis is regulated through Zn transporters, permeable channels, and metallothioneins. Recent studies highlight Zn's dynamic activity and its role as a signaling mediator. Zn acts as an intracellular signaling molecule, capable of communicating between cells, converting extracellular stimuli to intracellular signals, and controlling intracellular events. We have proposed that intracellular Zn signaling falls into two classes, early and late Zn signaling. This review addresses recent findings regarding Zn signaling and its role in physiological processes and pathogenesis.
• SIRP alpha/CD172a Regulates Eosinophil Homeostasis

  Noel Verjan Garcia, Eiji Umemoto, Yasuyuki Saito, Mikako Yamasaki, Erina Hata, Takashi Matozaki, Masaaki Murakami, Yun-Jae Jung, So-Youn Woo, Ju-Young Seoh, Myoung Ho Jang, Katsuyuki Aozasa, Masayuki Miyasaka

  JOURNAL OF IMMUNOLOGY 5 187 (5) 2268 - 2277 0022-1767 2011/09 [Refereed][Not invited]
   

  Eosinophils are abundant in the lamina propria of the small intestine, but they rarely show degranulation in situ under steady-state conditions. In this study, using two novel mAbs, we found that intestinal eosinophils constitutively expressed a high level of an inhibitory receptor signal regulatory protein alpha (SIRP alpha)/CD172a and a low, but significant, level of a tetraspanin CD63, whose upregulation is closely associated with degranulation. Cross-linking SIRP alpha/CD172a on the surface of wild-type eosinophils significantly inhibited the release of eosinophil peroxidase induced by the calcium ionophore A23187, whereas this cross-linking effect was not observed in eosinophils isolated from mice expressing a mutated SIRP alpha/CD172a that lacks most of its cytoplasmic domain (SIRP alpha Cyto(-/-)). The SIRP alpha Cyto(-/-) eosinophils showed reduced viability, increased CD63 expression, and increased eosinophil peroxidase release with or without A23187 stimulation in vitro. In addition, SIRP alpha Cyto(-/-) mice showed increased frequencies of Annexin V-binding eosinophils and free MBP(+)CD63(+) extracellular granules, as well as increased tissue remodeling in the small intestine under steady-state conditions. Mice deficient in CD47, which is a ligand for SIRP alpha/CD172a, recapitulated these phenomena. Moreover, during Th2-biased inflammation, increased eosinophil cell death and degranulation were obvious in a number of tissues, including the small intestine, in the SIRP alpha Cyto(-/-) mice compared with wild-type mice. Collectively, our results indicated that SIRP alpha/CD172a regulates eosinophil homeostasis, probably by interacting with CD47, with substantial effects on eosinophil survival. Thus, SIRP alpha/CD172a is a potential therapeutic target for eosinophil-associated diseases. The Journal of Immunology, 2011, 187: 2268-2277.
• Expression and subcellular localization of apical junction proteins in canine duodenal and colonic mucosa

  Hiroshi Ohta, Tomoki Yamaguchi, B. K. Wickramasekara Rajapakshage, Masahiro Murakami, Noboru Sasaki, Kensuke Nakamura, Shiang-Jyi Hwang, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  AMERICAN JOURNAL OF VETERINARY RESEARCH 72 (8) 1046 - 1051 0002-9645 2011/08 [Refereed][Not invited]
   

  Objective-To examine the expression and distribution of tight junction (TJ) and adherens junction (AM proteins in canine duodenal and colonic mucosa. Sample-Mucosa obtained from 4 healthy Beagles. Procedures-Biopsy specimens of the duodenum and colon were obtained via endoscopy from 4 healthy dogs. The expression patterns and subcelluar localization of claudin-1, -2, -3, -4, -5, -7, and -8; E-cadherin; and beta-catenin in the duodenum and colon were analyzed by use of immunoblotting and immunofluorescence microscopy. Results-In the duodenum, there was clear expression of claudin-3 and -5, E-cadherin, and beta-catenin proteins and weak expression of claudin-7 protein. In contrast, there was clear expression of claudin-2 and -3, E-cadherin, and beta-catenin proteins and weak expression of claudin-5 and -7 proteins in the colon, as determined by use of immunoblotting. As determined by the use of immunofluorescence microscopy, the duodenum and colon had staining for claudin-3 and -5, E-cadherin, and beta-catenin in the most apical region and staining for claudin-7 in the basolateral region. Staining for claudin-2 was also observed in the colon. Conclusions and Clinical Relevance-Information was provided about the expression patterns of TJ and AJ proteins in the duodenum and colon of clinically normal dogs. These results may provide valuable information for use in evaluating the importance of these TJ and AJ proteins in the pathogenesis of inflammatory bowel disease in dogs. (Am J Vet Res 2011;72:1046-1051)
• Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

  Masahiro Yamasaki, Mikiko Ishida, Kensuke Nakamura, Noboru Sasaki, Masahiro Murakami, Wickramasekara Rajapakshage Bandula Kumara, Yu Tamura, Sue Yee Lim, Hiroshi Ohta, Mitsuyoshi Takiguchi

  VETERINARY PARASITOLOGY 180 (3-4) 215 - 225 0304-4017 2011/08 [Refereed][Not invited]
   

  Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody. (C) 2011 Elsevier B.V. All rights reserved.
• A four-step model for the IL-6 amplifier, a regulator of chronic inflammations in tissue-specific MHC class II-associated autoimmune diseases

  Masaaki Murakami, Toshio Hirano

  FRONTIERS IN IMMUNOLOGY 2 22  1664-3224 2011 [Refereed][Not invited]
   

  It is commonly thought that autoimmune diseases are caused by the breakdown of self-tolerance, which suggests the recognition of specific antigens by autoreactive CD4+ T cells contribute to the specificity of autoimmune diseases (Marrack et al., 2001; Mathis and Benoist, 2004). In several cases, however, even for diseases associated with class II major histocompatibility complex (MHC) alleles, the causative tissue-specific antigens recognized by memory/activated CD4+ T cells have not been established (Mocci et al., 2000; Skapenko et al., 2005). Rheumatoid arthritis (RA) and arthritis in F759 knock-in mice (F759 mice) are such examples (Atsumi et al., 2002; Brennan et al., 2002; Falgarone 2009). These include associations with class II MHC and CD4 molecules; increased numbers of memory/activated CD4+ T cells; and improved outcomes in response to sup-pressions and/or deficiencies in class II MHC molecules, CD4+ T cells, and the T cell survival cytokine IL-7 Regarding the development of arthritis in F759 mice, it is not only the immune system, but also non-immune tissue that are involved, indicating that the importance of their interactions (Sawa et al., 2006, 2009; Ogura et al., 2008; Hirano, 2010; Murakami et al., 2011). Furthermore, we have shown that local events such as microbleeding together with an accumulation of activated CD4+ T cells in a manner independent of tissue antigen-recognitions induces arthritis in the joints of F759 mice (Murakami et al., 2011). For example, local microbleeding-mediated CCL20 expression induce such an accumulation, causing arthritis development via chronic activation of an IL-17A-dependent IL-6 signaling amplification loop in type 1 collagen+ cells that is triggered by CD4+ T cell-derived cytokine(s) such as IL-17A, which leads to the synergistic activation of STAT3 and NF kappa B in non-hematopoietic cells in the joint (Murakami et al., 2011). We named this loop the IL-6-mediated inflammation amplifier, or IL-6 amplifier for short (Ogura et al., 2008; Hirano, 2010; Murakami et al.,). Thus, certain class II MHC-associated, tissue-specific autoimmune diseases, including some RA subtypes, may be induced by local events that cause an antigen-independent accumulation of effector CD4+ T cells followed by the induction of the IL-6 amplifier in the affected tissue. In other words, in certain cases, the target tissue itself may determine the specificity of the autoimmune disease via activation of the IL-6 amplifier. To explain this hypothesis, we have proposed a four-step model for MHC class II-associated autoimmune diseases (Murakami et al., 2011): (1)T cell activation regardless of antigen specificity; (2) local events inducing a tissue-specific accumulation of activated T cells; (3) transient activation of the IL-6 amplifier; and (4) enhanced sensitivity to cytokines in the target tissue. The interaction of these events results in chronic activation of the IL-6 amplifier and subsequent manifestation of autoimmune diseases. Thus, the IL-6 amplifier, which is chronically activated by these four events, is a critical regulator of chronic inflammations in tissue-specific MHC class II-associated autoimmune diseases.
• Local microbleeding facilitates IL-6- and IL-17-dependent arthritis in the absence of tissue antigen recognition by activated T cells

  Masaaki Murakami, Yuko Okuyama, Hideki Ogura, Shogo Asano, Yasunobu Arima, Mineko Tsuruoka, Masaya Harada, Minoru Kanamoto, Yukihisa Sawa, Yoichiro Iwakura, Kiyoshi Takatsu, Daisuke Kamimura, Toshio Hirano

  JOURNAL OF EXPERIMENTAL MEDICINE 208 (1) 103 - 114 0022-1007 2011/01 [Refereed][Not invited]
   

  Cognate antigen recognition by CD4(+) T cells is thought to contribute to the tissue specificity of various autoimmune diseases, particularly those associated with class II MHC alleles. However, we show that localized class II MHC-dependent arthritis in F759 mice depends on local events that result in the accumulation of activated CD4(+) T cells in the absence of cognate antigen recognition. In this model, transfer of in vitro polarized Th17 cells combined with the induction of experimental microbleeding resulted in CCL20 production, the accumulation of T cells in the joints, and local production of IL-6. Disease induction required IL-17A production by transferred T cells, IL-6 and CCL20 expression, and STAT3 signaling in type I collagen-expressing cells. Our data suggest a model in which the development of autoimmune disease in F759 mice depends on four events: CD4(+) T cell activation regardless of antigen specificity, local events that induce T cell accumulation, enhanced sensitivity to T cell-derived cytokines in the tissue, and activation of IL-6 signaling in the tissue. This model provides a possible explanation for why tissue-specific antigens recognized by activated CD4(+) T cells have not been identified in many autoimmune diseases, especially those associated with class II MHC molecules.
• Babesia gibsoni: Detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis

  Masahiro Yamasaki, Yusuke Kobayashi, Kensuke Nakamura, Noboru Sasaki, Masahiro Murakami, Bandula Kumara Wickramasekara Rajapakshage, Hiroshi Ohta, Osamu Yamato, Yoshimitsu Maede, Mitsuyoshi Takiguchi

  EXPERIMENTAL PARASITOLOGY 127 (1) 119 - 126 0014-4894 2011/01 [Refereed][Not invited]
   

  In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis. (C) 2010 Elsevier Inc. All rights reserved.
• Reduced transcript levels of the heat shock protein 70 gene in diminazene aceturate-resistant Babesia gibsoni variants under low concentrations of diminazene aceturate

  Shiang-Jyi Hwang, Masahiro Yamasaki, Kensuke Nakamura, Noboru Sasaki, Masahiro Murakami, Bandula Kumara Wickramasekara Rajapakshage, Hiroshi Ohta, Yoshimitsu Maede, Mitsuyoshi Takiguchi

  JAPANESE JOURNAL OF VETERINARY RESEARCH 58 (3-4) 155 - 164 0047-1917 2010/11 [Refereed][Not invited]
   

  In our previous report, we developed a diminazene aceturate (DA)-resistant Babesia gibsoni strain that was maintained in culture with 200 ng/ml DA. While developing this strain, we also obtained DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks. Because heat shock protein 70 (Hsp70) seems to play important roles in adaptation to a stress environment in protozoan parasites, in the present study, we examined the copy number of B. gibsoni Hsp70 (BgHsp70) transcripts of those DA-resistant variants using quantitative real-time reverse transcription-polymerase chain reaction. We found that when wild-type B. gibsoni was exposed to 1 ng/ml DA, the level of BgHsp70 transcripts was decreased at day 14. The copy number of BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni, while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, although they were lower than in wild-type B. gibsoni. However, those in DA-resistant variants cultured with > 125 ng/ml DA were almost the same as wild-type B. gibsoni. These results indicated that the transcript levels of the BgHsp70 gene might be reduced when the parasites are exposed to a low concentration of DA, and then might recover to the normal level after achieving resistance against DA. We expect that further study of the function of BgHsp70 will elucidate the mechanism of drug resistance against DA in B. gibsoni.
• Development and Characterization of a Strain of Babesia gibsoni Resistant to Diminazene Aceturate In Vitro

  Shiang-Jyi Hwang, Masahiro Yamasaki, Kensuke Nakamura, Noboru Sasaki, Masahiro Murakami, Bandula Kumara Wickramasekara Rajapakshage, Hiroshi Ohta, Yoshimitsu Maede, Mitsuyoshi Takiguchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 72 (6) 765 - 771 0916-7250 2010/06 [Refereed][Not invited]
   

  We attempted to develop a strain of Babesia gibsoni resistant to diminazene aceturate (DA), an anti-babesial drug, in vitro. Since the DA-sensitive B. gibsoni strain could survive and proliferate in culture medium containing 1 ng/ml DA, the concentration of DA was gradually increased from 1 to 200 ng/ml. The results showed that the parasites could survive and proliferate in the medium containing 200 ng/ml DA, which was much higher than the 50% inhibitory concentration (IC(50)) of DA for B. gibsoni. Subsequently, these parasites were removed from erythrocytes and exposed directly to 200 ng/ml DA. They were able to survive and invade fresh erythrocytes, though the DA-sensitive B. gibsoni strain did not survive. Based on these results, the parasites cultured within 200 ng/ml DA were determined to be a DA-resistant B. gibsoni strain. In addition, the IC(50) levels of clindamycin, doxycycline and pentamidine for the DA-resistant B. gibsoni strain were determined. The IC(50) levels of clindamycin, doxycycline and pentamidine for the DA-resistant strain were higher than those for the DA-sensitive strain. The IC(50) of pentamidine for the resistant strain was much greater than that for the DA-sensitive strain. These results indicated that the DA-resistant B. gibsoni strain could have resistance not only to DA, but also to other anti-babesial drugs. In conclusion, we successfully developed a DA-resistant B. gibsoni strain in vitro.
• Zinc suppresses T(h)17 development via inhibition of STAT3 activation

  Chika Kitabayashi, Toshiyuki Fukada, Minoru Kanamoto, Wakana Ohashi, Shintaro Hojyo, Toru Atsumi, Naoko Ueda, Ichiro Azuma, Hiroshi Hirota, Masaaki Murakami, Toshio Hirano

  INTERNATIONAL IMMUNOLOGY 22 (5) 375 - 386 0953-8178 2010/05 [Refereed][Not invited]
   

  Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.
• Impact of Olmesartan on Progression of Coronary Atherosclerosis. A Serial Volumetric Intravascular Ultrasound Analysis From the OLIVUS (Impact of OLmesarten on progression of coronary atherosclerosis: evaluation by IntraVascular UltraSound) Trial

  Atsushi Hirohata, Keizo Yamamoto, Toru Miyoshi, Kunihiko Hatanaka, Satoshi Hirohata, Hitoshi Yamawaki, Issei Komatsubara, Masaaki Murakami, Eiki Hirose, Shinji Sato, Keisuke Ohkawa, Makoto Ishizawa, Hirosuke Yamaji, Hiroshi Kawamura, Shozo Kusachi, Takashi Murakami, Kazuyoshi Hina, Tohru Ohe

  Journal of the American College of Cardiology 55 (10) 976 - 982 0735-1097 2010/03/09 [Refereed][Not invited]
   

  Objectives: The aim of this study was to evaluate the impact of olmesartan on progression of coronary atherosclerosis. Background: Prior intravascular ultrasound (IVUS) trial results suggest slowing of coronary atheroma progression with some medicines but have not shown convincing evidence of regression with angiotension-II receptor blocking agents. Methods: A prospective, randomized, multicenter trial-OLIVUS (Impact of OLmesartan on progression of coronary atherosclerosis: evaluation by IntraVascular UltraSound)-was performed in 247 stable angina pectoris patients with native coronary artery disease. When these patients underwent percutaneous coronary intervention for culprit lesions, IVUS was performed in their nonculprit vessels (without angiographically documented coronary stenosis [< 50%]). Patients were randomly assigned to receive 10 to 40 mg of olmesartan or control and treated with a combination of beta-blockers, calcium channel blockers, diuretics, nitrates, glycemic control agents, and/or statins per physician's guidance. Serial IVUS examinations (baseline and 14-month follow-up) were performed to assess coronary atheroma volume. Volumetric IVUS analyses included lumen, plaque, vessel volume, percent atheroma volume (PAV), percent change in total atheroma volume (TAV) and PAV. Results: Patient characteristics and blood pressure control were identical between the 2 groups. However, follow-up IVUS showed significantly decreased TAV and percent change in PAV in the olmesartan group (5.4% vs. 0.6 % for TAV and 3.1% vs. -0.7% for percent change in PAV, control vs. olmesartan, p < 0.05 for all). Conclusions: These observations suggest a positive role in a potentially lower rate of coronary atheroma progression through the administration of olmesartan, an angiotension-II receptor blocking agent, for patients with stable angina pectoris. © 2010 American College of Cardiology Foundation.
• IL-6 positively regulates Foxp3(+)CD8(+) T cells in vivo

  Takayuki Nakagawa, Mineko Tsuruoka, Hideki Ogura, Yuko Okuyama, Yasunobu Arima, Toshio Hirano, Masaaki Murakami

  INTERNATIONAL IMMUNOLOGY 22 (2) 129 - 139 0953-8178 2010/02 [Refereed][Not invited]
   

  Although recent studies have identified regulatory roles for Foxp3(+)CD8(+) T cells, the mechanisms that induce their development and underlie their functions in vivo have not been elucidated. Here, we show that IL-6 positively regulates the Foxp3(+)CD8(+) T-cell development and function. The Foxp3(+)CD8(+) T cells that differentiated in vitro in the presence of IL-6 suppressed autoimmune colitis and arthritis in vivo. Moreover, Foxp3(+)CD8(+) T cells that developed in vivo in the presence of enhanced IL-6 signaling suppressed the development of a spontaneous T(h)17 cell-mediated autoimmune arthritis. Thus, we concluded that Foxp3(+)CD8(+) T cells develop in response to IL-6 and regulate chronic inflammation in T(h)17 cell-mediated F759 autoimmune arthritis. These results suggested that Foxp3(+)CD8(+) T cells may develop in response to IL-6 under certain inflammatory conditions in vivo and may regulate some other chronic inflammation diseases.
• CONTRAST-ENHANCED ULTRASONOGRAPHY FOR CHARACTERIZATION OF CANINE FOCAL LIVER LESIONS

  Kensuke Nakamura, Satoshi Takagi, Noboru Sasaki, Wickramasekara Rajapakshage Bandula Kumara, Masahiro Murakami, Hiroshi Ohta, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  VETERINARY RADIOLOGY & ULTRASOUND 51 (1) 79 - 85 1058-8183 2010/01 [Refereed][Not invited]
   

  In six normal beagles and 27 dogs with spontaneous focal or multifocal liver lesions, contrast-enhanced ultrasonography using Sonazoid (R) was performed. Sonazoid (R) is a newly developed second-generation contrast agent with the ability to be used for real-time contrast imaging along with parenchymal imaging. An appropriate protocol for the evaluation of all three phases (arterial, portal, and parenchymal) was established based on the results for normal beagles. By evaluation of the echogenicity of hepatic nodules during the arterial and parenchymal phases it was possible to differentiate malignant tumors from benign nodules with very high accuracy. In 15 of 16 dogs diagnosed as malignant tumors, nodules were clearly hypoechoic to the surrounding normal liver during the parenchymal phase. Additionally, malignant tumors had different echogenicity compared with the surrounding normal liver during the arterial phase in 14 of 15 dogs. In the portal phase, there were no characteristic findings. Contrast-enhanced ultrasonography with Sonazoid (R) appears to improve the characterization of canine focal and multifocal hepatic lesions.
• Primary Splenic Torsion in a Boston Terrier

  Hiroshi Ohta, Satoshi Takagi, Masahiro Murakami, Noboru Sasaki, Muneyoshi Yoshikawa, Kensuke Nakamura, Shiang-Jyi Hwang, Masahiro Yamasaki, Mitsuyoshi Takiguchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (11) 1533 - 1535 0916-7250 2009/11 [Refereed][Not invited]
   

  A 7-year-old female Boston terrier was referred to Hokkaido University Veterinary Teaching Hospital with a history of hemoglobinuria and anemia for several days. Abdominal radiographs showed splenomegaly, and Ultrasonography revealed a hypoechoic splenic parenchyma with interspersed linear echoes consistent with the ultrasonographic appearance of splenic torsion. Ultrasonography and computed tomography (CT) indicated a C-shaped spleen. Exploratory laparotomy confirmed the diagnosis of splenic torsion. A splenectomy was performed, and the dog recovered well Without complications. This is the first report of splenic torsion in Boston terriers, and the usefulness of ultrasonographic and CT findings of the splenic torsion was also confirmed.
• Hepatic Interleukin-7 Expression Regulates T Cell Responses

  Yukihisa Sawa, Yasunobu Arima, Hideki Ogura, Chika Kitabayashi, Jing-Jing Jiang, Toru Fukushima, Daisuke Kamimura, Toshio Hirano, Masaaki Murakami

  IMMUNITY 30 (3) 447 - 457 1074-7613 2009/03 [Refereed][Not invited]
   

  Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4(+) T cell and CD8(+) T cell survival, augmented CD8(+) T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.
• IFN-gamma expression in CD8(+) T cells regulated by IL-6 signal is involved in superantigen-mediated CD4(+) T cell death

  Toru Atsumi, Masae Sato, Daisuke Kamimura, Arisa Moroi, Yoichiro Iwakura, Ulrich A. K. Betz, Akihiko Yoshimura, Mika Nishihara, Toshio Hirano, Masaaki Murakami

  INTERNATIONAL IMMUNOLOGY 21 (1) 73 - 80 0953-8178 2009/01 [Refereed][Not invited]
   

  Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4(+) T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4(+) T cell death. Sag-induced CD4(+) T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-gamma was inversely correlated with the magnitude of IL-6 signaling, and IFN-gamma deficiency inhibited Sag-induced activated CD4(+) T cell death, suggesting that IL-6 suppresses CD4(+) T cell death via IFN-gamma expression. Interestingly, depletion of activated CD8(+) T cells inhibited Sag-mediated increases in IFN-gamma expression in IL-6 KO mice as well as the augmented CD4(+) T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8(+) T cells contributes to Sag-induced CD4(+) T cell death via IFN-gamma expression, highlighting this signaling axis in CD8(+) T cells as a potential therapeutic target for Sag-related syndromes.
• Successful cisplatin chemotherapy and radiotherapy in a dog with ovarian dysgerminoma.

  Nakayama M, Mori T, Iwatani N, Sakai H, Murakami M, Sato A, Maruo K

  J Jpn Vet Med Assoc 62 (5) 395 - 397 2009 [Not refereed][Not invited]
  
• 【樹状細胞による免疫制御と臨床応用 T細胞制御機構の理解から、樹状細胞療法の開発、自己免疫疾患・感染症の病態解明とその治療まで】樹状細胞の生物学と生理学(分化・サブセット活性化・動態) 樹状細胞による抗原提示の制御機構

  北條 慎太郎, 村上 正晃

  実験医学 (株)羊土社 26 (20) 3158 - 3168 0288-5514 2008/12 

  抗原提示とは、樹状細胞をはじめとする抗原提示細胞が細胞内で内在性あるいは外来性抗原を分解し、その抗原ペプチドをT細胞に提示する機構である。ペプチドの提示にはMHCが必要であり、一般的に内在性抗原由来のペプチドはMHCクラスI(MHC I)、外来性抗原抗原由来のペプチドはMHCクラスII(MHC II)によってCD8+T細胞あるいはCD4+T細胞に提示される。免疫寛容あるいは自己免疫応答は主としてMHCを介した樹状細胞とT細胞の相互作用に端を発し、不適当な免疫応答を回避するためにもMHCによる抗原提示機構は厳密に制御されていなければならない。MHCの抗原提示機構の研究はこれまで精力的に行われてきており、将来的に有用な医薬品の開発やワクチン製造に繋がる期待からもこれからますます活発になっていく分野であると言える。事実、近年ではERAAPなどの抗原ペプチドトリミングやその他抗原分解に携わる酵素が同定され、また内在性抗原の提示に限局していたMHC I抗原提示機構においても、従来の古典的経路の概念に当てはまらない複数の新しい外来性抗原のクロスプレゼンテーション経路が発見され脚光を浴びている。本稿では抗原提示の制御機構について最近の知見を含めて概説するとともに、これまでにわれわれが独自に得た抗原提示制御機構、(1)TRIF-GEFH1-RhoBシグナルによるMHC II輸送の制御機構、(2)亜鉛によるMHC II輸送の制御機構、(3)IL-6による樹状細胞成熟化制御についての知見を紹介する。(著者抄録)
• Interleukin-17 Promotes Autoimmunity by Triggering a Positive-Feedback Loop via Interleukin-6 Induction

  Hideki Ogura, Masaaki Murakami, Yuko Okuyama, Mineko Tsuruoka, Chika Kitabayashi, Minoru Kanamoto, Mika Nishihara, Yoichiro Iwakura, Toshio Hirano

  IMMUNITY 29 (4) 628 - 636 1074-7613 2008/10 [Refereed][Not invited]
   

  Dysregulated cytokine expression and signaling are major contributors to a number of autoimmune diseases. Interleukin-17A (IL-17A) and IL-6 are important in many disorders characterized by immune self-recognition, and IL-6 is known to induce the differentiation of T helper 17 (Th17) cells. Here we described an IL-17A-triggered positive-feed back loop of IL-6 signaling, which involved the activation of the transcription factors nuclear factor (NF)-kappa B and signal transducer and activator of transcription 3 (STAT3) in fibroblasts. Importantly, enhancement of this loop caused by disruption of suppressor of cytokine signaling 3 (SOCS3)-dependent negative regulation of the IL-6 signal transducer gp130 contributed to the development of arthritis. Because this mechanism also enhanced experimental autoimmune encephalomyelitis (EAE) in wild-type mice, it may be a general etiologic process underlying other Th17 cell-mediated autoimmune diseases.
• c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130

  Yoshinori Tanaka, Nobuyuki Tanaka, Yasushi Saeki, Keiji Tanaka, Masaaki Murakami, Toshio Hirano, Naoto Ishii, Kazuo Sugamura

  MOLECULAR AND CELLULAR BIOLOGY 28 (15) 4805 - 4818 0270-7306 2008/08 [Refereed][Not invited]
   

  Interieukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding alpha subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cb1, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.
• Intracellular zinc homeostasis and zinc signaling

  Masaaki Murakami, Toshio Hirano

  CANCER SCIENCE 99 (8) 1515 - 1522 1347-9032 2008/08 [Refereed][Not invited]
   

  Zinc (Zn) is an essential heavy metal that is incorporated into a number of human Zn metalloproteins. Zn plays important roles in nucleic acid metabolism, cell replication, and tissue repair and growth. Zn deficiency is associated with a range of pathological conditions, including impaired immunity, retarded growth, brain development disorders and delayed wound healing. Moreover, many reports have suggested that Zn is involved in cancer development and levels of Zn in serum and malignant tissues of patients with various types of cancer are abnormal. Zn may directly affect tumor cells by regulating gene expression profiles and/or cell viability, both of which are mediated in part by tumor-induced changes in Zn transporter expression. On the other hand, Zn may indirectly influence tumor cells by affecting processes within the cancer microenvironment, including immune responses; the functions and/or activity levels of immune cells that attack tumor cells are influenced by the intracellular Zn concentrations within those cells. In both cases, Zn contributes to intracellular metal homeostasis and/or signal transduction in tumor and immune cells. In this review article, we will summarize the current understanding of the roles of Zn homeostasis and signaling primarily in immune cells, with a discussion of the contributions of these processes to oncogenesis. (Cancer Sci 2008; 99: 1515-1522)
• Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

  Gui-Hua Jin, Toshio Hirano, Masaaki Murakami

  INTERNATIONAL IMMUNOLOGY 20 (6) 783 - 789 0953-8178 2008/06 [Refereed][Not invited]
   

  Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.
• Higher incidence and serum levels of minor cardiac biomarker elevation in sirolimus-eluting stent (Cypher) than bare metal stent implantations

  Tetsushi Seitou, Masaaki Murakami, Issei Komatsubara, Hiroshi Kawamura, Keizo Yamamoto, Kazuyoshi Hina, Satoshi Hirohata, Ryoko Shinohata, Yoshifumi Ninomiya, Shozo Kusachi

  Coronary Artery Disease 19 (2) 63 - 69 0954-6928 2008/03 [Refereed][Not invited]
   

  OBJECTIVES: Minor cardiac biomarker elevation after percutaneous coronary intervention has long-term prognostic significance. The sirolimus-eluting stent (Cypher) has been reported to require high postinflation pressure for optimal implantation. We examined the incidence of minor cardiac biomarker elevation induced by Cypher implantation. METHODS: We measured the serum concentration of cardiac troponin-I (cTnI) 24 h after stenting and those of creatine kinase isoenzyme MB and creatine kinase before, immediately after, and 6, 12 and 24 h after implantation in patients who underwent Cypher stent (CS group n=53) or bare metal stent (BMS group n=57) implantation. RESULTS: No significant difference in clinical background was observed between the two groups. When a cutoff cTnI value of 0.50 ng/ml was used, the CS group showed a significantly higher incidence of cTnI elevation (35.8%, 19/53) than the BMS group (14.0%, 8/57) (P< 0.05). Similarly, the incidence of cTnI 0.03 ng/ml tended to be higher in the CS group (88.7%, 47/53) than in the BMS group (73.7%, 42/57: 0.05< P< 0.10). Furthermore, the CS group showed significantly higher cTnI levels than the BMS group (1.32±2.38 vs. 0.34±0.91 ng/ml. P< 0.001). Essentially, similar results were obtained for serum creatine kinase isoenzyme MB and creatine kinase. Among clinical, lesion and procedural characteristics, postinflation pressure for stenting was significantly higher only in the CS group (18.2±2.8 atm) than in the BMS group (14.0±2.7 atm) (P< 0.001). CONCLUSIONS: The results demonstrated that CS implantation increases the incidence of minor cardiac biomarker elevation compared with BMS. The difference in postinflation pressure could account for the results. © 2008 Lippincott Williams & Wilkins, Inc.
• Roles of zinc and zinc signaling in immunity: Zinc as an intracellular signaling molecule

  Toshio Hirano, Masaaki Murakami, Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, Tomoyuki Suzuki

  ADVANCES IN IMMUNOLOGY, VOL 97 97 149 - 176 0065-2776 2008 [Refereed][Not invited]
   

  Zinc (Zn) is an essential nutrient required for cell growth, differentiation, and survival, and its deficiency causes growth retardation, immunodeficiency, and other health problems. Therefore, Zn homeostasis must be tightly controlled in individual cells. Zn is known to be important in the immune system, although its precise roles and mechanisms have not yet been resolved. Zn has been suggested to act as a kind of neurotransmitter. In addition, Zn has been shown to bind and affect the activity of several signaling molecules, such as protein tyrosine phosphatases (PTPs). However, it has not been known whether Zn itself might act as an intracellular signaling molecule, that is, a molecule whose intracellular status is altered in response to an extracellular stimulus, and that is capable of transducing the extracellular stimulus into an intracellular signaling event. Here we propose that Zn acts as a signaling molecule and that there are at least two kinds of Zn signaling: "late Zn signaling," which is dependent on a change in the expression profile of Zn transporters, and "early Zn signaling," which involves a "Zn wave" and is directly induced by an extracellular stimulus. We also review recent progress in uncovering the roles of Zn in the immune system.
• Association of corrected QT dispersion with symptoms improvement in patients receiving cardiac resynchronization therapy

  Kazuyoshi Hina, Hiroshi Kawamura, Takashi Murakami, Keizo Yamamoto, Hirosuke Yamaji, Masaaki Murakami, Satoshi Hirohata, Hiroko Ogawa, Kohsuke Sakane, Shozo Kusachi

  Heart and Vessels 23 (5) 325 - 333 0910-8327 2008 [Refereed][Not invited]
   

  Cardiac resynchronization therapy (CRT) is theoretically expected to affect repolarization as well as depolarization. We studied the effects of CRT on corrected QT (QTc) dispersion in association with symptomatic improvement. QTc dispersion was analyzed in 26 consecutive patients (67 ± 6 years old, 18 men and 8 women) who underwent CRT. CRT responders and nonresponders were defined as patients showing and not showing ≥1 class New York Heart Association symptomatic improvement 3 months after CRT, respectively. QTc interval, QRS width, and QTc dispersion were measured automatically from digital data using an analyzing system. There were 18 CRT responders and 8 nonresponders among the patients. CRT responders showed significantly larger QTc dispersion than CRT nonresponders before CRT (102 ± 26 vs 40 ± 12 ms, P < 0.01). A significant decrease in QTc dispersion by CRT was observed in responders (102 ± 26 to 52 ± 15 ms, P < 0.01). In contrast, QTc dispersion was not decreased by CRT in nonresponders (40 ± 12 to 39 ± 11 ms, not significant). The difference observed before CRT was thus abolished after CRT (52 ± 15 vs 39 ± 11 ms, not significant). Baseline values and changes in QRS width or QTc, as well as asynchrony of wall motion determined by tissue Doppler imaging, were not different between CRT responders and nonresponders before CRT. The present study with a small number of patients shows the potential utility of QTc dispersion for distinguishing CRT responders from CRT nonresponders before CRT, and warrants further study with a greater number of patients. © Springer Japan 2008.
• Prone position is essential for detection of pulmonary vein pseudostenosis by enhanced multidetector computed tomography in patients who undergo pulmonary vein isolation

  Hirosuke Yamaji, Kazuyoshi Hina, Hiroshi Kawamura, Takashi Murakami, Masaaki Murakami, Keizo Yamamoto, Atsushi Hirohata, Toru Miyoshi, Satoshi Hirohata, Shozo Kusachi

  Circulation Journal 72 (9) 1460 - 1464 1346-9843 2008 [Refereed][Not invited]
   

  Background: Pulmonary vein (PV) stenosis is a major complication of PV isolation (PVI) by catheter ablation, so in the present study the optimal position for detecting PV stenosis on enhanced multidetector computed tomography (MDCT) image acquisition was determined. Methods and Results: The 64-slice enhanced MDCT was carried out before and after PVI in 116 consecutive patients with atrial fibrillation while they were in the prone position, as well as while supine. The supine position MDCT image showed > 50% diameter stenosis of the PV in 11 (9%) patients before PVI (% diameter stenosis: mean 55±4%, range 51-65%). Greater than 50% diameter stenosis was seen in the left inferior PV in all 11 patients. The prone position attenuated the PV stenosis findings in the MDCT images in all 11 patients (mean 9±6%, range 2-18%). Stenosis visualized on images acquired in the supine position was, therefore, concluded to be pseudostenosis caused by descending aorta compression. At 3 months after PVI, no significant changes in PV diameter were observed in these 11 patients. Conclusion: The present study demonstrated that the prone position is essential for eliminating PV pseudostenosis observed on supine-position enhanced MDCT images. The results also indicate that preexisting PV organic stenosis is rare.
• 基礎医学から 免疫機構における亜鉛の役割

  深田 俊幸, 山崎 哲, 北條 慎太郎, 西田 圭吾, 村上 正晃, 平野 俊夫

  日本医事新報 (株)日本医事新報社 (4343) 63 - 69 0385-9215 2007/07
• IL-6-gp130-STAT3 in T cells directs the development of IL-17+T-h with a minimum effect on that of Treg in the steady state

  Mika Nishihara, Hideki Ogura, Naoko Ueda, Mineko Tsuruoka, Chika Kitabayashi, Fumio Tsuji, Hiroyuki Aono, Katsuhiko Ishihara, Eric Huseby, Ulrich A. K. Betz, Masaaki Murakami, Toshio Hirano

  INTERNATIONAL IMMUNOLOGY 19 (6) 695 - 702 0953-8178 2007/06 [Refereed][Not invited]
   

  IL-17-producing T-h (T(h)17) comprise a distinct lineage of pro-inflammatory T-h that are major contributors to autoimmune diseases. Treatment with IL-6 and transforming growth factor beta (TGF beta) induces naive CD4(+) T cells to generate Th17, which also requires expression of the IL-6/TGF[3 target ROR gamma t. We reported that IL-6 transduces two signaling pathways via tyrosine redidues of the signal transducer gp130: one depends on signal transducers and activators of transcription (STAT)-3 activation and the other on Src homology region 2 domain-containing phosphatase 2 (SHP2)/Grb2 associated binder (Gab)/mitogen-activated protein kinase (MAPK) activation. Here, we showed that CD4+ T cells carrying a mutant gp130 that transduces the SHP2/Gab/MAPK pathway but not the STAT3-mediated one failed to develop into Th17, while CD4+ T cells whose mutant gp130 transduces the STAT3 signal only generated Th17, indicating that IL-6 acts directly on T cells through the tyrosine residues of gp130 required for STAT3 activation to promote the development of Th17. Moreover, we found that gp130-STAT3 pathway is essential for Th17 development and for the expression of ROR gamma t by using T cells specifically lacking gp130 and STAT3. Noteworthy is that the regulatory T cell (Treg) percentages and numbers were comparable between all mutant mice we tested in vivo, although we showed that IL-6-gp130-STAT3 pathway suppressed Treg development in vitro. Thus, we conclude that IL-6 acts directly to promote the development of Th17 by activating the T cell gp130-STAT3 pathway but has a minimum effect on Treg development at least in the steady state in vivo. Therefore, blockade of IL-6-gp130-STAT3 pathway in CD4+ T cells could be a good target for controlling unwanted T(h)17-mediated immune responses including autoimmune diseases.
• Homeostatically proliferating CD4(+) T cells are involved in the pathogenesis of an Omenn syndrome murine model

  Khie Khiong, Masaaki Murakami, Chika Kitabayashi, Naoko Ueda, Shin-ichiro Sawa, Akemi Sakamoto, Brian L. Kotzin, Stephen J. Rozzo, Katsuhiko Ishihara, Marileila Verella-Garcia, John Kappler, Philippa Marrack, Toshio Hirano

  JOURNAL OF CLINICAL INVESTIGATION 117 (5) 1270 - 1281 0021-9738 2007/05 [Refereed][Not invited]
   

  Patients with Omenn syndrome (OS) have hypomorphic RAG mutations and develop varying manifestations of severe combined immunodeficiency. It is not known which symptoms are caused directly by the RAG mutations and which depend on other polymorphic genes. Our current understanding of OS is limited by the lack of an animal model. In the present study, we identified a C57BL/10 mouse with a spontaneous mutation in, and reduced activity of, RAG1. Mice bred from this animal contained high numbers of memory-phenotype T cells and experienced hepatosplenomegaly and eosinophilia, had oligoclonal T cells, and demonstrated elevated levels of IgE, major symptoms of OS. Depletion of CD4(+) T cells in the mice caused a reduction in their IgE levels. Hence these "memory mutant" mice are a model for human OS; many symptoms of their disease were direct results of the Rag hypomorphism and some were caused by malfunctions of their CD4(+) T cells.
• Homeostatically proliferating CD4+ T cells are involved in the pathogenesis of an Omenn syndrome murine model

  Khie Khiong, Masaaki Murakami, Chika Kitabayashi, Naoko Ueda, Shin-Ichiro Sawa, Akemi Sakamoto, Brian L. Kotzin, Stephen J. Rozzo, Katsuhiko Ishihara, Marileila Verella-Garcia, John Kappler, Philippa Marrack, Toshio Hirano

  Journal of Clinical Investigation 117 (5) 1270 - 1281 0021-9738 2007/05/01 [Refereed][Not invited]
   

  Patients with Omenn syndrome (OS) have hypomorphic RAG mutations and develop varying manifestations of severe combined immunodeficiency. It is not known which symptoms are caused directly by the RAG mutations and which depend on other polymorphic genes. Our current understanding of OS is limited by the lack of an animal model. In the present study, we identified a C57BL/10 mouse with a spontaneous mutation in, and reduced activity of, RAG1. Mice bred from this animal contained high numbers of memory-phenotype T cells and experienced hepatosplenomegaly and eosinophilia, had oligoclonal T cells, and demonstrated elevated levels of IgE, major symptoms of OS. Depletion of CD4+ T cells in the mice caused a reduction in their IgE levels. Hence these "memory mutant" mice are a model for human OS many symptoms of their disease were direct results of the Rag hypomorphism and some were caused by malfunctions of their CD4+ T cells.
• Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function

  Hidemitsu Kitamura, Hideyuki Morikawa, Hokuto Kamon, Megumi Iguchi, Shintaro Hojyo, Toshiyuki Fukada, Susumu Yamashita, Tsuneyasu Kaisho, Shizuo Akira, Masaaki Murakami, Toshio Hirano

  NATURE IMMUNOLOGY 7 (9) 971 - 977 1529-2908 2006/09 [Refereed][Not invited]
   

  Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.
• CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8(+) T cells in a manner dependent on IL-4

  Naoko Ueda, Hiroko Kuki, Daisuke Kamimura, Shinichiro Sawa, Kenichiro Seino, Takuya Tashiro, Ken-ichi Fushuku, Masaru Taniguchi, Toshio Hirano, Masaaki Murakami

  INTERNATIONAL IMMUNOLOGY 18 (9) 1397 - 1404 0953-8178 2006/09 [Refereed][Not invited]
   

  CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GaICer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GaICer enhanced homeostatic proliferation of CD8(+) but not CD4(+) T cells and this effect of alpha-GaICer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8(+) T cell homeostatic proliferation. Importantly, the expression of IL-4R alpha and STAT6 in CD8(+) T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8(+) T cells. Consistent with this, the level of IL-4R alpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4(+) T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8(+) T cell homeostatic proliferation in vivo.
• TRIF-GEFH1-RhoB pathway is involved in MHCII expression on dendritic cells that is critical for CD4 T-cell activation

  Hokuto Kamon, Takaya Kawabe, Hidemitsu Kitamura, Jihye Lee, Daisuke Kamimura, Tsuneyasu Kaisho, Shizuo Akira, Akihiro Iwamatsu, Hisashi Koga, Masaaki Murakami, Toshio Hirano

  EMBO JOURNAL 25 (17) 4108 - 4119 0261-4189 2006/09 [Refereed][Not invited]
   

  Dendritic cells (DC) play a central role in immune responses by presenting antigenic peptides to CD(4+) T cells through MHCII molecules. Here, we demonstrate a TRIF-GEFH1-RhoB pathway is involved in MHCII surface expression on DC. We show the TRIF (TIR domain-containing adapter inducing IFN beta)- but not the myeloid differentiation factor 88 (MyD88)-dependent pathway of lipopolysaccharide (LPS)-signaling in DC is crucial for the MHCII surface expression, followed by CD(4+) T-cell activation. LPS increased the activity of RhoB, but not of RhoA, Cdc42, or Rac1/2 in a manor dependent on LPS-TRIF- but not LPS-Myd88-signaling. RhoB colocalized with MHCII+ lysosomes in DC. A dominant-negative (DN) form of RhoB (DN-RhoB) or RhoB's RNAi in DC inhibited the LPS-induced MHCII surface expression. Moreover, we found GEFH1 associated with RhoB, and DN-GEFH1 or GEFH1's RNAi suppressed the LPS-mediated RhoB activation and MHCII surface expression. DN-RhoB attenuated the DC's CD(4+) T-cell stimulatory activity. Thus, our results provide a molecular mechanism relating how the MHCII surface expression is regulated during the maturation stage of DC. The activation of GEFH1-RhoB through the TRIF-dependent pathway of LPS in DC might be a critical target for controlling the activation of CD4+ T cells.
• Signaling of vascular endothelial growth factor receptor-1 tyrosine kinase promotes rheumatoid arthritis through activation of monocytes/macrophages

  Masato Murakami, Shinobu Iwai, Sachie Hiratsuka, Mai Yamauchi, Kazuhide Nakamura, Yoichiro Iwakura, Masabumi Shibuya

  BLOOD 108 (6) 1849 - 1856 0006-4971 2006/09 [Refereed][Not invited]
   

  Vascular endothelial growth factor (VEGF) and VEGF receptor-1 (VEGFR-1/Flt-1) were shown to be involved in pathological angiogenesis, particularly rheumatoid arthritis (RA). However, the molecular basis of their actions is not fully understood. Here we report that in a murine model of RA, deletion of the tyrosine kinase (TK) domain of VEGFR-1 decreased the incidence and clinical symptoms of RA. Pathological symptoms, such as synovial hyperplasia, inflammatory infiltrates, pannus formation, and cartilage/bone destruction, became milder in Vegfr-1 tk(-/-) mice compared with wild-type (Wt) mice in the human T-cell leukemia virus-1 (HTLV-1) pX-induced chronic models. VEGFR-1 TK-deficient bone marrow cells showed a suppression of multilineage colony formation. Furthermore, macrophages induced to differentiate in vitro showed a decrease in immunologic reactions such as phagocytosis and the secretion of interleukin-6 (IL-6) and VEGF-A. Treatment of this RA model with a small molecule inhibitor for VEGFR TK, KRN951, also attenuated the arthritis. These results indicate that the VEGFR-1 TK signaling modulates the proliferation of bone marrow hematopoietic cells and immunity of monocytes/macrophages and promotes chronic inflammation, which may be a new target in the treatment of RA.
• IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb

  D Kamimura, Y Sawa, M Sato, E Agung, T Hirano, M Murakami

  JOURNAL OF IMMUNOLOGY 177 (1) 306 - 314 0022-1767 2006/07 [Refereed][Not invited]
   

  IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 eDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8(+) T and NK1.1(high) cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44(high)CD8(+) population, and the increased population was maintained for > 300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.
• Autoimmune arthritis associated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/IL-7-dependent homeostatic proliferation of CD4(+) T cells

  S Sawa, D Kamimura, GH Jin, H Morikawa, H Kamon, M Nishihara, K Ishihara, M Murakami, T Hirano

  JOURNAL OF EXPERIMENTAL MEDICINE 203 (6) 1459 - 1470 0022-1007 2006/06 [Refereed][Not invited]
   

  Mice homozygous for the F759 mutation in the gp130 interleukin ( IL)-6 receptor subunit have enhanced gp130-mediated signal transducer and activator of transcription ( STAT)3 activation and spontaneously developed a lymphocyte-mediated rheumatoid arthritis-like joint disease. Here, we show that the development of the disease is dependent on both major histocompatibility complex ( MHC)II-restricted CD4(+) T cells and IL-6 family cytokines. In spite of the necessity for CD4(+) T cells, the gp130 mutation was only required in nonhemtopoietic cells for the disease. The gp130 mutation resulted in enhanced production of IL-7. Conditional knockout of STAT3 in nonlymphoid cells showed that the enhancement of IL-7 production was dependent on STAT3 activation by IL-6 family cytokines. Homeostatic proliferation of CD4(+) T cells was enhanced in gp130 mutant mice and acceleration of homeostatic proliferation enhanced the disease, whereas the inhibition of homeostatic proliferation suppressed the disease. Anti-IL-7 antibody treatment inhibited not only the enhanced homeostatic proliferation, but also the disease in gp130 mutant mice. Thus, our results show that autoimmune disease in gp130 mutant mice is caused by increased homeostatic proliferation of CD4(+) T cells, which is due to elevated production of IL-7 by nonhematopoietic cells as a result of IL-6 family cytokine-gp130-STAT3 signaling.
• Grasp a pTyr-peptide by its SOCS

  T Hirano, M Murakami

  DEVELOPMENTAL CELL 10 (5) 542 - 544 1534-5807 2006/05 [Refereed][Not invited]
   

  Signaling via suppressors of cytokine signaling (SOCS) is an important negative feedback system for cytokine-mediated signal transduction. Recently in Molecular Cell, Babon et al. (2006) described the tertiary structure of SOCS3 in complex with a phosphotyrosine-containing peptide from the IL-6 receptor sub-unit gp130, and they identified the specific amino acids that are critical for binding.
• Coronary pressure measurement to identify the lesion requiring percutaneous coronary intervention in equivocal tandem lesions

  Minoru Hirota, Kohichiro Iwasaki, Keizo Yamamoto, Shozo Kusachi, Kazuyoshi Hina, Satoshi Hirohata, Masaaki Murakami, Shigeshi Kamikawa, Takashi Murakami, Yasushi Shiratori

  Coronary Artery Disease 17 (2) 181 - 186 0954-6928 2006/03 [Refereed][Not invited]
   

  OBJECTIVES: No reliable methods are available for determining application of percutaneous coronary intervention for treatment of equivocal tandem lesions. We investigated whether coronary pressure measurement is useful for determining the lesion that requires percutaneous coronary intervention in tandem lesions. METHODS: We measured coronary pressure in 72 consecutive patients with tandem lesions. Myocardial fractional flow reserve (FFRmyo) was obtained as the ratio of coronary pressure distal to the lesion/aortic pressure under maximal hyperemia. If the FFRmyo across the tandem lesions was ≥ 0.75, we deferred percutaneous coronary intervention for the lesion. When the tandem lesions showed FFRmyo< 0.75, percutaneous coronary intervention was performed on the lesion that showed angiographically higher stenosis. When FFRmyo was < 0.75 after one-lesion percutaneous coronary intervention, this intervention was carried out on the remaining lesion. RESULTS: We deferred percutaneous coronary intervention for 26 patients (36.1%), and performed percutaneous coronary intervention in 46 patients (63.8%). We performed percutaneous coronary intervention for one lesion in 19 patients (26.4%) and for both lesions in 27 patients (37.5%). Among patients in whom percutaneous coronary intervention was deferred, only two patients (7.7%) required target lesion revascularization during the follow-up period. This rate was not higher than that in the 46 patients who underwent percutaneous coronary intervention for one or two lesions (six patients, 13.0%). Similarly, the target lesion revascularization in lesions with initially deferred percutaneous coronary intervention (5.6%, 4/71 lesions) was not higher than that in lesions with percutaneous coronary intervention (15.1%, 11/73 lesions). Major cardiac events, cardiac death and acute myocardial infarction, did not occur in patients with deferred percutaneous coronary intervention and in those with percutaneous coronary intervention during the follow-up period. CONCLUSION: Our results clearly showed that coronary pressure measurement was clinically useful for identifying equivocal tandem lesions requiring percutaneous coronary intervention. © 2006 Lippincott Williams & Wilkins.
• Nicorandil reduces the incidence of minor cardiac marker elevation after coronary stenting

  Masaaki Murakami, Kohichiro Iwasaki, Shozo Kusachi, Kazuyoshi Hina, Minoru Hirota, Satoshi Hirohata, Shigeshi Kamikawa, Mutsuko Sangawa, Keizo Yamamoto, Yasushi Shiratori

  International Journal of Cardiology 107 (1) 48 - 53 0167-5273 2006/02/08 [Refereed][Not invited]
   

  Background: Minor cardiac marker elevation after percutaneous coronary intervention has long-term prognostic significance. We examined whether nicorandil, a nicotinamide-nitrate ester, reduces the incidence of minor cardiac marker elevation after coronary stenting. Methods: Patients (n = 192) undergoing coronary stenting were randomly assigned to receive nicorandil (nicorandil group, n = 91) or vehicle (control group, n = 101). Nicorandil (2 μg/kg/min, intravenously) was administered immediately after the patients were transferred into the catheterization laboratory and continued for 6 h. We measured the serum concentrations of creatine kinase isoenzyme MB (CK-MB) before, immediately after, and 6, 12, and 24 h after the procedure, and those of cardiac troponin T (cTnT) 24 h after the procedure. Results: There was no significant difference in clinical background between the 2 groups. The nicorandil group showed a significantly lower incidence of CK-MB elevation (> 1 × upper limit of control range, 20 IU/l) than the control group (8.8% vs 21.8%, p < 0.05). The levels of serum CK-MB in the nicorandil group were significantly lower than those in the control group (13.4 ± 5.7 vs 16.5 ± 9.7 IU/l, p < 0.01). Similarly, the nicorandil group showed a significantly lower incidence of cTnT elevation [> 1 × (0.1 ng/ml) or > 2 × (0.2 ng/ml)] upper limit of control range than the control group (14.3% vs 26.7%, p < 0.05, or 7.7% vs 17.8%, p < 0.05). Serum cTnT levels were also significantly lower in the nicorandil group than in the control group (0.05 ± 0.10 vs 0.15 ± 0.36 ng/ml, p < 0.05). Conclusions: The results demonstrated that nicorandil reduces minor cardiac marker elevation after coronary stenting. © 2005 Elsevier Ireland Ltd. All rights reserved.
• IL-6-STAT3 controls intracellular MHC class II alpha beta dimer level through cathepsin S activity in dendritic cells

  H Kitamura, H Kamon, SI Sawa, SJ Park, N Katunuma, K Ishihara, M Murakami, T Hirano

  IMMUNITY 23 (5) 491 - 502 1074-7613 2005/11 [Refereed][Not invited]
   

  We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alpha beta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alpha beta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alpha beta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alpha beta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alpha beta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alpha beta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.
• IL-6/IL-12関連サイトカインIL-27の機能発現におけるSTAT3の役割

  善本 隆之, 大脇 敏之, 浅川 正幸, 森嶋 紀子, 竹田 潔, 村上 正晃, 平野 敏夫, 水口 純一郎

  日本免疫学会総会・学術集会記録 (NPO)日本免疫学会 35 283 - 283 0919-1984 2005/11
• Coronary pressure measurement to determine treatment strategy for equivocal left main coronary artery lesions

  Shunji Suemaru, Kohichiro Iwasaki, Keizo Yamamoto, Shozo Kusachi, Kazuyoshi Hina, Satoshi Hirohata, Minoru Hirota, Masaaki Murakami, Shigeshi Kamikawa, Takashi Murakami, Yasushi Shiratori

  Heart and Vessels 20 (6) 271 - 277 0910-8327 2005/11 [Refereed][Not invited]
   

  It is often hard to select a treatment strategy for equivocal left main coronary artery (LMCA) disease. We investigated the usefulness of coronary pressure (CP) measurement for determining the treatment strategy in intermediate LMCA disease. We measured CP in 15 consecutive patients with equivocal LMCA disease (age 67.6 ± 7.5 years, 14 males). Myocardial fractional flow reserve (FFRmyo) was obtained as the ratio of CP distal to the lesion/aortic pressure under maximal coronary dilation. Patients with FFRmyo ≥0.75 and < 0.75 received medical therapy and coronary artery bypass grafting (CABG), respectively, and were followed up for 32.5 ± 9.7 (20-47) months. Eight patients received medical therapy and 7 patients underwent CABG in accordance with the FFRmyo criteria noted above. FFRmyo of the LMCA was 0.91 ± 0.01 and 0.61 ± 0.03 in patients who received medical and surgical therapy, respectively. Neither reference vessel diameter, minimal lumen diameter, nor percent diameter stenosis was significantly different between patients who received medical and surgical therapy. During the follow-up period, no patients with medical therapy showed symptoms due to the LMCA lesion. Similarly, 5 of 7 patients with CABG showed improvement of symptoms and the remaining 2 patients were hospitalized with congestive heart failure. No cardiac death was recorded in the patients with medical or surgical therapy. In conclusion, the present results clearly demonstrated that CP is clinically useful for determining the treatment strategy for equivocal LMCA lesions but coronary angiography is not. © Springer-Verlag Tokyo 2005.
• Hyperactivation of gp130-mediated STAT3 signaling induces a rheumatoid arthritis-like disease that is dependent on MHC class II restricted CD4+ T cells

  Masaaki Murakami, Shin-ichiro Sawa, Daisuke Kamimura, Hokuto Kamon, Hidemitsu Kitamura, Takaya Kawabe, Park Sung-Joo, Katsuhiko Ishihara, Toshio Hirano

  International Congress Series 1285 207 - 211 0531-5131 2005/11 [Refereed][Not invited]
   

  Mice having a point mutation of IL-6 signal transducer, gp130, named gp130F759 induced a rheumatoid arthritis-like disease. The disease induction is totally dependent on mature lymphocytes, since no arthritis-like disease induced in a double mutant between gp130F759 and RAG2KO mice. We prepared several other double mutants including gp130F759/IgMKO, gp130F759/CD8KO, gp130F759/CIITAKO and gp130F759/CD4KO and analyzed the development of the arthritis. We found that development of the disease attenuated in gp130F759/CIITAKO and gp130F759/CD4KO, suggesting the MHC class II-restricted CD4+ T cells are important for the disease development. We showed memory/activated phenotype of CD4+ T cells increased in gp130F759 mice. Since activation of CD4+ T cells is mainly controlled by dendritic cells (DC) in vivo, we investigated this feature of DC in gp130F759 mice. We isolated DCs from superficial lymph nodes and observed that IL-6-mediated signaling suppresses maturation of DCs in vivo. However, total number of DCs in vivo significantly increased in gp130F759 mice compared with wild type controls. Thus, we hypothesize that the rheumatoid arthritis-like disease in gp130F759 mice is induced by an interaction between CD4+ T cells and DC under gp130F759 signal regulation. © 2005.
• Significant correlation of recruitable coronary collateral blood flow determined by coronary wedge pressure with ST-segment elevation during coronary occlusion

  Shigeshi Kamikawa, Kohichiro Iwasaki, Keizo Yamamoto, Shozo Kusachi, Kazuyoshi Hina, Satoshi Hirohata, Masaaki Murakami, Minoru Hirota, Takashi Murakami, Yasushi Shiratori

  Coronary Artery Disease 16 (4) 231 - 236 0954-6928 2005/06 [Refereed][Not invited]
   

  Objectives: Quantitative assessment of coronary collateral blood flow can be archived by measuring coronary pressure. We studied the relationships between recruitable coronary collateral blood flow and electrocardiographic changes during percutaneous coronary intervention (PCI). Methods: We measured coronary pressure during coronary occlusion with PCI in 119 patients with left anterior descending coronary artery stenosis. During balloon inflation, the electrocardiogram was continuously recorded. The ST-segment elevation in the most elevated lead was defined as MaxST and the sum of the maximal ST elevation in leads V2-V4 was defined as ΣST. Fractional collateral flow (Qc/Q N) was calculated as the coronary wedge pressure divided by the mean aortic pressure. Myocardial ischemia was defined as an ST-segment shift > 0.1 mV in any of the V2, V3 or V4 leads. Results: A significant relationship between Qc/QN and MaxST was observed (r= -0.455, P< 0.0001). Similarly, Qc/QN was significantly correlated with ΣST (r= -0.477, P< 0.0001). The receiver operating characteristic curve showed that a cut-off value of 0.27 for Qc/QN, with sensitivity of 71.4% and specificity of 76.2%, was an indicator of electrophysiologically sufficient recruitable coronary collateral blood flow for prevention of ischemia during coronary obstruction. Qc/QN values during the first, second, third and fourth inflation were not significantly different. Conclusions: Qc/QN could be clinically useful for determining whether there is electrophysiologically sufficient recruitable coronary collateral blood flow for prevention of ischemia during coronary obstruction. Repeat transient coronary occlusion during PCI did not lead to increased collateral blood flow. © 2005 Lippincott Williams & Wilkins.
• [Mechanisms for generation and maintenance of memory CD8 T cells].

  Kamimura D, Murakami M

  Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 4 369 - 374 0047-1852 2005/04 [Refereed][Not invited]
  
• Mechanism of macrophage activation by chitin derivatives

  T Mori, M Murakami, M Okumura, T Kadosawa, T Uede, T Fujinaga

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (1) 51 - 56 0916-7250 2005/01 [Refereed][Not invited]
   

  In order to analyze the detailed mechanisms responsible for macrophage activation by chitin derivatives, resident peritoneal macrophages were prepared and stimulated with chitin, chitosan and low-molecular weight chitosan. Our findings were as follows: (i) chitosan induced apoptosis of peritoneal macrophages, but this did not occur when chitin or water soluble low-molecular weight chitosan were used; (ii) chitosan treatment induced activation markers, such as the major histocompatibility complex (MHC) class I, class II, Fc receptors, transferrin receptor, mannose receptor, Fas, and macrophage inflammatory protein (MIP)-2, whereas chitin and low molecular weight soluble chitosan induced only the expression of MHC class I and 11 molecules; (iii) apoptosis induced by chitosan was mediated by the Fas signaling pathway, in response to phagocytosis via the mannose receptor. We conclude that since chitosan activates macrophages, this may be the mechanism by which it accelerates wound healing.
• Evidence of a novel IL-2/15R beta-targeted cytokine involved in homeostatic proliferation of memory CD8(+) T cells

  D Kamimura, N Ueda, Y Sawa, S Hachida, T Atsumi, T Nakagawa, SI Sawa, GH Jin, H Suzuki, K Ishihara, M Murakami, T Hirano

  JOURNAL OF IMMUNOLOGY 173 (10) 6041 - 6049 0022-1767 2004/11 [Refereed][Not invited]
   

  The homeostasis of memory CD8(+) T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8(+) T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25(+)CD4(+) T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8(+) T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44(high)IL-2/15Rbeta(high) CD8(+) T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15Rbeta signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8(+) T cells, although the only cytokines known to act through IL-2/15Rbeta are IL-2 and IL-15; and 4) the expression of IL-2/15Rbeta molecules on memory CD8(+) T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8(+) T cell division by an IL-15-independent but by an IL-2/15Rbeta-dependent mechanism, suggesting the existence of a novel IL-2/15Rbeta-utilizing cytokine that acts directly on memory CD8(+) T cells to promote cell division.
• IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

  SJ Park, T Nakagawa, H Kitamura, T Atsumi, H Kamon, S Sawa, D Kamimura, N Ueda, Y Iwakura, K Ishihara, M Murakami, T Hirano

  JOURNAL OF IMMUNOLOGY 173 (6) 3844 - 3854 0022-1767 2004/09 [Refereed][Not invited]
   

  Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130 (F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.
• New IL-6 (gp130) family cytokine members, CLC/NNT1/BSF3 and IL-27

  M Murakami, D Kamimura, T Hirano

  GROWTH FACTORS 22 (2) 75 - 77 0897-7194 2004/06 [Refereed][Not invited]
  
• The point mutation of tyrosine 759 of the IL-6 family cytokine receptor gp130 synergizes with HTLV-1 pX in promoting rheumatoid arthritis-like arthritis

  K Ishihara, S Sawa, H Ikushima, S Hirota, T Atsumi, D Kamimura, SJ Park, M Murakami, Y Kitamura, Y Iwakura, T Hirano

  INTERNATIONAL IMMUNOLOGY 16 (3) 455 - 465 0953-8178 2004/03 [Refereed][Not invited]
   

  Rheumatoid arthritis (RA) is a polygenic autoimmune disease. The autoimmunity develops from synergistic actions of genetic and environmental factors. We generated a double-mutant mouse by crossing two murine models of RA, a gp130 mutant knock-in mouse (gp130(F759/F759)) and an HTLV-1 pX transgenic mouse (pX-Tg), in a C57BL/6 background, which is resistant to arthritis. The mice spontaneously developed severe arthritis with a much earlier onset than the gp130(F759/F759) mice and with a much higher incidence than did the pX-Tg mice. The symptoms of gp130(F759/F759) mice, including lymphoadenopathy, splenomegaly, hyper-gamma-globulinemia, autoantibody production, increases in memory/activated T cells and granulocytes in the peripheral lymphoid organs, and a decrease in the class II MHCbright CD11c(+) population, were augmented in the double mutants. Marked reductions in incidence, severity and immunological abnormalities were seen in the triple mutant, IL-6(-/-)/gp130(F759/F759)/pX-Tg, indicating that the arthritis in the double mutant is IL-6 dependent. gp130(F759/F759)/pX-Tg is a unique mouse model for RA.
• Membrane dynamics and cell signaling as studied by single molecule imaging

  T Kobayashi, M Murakami, Y Takeda, A Kusumi, A Yoshimura

  SEIKAGAKU 76 (2) 91 - 100 0037-1017 2004/02 [Refereed][Not invited]
  
• Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

  K Yamashita, T Masunaga, N Yanagida, M Takehara, T Hashimoto, T Kobayashi, H Echizenya, N Hua, M Fujita, M Murakami, H Furukawa, T Uede, S Todo

  TRANSPLANTATION 76 (7) 1089 - 1096 0041-1337 2003/10 [Refereed][Not invited]
   

  Background We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. Methods. Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1(av1)) to Lewis (LEW, RT1(1)) rats. Vector (1x10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTIA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. Results. Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. Conclusion. Simultaneous blockade of CD28 and CD154 path-ways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This:; strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.
• IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages

  H Yasukawa, M Ohishi, H Mori, M Murakami, T Chinen, D Aki, T Hanada, K Takeda, S Akira, M Hoshijima, T Hirano, KR Chien, A Yoshimura

  NATURE IMMUNOLOGY 4 (6) 551 - 556 1529-2908 2003/06 [Refereed][Not invited]
   

  Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling.
• Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis

  N Tosa, M Murakami, WY Jia, M Yokoyama, T Masunaga, C Iwabuchi, M Inobe, K Iwabuchi, T Miyazaki, K Onoe, M Iwata, T Uede

  INTERNATIONAL IMMUNOLOGY 15 (6) 741 - 749 0953-8178 2003/06 [Refereed][Not invited]
   

  Transcriptional expression of a gene or genes is absolutely required for induction of glucocorticold-induced thymocyte apoptosis. We have previously shown that expression of T cell death-associated gene 8 (TDAG8) is quickly induced exclusively in the thymus after dexamethasone (DEX) treatment. Here, we present data that TDAG8 expression is induced prior to induction of DEX-mediated apoptosis. In contrast, TDAG8 expression in thymocytes was not induced in the process of gamma-irradiation-mediated apoptosis. TDAG8 expression accelerated only DEX-induced, but not TCR-mediated or gamma-irradiation-induced, thymocyte apoptosis in transgenic mice overexpressing TDAG8. Interestingly, these effects were specifically detected in CD4(+)CD8(+) double-positive thymocytes. Moreover, activation of caspase-3, -8 and -9 was enhanced in thymocytes of TDAG8 transgenic mice after DEX stimulation. In conclusion, TDAG8 expression is involved in glucocorticold-induced signals to activate caspase-9, -8 and -3 for subsequent apoptosis induction in CD4(+)CD8(+) double-positive thymocytes.
• Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model

  K Kanaya, Y Tsuchida, M Inobe, M Murakami, T Hirose, S Kon, S Kawaguchi, T Wada, T Yamashita, S Ishii, T Uede

  TRANSPLANTATION 75 (3) 275 - 281 0041-1337 2003/02 [Refereed][Not invited]
   

  Background. The blockade of costimulatory signal pathway by anti-CD40 ligand antibody or cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) prolongs allograft survival in various vascularized organ transplantations. Because of the short half life of these agents, repeated administration of proteins is required to achieve significant graft survival. Furthermore, there is limited information regarding the effect of cosimulatory blockade on the survival of composite tissue allografts. Therefore, we examined the effect of adenovirus-mediated gene transfer of CTLA4Ig or CD40Ig gene or both in composite tissue allotransplantation. Methods. The hind limbs removed from male ACI rats (RT1(a)) were transplanted into female Lewis rats (RT1(1)) heterotopically. The recombinant adenovirus carrying CTI.A4Ig (AxCTLA4Ig) or CD40Ig (AxCD40Ig) was intravenously administered after limb transplantation. Results. Limb allograft survival was significantly prolonged by either AxCTLA4Ig or AxCD40Ig treatment at 1 x 10(9) plaque forming unit (mean survival time [MST] of 39.4 +/- 6.0 and 13.0 +/- 2.9, respectively) compared with the adenovirus vector containing beta-galactosidase-treated group (MST of 4.8 +/- 0.8). Combination of AxCTLA4Ig and AxCD40Ig led to significant prolongation of graft survival (MST of 49.2 +/- 6.6). Serum levels of CD40Ig were higher in rats treated with combination therapy than those treated with AxCD40Ig alone, whereas the serum levels of CTLA4Ig in rats treated with AxCTLA4Ig alone and AxCTLA4Ig and AxCD40Ig combined were very similar. Conclusion. This study indicates that an adenovirus-mediated gene therapy of CTLA4Ig or CD40Ig has a therapeutic potential for preventing rejection in composite tissue transplantation. Furthermore, a combination therapy of AxCTLA4Ig and AxCD40Ig was even more effective in preventing acute rejection and prolonging the survival of allografted limbs without apparent complication.
• [Role of cytokines for maintenance of memory CD8+ T cells].

  Kamon H, Murakami M

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 16 Suppl 47 2313 - 2317 0039-9450 2002/12 [Refereed][Not invited]
  
• RANTES production by memory phenotype T cells is controlled by a posttranscriptional, TCR-dependent process

  BJ Swanson, M Murakami, TC Mitchell, J Kappler, P Marrack

  IMMUNITY 17 (5) 605 - 615 1074-7613 2002/11 [Refereed][Not invited]
   

  An examination of differences in gene expression between memory and naive phenotype T cells revealed elevated levels of mRNA for several chemokines, especially RANTES, in memory phenotype T cells. Although RANTES mRNA is spliced and cytoplasmic, these cells do not contain or secrete significant amounts of RANTES protein without TCR stimulation. This secretion is independent of transcription, but requires translation. In vivo, CD8+ memory T cells proliferate continuously and slowly in response to IL-15; however, IL-15 does not stimulate RANTES secretion. These results show that memory phenotype CD8+ T cells use preexisting mRNA to produce and secrete RANTES rapidly following TCR stimulation. Such storage of preformed mRNAs for important inflammatory mediators may contribute to the speed of secondary immune responses.
• Intrinsic and extrinsic manipulation of B7/CTLA-4 interaction for induction of anti-tumor immunity against osteosarcoma cells

  M Nagamori, S Kawaguchi, M Murakami, T Wada, S Nagoya, T Yamashita, M Inobe, T Uede

  ANTICANCER RESEARCH 22 (6A) 3223 - 3227 0250-7005 2002/11 [Refereed][Not invited]
   

  Background: B7 family members play a central costimulatory role in T cell activation. We identified B7-1alpha, an alternatively spliced form of B7-1. The therapeutic efficacy of B7-1alpha-expressing tumor vaccine remains uncertain. Materials and Methods: The murine osteosarcoma cell line, LM8, was engineered to express equivalent levels of B7-1 (B7-1-LM8) and B7-1alpha (B7-1alpha-LM8). The therapeutic efficacy of B7-transfected cells and anti-CTLA-4 blocking mAb was evaluated by the mixing experiments on the primary tumor, pulmonary metastasis and survival time. Results: (i) a mixture of B7-1-LM8 or B7-1alpha-LM8 cells inhibited growth of the subcutaneous LM8 tumors and augmented the therapeutic effects of anti-CTLA-4 mAb and (ii) a combination of B7-1alpha-LM8 cells and anti-CTLA-4 mAb most significantly eradicated pulmonary metastasis and prolonged the survival time of mice. Conclusion: These findings suggest that intrinsic (lack of IgC-like domain in B7-1alpha) and extrinsic (anti-CTLA-4 mAb) manipulations of B7/CTLA-4 interaction synergistically improve the therapeutic efficacy of B7-based osteosarcoma vaccines.
• In vivo immunogenicity of osteosarcoma cells that express B7-1a, an alternatively spliced form of B7-1

  M Nagamori, S Kawaguchi, M Murakami, T Wada, M Inobe, S Ishii, T Uede

  ANTICANCER RESEARCH 22 (4) 2009 - 2013 0250-7005 2002/07 [Refereed][Not invited]
   

  Background: B7 family members play a central costimulatory role in T cell activation. We have previously identified B7-1a, an. alternatively spliced form of B7-1. The function of B7-1a in induction of anti-tumor immunity remains uncertain. Materials and Methods: The cDNAs of murine B7-1, B7-1a and B7-2 were introduced into a murine osteosarcoma cell line, LM8. The ability of B7 transfectants to elicit in vivo anti-tumor immunity was comparatively analyzed with respect to tumorigenecity, pulmonary metastasis and survival time. Results: LMS cells expressing B7-1, B7-1a, or B7-2 all elicited immunological responses in immunocompetent C3H/He mice. Notably, the anti-tumor effects were most obvious in mice inoculated with B7-1a-transfected LM8 cells. Such a difference among B7-transfectants became indistinguishable in immunodeficient nude mice. Conclusion: These findings indicate that B7-1a serves as a more efficacious costimulatory molecule than B7-1 or B7-2 in the induction and maintenance of anti-tumor immune responses against a poorly immunogenic osteosarcoma cell line.
• Overexpression of the Wilms' tumor gene WT1 in de novo lung cancers

  Y Oji, S Miyoshi, H Maeda, S Hayashi, H Tamaki, SI Nakatsuka, M Yao, E Takahashi, Y Nakano, H Hirabayashi, Y Shintani, Y Oka, A Tsuboi, N Hosen, M Asada, T Fujioka, M Murakami, K Kanato, M Motomura, EH Kim, M Kawakami, K Ikegame, H Ogawa, K Aozasa, Kawase, I, H Sugiyama

  INTERNATIONAL JOURNAL OF CANCER 100 (3) 297 - 303 0020-7136 2002/07 [Refereed][Not invited]
   

  Expression of the Wilms' tumor gene WTI in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WTI gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WTI protein with an anti-WTI antibody. Overexpression of the WTI gene was also demonstrated in 516 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WTI gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the non-mutated, wild-type WTI gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WTI gene and its products. (C) 2002 Wiley-Liss, Inc.
• Combination treatment with FTY720 and CTLA4IgG preserves the respiratory epithelium and prevents obliterative disease in a murine airway model

  K Konishi, M Inobe, A Yamada, M Murakami, S Todo, T Uede

  JOURNAL OF HEART AND LUNG TRANSPLANTATION 21 (6) 692 - 700 1053-2498 2002/06 [Refereed][Not invited]
   

  Background: Mouse heterotopic tracheal transplantation offers a reproducible model of obliterative bronchiolitis after lung transplantation. CTLA4IgG inhibits signaling of the CD28/B7 pathway and induces antigen-specific T-cell unresponsiveness. FTY720 induces T-cell apoptosis and sequestration of circulating mature lymphocytes. We previously found that CTLA4IgG could prevent the development of obliterative airway disease but could not preserve the respiratory epithelium of grafted tracheae. We evaluated whether treatment with either FTY720 or CTLA4IgG, or with combination FTY720 and CTLA4IgG could preserve the respiratory epithelium and inhibit the development of obliterative airway disease. Methods: Tracheae with main bronchi from C3H/He mice were transplanted heterotopically into BALB/C mice and harvested on Day 35. Recipient mice received either no treatment or treatment with intraperitoneal FTY720, CTLA4IgG, or the combination of the 2. Results: Either FTY720 or CTLA4IgG alone significantly inhibited the development of obliterative airway disease. However, normal ciliated columnar respiratory epithelial cells were lost in the allografts. In contrast, combination therapy preserved the respiratory epithelium of the allografted tracheae. FTY720 concentration in the tissue was very high; treatment with FTY720 inhibited mixed lymphocyte reactions and augmented T-cell apoptosis. Conclusion: Combination treatment with FTY720 and CTLA4IgG may prevent obliterative airway disease.
• CD25+CD4+T cells contribute to the control of memory CD8+T cells

  M Murakami, A Sakamoto, J Bender, J Kappler, P Marrack

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 (13) 8832 - 8837 0027-8424 2002/06 [Refereed][Not invited]
   

  Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. in animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.
• Induction of donor-specific tolerance by adenovirus-mediated CD40Ig gene therapy in rat liver transplantation

  M Nomura, K Yamashita, M Murakami, M Takehara, H Echizenya, M Sunahara, N Kitagawa, M Fujita, H Furukawa, T Uede, S Todo

  TRANSPLANTATION 73 (9) 1403 - 1410 0041-1337 2002/05 [Refereed][Not invited]
   

  Background. Blockade of CD40-CD40 ligand (CD154) costimulatory pathway with anti-CD154 antibody (Ab) prolongs allograft survival in experimental organ transplantations; however, repeated agent administration is needed to provide an adequate immunosuppression. Seeking for simple and effective approach to interfere this signaling, we applied adenovirus-mediated gene therapy by encoding CD40Ig gene (AdCD40Ig). Methods. Liver graft from ACI (RT1(av1)) rat was transplanted orthotopically into LEW (RT1(1)) rat, and AdCD40Ig was given to animals via the penile vein immediately after grafting (n=6). Results. A single treatment with AdCD40Ig at 1 x 10(9) plaque forming units induced specific expression of CD40Ig gene on allograft liver, produced substantial amount of the protein in the sera, and allowed indefinite graft survival. Whereas, LEW recipients given no treatment or control adenovirus vector (AdLacZ) promptly rejected ACI liver. In addition, AdCD40Ig-treated, long-term survivors accepted skin graft from the donor strain but not the third party graft. Histopathology revealed that liver structure of the long-term surviving animals was completely preserved in normal with no infiltration of mononuclear cells. Conclusion. Blockade of CD40-CD154 pathway by CD40Ig gene therapy is a potent alloantigen-specific immunosuppressive strategy to induce permanent acceptance of liver allograft and would be a new therapeutic candidate in a clinical liver transplantation.
• Development of atherosclerosis in osteopontin transgenic mice

  S Chiba, H Okamoto, S Kon, C Kimura, M Murakami, M Inobe, Y Matsui, T Sugawara, T Shimizu, T Uede, A Kitabatake

  HEART AND VESSELS 16 (3) 111 - 117 0910-8327 2002/03 [Refereed][Not invited]
   

  Osteopontin (OPN), a noncollagenous adhesive protein, may possibly be implicated in atherosclerosis, in which macrophages and activated T lymphocytes could have higher OPN levels within the atherosclerotic plaques. However, it is not known whether a higher OPN level is a cause or a result of atherosclerosis or whether it has a promoting or inhibitory effect on atherosclerosis, To clarify the role of OPN in atherosclerosis, we developed a transgenic mouse (OPN-TG) in which the exogenous OPN gene was designed to be expressed by hematopoietic cells, expressing OPN, which carried the immunoglobulin enhancer (Emu)/SV40 promoter. In OPN-TG, the expression of exogenously transfected OPN RNA was found in lymphoid organs, such as the thymus and spleen, and the kidney. In the present study, OPN-TG mice were assigned into two groups, an atherogenic diet group (15% fat, 1.25% cholesterol) for 3 months or a standard diet group (4% fat), and both groups were compared with wild-type C57BL/6 mice to investigate the relationship between osteopontin and the atherosclerotic lesion. In wild-type mice, OPN mRNA was detected in kidney, but not in lymphoid tissues. In both OPN-TG and wild-type mice fed with control diets, atherosclerotic lesions were not found in the aortic sinus or the thoracic and abdominal aorta. In both OPN-TG and wildtype mice fed with atherogenic diets, a high incidence of atherosclerotic lesions was noted in the aortic sinus. The atherosclerotic lesions were significantly larger in OPN-TG as compared with those in control littermate mice (size: 33.8% +/- 23.4% vs 10.9% +/- 20.4%, respectively, P < 0.05). Activated foamy macrophages within atherosclerotic plaque in OPN-TG expressed a considerably larger amount of OPN compared with such macrophages in control mice. The OPN protein detected in the atherosclerotic lesions was not due to the deposition of serum OPN., but mainly due to in situ production by the infiltrating macrophages. Thus, these results suggest that OPN is atherogenic and that macrophages expressing OPN can be easily activated and thus promote atheromatous lesions if a high fat diet is consumed.
• Feasibility of immunosuppression in composite tissue allografts by systemic administration of CTLA4Ig

  N Iwasaki, T Gohda, C Yoshioka, A Murakami, M Inobe, A Minami, T Uede

  TRANSPLANTATION 73 (3) 334 - 340 0041-1337 2002/02 [Refereed][Not invited]
   

  Background. Although recent experimental studies have demonstrated CTLA4Ig to be a potent immunosuppressant in vascularized solid organ allografts, little attention has been given to the effect of this soluble recombinant fusion protein on immunosuppression in composite tissue allografts (CTAs). Using a rat hind limb allograft model, we examined the efficacy of CTLA4Ig against the allograft rejection of composite tissue. Methods. The hind limbs of ACI rats (RT1(a)) were heterotopically transplanted to Lewis rats (RT1(1)). Controls received no immunotherapy. Experimental recipients were treated with a single i.p. injection of either human immunoglobulin (Ig)G (0.5 mg/body) or CTLA4Ig (0.5 mg/body) according to different time schedules. Graft survival time and histopathological changes for each experimental group were evaluated and statistically compared. Results. Graft survival times were prolonged significantly in rats treated with CTLA4Ig on day 1 and day 2 after transplantation, compared with survival times of controls. In particular, the most significant prolongation was found in rats treated on day 2. At 7 days after transplantation, moderate-to-severe histological rejection occurred in all tissues in control rats. On the other hand, in rats treated with CTLA4Ig, all tissues showed significantly better preservation. Among these treated rats, the rats treated on day 2 showed excellent histopathological conditions in each tissue. Conclusions. This study supports the feasibility of using CTLA4Ig for preventing acute rejection in CTA. On the basis of the current results, the administration of CTLA4Ig for CTA is more effective at 24-48 hr after transplantation, after the initial immune response has been allowed to begin.
• Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages

  H Ueno, F Nakamura, M Murakami, M Okumura, T Kadosawa, T Fujinaga

  BIOMATERIALS 22 (15) 2125 - 2130 0142-9612 2001/08 [Refereed][Not invited]
   

  Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors. (C) 2001 Elsevier Science Ltd. All rights reserved.
• Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis

  K Ijima, M Murakami, H Okamoto, M Inobe, S Chikuma, Saito, I, Y Kanegae, Y Kawaguchi, A Kitabatake, T Uede

  HUMAN GENE THERAPY 12 (9) 1063 - 1077 1043-0342 2001/06 [Refereed][Not invited]
   

  We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 mug/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 mug/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.
• Chitosan accelerates the production of osteopontin from polymorphonuclear leukocytes

  H Ueno, M Murakami, M Okumura, T Kadosawa, T Uede, T Fujinaga

  BIOMATERIALS 22 (12) 1667 - 1673 0142-9612 2001/06 [Refereed][Not invited]
   

  Chitosan is a copolymer of beta (1 --> 4) glucosamine and N-acetyl-D-glucosamine, which accelerates the infiltration of polymorphonuclear leukocytes (PMN) in the early phase of wound healing. In the granulation tissue treated with chitosan in canine experimental wound, osteopontin (OPN) was strongly positive in PMN immunohistochemically. OPN is a glycosylated phosphoprotein and promotes the attachment or spread of a variety of cell types. In addition, OPN may play a role in granulomatous inflammation. Production of OPN in PMN was therefore investigated in vitro using human PMN in this study. PMN stimulated with granulocyte-colony stimulating factor (G-CSF) and chitosan accumulated OPN mRNA, and released OPN into their culture supernatants. These findings suggest that OPN is synthesized by migrating PMN which plays the novel role of regulating the evolution of wound healing with chitosan treatment at the early phase of healing, (C) 2001 Elsevier Science Ltd. All rights reserved.
• Long-term acceptance of allografts by in vivo gene transfer of regulatable adenovirus vector containing CTLA4IgG and loxP

  M Takehara, M Murakami, M Inobe, K Tanaka, S Chikuma, Saito, I, Y Kanegae, Y Yasunami, M Nakano, K Yamashita, S Todo, T Uede

  HUMAN GENE THERAPY 12 (4) 415 - 426 1043-0342 2001/03 [Refereed][Not invited]
   

  CTLA4IgG was shown to inhibit the costimulatory signal for T cell activation by interfering with the ligation of CD28 and B7-1 or B7-2. To inhibit various immune responses including acute cellular rejection of allografts, a certain level of serum CTLA4IgG should be maintained for an appropriate period. We previously reported on an adenovirus vector containing CTLA4IgG, which we designated Adex1CACTLA4IgG. Adex1CACTLA4IgG was able to maintain a significant level of serum CTLA4IgG for a long period on intravenous injection, which in turn inhibited various immune responses including protective immunity against infectious agents. To overcome the inhibitory effect, we constructed a new adenovirus vector, Adex1CALoxCTLA4IgGLox, by cloning CTLA4IgG cDNA between two loxP sequences under the control of the CAG promoter. We demonstrated that the administration of adenovirus vector containing Cre recombinase gene (Adex1CACre) at the desired time induced Cre-mediated recombination within a gene derived from Adex1CALoxCTLA4IgGLox vector, and the cDNA of CTLA4IgG was excised from the transduced gene and terminated the expression of CTLA4IgG in vitro and in vivo. More importantly, we also demonstrated that the long-term acceptance of allografts was achieved after the termination of CTLA4IgG expression, while the immune response against adenovirus was restored.
• Novel CD40-IgG adenovirus-mediated gene therapy as a potent immunosuppressive treatment for liver transplantation in rats

  M Nomura, K Yamashita, M Murakami, M Takehara, M Konishi, H Echizenya, N Yanagida, M Sunahara, N Kitagawa, H Furukawa, T Uede, S Todo

  TRANSPLANTATION PROCEEDINGS 33 (1-2) 189 - 189 0041-1345 2001/02 [Refereed][Not invited]
  
• Tolerance induction by a single donor pretreatment with the adenovirus vector encoding CTLA4Ig gene in rat orthotopic liver transplantation

  N Yanagida, M Nomura, K Yamashita, M Takehara, M Murakami, H Echizenya, K Konishi, N Kitagawa, H Furukawa, T Uede, S Todo

  TRANSPLANTATION PROCEEDINGS 33 (1-2) 573 - 574 0041-1345 2001/02 [Refereed][Not invited]
  
• CTLA4IgG gene delivery prevents autoantibody production and lupus nephritis in MRL/lpr mice

  M Takiguchi, M Murakami, Nakagawa, I, Saito, I, A Hashimoto, T Uede

  LIFE SCIENCES 66 (11) 991 - 1001 0024-3205 2000/02 [Refereed][Not invited]
   

  MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune syndrome closely resembling systemic lupus erythematosus (SLE) in humans, characterized by hypergammaglobulinemia, various autoantibody production, and the development of fatal glomerulonephritis. We have previously demonstrated that systemic administration of soluble form of CTLA4IgG prevented autoantibody-related diseases in MRL/lpr mice. To test the potential protective effects of CTLA4IgG gene delivery on the development of lupus nephritis, we injected MRL/lpr mice with, a recombinant adenovirus vector containing CTLA4IgG gene, Adex1CACTLA4IgG (AdCTLA4IgG). It was demonstrated that a single administration of intravenous injection of AdCTLA4IgG into MRL/lpr mice resulted in almost complete amelioration of lupus nephritis.
• ラット脾臓より抽出した樹状突起細胞のアロ抗原特異的T細胞刺激機序の解析

  臼木 智哲, 原田 浩, 竹内 一郎, 小柳 知彦, 猪部 学, 村上 正晃, 上出 利光

  日本泌尿器科学会雑誌 一般社団法人 日本泌尿器科学会 91 (3) 217 - 217 2000
• Janus Kinase 2 is associated with a box 1-like motif and phosphorylates a critical tyrosine residue in the cytoplasmic region of cytotoxic T lymphocyte associated molecule-4

  Shunsuke Chikuma, Masaaki Murakami, Kumiko Tanaka, Toshimitsu Uede

  Journal of Cellular Biochemistry 78 (2) 241 - 250 0730-2312 2000 [Refereed][Not invited]
   

  It is a consensus that a cytotoxic T lymphocyte associated molecule-4 (CTLA-4) transduces inhibitory signal for T cell activation under physiological condition, indicating that this molecule is an important regulator of T cell homeostasis in vivo. It has been reported that phosphorylation and dephosphorylation of tyrosine residue Y-165 in the cytoplasmic region of CTLA-4 play an important role in its negative signaling and cell surface expression. Some signaling molecules such as Src homology 2 protein tyrosine phosphatase 2 (SHP-2) and the p85 subunit of phosphatidylinositol 3 kinase (P13 kinase) associate with phosphorylated tyrosine residue Y-165, through Src homology 2 (SH2) domains. On the other hand, the adapter complex proteins, AP-2 and AP-50 interact with the same tyrosine residue when unphosphorylated, resulting in clathrin-mediated endocytosis of CTLA-4 molecules. The objective of this study is to identify a tyrosine kinase that can directly bind and phosphorylate the critical tyrosine residue, Y-165 in the cytoplasmic domain of CTLA-4. Here, we demonstrated that 1) Janus Kinase 2 (Jak2) was directly associated with a box 1-like motif in the cytoplasmic tail of CTLA-4 molecule, 2) Jak2 phosphorylated Y-165 residue in the cytoplasmic region of CTLA-4 molecule, and 3) Jak2 was associated with CTLA-4 in HUT 78 T cell lines. (C) 2000 Wiley-Liss, Inc.
• Abortive alloantigen presentation by donor dendritic cells leads to donor-specific tolerance: a study with a preoperative CTLA4Ig inoculation

  H Harada, H Ishikura, Nakagawa, I, J Shindou, M Murakami, T Uede, T Koyanagi, T Yoshiki

  UROLOGICAL RESEARCH 28 (1) 69 - 74 0300-5623 2000/01 [Refereed][Not invited]
   

  Donor dendritic cells (DCs) within allografts initiate the induction of an allospecific T cell response, while an abortive alloantigen presentation by DCs may induce allospecific unresponsiveness. We thus investigated the tolerogenic effect of donor DCs that were made incompetent in alloantigen presentation by treatment of CTLA4Ig. When we treated rats with donor DCs (2 x 10(6)/rat i.v.) on the preoperative day, nine rejected allografts in an accelerated manner (5.0 +/- 2.2 vs. 8.2 +/- 1.6 days in the control group). Preoperative inoculation of DCs pulsed with CTLA4Ig, a procedure which suppresses an allogeneic mixed lymphocyte reaction (MLR), also provoked an accelerated rejection (5.6 +/- 1.7 days). When DCs and CTLA4Ig (500 mu g/rat i.p. on days -9, -7 and -5) were concomitantly inoculated, allograft survival was significantly prolonged (>38.7 +/- 40.0 days); a preoperative CTLA4Ig inoculation alone failed to do so (7.5 +/- 1.2 days). Long-term graft survivors tolerated skin grafts from the donor but not from those from a third party. These results indicate that abortive alloantigen presentation by donor DCs, upon which an accessory signal pathway is suppressed by CTLA4Ig, leads to prolonged graft survival and donor-specific tolerance.
• Involvement of CD28/CTLA4-B7 costimulatory pathway in the development of lymphadenopathy and splenomegaly in MRL/lpr mice

  M Takiguchi, M Murakami, Nakagawa, I, MM Rashid, N Tosa, S Chikuma, A Hashimoto, T Uede

  JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (1) 29 - 36 0916-7250 2000/01 [Refereed][Not invited]
   

  MRL/lpr mouse is an established animal model which develops autoimmune diseases including glomerulonephritis, sialoadenitis, hepatitis and inflammatory lung disease. Additionally, it has been reported that lpr strains uniquely accumulate CD3(+)CD4(-)CD8(-)B220(+) (double negative, DN) T cells in lymphoid organs leading to lymphadenopathy and splenomegaly. To investigate the role of CD28/CTLA4-B7 pathway in the development of lymphadenopathy and splenomegaly, MRL/lpr mice were treated with soluble form of CTLA4 molecules. CTLA4IgG, which efficiently blocks this pathway. It was demonstrated that (i) the development of DN T cells was independent of the CD28/CTLA4-B7 pathway, (ii) the CD28/CTLA4-B7 pathway was required for the development of lymphadenopathy and splenomegaly, (iii) the CD28/CTLA4-B7 pathway was important for the accumulation of various cell populations in the lymph node and spleen, (iv) composition of the accumulating cell populations was not altered by CTLA4IgG treatment, and (v) activation of conventional T cells and IL-4 production from conventional T cells were the CD28/CTLA4-B7 pathway dependent. Thus, we concluded that the CD28/CTLA4-B7 pathway was required for the development of full-blown lymphadenopathy and splenomegaly in MRL/lpr mice.
• The role of osteopontin in the development of granulomatous lesions in lung.

  S Chiba, M M Rashid, H Okamoto, H Shiraiwa, S Kon, M Maeda, M Murakami, M Inobe, A Kitabatake, A F Chambers, T Uede

  Microbiology and immunology 44 (4) 319 - 32 0385-5600 2000 [Refereed][Not invited]
   

  Osteopontin (OPN) has been shown to be expressed by cells in granulomas of various origins, but whether it plays a functional role in granuloma formation is not known. Here we used a cardiomyopathic hamster (TO2) model, to test the hypothesis that OPN contributes functionally to granuloma development. We immunized cardiomyopathic and normal hamsters by subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, and assessed various tissues for both OPN RNA expression and granuloma formation. Cardiomyopathic hamsters expressed OPN, and formed granulomatous lesions, in heart tissue in both immunized and untreated animals. In addition, immunization induced expression of OPN in lung and lymph nodes of cardiomyopathic (but not normal) hamsters, and also induced granuloma formation in these organs. To test whether OPN expression could play a functional role in inducing granulomas, we produced an adenoviral vector containing the murine OPN gene, and introduced this vector intratracheally into the lungs of normal hamsters. The OPN-containing vector, but not the control vector, induced pulmonary granuloma formation. These studies provided direct in vivo evidence that OPN can contribute functionally to the formation of granulomatous lesions, and suggest that OPN expression may be a common factor involved in formation of granulomas of various origin.
• アデノウイルスベクターを用いた CTLA 4 Ig 遺伝子導入による移植後免疫反応の制御

  上出 利光, 村上 正晃, 竹原 めぐみ, 藤堂 省

  日本臨床免疫学会会誌 = Japanese journal of clinical immunology 日本臨床免疫学会 22 (6) 417 - 417 0911-4300 1999/12/31
• Topical CTLA4-lg suppresses ongoing mucosal immune response in presensitized murine model of allergic rhinitis

  J Sato, K Asakura, M Murakami, T Uede, A Kataura

  INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY 119 (3) 197 - 204 1018-2438 1999/07 [Refereed][Not invited]
   

  Allergic rhinitis is thought to be mediated by CD4+ T cells producing Th2-associated cytokines. Optimal Ag-specific T-cell activation requires the engagement of T-cell receptor with antigen (Ag) in the context of MHC, and the engagement of appropriate costimulatory molecules. One of the most well-characterized costimulatory pathways is the interaction of B7/CD28-CTLA4 molecules. Recent studies have suggested that the costimulatory pathway may influence the development of Th2 immune responses. The objective of this study was the examination of the role of B7/CD28-CTLA4 costimulatory pathway in the pathogenesis of ovalbumin (OVA)induced immune response in presensitized murine model of allergic rhinitis, Systemically presensitized BALB/c mice significantly developed Ag-induced early phase nasal symptoms, nasal hyperresponsiveness to histamine, nasal eosinophilia, serum levels of OVA-specific IgE and Th2-associated cytokines following repeated topical Ag challenges. Topical administration of CTLA4-Ig during nasal challenges inhibited Ag-induced nasal symptoms and histamine hyperresponsiveness. We also found a significant reduction in nasal lavage eosinophilia and serum levels of OVA-specific IgE, Furthermore, CTLA4-Ig treatment significantly decreased interleukin (IL)-4 content in nasal tissue, while there was no significant change in IL-5 or IFN-gamma levels. These results suggest that B7/CD28-CTLA4 costimulatory pathway mediates the development of ongoing Th2 immune responses and plays a major role in regulating allergic disease, such as allergic rhinitis.
• Blockade of CD28/CTLA4-B7 pathway prevented autoantibody-related diseases but not lung disease in MRL/lpr mice

  M Takiguchi, M Murakami, Nakagawa, I, A Yamada, S Chikuma, Y Kawaguchi, A Hashimoto, T Uede

  LABORATORY INVESTIGATION 79 (3) 317 - 326 0023-6837 1999/03 [Refereed][Not invited]
   

  We studied the role of CD28/CTLA4 costimulatory T-cell activation pathway on the pathogenesis of MRL/Ipr mice. Administration of CTLA4IgG from day 0 significantly inhibited autoantibody production such as anti-double-stranded DNA antibody and rheumatoid factor. In addition, end-organ diseases in kidney, salivary gland, and liver were significantly improved. Improvement of pathologic findings coincided with a significant improvement in survival. At 350 days of age, 90% of mice treated with CTLA4IgG from day 0 were still alive, compared with none of mice treated with hIgG. As expected, activation of conventional T cells was significantly inhibited after CTLA4IgG treatment. However, lung disease that was characterized by perivascular accumulation and interstitial infiltration of lymphocytes and macrophages was not inhibited. Even after CTLA4IgG treatment from day 0, pathologic findings of lung disease were not improved. Additionally, the expression of activation markers such as B7-1, B7-2, CD71, ICAM1, and LFA1 on Mac1(+) fraction in both spleen and lung and the concentration of TNF alpha in bronchoalveolar lavage fluid were not significantly suppressed. These results demonstrated that lung disease was independent of the CD28/CTLA4-B7 pathway. Thus, this study emphasizes the differential dependence of the CD28/CTLA4-B7 pathway in development of diseases in MRL/Ipr mice.
• CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts

  K Tarumi, M Murakami, A Yagihashi, Nakagawa, I, K Hirata, T Uede

  TRANSPLANTATION 67 (4) 520 - 525 0041-1337 1999/02 [Refereed][Not invited]
   

  Background. CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. Methods. Small bowels of Brown-Norway rats (RT1(n)) were heterotopically transplanted into Lewis rats (RT1(l)). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. Results. kong-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1(b)) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 mu g/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 mu g/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Th1 to Th2 cytokine production.
• Suppressive effects of CTLA4-IG on nasal allergic reactions in presensitized murine model

  J Sato, K Asakura, M Murakami, T Uede, A Kataura

  LIFE SCIENCES 64 (9) 785 - 795 0024-3205 1999/01 [Refereed][Not invited]
   

  Ag-specific T cell activation requires the engagement of T cell receptor (TCR) with antigen in the context of MHC, and the engagement of appropriate costimulatory molecules. It is well established that B7/CD28-CTLA4 costimulatory pathway plays an important role in the induction of T helper (Th) cells in T-cell dependent immune reactions. In this study, we evaluated the effects of blocking the costimulatory pathway by systemic administration of CTLA4-Ig during repeated nasal antigen challenges in systemically presensitized mouse. The antigen-induced early phase nasal symptoms, nasal hyperresponsiveness to histamine and nasal eosinophilia were significantly suppressed by CTLA4-Ig treatment. Elevation of serum level of antigen-specific IgE, but not IgG1 or IgG2a was inhibited by the treatment. In relation to cytokine levels in the tissue extracts of the nasal mucosa, an up-regulation of IL-4 was significantly inhibited, however, the levels of IL-5 and IFN-gamma were not affected by the treatment. These results suggest that B7/CD28-CTLA4 costimulatory pathway plays an important role in on-going Th2-related allergic reactions in the nose.
• CD44 variants but not CD44s cooperate with beta 1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis

  YU Katagiri, J Sleeman, H Fujii, P Herrlich, H Hotta, K Tanaka, S Chikuma, H Yagita, K Okumura, M Murakami, Saiki, I, AF Chambers, T Uede

  CANCER RESEARCH 59 (1) 219 - 226 0008-5472 1999/01 [Not refereed][Not invited]
   

  The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility, First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycine-aspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants, Antibodies directed against the integrin beta 1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies, This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta 1 antibodies. Finally, we show that OPN binding by CD44 variants/beta 1-containing integrins promotes cell spreading, motility, and chemotactic behavior.
• Introduction of an osteopontin gene confers the increase in B1 cell population and the production of anti-DNA autoantibodies

  Junko Iizuka, Yohko Katagiri, Norihiro Tada, Masaaki Murakami, Tohru Ikeda, Masahiro Sato, Katsuiku Hirokawa, Seiji Okada, Masahiko Hatano, Takeshi Tokuhisa, Toshimitsu Uede

  Laboratory Investigation 78 (12) 1523 - 1533 0023-6837 1998/12 [Refereed][Not invited]
   

  Osteopontin (OPN) is an Arg-Gly-Asp-containing phosphoprotein that is secreted by activated T cells. The concentration of serum OPN protein is elevated in autoimmune-prone MRL-lpr mice as well as in patients with systemic lupus erythematosus. Previously, it was shown that OPN induces the polyclonal activation of B cells, resulting in the augmented production of immunoglobulin, indicating that OPN plays some role in the development of autoimmune disease. However, the link between OPN and development of autoimmune disease remains unclear. To analyze the role of OPN in immune system and autoimmune diseases, we have generated two kinds of transgenic mice: one carries the immunoglobulin (Ig) enhancer/SV40 promoter and the other carries the cytomegalovirus enhancer/chicken β-actin (CAG) promoter. In both groups of transgenic mice, the B1 cell population in peritoneal cavity was markedly increased and titer of IgM and IgG3 antibodies in the serum was considerably higher than that in wild-type mice. Most important, the titer of the IgM class of anti-double-stranded DNA antibody was significantly elevated in transgenic mice. These results strongly suggest that OPN may have an important role in the propagation and differentiation of B1 cells and production of autoantibodies.
• The mechanism by which proteolysis enhances the ligand-binding activity of guinea pig type II Fc receptor for IgG (Fc gamma RIIB)

  Y Isashi, T Yamashita, S Nagasawa, K Tanaka, M Murakami, T Uede

  JOURNAL OF BIOCHEMISTRY 123 (5) 959 - 967 0021-924X 1998/05 [Refereed][Not invited]
   

  We have previously shown that the ligand-binding activity of type II Fc receptor for IgG (Fc gamma RIIB) on guinea pig peripheral blood polymorphonuclear leukocytes is very low and dramatically increases after treatment of the cells with proteolytic enzymes. In the present study, we analyzed the mechanism of this augmentation. We found that the protease treatment failed to enhance the binding of monomeric IgG to Fc gamma RIIB, increased the binding of small immune complexes (IC) prepared under antigen-excess conditions only modestly, but markedly enhanced the binding of large IC prepared under antibody-excess conditions. These results suggest that proteolysis increases the ligand-binding avidity but not the intrinsic affinity of Fc gamma RIIB, Confocal laser scanning microscopy revealed that the mobility of Fc gamma RIIB on the cell surface was increased after protease treatment. In addition, transfection experiments indicated that the effect of proteolysis on IC binding to CHO cells expressing guinea pig Fc gamma RIIB was strongly dependent on the receptor density. Finally, we demonstrated that the transmembrane and cytoplasmic domains of Fc gamma RIIB were not involved in the proteolysis-induced augmentation of IC binding. Together our results suggest that the mobility of Fc gamma RIIB, which may be restricted due to the association of the ectodomain of the receptor with unknown membrane proteins, is enhanced by proteolysis, allowing the receptors to bind multivalent ligands more readily and hence with higher avidity.
• Expression and co-stimulatory function of B7-2 on murine CD4+ T cells

  Rie Hakamada-Taguchi, Takuma Kato, Hiroshi Ushijima, Masaaki Murakami, Toshimitsu Uede, Hideo Nariuchi

  European Journal of Immunology 28 (3) 865 - 873 0014-2980 1998/03 [Refereed][Not invited]
   

  Co-stimulatory signals mediated by the interaction of B7-1/B7-2 with CD28 are important for the activation of CD4+ T cells stimulated with antigen on antigen-presenting cells. There are controversies about the expression and function of B7-1/B7-2 on CD4+ T cells. The aim of this study was to analyze the expression of B7-1/B7-2 on naive and memory CD4+ T cells and the co-stimulatory function in the activation of naive CD4+ T cells stimulated by TCR ligation. Present results indicate that memory CD4+ T cells express B7-2 molecules on their surface, whereas naive CD4+ T cells do not. Neither memory nor naive CD4+ T cells expressed B7-1 molecule on their surface, although B7-1 mRNA was faintly expressed in memory T cells. B7-2 molecules expressed on memory T cells co-stimulated CD4+ naive T cells stimulated with plate-coated anti-CD3 to produce IL-2. Naive CD4+ T cells were shown to express B7-2 after co-stimulation with B7-2 and TCR ligation, because the naive T cells stimulated with anti-CD3 and B7-2CHO expressed B7-2 on their surface, although it remained to be studied whether the co-stimulation with B7-2 directly induced B7-2 expression on naive T cells. Our present results indicate that memory CD4+ T cells play some role in the activation of naive CD4+ T cells through the co-stimulation with B7-2 molecules.
• Vaccination with B7-1(+) tumor and anti-adhesion therapy with RGD pseudo-peptide (FC-336) efficiently induce anti-metastatic effect

  H Fujii, M Inobe, Y Hayakawa, F Kimura, M Murakami, Y Onishi, Azuma, I, T Uede, Saiki, I

  CLINICAL & EXPERIMENTAL METASTASIS 16 (2) 141 - 148 0262-0898 1998/03 [Refereed][Not invited]
   

  We have previously shown that expression of costimulatory ligand B7-1 on MHC class I+ tumor cells (B16-BL6 melanoma) resulted in marked reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice and led to induction of immunity to the challenge by the parental B7-1 negative tumor. Here we investigated the effectiveness of irradiated B7-1 transfected tumor cells as a vaccine on established tumor metastasis and whether or not expression of B7-1 molecule on tumor cells in combination with administration of anti-adhesion peptide FC-336 can augment the antimetastatic efficacy. Immunization with X-irradiated B7-1 transfectants after i.v. injection of B7-1(-) parental B16-BL6 cells was effective in inhibiting lung metastasis. We also found that vaccination with irradiated B7-1 transfectants after excision of primary tumor on day 21 resulted in significant inhibition of spontaneous lung metastasis by intrafootpad injection of viable parental B16-BL6 melanoma, as compared with the untreated control. However, immunizing twice with mock transfectants did not affect inhibition of spontaneous lung metastasis of wild-type tumors. On the other hand, multiple administration of a pseudo-peptide of RGD sequence (FC-336) after tumor inoculation inhibited spontaneous lung metastasis through the interference of tumor invasion, migration and adhesion. Combined treatment of B7-1 transfected tumor vaccine and anti-adhesive therapy with FC-336 led to the augmentation of the antimetastatic effect in both experimental and spontaneous metastasis models, as compared with either treatment alone. B7-1- and FC-336-mediated inhibition of tumor metastasis may be mediated by different mechanisms at various steps of metastasis, based on the regulation (promotion or inhibition) of tumor interaction with host cells and components. (C) 1998 Rapid Science Ltd.
• Prolongation of rat small bowel allograft survival of CTLA-4 IG

  K. Tarumi, A. Yagihashi, M. Murakami, T. Uede, K. Hirata

  Transplantation Proceedings 30 (6) 2596 - 2599 0041-1345 1998 [Refereed][Not invited]
  
• The functional role of B7 molecules on the induction of thymocyte activation and apoptosis

  N Aoki, M Inobe, M Murakami, R Abe, H Iizuka

  MICROBIOLOGY AND IMMUNOLOGY 42 (8) 555 - 565 0385-5600 1998 [Refereed][Not invited]
   

  To evaluate the role of B7 on thymocyte activation and apoptosis, we took advantage of TCR transgenic mice in which the majority of thymocytes express a uniform TCR that is specific for ovalbumin, We also prepared Chinese hamster ovary (CHO) cells expressing B7 and appropriate class II molecules. We found that the apoptosis of double-positive thymocytes by TCR-mediated signaling, which presumably represents negative selection, requires a costimulatory signal provided by B7-1 or B7-2. The requirement of B7-1 costimulation for the apoptosis of thymocytes does not change in either low or high antigenic peptide loading. We also demonstrated that two signals through TCR and CD28 augmented the proliferation of thymocytes, and the requirement of CD28-mediated signal by B7-1 or B7-2 for thymocyte proliferation became less evident when high doses of antigenic peptide were loaded, indicating that the intensity of TCR-mediated signal determines the requirement of B7-mediated second signal for thymocyte proliferation.
• B7-1-transfected tumor vaccine counteracts chemotherapy-induced immunosuppression and prolongs the survival of rats bearing highly metastatic osteosarcoma cells

  M Hayakawa, S Kawaguchi, S Ishii, M Murakami, T Uede

  INTERNATIONAL JOURNAL OF CANCER 71 (6) 1091 - 1102 0020-7136 1997/06 [Refereed][Not invited]
   

  To investigate the therapeutic efficacy of B7-1-expressing tumor vaccine on metastatic osteosarcoma, we introduced mouse B7-1 cDNA into a rat osteosarcoma cell line, MSK-8G. Flow cytometric analysis confirmed that the transfectants designated as B7-1-8G 10-1 and B7-1-8G 15-5 stably expressed B7-1 molecules on the cell surface. B7-1 transfectants were not only rejected by immunocompetent F344 rats but also conferred systemic immunity that protected against challenge with B7-negative parental osteosarcoma cells. In contrast, T-cell-deficient nude rats failed to reject B7-1 transfectants, indicating that T cells play a major role in the development of systemic immunity. We then conducted experimental therapies using irradiated B7-1-transfected tumor vaccine and methotrexate in an orthotopic implantation model. B7-1-transfected tumor vaccine significantly reduced the number of pulmonary metastatic nodules. Moreover, the combination of methotrexate and tumor vaccine further decreased the number of pulmonary metastatic nodules. Most important, the combined therapy with methotrexate and tumor vaccine resulted in a tumor-free condition as judged by the histopathological absence of tumors and survival of rats for more than 180 days. Furthermore, B7-1-transfected tumor vaccine could counteract the immunosuppressive effect of methotrexate. These findings strongly suggest that the B7-1-transfected tumor vaccine may be of clinical value for patients with metastatic osteosarcoma who exhibit immunosuppression due to chemotherapy. (C) 1997 Wiley-Liss, Inc.
• Vascularized bone marrow allotransplantation in rats prolongs a simultaneous skin allograft

  Y Tsuchida, M Usui, M Murakami, T Uede

  TRANSPLANTATION PROCEEDINGS 29 (3) 1732 - 1733 0041-1345 1997/05 [Refereed][Not invited]
  
• Involvement of a common 10-amino-acid segment in the cytoplasmic region of CD40 but different MAP kinases in different CD40-mediated responses

  Toyomichi Hara, Masaaki Murakami, Hiroaki Maeda, Masahiko Hibi, Toshimitsu Uede

  Biochemical and Biophysical Research Communications 233 (1) 187 - 192 0006-291X 1997/04/07 [Refereed][Not invited]
   

  CD40-mediated signals can induce cell aggregation, proliferation and rescue from apoptosis in WEHI231. To define which segment of cytoplasmic domain of CD40 and how signals are involved in those events, we generated mutant CD40 transfectants. We demonstrated the same 10 amino acid segment that could bind to tumor necrosis factor receptor associated factor-2 and -3 mediated all those responses. However, activation pattern of mitogen activated protein kinases was different. Immunoglobulin M-mediated apoptosis was inhibited by CD40-mediated signal that activated c-Jun aminoterminal kinase synergistically. While, CD40 stimulus through the 10 amino acid segment alone that induced cell aggregation and proliferation resulted in activation of extracellular signal-regulated protein kinase 2.
• Limb allografts in rats treated with anti-ICAM-1 and anti-LFA-1 monoclonal antibodies

  Yoshihiko Tsuchida, Masamichi Usui, Takafumi Naitoh, Terukazu Takahashi, Masaaki Murakami, Toshimitsu Uede

  Journal of Reconstructive Microsurgery 13 (2) 107 - 110 0743-684X 1997 [Refereed][Not invited]
   

  This study examined whether administration of anti-ICAM-1 mAb (monoclonal antibody) and anti-LFA-1 mAb were effective for inhibition of the acute rejection reaction in a rat-limb allograft model. The grafts were carried out using major histocompatibility complex mismatch pairs of rats: ACl rats as the donors and Lewis rats as the recipients. The subjects were of species with completely different major histocompatibility complexes. The average length of survival in this combination was 4.7 ± 0.6 days. When anti-ICAM-1, anti-LFA-1 mAb, or a combination of both were administered intraperitoneally at 1 mg/kg body weight daily for 7 days after the graft, the mean survival times were 4.3 ± 0.6, 4.7 ± 0.6, or 4.7 ± 0.6 days, respectively. The agents were found to exercise no beneficial effects for transplants. Transplants were then carried out with a single intramuscular administration of FK506 at 3mg/kg body weight on the day of the operation, to observe the effect of this adjunct therapy. The mean survival time averaged 16.0 ± 5.5 days with FK506 alone. Administration of anti-ICAM-1, anti-LFA- 1, and both antibodies together with FK506 resulted in mean survival times of 15.4 ± 3.0, 25.0 ± 5.8 and 13.2 ± 5.8 days, respectively. Thus, a significant prolongation of survival time was observed when the combination of anti-LFA-1 mAb and FK506 was used. In contrast, anti-ICAM-1 mAb and FK506 or both mAb and FK506 showed no beneficial effects.
• Molecular cloning and characterization of guinea pig Fc gamma RIII: Expression but not function is independent of the gamma chain of Fc epsilon RI

  Y Isashi, T Yamashita, S Nagasawa, M Murakami, T Uede

  INTERNATIONAL IMMUNOLOGY 8 (9) 1335 - 1346 0953-8178 1996/09 [Refereed][Not invited]
   

  We have isolated two cDNA clones encoding the guinea pig receptor for the Fc portion of IgG2 (Fc gamma 2R) from a guinea pig peritoneal macrophage cDNA library. Analysis of the predicted amino acid sequence of the one cDNA clone indicated that the guinea pig Fc gamma 2R is a type I transmembrane protein and has similar to 72% DNA sequence homology and similar to 57% protein sequence homology with the human Fc gamma RIII. Therefore, we propose that the guinea pig Fc gamma 2R is referred to as guinea pig Fc gamma RIII. The most important finding in this report is that the obtained cDNA directed the cell surface expression of the Fc gamma 2R on COS-7 cells without association with the gamma chain of the high-affinity IgE receptor (Fc epsilon RI gamma) which is required for human and mouse Fc gamma RIII to be expressed on the cell surface. Furthermore, we demonstrated that the endocytosis activity of Fc gamma RIII is dependent upon the association with Fc epsilon RI gamma, suggesting that Fc epsilon RI gamma is involved in the functions of guinea pig Fc gamma RIII. The other clone was found to lack the sequence encoding transmembrane and cytoplasmic domains, suggesting the presence of a soluble form of guinea pig Fc gamma RIII. Northern blot analysis and RT-PCR showed that a transmembrane form of guinea pig Fc gamma RIII was expressed in peritoneal macrophages, but not in neutrophils in spite of the fact that they express Fc gamma 2R, indicating that the Fc gamma 2R on neutrophils is a product of a distinct gene.
• Non-RGD domains of osteopontin promote cell adhesion without involving αv integrins

  Yohko U. Katagiri, Masaaki Murakami, Kiyoshi Mori, Junko Iizuka, Toyomichi Hara, Kumiko Tanaka, Wen-Yi Jia, Ann F. Chambers, Toshimitsu Uede

  Journal of Cellular Biochemistry 62 (1) 123 - 131 0730-2312 1996/07 [Refereed][Not invited]
   

  Osteopontin (OPN) is an integrin-binding secreted protein that contains an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell types via RGD-mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N-terminal or C-terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16-BL6 cells was not inhibited by either GRGDS peptides or the anti-αv antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C-terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti-αv antibody. Together these results indicate that B16-BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN.
• The role of the B7-1a molecule, an alternatively spliced form of murine B7-1 (CD80), on T cell activation

  M Inobe, N Aoki, PS Linsley, JA Ledbetter, R Abe, M Murakami, T Uede

  JOURNAL OF IMMUNOLOGY 157 (2) 582 - 588 0022-1767 1996/07 [Refereed][Not invited]
   

  B7 molecules (CD80 (B7-1) and CD86 (B7-2/B70)) on APCs provide costimulatory signals for T cell proliferation. We previously described the presence of an alternatively spliced form of murine CD80 (previously termed MB7-2 and renamed as B7-1a) that completely lacks the second Ig-like domain coded by exon 3 in activated murine B cells. In this study, we first examined whether B7-1a mRNA can be detected in vivo by RNase protection assay. The expression of B7-1a mRNA was only detected in lymphoid organs although the level of expression was lesser than that of CD80 mRNA. However, we demonstrated that the expression of B7-1a mRNA like CD80 mRNA was considerably augmented in spleen cells treated with either LPS in vitro or OVA/CFA conjugate in vivo. We next determined the functional activity of B7-1a using Chinese hamster ovary (CHO) cells transfected by B7 genes. When resting T cells were cocultured with CHO cells expressing B7-1a molecules in the presence of PMA/ionomycin, T cell proliferation was not detected, while CHO cells either expressing CD80 or CD86 could promote the proliferation of resting T cells. In contrast to resting T cells, CHO cells expressing B7-1a could support the proliferation of activated T cells. Thus, costimulatory activity of B7-1a molecules was dependent upon the activation stage of T cells. Therefore the IgV-like region of CD80 contains a critical region for functional interaction with its ligands and can transduce a costimulatory signal for T cell proliferation.
• Identification and characterization of an alternative cytotoxic T lymphocyte-associated protein 4 binding molecule on B cells

  M Murakami, Y Takahashi, Y Isashi, S Kon, WY Jia, M Inobe, R Abe, T Uede

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 93 (15) 7838 - 7842 0027-8424 1996/07 [Refereed][Not invited]
   

  To determine whether alternative cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins exist on B cells, we constructed (i) mCTLA4hIgG consisting of the extracellular region of a mouse CTLA4 molecule and the Fc portion of a human IgG1 molecule and (ii) PYAAhIgG, a mutant mCTLA4hIgG, having two amino acid substitutions on the conserved MYPPPY motif in the complementarity-determining region 3-like region and lacking detectable binding to both B7-1 and B7-2 molecules. Using these fusion proteins (mCTLA4hIgG and PYAAhIgG), we demonstrated that a mouse immature B-cell line, WEH1231 cells, expressed alternative CTLA4 binding molecules (ACBMs) that were distinct from both B7-1 and B7-2. ACBMs were 130-kDa disulfide-linked proteins. More importantly, ACBMs were able to provide costimulatory signal for T-cell proliferation in the presence of anti-CD3 monoclonal antibodies. In addition, we demonstrated that more than 20% of B220(+) cells obtained from normal mouse spleen expressed ACBMs.
• Vaccination of tumor cells transfected with the B7-1 (CD80) gene induces the anti-metastatic effect and tumor immunity in mice

  H Fujii, M Inobe, F Kimura, J Murata, M Murakami, Y Onishi, Azuma, I, T Uede, Saiki, I

  INTERNATIONAL JOURNAL OF CANCER 66 (2) 219 - 224 0020-7136 1996/04 [Refereed][Not invited]
   

  The present study demonstrates that the transfection of B7-1 or its variant MB7-2 genes into MHC class 1(+) tumor cells (B16-BL6 or K1735-M2 melanoma) resulted in the remarkable reduction of lung metastasis caused by i.v. injection into immunocompetent syngeneic mice. However, i.v. injection of the transfectants into T cell-deficient nude mice did not affect reduction of lung tumor colonies as compared with parental wild-type tumors, suggesting that such an inhibitory effect was closely associated with T cell-mediated responses. The reduced metastasis of B7(+) tumor cells consequently led to the significant prolongation of survival. Expression of B7 on tumor cells did not influence the tumorigenicity in vivo and tumor cell invasion into basement membrane Matrigel in vitro. We also found that immunization of X-irradiated by transfectants was effective as a tumor vaccine for preventing lung metastasis caused by i.v. injection of B7(-) parental B16-BL6 cells but not against other syngeneic 3LL tumors. Thus, the by-mediated anti-metastatic effect was tumor-specific. Vaccinations of irradiated B7(+) tumor cells before and after surgical excision of the s.c. inoculated primary B7(-) tumors on day 21 achieved effectively the prevention of spontaneous lung metastasis. Our report that vaccination of irradiated B7(+) tumor cells led to a therapeutic effect in an established tumor metastasis model clearly expands and confirms previous related observations. (C) 1996 Wiley-Liss, Inc.
• CD28 co-stimulatory signals induce IL-2 receptor expression on antigen-stimulated virgin T cells by an IL-2-independent mechanism

  K Toyooka, S Maruo, T Iwahori, N Yamamoto, XG Tai, R Abe, Y Takahama, M Murakami, T Uede, T Hamaoka, H Fujiwara

  INTERNATIONAL IMMUNOLOGY 8 (2) 159 - 169 0953-8178 1996/02 [Refereed][Not invited]
   

  Intravenous sensitization of C57BL/6 (B6) mice with class II H-2-disparate B6-C-H-2(bm12) (bm12) resting B cells induced anti-bm12 CD4(+) T cell tolerance as shown by hyporesponsiveness in the anti-bm12 mixed lymphocyte reaction (MLR). The present study investigated the mechanism(s) of the failure of bm12 B cells to stimulate the proliferation of B6 anti-bm12 CD4(+) T cells. While stimulation in vitro of B6 splenic T cells with bm12 antigen-presenting cells (APC) induced IL-2 mRNA expression and IL-2 production, T cells stimulated with bm12 B cells expressed much less IL-2 mRNA and secreted very low but detectable levels of IL-2. Moreover, the T cells stimulated with the bm12 B cells did not proliferate and this was not corrected by the addition of rIL-2. These results suggest that T cells stimulated with bm12 B cells fail to generate IL-2 responsiveness. Further, whereas IL-2 receptor (IL-2R) alpha chain expression was significantly induced on B6 T cells stimulated with bm12 APC; stimulation with bm12 B cells did not induce IL-2R expression over background levels. However, virgin T cells stimulated with both bm12 B cells and anti-CD28 mAb proliferated and displayed a dramatic increase in IL-2 production as well as IL-2R expression to levels commensurate with those resulting from bm12 APC stimulation. IL-2R expression was induced by stimulation with bm12 APC or bm12 B cells plus anti-CD28 mAb even in the presence of sufficient amounts of anti-IL-2 mAb for neutralizing produced IL-2; while levels of IL-2R were significantly lower compared to those induced in the absence of anti-IL-2 mAb, increased frequencies of IL-2R(+) cells were comparable. Conversely, IL-2R was not induced by bm12 B cell stimulation in the presence of IL-2. Moreover, IL-2R expression and proliferation induced by stimulation with bm12 APC was inhibited by CTLA-4-Ig, a soluble recombinant fusion protein capable of blocking the CD28 co-stimulatory pathway. Thus, these results indicate that the failure of resting B cells to induce full activation of alloreactive virgin T cells is due to ineffective CD28 co-stimulation and that the CD28 co-stimulatory signals not only stimulate IL-2 production but also induce IL-2R expression by an IL-2-independent mechanism.
• Long-term acceptance of major histocompatibility complex-mismatched cardiac allograft induced by a low dose of CTLA4IgM plus FK506

  A Yamada, M Murakami, K Ijima, H Yagita, K Okumura, S Komatsu, T Uede

  MICROBIOLOGY AND IMMUNOLOGY 40 (7) 513 - 518 0385-5600 1996 [Not refereed][Not invited]
   

  The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fe portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2(b)) neonatal hearts were transplanted to CBA/J (H-2(k)) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.
• Functional Analysis of the Osteopontin Molecule

  YOHKO KATAGIRI, KIYOSHI MORI, TOYOMICHI HARA, KUMIKO TANAKA, MASAAKI MURAKAMI, TOSHIMITSU UEDE

  Annals of the New York Academy of Sciences 760 (1) 371 - 374 1749-6632 1995 [Refereed][Not invited]
  
• The rat neutrophil low-affinity Fc receptor for IgG: molecular cloning and functional characterization

  Yasuhiro Isashi, Masatada Tamakoshi, Yumiko Nagai, Tetsuo Sudo, Masaaki Murakami, Toshimitsu Uede

  Immunology Letters 46 (1-2) 157 - 163 0165-2478 1995 [Refereed][Not invited]
   

  A complementary DNA (cDNA) clone encoding rat Fcγ receptor II (FcγRII) was isolated from rat neutrophils and characterized. The cDNA encodes a type I transmembrane protein with 285 amino acids having an extracellular domain consisting of two immunoglobulinlike domains (179 amino acids), a transmembrane domain (26 amino acids), and a cytoplasmic domain (47 amino acids). The nucleotide sequences are identical to that of recently cloned FcγRII from rat mast cells. This protein was expressed on FcR-negative Chinese hamster ovary (CHO) cells. The characterization of cDNA-transfected CHO cells clearly indicated that the protein encoded by the cDNA clone binds guinea-pig IgG1 and IgG2 complexes and unexpectedly binds monomeric rat IgG1, but not IgG2. Furthermore, the affinity for immune complexes was significantly augmented by protease treatment of transfectants. In addition, endocytosis of immune complex was noted in transfectants. © 1995.
• Neutrophil-independent myocardial dysfunction during an early stage of global ischemia and reperfusion of isolated hearts

  Yasuhiro Kamikubo, Masaaki Murakami, Michiaki Imamura, Toshifumi Murashita, Keishu Yasuda, Toshimitsu Uede

  Immunopharmacology 29 (3) 261 - 271 0162-3109 1995 [Refereed][Not invited]
   

  The effect of global ischemia and reperfusion on the expression of cytokine genes and cell adhesion molecules by myocardial tissues and neutrophils was studied by using the Langendorff model. Although cardiac function deteriorated after reperfusion of ischemic hearts, there was no evidence of inflammation and myocardial degeneration, which is in contrast to previous findings that neutrophil-mediated inflammation is a critical step in post-ischemic reperfusion injury in regional ischemia. Flow cytometry analysis demonstrated that the global ischemia and reperfusion did not affect the expression of adhesion molecules on neutrophils. We also examined the expression of various cytokines which are involved in inflammatory responses. Only interleukin 1α was induced after the reperfusion of the ischemic hearts. These results suggest that neutrophils barely contribute to the myocardial dysfunction and IL-1α may play a role in post-ischemic myocardial dysfunction during the early stage of reperfusion. © 1995 Elsevier Science B.V. All rights reserved.
• IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase

  Masaaki Murakami, Masahiko Hibi, Naoko Nakagawa, Toshimasa Nakagawa, Kiyoshi Yasukawa, Koichi Yamanishi, Tetsuya Taga, Tadamitsu Kishimoto

  Science 260 (5115) 1808 - 1810 0036-8075 1993 [Refereed][Not invited]
   

  The biological functions of interleukin-6 (IL-6) are mediated through a signal-transducing component of the IL-6 receptor, gp130, which is associated with the ligand-occupied IL-6 receptor (IL-6R) protein. Binding of IL-6 to IL-6R induced disulfide-linked homodimerization of gp130. Tyrosine kinase activity was associated with dimerized but not monomeric gp130 protein. Substitution of serine for proline residues 656 and 658 in the cytoplasmic motif abolished tyrosine kinase activation and cellular responses but not homodimerization of gp130. The IL-6-induced gp130 homodimer appears to be similar in function to the heterodimer formed between the leukemia inhibitory factor (LIF) receptor (LIFR) and gp130 in response to the LIF or ciliary neurotrophic factor (CNTF). Thus, a general first step in IL-6-related cytokine signaling may be the dimerization of signal-transducing molecules and activation of associated tyrosine kinases.
• INTERLEUKIN-6 RECEPTOR AND SIGNALS

  T TAGA, M HIBI, M MURAKAMI, M SAITO, H YAWATA, M NARAZAKI, Y HIRATA, T SUGITA, K YASUKAWA, T HIRANO, T KISHIMOTO

  CHEMICAL IMMUNOLOGY 51 181 - 204 1015-0145 1992 [Refereed][Not invited]
  
• Regulation of IL-6 receptor and GP130 expression on human cell lines of lymphoid and myeloid origin

  Andras Falus, Tetsuya Taga, Masahiko Hibi, Masaaki Murakami, Tadamitsu Kishimoto

  Cytokine 4 (6) 495 - 499 1043-4666 1992 [Refereed][Not invited]
   

  The expression of 80 kDa interleukin-6 receptor (IL-6R) and the associated molecule gp130 has been studied on human cell lines by FACS- and Northern blot analysis. The effects of dexamethasone, dibutyric-(DB)-cAMP and phorbol-12-myristate-13-acetate (TPA) have been studied on plasmacytoma cell line U266, B cell line BMNH and monocytoid cell line U937. Our data show a definite downregulation of IL-6R and gp130 expression by TPA in U266 and BMNH at both mRNA and cell surface protein levels. In U937 TPA inhibits only the IL-6R expression, without affecting that of gp130. DB-cAMP decreases the expression of both proteins in U937, slightly inhibits the IL-6R expression in U266, but is uneffective in BMNH. Dexamethasone induces considerable upregulation of gp130 only in U266. Our findings suggest separate regulation of IL-6R and gp130 on U266, BMNH and U937 cell lines. © 1992.
• Critical cytoplasmic region of the interleukin 6 signal transducer gp130 is conserved in the cytokine receptor family

  Masaaki Murakami, Masashi Narazaki, Masahiko Hibi, Hideo Yawata, Kiyoshi Yasukawa, Michinari Hamaguchi, Tetsuya Taga, Tadamitsu Kishimoto

  Proceedings of the National Academy of Sciences of the United States of America 88 (24) 11349 - 11353 0027-8424 1991 [Refereed][Not invited]
   

  Interleukin 6 (IL-6) signal is transduced through gp130 that associates with a complex of IL-6 and IL-6 receptor. Truncations or amino acid substitutions were introduced in the cytoplasmic region of human gp130, and the mutant cDNAs were transfected into murine interleukin 3-dependent cells to determine amino acid residues critical for generating the IL-6-mediated growth signal. In the 277-amino acid cytoplasmic region of gp130, a 61-amino acid region proximal to the transmembrane domain was sufficient for generating the growth signal. In this region, two short segments were significantly homologous with other cytokine-receptor family members. One segment is conserved in almost all members of the family, and the other is found especially in granulocyte colony-stimulating factor receptor, interleukin 2 receptor β chain, erythropoietin receptor, KH97 (a granulocyte/macrophage colony-stimulating factor receptor-associated molecule), and interleukin 3 receptor. gp130 molecules with mutations in either of these two segments could not transduce growth signal. Loss of signal-transducing ability of gp130 with such a mutation coincided with disappearance of IL-6-induced tyrosine phosphorylation of gp130.
• MOLECULAR-CLONING AND EXPRESSION OF AN IL-6 SIGNAL TRANSDUCER, GP130

  M HIBI, M MURAKAMI, M SAITO, T HIRANO, T TAGA, T KISHIMOTO

  CELL 63 (6) 1149 - 1157 0092-8674 1990/12 [Refereed][Not invited]
   

  Interleukin-6 (IL-6) signal is transduced through a membrane glycoprotein, gp130, which associates with IL-6 receptor (IL-6-R). A cDNA encoding human gp130 has been cloned, revealing that it consists of 918 amino acids with a single transmembrane domain. The extracellular region comprises six units of a fibronectin type III module, and part of this region of approximately 200 amino acids has features typical of a cytokine receptor family. A cDNA-expressed gp130 showed no binding property to IL-6 or several other cytokines. Although a transfectant with an IL-6-R cDNA expressed mainly low affinity IL-6 binding sites, an increase in high affinity binding sites was observed after cotransfection with a gp130 cDNA. This confirmed that a gp130 is involved in the formation of high affinity IL-6 binding sites. A cloned gp130 could associate with a complex of IL-6 and soluble IL-6-R and transduce the growth signal when expressed in a murine IL-3-dependent growth signal when expressed in a murine IL-3-dependent cell line.
• INTERLEUKIN-6 (IL-6) AND ITS RECEPTOR (IL-6R) IN MYELOMA PLASMACYTOMA

  S SUEMATSU, M HIBI, T SUGITA, M SAITO, M MURAKAMI, T MATSUSAKA, T MATSUDA, T HIRANO, T TAGA, T KISHIMOTO

  CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY 166 13 - 22 0070-217X 1990 [Refereed][Not invited]
  
• Interleukin 6 and its receptor in the immune response and hematopoiesis

  Toshio Hirano, Tetsuya Taga, Tadashi Matsuda, Masahiko Hibi, Sachiko Suematsu, Bo Tang, Masaaki Murakami, Tadamitsu Kishimoto

  The International Journal of Cell Cloning 8 (1 S) 155 - 167 1549-4918 1990 [Refereed][Not invited]
   

  Interleukin 6 (IL‐6) plays critical roles in the immune response and hematopoiesis. It is a potent B cell differentiation factor inducing antibody‐forming plasma cells. It enhances interleukin 3‐induced proliferation of hematopoietic stem cells. Furthermore, IL‐6 induces maturation of megakaryocytes. In IL‐6 transgenic mice, a massive polyclonal plasmacytosis and an increase in the number of mature megakaryocytes in the bone marrow were observed. The data indicated that deregulated expression of the IL‐6 gene induced a polyclonal plasmacytosis and could be involved in the oncogenesis of plasma cell neoplasias. IL‐6 receptor (IL‐6R) was molecularly cloned and found to be an immunoglobulin superfamily having an MW of 80 kDa. Upon the binding of EL‐6 to its 80 kDa IL‐6R, a second non‐binding molecule, gp130 was shown to associate with IL‐6R. The complex of IL‐6 and soluble IL‐6R lacking both transmembrane and cytoplasmic domains could bind gp130 and transduce the signal. The results indicate that the IL‐6R system consists of two polypeptide chains: one is an 80 kDa ligand‐binding molecule and the other is a possible signal transducer, gp130. Copyright © 1990 AlphaMed Press
• IL-6 AND IL-6 RECEPTOR

  M MURAKAMI, T HIRANO

  SEIKAGAKU 62 (1) 32 - 35 0037-1017 1990/01 [Refereed][Not invited]
  
• Massive subcutaneous hemangiosarcoma in a dog.

  藤永徹, 諏訪隆彦, 成清美智代, 奥村正裕, 井上久美子, 滝口満喜, 村上正晃, 田島誉士, 大友勘十郎

  日本獣医師会雑誌 42 (7) 491 - 494 0446-6454 1989 [Refereed][Not invited]
  

Conference Activities & Talks

• 血液脳関門への免疫細胞の侵入口の形成—炎症アンプのヒト病気への関連と定常時の機能  [Invited]

  村上 正晃

  第13回神経-筋の免疫疾患を考える会  2013
• EAE investigation by using the ultra-high field MRI.  [Invited]

  Masaaki Murakami

  Osaka University Live Immuno-Imaging Facility Opening workshop  2013
• How is a gateway of immune cells created in the brain-blood barrier?  [Invited]

  Masaaki Murakami

  Neuro2013  2013
• A gravity-mediated gateway for autoreactive CD4+ T cells in the brain-blood barrier.  [Invited]

  Masaaki Murakami

  34th International Society for Gravitational Physiology  2013
• 炎症アンプによる病気、病態の制御  [Invited]

  村上 正晃

  次世代重点研究プログラムセミナー  2013
• 炎症の慢性化機構の解明と制御  [Invited]

  村上 正晃

  第18回日本病態プロテアーゼ学会学術集会  2013
• 慢性炎症の誘導機構”炎症アンプ”の活性化と病態形成  [Invited]

  村上 正晃

  第６回Symphony  2013
• 炎症アンプの活性化と関節炎の発症  [Invited]

  村上 正晃

  小児リウマチ学会  2013
• 炎症の慢性化誘導機構『IL-6アンプ』と関節炎  [Invited]

  村上 正晃

  第56回日本リウマチ学会学術集会  2012
• 神経刺激による血液脳関門の血管の制御：自己反応性T細胞の侵入口の形成  [Invited]

  村上 正晃

  第46回北摂循環器研究会  2012
• 非免疫細胞に存在する炎症誘導機構、 IL-6アンプ  [Invited]

  村上 正晃

  北海道大学遺伝子病制御研究所セミナー  2012
• 神経刺激による血液脳関門への免疫細胞の侵入口の形成  [Invited]

  村上 正晃

  第22回サイトメトリー学会学術集会  2012
• 局所の神経活性化による病原性T細胞の中枢移行メカニズム  [Invited]

  村上 正晃

  病態に根ざしたALSの新規治療法開発分科班ワークショップ  2012
• 局所の神経活性化による自己免疫病の制御  [Invited]

  村上 正晃

  名古屋大学大学院医学系研究科GCOEニューロサイエンスコースセミナー  2012
• IL-6アンプと慢性炎症  [Invited]

  村上 正晃

  第27回日本整形外科学会基礎学術集会  2012
• 病原T細胞の中枢神経系への侵入口の形成メカニズム—非免疫細胞の炎症誘導機構“IL-6アンプ”の役割—  [Invited]

  村上 正晃

  北海道大学獣医学研究科学術交流基金群講演  2012
• 重力刺激による中枢神経系への免疫細胞侵入口の形成  [Invited]

  村上 正晃

  第58回日本宇宙航空環境医学会  2012
• 活性化T細胞と中枢神経系の病気、病態  [Invited]

  村上 正晃

  第41回日本免疫学会学術集会  2012
• Local neural pathway and a gateway for pathogenic T cells in the CNS  [Invited]

  Masaaki Murakami

  2012 Annual Meeting of the Japanese Society for Immunology  2012
• Regional neural activation defines a gateway in the blood brain barrier for immune cells  [Invited]

  Masaaki Murakami

  IFReC-SIgN Winter School  2012
• サイトカインと慢性炎症—“IL-6アンプ”の役割  [Invited]

  村上 正晃

  富山大学大学院医学薬学研究部特別セミナー・特別講演  2012
• 血液脳関門への免疫細胞の侵入口の形成と自己免疫疾患  [Invited]

  村上 正晃

  第12回東北大学がん・エピゲノム研究会  2012
• 中枢神経系への免疫細胞の侵入メカニズム  [Invited]

  村上 正晃

  第40回日本臨床免疫学会総会・シンポジウム  2012
• 免疫細胞の中枢神経系への侵入口と仕組みについて  [Invited]

  村上 正晃

  第17回グリア研究会・招待セミナー  2012
• IL-17A-triggered positive-feedback for IL-6-signaling, a key player for inflammation, is associated with various human diseases.  [Invited]

  Masaaki Murakami

  RCAI-CGM Joint Meeting  2011
• Inflammation and Zinc signaling.  [Invited]

  Masaaki Murakami

  The 5th International Conference on Metals and Genetics  2011
• The IL-6 amplifier activation, a key player of inflammation, in human diseases and disorders.  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology・International symposium  2011
• Zinc and CD4+ T cell-mediated autoimmune diseases.  [Invited]

  Masaaki Murakami

  The 60th Fujihara Seminar Zinc Signaling and Cellular Functions  2010
• How does IL-17 induce Autoimmune Diseases?  [Invited]

  Masaaki Murakami

  The 2nd International Symposium of WPI-IFReC  2009
• Liver as a regulator of T cells.  [Invited]

  Masaaki Murakami

  The 5th International Workshop of Kyoto T Cell Conference 2009  2009
• Is the recognition of cognate antigens always required for the determination of the tissue specificity of autoimmune diseases associated with MHC II?”  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2009
• 抗原提示におけるサイトカインとカテプシンの関係  [Invited]

  村上 正晃

  徳島文理大学・健康科学研究所・私学助成研究発表会  2008
• Foxp3+抑制性CD8+ T細胞による免疫反応の制御  [Invited]

  村上 正晃

  第18回日本サイトメトリー学会学術総会  2008
• 細胞内亜鉛信号による細胞活性化の制御  [Not invited]

  村上 正晃

  日本薬学会第128回年会  2008
• An IL-17-Triggered Positive Feedback Loop of IL-6 Signaling in Autoimmune Diseases.  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2008
• Hepatic IL-7 controls T cell responses.  [Invited]

  Masaaki Murakami

  WPI-IFReC Third Seminar  2008
• サイトカイン発現ネットワークと自己免疫疾患  [Invited]

  村上 正晃

  第27回フロンティアバイオサイエンスコロキュウム  2007
• 非免疫系細胞を起点とするサイトカインカスケードの異常と自己免疫疾患  [Invited]

  村上 正晃

  かずさDNA研究所セミナー  2007
• 網羅的に分子の挙動を捉える技術−cDNAや抗体というリソースを活用した新しい方法  [Invited]

  村上 正晃

  BMB2007（第30回日本分子生物学会年会・第80回日本生化学会大会 合同大会）  2007
• IL-17 derived from homeostatic proliferating memory/activated CD4+ T cells develops arthritis in F759 mice  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2007
• 免疫応答における亜鉛の役割：亜鉛は新しいセカンドメッセンジャーである  [Invited]

  村上 正晃

  メタロチオンネインおよびメタルバイオサイエンス研究会  2007
• Zinc signaling in dendritic cells  [Invited]

  Masaaki Murakami

  The 20th Naito Conference on Innate Immunity in Medicine and Biology  2007
• 免疫分野における高速セルソーターの応用  [Invited]

  村上 正晃

  第37回日本免疫学会学術集会  2007
• 免疫分野における高速セルソーターの応用例  [Invited]

  村上 正晃

  第36回日本免疫学会学術集会  2006
• Non-hematopoietic cell populations play a role for IL-6-mediated enhancement of CD4+ T cell activation  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2006
• サイトカイン信号によるCD4+ T細胞の恒常性維持の破綻と免疫異常  [Invited]

  村上 正晃

  第28回北海道大学獣医学学術交流基金群講演会  2006
• IL-2ファミリーサイトカインによるメモリーCD8+ T細胞の維持機構の解析  [Invited]

  村上 正晃

  第14回日本癌病態治療研究会  2005
• Hyperactivation of gp130-Mediated STAT3 signaling induces rheumatoid arthritis like disease.  [Invited]

  Masaaki Murakami

  第8回上原記念生命科学財団  2005
• IL-2ファミリーサイトカインによるメモリーCD8+ T細胞の維持機構の解析  [Invited]

  村上 正晃

  第１３回日本アポトーシス研究会学術集会  2004
• Anti-IL-2-meidated effecter CD8+ T cell activation.  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2003
• 生体内でのメモリーT細胞の維持機構  [Invited]

  村上 正晃

  東京大学医学部免疫学教室セミナー  2002
• The role of CD25+CD4+ T cells in control of memory CD8+ T cells.  [Invited]

  Masaaki Murakami

  Annual Meeting of the Japanese Society for Immunology  2002
• Homeostasis of memory T cells.  [Invited]

  Masaaki Murakami

  The 2001 AAI Advanced Course in Immunology  2001
• The maintenance of memory T cells in vivo.  [Invited]

  Masaaki Murakami

  Annual Scientific Meeting of the Gerontological Society of America  2000

Works

• 炎症ストローマの人為的コントロールによる難治性慢性炎症疾患群の制御

  2010
• 急性期蛋白による自己免疫疾患・感染症の制御

  2010
• 関節リウマチ関連遺伝子の探索とその機能解析

  2006

MISC

• 【BBB-単なる障壁ではない】正常におけるメカニズム BBBにおける免疫細胞の侵入口形成機構,ゲートウェイ反射について

  西 李依子, 村上 薫, 長谷部 理絵, 村上 正晃  Clinical Neuroscience  40-  (12)  1544  -1548  2022/12
• サイトカインストームとIL-6アンプ—特集 コロナウイルス感染の免疫学

  村上 薫, 北條 慎太郎, 田中 くみ子, 村上 正晃  炎症と免疫 / 「炎症と免疫」編集委員会 編  30-  (1)  18  -27  2022/01
• Gateway Reflex and Mechanotransduction

  Shiina Matsuyama, Yuki Tanaka, Rie Hasebe, Shintaro Hojyo, Masaaki Murakami  Frontiers in Immunology  12-  2021/12/22  

  The gateway reflex explains how autoreactive CD4+ T cells cause inflammation in tissues that have blood-barriers, such as the central nervous system and retina. It depends on neural activations in response to specific external stimuli, such as gravity, pain, stress, and light, which lead to the secretion of noradrenaline at specific vessels in the tissues. Noradrenaline activates NFkB at these vessels, followed by an increase of chemokine expression as well as a reduction of tight junction molecules to accumulate autoreactive CD4+ T cells, which breach blood-barriers. Transient receptor potential vanilloid 1 (TRPV1) molecules on sensory neurons are critical for the gateway reflex, indicating the importance of mechano-sensing. In this review, we overview the gateway reflex with a special interest in mechanosensory transduction (mechanotransduction).
• ループスモデルマウスにおける慢性ストレス誘導性IL-12/23p40による精神神経病態への影響

  阿部 靖矢, 藤枝 雄一郎, 奥 健志, 河野 通仁, 田中 勇希, 加藤 将, 有沼 良幸, 佐藤 和貴郎, 村上 正晃, 渥美 達也  日本臨床免疫学会総会プログラム・抄録集  49回-  73  -73  2021/10
• Identification of pathogenic Single Nucleotide Polymorphism in Inflammatory Colorectal Polyps of Miniature Dachshunds(和訳中)

  Bin Teoh Yong, 永田 矩之, 大田 寛, 佐々木 東, 中村 健介, 村上 正晃, 滝口 満喜  日本獣医学会学術集会講演要旨集  164回-  [HSO  -32]  2021/09
• Regulation of autoimmune disease by the Gateway Reflex, a potential therapeutic target for neuromodulation

  長谷部理絵, 村上正晃  医学のあゆみ  277-  (9)  2021
• ATPによる感覚神経-介在神経回路の活性化は遠隔部位での炎症反応を誘導する

  長谷部理絵, 田中勇希, 北條慎太郎, 村上正晃  日本インターフェロン・サイトカイン学会学術集会抄録集  85th (CD-ROM)-  2021
• ショック管理 2021-’22-ガイドライン,スタンダード,論点そして私見-VII.ショックと新たな知見/治療法 新たな神経系と免疫系の相互作用:ゲートウェイ反射と炎症反射

  久保田晋平, 久保田晋平, 長谷部理絵, 村上正晃, 村上正晃  救急・集中治療  33-  (3)  2021
• Orosomucoid 1 is involved in the development of chronic allograft rejection after kidney transplantation.

  Haruka Higuchi, Daisuke Kamimura, Jing-Jing Jiang, Toru Atsumi, Daiki Iwami, Kiyohiko Hotta, Hiroshi Harada, Yusuke Takada, Hiromi Kanno-Okada, Kanako C Hatanaka, Yuki Tanaka, Nobuo Shinohara, Masaaki Murakami  International immunology  32-  (5)  335  -346  2020/05/08  [Not refereed][Not invited]
   

  Chronic allograft rejection is the most common cause of long-term allograft failure. One reason is that current diagnostics and therapeutics for chronic allograft rejection are very limited. We here show that enhanced NFκB signaling in kidney grafts contributes to chronic active antibody-mediated rejection (CAAMR), which is a major pathology of chronic kidney allograft rejections. Moreover, we found that urinary orosomucoid 1 (ORM1) is a candidate marker molecule and therapeutic target for CAAMR. Indeed, urinary ORM1 concentration was significantly higher in kidney transplant recipients pathologically diagnosed with CAAMR than in kidney transplant recipients with normal histology, calcineurin inhibitor toxicity, or interstitial fibrosis and tubular atrophy. Additionally, we found that kidney biopsy samples with CAAMR expressed more ORM1 and had higher NFκB and STAT3 activation in tubular cells than samples from non-CAAMR samples. Consistently, ORM1 production was induced after cytokine-mediated NFκB and STAT3 activation in primary kidney tubular cells. The loss- and gain-of-function of ORM1 suppressed and promoted NFκB activation, respectively. Finally, ORM1-enhanced NFκB-mediated inflammation development in vivo. These results suggest that an enhanced NFκB-dependent pathway following NFκB and STAT3 activation in the grafts is involved in the development of chronic allograft rejection after kidney transplantation and that ORM1 is a non-invasive candidate biomarker and possible therapeutic target for chronic kidney allograft rejection.
• マウスモデルにおける抗リン脂質抗体の精神神経症状や中枢神経系細胞への影響

  阿部靖矢, 阿部靖矢, 奥健志, 狩野皓平, 河野通仁, 藤枝雄一郎, AMENGUAL Olga, 田中勇希, 上村大輔, 保田晋助, 村上正晃, 渥美達也  日本臨床免疫学会総会プログラム・抄録集  47th-  74  -74  2019
• デング熱重症化マウスモデルでは,ガンマ・デルタT細胞から産生されたIL-17AがIL-6産生を増強し,血漿漏出を伴う致死的感染を引き起こしている

  黒須剛, 奥崎大介, PHANTHANAWIBOON Supranee, 吉河智城, 下島昌幸, 村上正晃, 上村大輔, 永田典代, 岩田奈織子, 西条政幸  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• 膵癌ドライバー変異はARF6-AMAP1経路を活性化し悪性度と免疫回避能を促進する(Pancreatic KRAS and TP53 oncogenes cooperatively activate ARF6-AMAP1 pathway to drive malignancy and immune evasion)

  橋本 あり, 橋本 茂, 古川 聖太郎, 蔦保 暁生, 小野寺 康仁, 大塚 勇太郎, 半田 悠, 及川 司, 水上 裕輔, 村上 正晃, 平野 聡, 佐邊 壽孝  日本癌学会総会記事  77回-  2219  -2219  2018/09  [Not refereed][Not invited]
  
• 軟骨細胞にはNF-κB Arthritis Inducer 1を介する炎症回路活性化機構が存在する

  太田 光俊, 田中 勇希, 有馬 康伸, 上村 大輔, 小野寺 智洋, 村上 正晃, 岩崎 倫政  北海道整形災害外科学会雑誌  60-  (1)  164  -164  2018/08  [Not refereed][Not invited]
  
• Photopic light intensity inhibits retinal inflammation via down-regulating local adrenergic system

  Daisuke Kamimura, Andrea Stofkova, Takuto Ohki, Yasunobu Arima, Masaaki Murakami  CYTOKINE  100-  51  -52  2017/12  [Not refereed][Not invited]
  
• Brain micro-inflammation at specific vessels establishes a new neural circuit, which dysregulates the gastrointestinal homeostasis under stress conditions

  Yasunobu Arima, Takuto Ohki, Naoki Nishikawa, Kotaro Higuchi, Junko Nio-Kobayashi, Stofkova Andrea, Toshihiko Iwanaga, Marco Prinz, Daisuke Kamimura, Masaaki Murakami  CYTOKINE  100-  52  -52  2017/12  [Not refereed][Not invited]
  
• Stress-gateway reflex: Chronic stress induces fatal organ dysfunctions through a new neural circuit

  Masaaki Murakami  Clinical and Experimental Neuroimmunology  8-  (4)  283  -284  2017/11/01  [Not refereed][Not invited]
  
• Existence of IL-6 and IL-17 Mediated Inflammation Amplifier Loop in Reactive Arthritis

  Ramnath Misra, Sandeep Kumar, Rajeev Singh, Abhisek Singh, Daisuke Kamimura, Smriti Chaurasia, Yasunobu Aria, Tori Austria, Ratnadeep Mukherjee, Balachandran Ravindran, Vikas Agarwal, Masaaki Murakami  ARTHRITIS & RHEUMATOLOGY  69-  2017/10  [Not refereed][Not invited]
  
• Targeting molecules involved in immune cell trafficking to the central nervous system for therapy in multiple sclerosis

  Mohamed Elfeky, Daisuke Kamimura, Yasunobu Arima, Masaaki Murakami, Lawrence Steinman  Clinical and Experimental Neuroimmunology  8-  (3)  183  -191  2017/08/01  [Not refereed][Not invited]
   

  The entry of immune cells into the central nervous system (CNS) for immune surveillance occurs during normal physiological conditions, although it is difficult to detect. However, during CNS autoimmune diseases, such as multiple sclerosis and its animal model experimental autoimmune encephalomyelitis, immune cells extensively invade the CNS. Understanding the mechanisms of immune cell migration and entry into the CNS provides a plan for the development of novel therapeutics. A goal would be to target specific molecules and/or pathways, and thereby modulate inflammatory cell migration into the CNS, which ideally could suppress disease progression without compromising beneficial immune surveillance. In the present review, we highlight the key mechanisms that directly or indirectly traffic and pass immune cells, particularly T cells to the CNS. We illuminate and focus on the following matters: T-cell licensing, regional neural stimulations, gut–CNS communications and integrin-mediated cell adhesion. We discuss currently approved drugs that target T-cell migration to the CNS.
• 軟骨細胞にはNF-κB arthritis inducer 1を介する炎症回路活性化機構が存在する

  太田 光俊, 田中 勇希, 蒋 菁菁, 中川 育磨, 樋口 はるか, 藤田 宗純, 有馬 康伸, 熱海 徹, 上村 大輔, 小野寺 智洋, 村上 正晃, 岩崎 倫政  日本整形外科学会雑誌  91-  (8)  S1728  -S1728  2017/08  [Not refereed][Not invited]
  
• Regulation of immune cell gateways in organs and a dementia including Alzheimer's diseases

  有馬 康伸, 上村 大輔, 村上 正晃  Dementia Japan : 日本認知症学会誌  31-  (2)  265  -273  2017/05
• Activation of Inflammation Amplifier in Kidney Graft and Urinary Biomarker for Chronic Rejection

  H. Higuchi, D. Iwami, K. Hotta, N. Shinohara, M. Murakami  AMERICAN JOURNAL OF TRANSPLANTATION  17-  720  -720  2017/04  [Not refereed][Not invited]
  
• THE ACTIVATION OF INFLAMMATION AMPLIFIER IN KIDNEY TRANSPLANT GRAFT AND URINARY BIOMARKER FOR CHRONIC REJECTION

  Haruka Higuchi, Daiki Iwami, Kiyohiko Hotta, Nobuo Shinohara, Masaaki Murakami  JOURNAL OF UROLOGY  197-  (4)  E76  -E76  2017/04  [Not refereed][Not invited]
  
• Gateway reflexes: A new paradigm of neuroimmune interactions

  Takuto Ohki, Daisuke Kamimura, Yasunobu Arima, Masaaki Murakami  Clinical and Experimental Neuroimmunology  8-  (1)  23  -32  2017/02/01  [Not refereed][Not invited]
   

  The immune system is affected considerably by the physical environment. The molecular mechanisms that regulate this connection are not well understood. Because neural activities sense the environment, it has been hypothesized that neural signals transduce environmental stimulations into immune responses. Recently, we discovered that regional neural activations change the dynamics of immune cell migration through the activity of endothelial cells by using a multiple sclerosis model, experimental autoimmune encephalomyelitis. More specifically, we identified the dorsal vessels of the fifth lumbar (L5) spinal cord as the initial entry site of pathogenic T cells. This entry site was defined by the activation of sensory neurons innervating from the soleus muscles, which are the main antigravity muscles. The resulting sensory neural activation created a gateway through the activation of regional sympathetic pathways to increase the expression of chemokines at the dorsal vessels of the L5 spinal cord, attracting autoreactive pathogenic T cells. In other experiments, we activated different regional neurons by electric pulses or pain sensation and discovered that gateways for immune cells, including autoreactive pathogenic T cells, are dependent on the site of the regional neural activation, particularly the sensory–sympathetic crosstalk. We termed these neuroimmune interactions as “gateway reflexes.” In the present review, we discuss details of the gateway reflex.
• Computer model analysis of the difference between F759 and wild type mice in rheumatoid-like arthritis emergence

  S. Yamada, A. Yoshimura, T. Atsumi, M. Murakami  EUROPEAN JOURNAL OF IMMUNOLOGY  46-  53  -53  2016/08  [Not refereed][Not invited]
  
• A novel regulator of NF kappa B signaling enhances I kappa B alpha ubiquitination and promotes inflammatory disease development

  Y. Okuyama, T. Atsumi, J. -J Jiang, A. Nakamura, H. Ogura, J. Meng, D. Kamimura, N. Shii, T. Hirano, M. Murakami  EUROPEAN JOURNAL OF IMMUNOLOGY  46-  410  -410  2016/08  [Not refereed][Not invited]
  
• Inflammation Amplifier, a Chemokine Inducer in Non-Immune Cells in Kidney Transplantation Graft.

  H. Higuchi, H. Bando, J. Jing-Jing, T. Atsumi, D. Iwami, K. Morita, N. Shinohara, M. Murakami  AMERICAN JOURNAL OF TRANSPLANTATION  16-  605  -605  2016/06  [Not refereed][Not invited]
  
• Regulation of Immune Cell Gateways in Organs

  上村 大輔, 有馬 康伸, 村上 正晃  最新医学  71-  (2)  176  -187  2016/02
• Pain is an inducer for relapse in multiple sclerosis models through a regional neural signal

  Yasunobu Arima, Kotaro Higuchi, Naoki Nishikawa, Andrea Stofkova, Takuto Ohki, Daisuke Kamimura, Masaaki Murakami  Clinical and Experimental Neuroimmunology  6-  (4)  343  -344  2015/11/01  [Not refereed][Not invited]
  
• Immune cell gateways in the central nervous system regulated by regional neural stimulations

  Daisuke Kamimura, Andrea Stofkova, Naoki Nishikawa, Toru Atsumi, Yasunobu Arima, Masaaki Murakami  Clinical and Experimental Neuroimmunology  6-  (2)  120  -128  2015/05/01  [Not refereed][Not invited]
   

  The central nervous system (CNS) is an immune-privileged tissue due to a specialized blood vessel structure, the blood-brain barrier. Indeed, the blood-brain barrier tightly limits cell migrations into the CNS. However, several immune cells, including T cells, can be found there. It is hypothesized that these cells have both beneficial and detrimental roles in immune surveillance and inflammation development, and that they can lead to multiple diseases, such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. The presence of immune cells within the CNS suggests a gateway that allows access from the peripheral blood stream. We recently identified dorsal vessels of the fifth lumbar spinal cord as a gateway for autoreactive T cells that opened with excessive chemokine expression. A chemokine inducer, the inflammation amplifier, which is hyperactivated by regional neural activations such as gravity, appears critical for formation of this fifth lumbar gateway. The gating of immune cells to the CNS can be artificially controlled by electric stimulations, at least in mice. We named this neural stimulation-dependent gating the "gateway reflex." Physical and/or chemical manipulations of the gateway reflex through the inflammation amplifier hold promising therapeutic value for neuroimmunological disorders.
• In Vivo Expansion of Donor-Specific Regulatory T Cells by a New Triazolopyrimidine Derivative and Donor-Specific Transfusion

  S. Emoto, R. Goto, S. Shibasaki, A. Nagatsu, H. Ono, R. Igarashi, M. Fukai, T. Shimamura, K. Saiga, M. Murakami, A. Taketomi, S. Todo, K. Yamashita  AMERICAN JOURNAL OF TRANSPLANTATION  15-  2015/05  [Not refereed][Not invited]
  
• In vivo MRI monitoring of inflammatory alterations and cellular dynamics in the central nervous system

  Yuki Mori, Yasunobu Arima, Ting Chen, Dasong Zhu, Yutaka Komai, Masaaki Murakami, Yoshichika Yoshioka, Tetsuya Fujisawa, Syoji Kobashi, Yutaka Hata  World Automation Congress Proceedings  355  -360  2014/10/24  [Refereed][Not invited]
   

  © 2014 TSI Press. This paper demonstrates the possibility of in vivo imaging for neuroimmunological assessments using ultra high-field magnetic resonance imaging (UHF-MRI). UHF-MRI provides a highly sensitive MR microimaging technique could be used to identify previously invisible pathologies and cellular dynamics in the central nervous system (CNS) of living animals. Our technique could reveal the mechanisms underlying the immune responses and cell dynamics during neuroinflammation, CNS diseases, and also in the normal state.
• Inflammation amplifier (IL-6 amplifier) and its targets and regulators

  小椋 英樹, 村上 正晃  臨床免疫・アレルギー科  59-  (3)  265  -273  2013/03
• IL-6アンプと神経-免疫クロストーク (特集 最新の免疫学 : 自然免疫・獲得免疫)

  有馬 康伸, 村上 正晃  医薬ジャーナル  49-  (2)  109  -116,11  2013/02  [Not refereed][Not invited]
  
• Autoimmune disorder phenotypes in HVCN1 gene deficient mice

  Mari Sasaki, Akihiro Tojo, Yoshifumi Okochi, Nana Miyawaki, Daisuke Kamimura, Akihito Yamaguchi, Masaaki Murakami, Yasushi Okamura  JOURNAL OF PHYSIOLOGICAL SCIENCES  63-  S192  -S192  2013  [Not refereed][Not invited]
  
• The gateway theory: bridging neural and immune interactions in the CNS

  Daisuke Kamimura, Moe Yamada, Masaya Harada, Lavannya Sabharwal, Jie Meng, Hidenori Bando, Hideki Ogura, Toru Atsumi, Yasunobu Arima, Masaaki Murakami  FRONTIERS IN NEUROSCIENCE  7-  2013  [Not refereed][Not invited]
   

  The central nervous system (CNS) is considered an immune-privileged tissue protected by a specific vessel structure, the blood-brain barrier (BBB). Upon infection or traumatic injury in the CNS, the BBB is breached, and various immune cells are recruited to the affected area. In the case of autoimmune diseases in the CNS like multiple sclerosis (MS), autoreactive T cells against some CNS-specific antigens can theoretically attack neurons throughout the CNS. The affected CNS regions in MS patients can be detected as multiple focal plaques in the cerebrum, thoracic cord, and other regions. Vision problems are often associated with the initial phase of MS, suggesting a disturbance in the optic nerves. These observations raise the possibility that there exist specific signals that direct autoreactive T cells past the BBB and into particular sites of the CNS. Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we recently defined the mechanism of the pathogenesis in which regional neural stimulations modulate the status of the blood vessel endothelium to allow the invasion of autoreactive T cells into specific sites of the CNS via the fifth lumbar cord. This gate for autoreactive T cells can be artificially manipulated by removing gravity forces on the hind legs or by electric pulses to the soleus muscles, quadriceps, and triceps of mice, resulting in an accumulation of autoreactive T cells in the intended regions via the activation of regional neurons. Gating blood vessels by regional neural stimulations, a phenomenon we call the gateway theory, has potential therapeutic value not only in preventing autoimmunity, but also in augmenting the effects of cancer immunotherapies.
• IL-6 amplifier, NF-kappa B-triggered positive feedback for IL-6 signaling, in grafts is involved in allogeneic rejection responses (vol 189, pg 1928, 2012)

  J. Lee, T. Nakagiri, T. Oto, M. Harada, E. Morii, Y. Shintani, M. Inoue, Y. Iwakura, S. Miyoshi, M. Okumura, T. Hirano, M. Murakami  JOURNAL OF IMMUNOLOGY  189-  (12)  5997  -5997  2012/12  [Not refereed][Not invited]
  
• Local microbleeding facilitates IL-6- and IL-17-dependent arthritis in the absence of tissue antigen recognition by activated T cells (vol 208, pg 103, 2011)

  Masaaki Murakami, Yuko Okuyama, Hideki Ogura, Shogo Asano, Yasunobu Arima, Mineko Tsuruoka, Masaya Harada, Minoru Kanamoto, Yukihisa Sawa, Yoichiro Iwakura, Kiyoshi Takatsu, Daisuke Kamimura, Toshio Hirano  JOURNAL OF EXPERIMENTAL MEDICINE  209-  (12)  2321  -2321  2012/11  [Not refereed][Not invited]
  
• Signal regulatory protein alpha (SIRP α)/CD172a regulates eosinophil homeostasis

  VERJAN GARCIA Noel, UMEMOTO Eiji, SAITO Yasuyuki, HATA Erina, MATOZAKI Takashi, MURAKAMI Masaaki, JANG Myoung Ho, MIYASAKA Masayuki  日本免疫学会総会・学術集会記録  40-  122  2011/11/07  [Not refereed][Not invited]
  
• Local microbleeding facilitates IL-6- and IL-17-dependent arthritis in the absence of tissue antigen recognition by activated T cells

  Masaaki Murakami, Yuko Okuyama, Hideki Ogura, Shogo Asano, Yasunobu Arima, Mineko Tsuruoka, Masaya Harada, Minoru Kanamoto, Yukihisa Sawa, Yoichiro Iwakura, Kiyoshi Takatsu, Daisuke Kamimura, Toshio Hirano  JOURNAL OF EXPERIMENTAL MEDICINE  208-  (1)  103  -114  2011/01  [Not refereed][Not invited]
   

  Cognate antigen recognition by CD4(+) T cells is thought to contribute to the tissue specificity of various autoimmune diseases, particularly those associated with class II MHC alleles. However, we show that localized class II MHC-dependent arthritis in F759 mice depends on local events that result in the accumulation of activated CD4(+) T cells in the absence of cognate antigen recognition. In this model, transfer of in vitro polarized Th17 cells combined with the induction of experimental microbleeding resulted in CCL20 production, the accumulation of T cells in the joints, and local production of IL-6. Disease induction required IL-17A production by transferred T cells, IL-6 and CCL20 expression, and STAT3 signaling in type I collagen-expressing cells. Our data suggest a model in which the development of autoimmune disease in F759 mice depends on four events: CD4(+) T cell activation regardless of antigen specificity, local events that induce T cell accumulation, enhanced sensitivity to T cell-derived cytokines in the tissue, and activation of IL-6 signaling in the tissue. This model provides a possible explanation for why tissue-specific antigens recognized by activated CD4(+) T cells have not been identified in many autoimmune diseases, especially those associated with class II MHC molecules.
• Local microbleeding facilitates IL-6- and IL-17-dependent arthritis in the absence of tissue antigen recognition by activated T cells

  Masaaki Murakami, Yuko Okuyama, Hideki Ogura, Shogo Asano, Yasunobu Arima, Mineko Tsuruoka, Masaya Harada, Minoru Kanamoto, Yukihisa Sawa, Yoichiro Iwakura, Kiyoshi Takatsu, Daisuke Kamimura, Toshio Hirano  JOURNAL OF EXPERIMENTAL MEDICINE  208-  (1)  103  -114  2011/01  [Not refereed][Not invited]
   

  Cognate antigen recognition by CD4(+) T cells is thought to contribute to the tissue specificity of various autoimmune diseases, particularly those associated with class II MHC alleles. However, we show that localized class II MHC-dependent arthritis in F759 mice depends on local events that result in the accumulation of activated CD4(+) T cells in the absence of cognate antigen recognition. In this model, transfer of in vitro polarized Th17 cells combined with the induction of experimental microbleeding resulted in CCL20 production, the accumulation of T cells in the joints, and local production of IL-6. Disease induction required IL-17A production by transferred T cells, IL-6 and CCL20 expression, and STAT3 signaling in type I collagen-expressing cells. Our data suggest a model in which the development of autoimmune disease in F759 mice depends on four events: CD4(+) T cell activation regardless of antigen specificity, local events that induce T cell accumulation, enhanced sensitivity to T cell-derived cytokines in the tissue, and activation of IL-6 signaling in the tissue. This model provides a possible explanation for why tissue-specific antigens recognized by activated CD4(+) T cells have not been identified in many autoimmune diseases, especially those associated with class II MHC molecules.
• ErratumCombination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation (vol 20, pg 783, 2008)

  Gui-Hua Jin, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  22-  (5)  411  -411  2010/05  [Not refereed][Not invited]
  
• Zinc suppresses T(h)17 development via inhibition of STAT3 activation

  Chika Kitabayashi, Toshiyuki Fukada, Minoru Kanamoto, Wakana Ohashi, Shintaro Hojyo, Toru Atsumi, Naoko Ueda, Ichiro Azuma, Hiroshi Hirota, Masaaki Murakami, Toshio Hirano  INTERNATIONAL IMMUNOLOGY  22-  (5)  375  -386  2010/05  [Not refereed][Not invited]
   

  Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.
• Zinc suppresses T(h)17 development via inhibition of STAT3 activation

  Chika Kitabayashi, Toshiyuki Fukada, Minoru Kanamoto, Wakana Ohashi, Shintaro Hojyo, Toru Atsumi, Naoko Ueda, Ichiro Azuma, Hiroshi Hirota, Masaaki Murakami, Toshio Hirano  INTERNATIONAL IMMUNOLOGY  22-  (5)  375  -386  2010/05  [Not refereed][Not invited]
   

  Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.
• IL-6 positively regulates Foxp3(+)CD8(+) T cells in vivo

  Takayuki Nakagawa, Mineko Tsuruoka, Hideki Ogura, Yuko Okuyama, Yasunobu Arima, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  22-  (2)  129  -139  2010/02  [Not refereed][Not invited]
   

  Although recent studies have identified regulatory roles for Foxp3(+)CD8(+) T cells, the mechanisms that induce their development and underlie their functions in vivo have not been elucidated. Here, we show that IL-6 positively regulates the Foxp3(+)CD8(+) T-cell development and function. The Foxp3(+)CD8(+) T cells that differentiated in vitro in the presence of IL-6 suppressed autoimmune colitis and arthritis in vivo. Moreover, Foxp3(+)CD8(+) T cells that developed in vivo in the presence of enhanced IL-6 signaling suppressed the development of a spontaneous T(h)17 cell-mediated autoimmune arthritis. Thus, we concluded that Foxp3(+)CD8(+) T cells develop in response to IL-6 and regulate chronic inflammation in T(h)17 cell-mediated F759 autoimmune arthritis. These results suggested that Foxp3(+)CD8(+) T cells may develop in response to IL-6 under certain inflammatory conditions in vivo and may regulate some other chronic inflammation diseases.
• IL-6 positively regulates Foxp3(+)CD8(+) T cells in vivo

  Takayuki Nakagawa, Mineko Tsuruoka, Hideki Ogura, Yuko Okuyama, Yasunobu Arima, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  22-  (2)  129  -139  2010/02  [Not refereed][Not invited]
   

  Although recent studies have identified regulatory roles for Foxp3(+)CD8(+) T cells, the mechanisms that induce their development and underlie their functions in vivo have not been elucidated. Here, we show that IL-6 positively regulates the Foxp3(+)CD8(+) T-cell development and function. The Foxp3(+)CD8(+) T cells that differentiated in vitro in the presence of IL-6 suppressed autoimmune colitis and arthritis in vivo. Moreover, Foxp3(+)CD8(+) T cells that developed in vivo in the presence of enhanced IL-6 signaling suppressed the development of a spontaneous T(h)17 cell-mediated autoimmune arthritis. Thus, we concluded that Foxp3(+)CD8(+) T cells develop in response to IL-6 and regulate chronic inflammation in T(h)17 cell-mediated F759 autoimmune arthritis. These results suggested that Foxp3(+)CD8(+) T cells may develop in response to IL-6 under certain inflammatory conditions in vivo and may regulate some other chronic inflammation diseases.
• 自己免疫性関節炎発しようにおけるTh17細胞の役割

  Medical Science Digest  36(29): 7-10-  2010  [Not refereed][Not invited]
  
• 亜鉛と亜鉛トランスポーター：サイトカイン／増殖因子の情報伝達と病態形成への関与

  2010  [Not refereed][Not invited]
  
• Interleukin 6 in autoimmune and inflammatory diseases.

  Physical and Biological Sciences  86(7); 717-730-  2010  [Not refereed][Not invited]
  
• Zinc is an intracellular signaling molecule: early and late signal

  2010  [Not refereed][Not invited]
  
• Zinc and Th17-mediated autoimmune diseases

  2010  [Not refereed][Not invited]
  
• Regulation of T cell Activation via a Metal Transporter

  2010  [Not refereed][Not invited]
  
• Zinc and its transporters: Their roles in intracellular signaling and diseases

  2010  [Not refereed][Not invited]
  
• IL-6 plays roles not only for developing Th17 but also for inducing an IL-6 amplifier in autoimmue diseases.

  2010  [Not refereed][Not invited]
  
• Hepatic IL-7, which is Induced by TLR–Type I IFN Signaling, Promotes T cell Responses.

  2010  [Not refereed][Not invited]
  
• Interleukin 6 in autoimmune and inflammatory diseases.

  Physical and Biological Sciences  86(7); 717-730-  2010  [Not refereed][Not invited]
  
• Combination of IL-2 plus anti-IL-2 mAbs reduces cancer metastasis through activation of NK cells.

  2010  [Not refereed][Not invited]
  
• Zinc is an intracellular signaling molecule: early and late signal

  2010  [Not refereed][Not invited]
  
• Regulation of T cell Activation via a Metal Transporter

  2010  [Not refereed][Not invited]
  
• Immune response controlled by hepatic IL-7

  村上 正晃, 澤 幸久  Clinical immunology & allergology  53-  (1)  79  -87  2010  [Not refereed][Not invited]
  
• Homeostatic proliferation of CD4+ T cells is involved in the pathogenesis of a mouse model of Omenn syndrome.

  2010  [Not refereed][Not invited]
  
• Specific antigen recognition by CD4+ T cells is not required for a MHC class II-associated autoimmune arthritis.

  2010  [Not refereed][Not invited]
  
• Zinc and CD4+ T cell-mediated autoimmune diseases

  2010  [Not refereed][Not invited]
  
• A zinc finger motif containing protein regulates the activation of the IL-6 amplifier, which is a critical for the development of an autoimmune arthritis.

  2010  [Not refereed][Not invited]
  
• Homeostatic proliferation of CD4+ T cells is involved in the pathogenesis of a mouse model of Omenn syndrome.

  2010  [Not refereed][Not invited]
  
• IFN-γ from CD8+ T cells, which is regulated by IL-6 signal, is involved in superantigen-mediated CD4+ T cell death.

  2010  [Not refereed][Not invited]
  
• Specific antigen recognition by CD4+ T cells is not required for a MHC class II-associated autoimmune arthritis.

  2010  [Not refereed][Not invited]
  
• Zinc and CD4+ T cell-mediated autoimmune diseases

  2010  [Not refereed][Not invited]
  
• A zinc finger motif containing protein regulates the activation of the IL-6 amplifier, which is a critical for the development of an autoimmune arthritis.

  2010  [Not refereed][Not invited]
  
• Zinc-mediated STAT3 suppression is involved in inhibition of autoimmune disease development via regulating Th17 differentiation.

  2010  [Not refereed][Not invited]
  
• Zinc and Th17-mediated autoimmune diseases

  2010  [Not refereed][Not invited]
  
• 免疫学 肝のIL-7による免疫系制御

  医学のあゆみ  232(2): 149-150-  2010  [Not refereed][Not invited]
  
• 医学のあゆみ【第5土曜特集】「サイトカインと疾患-あらたな病態モデルから治療へ」

  医歯薬出版(株)  2010  [Not refereed][Not invited]
  
• IFN-γ from CD8+ T cells, which is regulated by IL-6 signal, is involved in superantigen-mediated CD4+ T cell death.

  2010  [Not refereed][Not invited]
  
• Hepatic IL-7, which is Induced by TLR–Type I IFN Signaling, Promotes T cell Responses.

  2010  [Not refereed][Not invited]
  
• Zinc-mediated STAT3 suppression is involved in inhibition of autoimmune disease development via regulating Th17 differentiation.

  2010  [Not refereed][Not invited]
  
• Combination of IL-2 plus anti-IL-2 mAbs reduces cancer metastasis through activation of NK cells.

  2010  [Not refereed][Not invited]
  
• IL-6 plays roles not only for developing Th17 but also for inducing an IL-6 amplifier in autoimmue diseases.

  2010  [Not refereed][Not invited]
  
• Hepatic Interleukin-7 Expression Regulates T Cell Responses

  Yukihisa Sawa, Yasunobu Arima, Hideki Ogura, Chika Kitabayashi, Jing-Jing Jiang, Toru Fukushima, Daisuke Kamimura, Toshio Hirano, Masaaki Murakami  IMMUNITY  30-  (3)  447  -457  2009/03  [Not refereed][Not invited]
   

  Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4(+) T cell and CD8(+) T cell survival, augmented CD8(+) T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.
• Hepatic Interleukin-7 Expression Regulates T Cell Responses

  Yukihisa Sawa, Yasunobu Arima, Hideki Ogura, Chika Kitabayashi, Jing-Jing Jiang, Toru Fukushima, Daisuke Kamimura, Toshio Hirano, Masaaki Murakami  IMMUNITY  30-  (3)  447  -457  2009/03  [Not refereed][Not invited]
   

  Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4(+) T cell and CD8(+) T cell survival, augmented CD8(+) T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.
• Hepatic Interleukin-7 Expression Regulates T Cell Responses

  Yukihisa Sawa, Yasunobu Arima, Hideki Ogura, Chika Kitabayashi, Jing-Jing Jiang, Toru Fukushima, Daisuke Kamimura, Toshio Hirano, Masaaki Murakami  IMMUNITY  30-  (3)  447  -457  2009/03  [Not refereed][Not invited]
   

  Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4(+) T cell and CD8(+) T cell survival, augmented CD8(+) T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.
• Hepatic Interleukin-7 Expression Regulates T Cell Responses

  Yukihisa Sawa, Yasunobu Arima, Hideki Ogura, Chika Kitabayashi, Jing-Jing Jiang, Toru Fukushima, Daisuke Kamimura, Toshio Hirano, Masaaki Murakami  IMMUNITY  30-  (3)  447  -457  2009/03  [Not refereed][Not invited]
   

  Systemic cytokine activity in response to Toll-like receptor (TLR) signaling induces the expression of various proteins in the liver after infections. Here we show that Interleukin-7 (IL-7), the production of which was thought to occur at a constant rate in vivo, was a hepatically expressed protein that directly controled T cell responses. Depletion of IL-7 expression in the liver abrogated several TLR-mediated T cell events, including enhanced CD4(+) T cell and CD8(+) T cell survival, augmented CD8(+) T cell cytotoxic activity, and the development of experimental autoimmune encephalitis, a Th17 cell-mediated autoimmune disease. Thus, T cell responses are regulated by hepatocyte-derived IL-7, which is expressed in response to TLR signaling in vivo. We suggested that TLR-induced IL-7 expression in the liver, which is an acute-phase response, may be a good diagnostic and therapeutic target for efficient vaccine developments and for conditions characterized by TLR-mediated T cell dysregulation, including autoimmune diseases.
• The recognition of specific antigens is not always required for the development of tissue-specific autoimmune diseases associated with MHC II.

  2009  [Not refereed][Not invited]
  
• Impaired STAT activations in an ENU mutant mouse carrying excess memory T cells

  2009  [Not refereed][Not invited]
  
• Hepatic IL-7 Regulates CD4+T Cell-mediated Autoimmune diseases.

  2009  [Not refereed][Not invited]
  
• IFN-gamma expression in CD8(+) T cells regulated by IL-6 signal is involved in superantigen-mediated CD4(+) T cell death

  Toru Atsumi, Masae Sato, Daisuke Kamimura, Arisa Moroi, Yoichiro Iwakura, Ulrich A. K. Betz, Akihiko Yoshimura, Mika Nishihara, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  21-  (1)  73  -80  2009/01  [Not refereed][Not invited]
   

  Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4(+) T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4(+) T cell death. Sag-induced CD4(+) T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-gamma was inversely correlated with the magnitude of IL-6 signaling, and IFN-gamma deficiency inhibited Sag-induced activated CD4(+) T cell death, suggesting that IL-6 suppresses CD4(+) T cell death via IFN-gamma expression. Interestingly, depletion of activated CD8(+) T cells inhibited Sag-mediated increases in IFN-gamma expression in IL-6 KO mice as well as the augmented CD4(+) T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8(+) T cells contributes to Sag-induced CD4(+) T cell death via IFN-gamma expression, highlighting this signaling axis in CD8(+) T cells as a potential therapeutic target for Sag-related syndromes.
• Liver as a regulator of T cells

  2009  [Not refereed][Not invited]
  
• Zinc prevents Th17 cell-mediated autoimmune disease by directly inhibiting STAT3

  2009  [Not refereed][Not invited]
  
• 関節局所でのIL-6 amplify loopの活性化がF759関節炎発症には重要である

  2009  [Not refereed][Not invited]
  
• The recognition of specific antigens is not always required for the development of tissue-specific autoimmune diseases associated with MHC II.

  2009  [Not refereed][Not invited]
  
• Foxp3+CD8+ T cells inhibit the development of F759 arthritis.

  2009  [Not refereed][Not invited]
  
• 亜鉛シグナルの標的分子の探索と解析

  2009  [Not refereed][Not invited]
  
• Zinc prevents Th17 cell-mediated autoimmune disease by directly inhibiting STAT3

  2009  [Not refereed][Not invited]
  
• Foxp3+CD8+ T cells inhibit the development of F759 arthritis.

  2009  [Not refereed][Not invited]
  
• Impaired STAT activations in an ENU mutant mouse carrying excess memory T cells

  2009  [Not refereed][Not invited]
  
• IFN-gamma expression in CD8(+) T cells regulated by IL-6 signal is involved in superantigen-mediated CD4(+) T cell death

  Toru Atsumi, Masae Sato, Daisuke Kamimura, Arisa Moroi, Yoichiro Iwakura, Ulrich A. K. Betz, Akihiko Yoshimura, Mika Nishihara, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  21-  (1)  73  -80  2009/01  [Not refereed][Not invited]
   

  Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4(+) T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4(+) T cell death. Sag-induced CD4(+) T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-gamma was inversely correlated with the magnitude of IL-6 signaling, and IFN-gamma deficiency inhibited Sag-induced activated CD4(+) T cell death, suggesting that IL-6 suppresses CD4(+) T cell death via IFN-gamma expression. Interestingly, depletion of activated CD8(+) T cells inhibited Sag-mediated increases in IFN-gamma expression in IL-6 KO mice as well as the augmented CD4(+) T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8(+) T cells contributes to Sag-induced CD4(+) T cell death via IFN-gamma expression, highlighting this signaling axis in CD8(+) T cells as a potential therapeutic target for Sag-related syndromes.
• Liver as a regulator of T cells

  2009  [Not refereed][Not invited]
  
• IL-6アンプと炎症性疾患

  細胞工学  28(11):1107-1112, 2009-  2009  [Not refereed][Not invited]
  
• Metallothionein I/II分子によるCD4+T細胞依存性の自己免疫疾患の抑制機序

  2009  [Not refereed][Not invited]
  
• Hepatic IL-7 Regulates CD4+T Cell-mediated Autoimmune diseases.

  2009  [Not refereed][Not invited]
  
• c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130

  Yoshinori Tanaka, Nobuyuki Tanaka, Yasushi Saeki, Keiji Tanaka, Masaaki Murakami, Toshio Hirano, Naoto Ishii, Kazuo Sugamura  MOLECULAR AND CELLULAR BIOLOGY  28-  (15)  4805  -4818  2008/08  [Not refereed][Not invited]
   

  Interieukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding alpha subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cb1, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.
• c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130

  Yoshinori Tanaka, Nobuyuki Tanaka, Yasushi Saeki, Keiji Tanaka, Masaaki Murakami, Toshio Hirano, Naoto Ishii, Kazuo Sugamura  MOLECULAR AND CELLULAR BIOLOGY  28-  (15)  4805  -4818  2008/08  [Not refereed][Not invited]
   

  Interieukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding alpha subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cb1, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.
• Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

  Gui-Hua Jin, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  20-  (6)  783  -789  2008/06  [Not refereed][Not invited]
   

  Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.
• Combination treatment with IL-2 and anti-IL-2 mAbs reduces tumor metastasis via NK cell activation

  Gui-Hua Jin, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  20-  (6)  783  -789  2008/06  [Not refereed][Not invited]
   

  Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.
• Roles of zinc in intracellular signal transduction

  村上 正晃, 平野 俊夫  Cell technology.  27-  (3)  237  -241  2008/03
• Roles of zinc and zinc signaling in immunity: Zinc as an intracellular signaling molecule

  Toshio Hirano, Masaaki Murakami, Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, Tomoyuki Suzuki  ADVANCES IN IMMUNOLOGY, VOL 97  97-  149  -176  2008  [Not refereed][Not invited]
   

  Zinc (Zn) is an essential nutrient required for cell growth, differentiation, and survival, and its deficiency causes growth retardation, immunodeficiency, and other health problems. Therefore, Zn homeostasis must be tightly controlled in individual cells. Zn is known to be important in the immune system, although its precise roles and mechanisms have not yet been resolved. Zn has been suggested to act as a kind of neurotransmitter. In addition, Zn has been shown to bind and affect the activity of several signaling molecules, such as protein tyrosine phosphatases (PTPs). However, it has not been known whether Zn itself might act as an intracellular signaling molecule, that is, a molecule whose intracellular status is altered in response to an extracellular stimulus, and that is capable of transducing the extracellular stimulus into an intracellular signaling event. Here we propose that Zn acts as a signaling molecule and that there are at least two kinds of Zn signaling: "late Zn signaling," which is dependent on a change in the expression profile of Zn transporters, and "early Zn signaling," which involves a "Zn wave" and is directly induced by an extracellular stimulus. We also review recent progress in uncovering the roles of Zn in the immune system.
• Dysregulation of an IL-17-Triggered Positive Feedback Loop of IL-6 Signaling in an Autoimmune Arthritis.

  Immunity  29: 628-636-  2008  [Not refereed][Not invited]
  
• Roles of zinc and zinc signaling in immunity: Zinc as an intracellular signaling molecule

  Toshio Hirano, Masaaki Murakami, Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, Tomoyuki Suzuki  ADVANCES IN IMMUNOLOGY, VOL 97  97-  149  -176  2008  [Not refereed][Not invited]
   

  Zinc (Zn) is an essential nutrient required for cell growth, differentiation, and survival, and its deficiency causes growth retardation, immunodeficiency, and other health problems. Therefore, Zn homeostasis must be tightly controlled in individual cells. Zn is known to be important in the immune system, although its precise roles and mechanisms have not yet been resolved. Zn has been suggested to act as a kind of neurotransmitter. In addition, Zn has been shown to bind and affect the activity of several signaling molecules, such as protein tyrosine phosphatases (PTPs). However, it has not been known whether Zn itself might act as an intracellular signaling molecule, that is, a molecule whose intracellular status is altered in response to an extracellular stimulus, and that is capable of transducing the extracellular stimulus into an intracellular signaling event. Here we propose that Zn acts as a signaling molecule and that there are at least two kinds of Zn signaling: "late Zn signaling," which is dependent on a change in the expression profile of Zn transporters, and "early Zn signaling," which involves a "Zn wave" and is directly induced by an extracellular stimulus. We also review recent progress in uncovering the roles of Zn in the immune system.
• Intracellular Zinc homeostasis and Zinc signaling.

  Cancer Science  99:1515-22-  2008  [Not refereed][Not invited]
  
• Intracellular Zinc homeostasis and Zinc signaling.

  Cancer Science  99:1515-22-  2008  [Not refereed][Not invited]
  
• Dysregulation of an IL-17-Triggered Positive Feedback Loop of IL-6 Signaling in an Autoimmune Arthritis.

  Immunity  29: 628-636-  2008  [Not refereed][Not invited]
  
• Th17 differentiation and cytokine signaling

  奥山 祐子, 村上 正晃  Clinical immunology & allergology  48-  (4)  347  -353  2007/10
• IL-6-gp130-STAT3 in T cells directs the development of IL-17+T-h with a minimum effect on that of Treg in the steady state

  Mika Nishihara, Hideki Ogura, Naoko Ueda, Mineko Tsuruoka, Chika Kitabayashi, Fumio Tsuji, Hiroyuki Aono, Katsuhiko Ishihara, Eric Huseby, Ulrich A. K. Betz, Masaaki Murakami, Toshio Hirano  INTERNATIONAL IMMUNOLOGY  19-  (6)  695  -702  2007/06  [Not refereed][Not invited]
   

  IL-17-producing T-h (T(h)17) comprise a distinct lineage of pro-inflammatory T-h that are major contributors to autoimmune diseases. Treatment with IL-6 and transforming growth factor beta (TGF beta) induces naive CD4(+) T cells to generate Th17, which also requires expression of the IL-6/TGF[3 target ROR gamma t. We reported that IL-6 transduces two signaling pathways via tyrosine redidues of the signal transducer gp130: one depends on signal transducers and activators of transcription (STAT)-3 activation and the other on Src homology region 2 domain-containing phosphatase 2 (SHP2)/Grb2 associated binder (Gab)/mitogen-activated protein kinase (MAPK) activation. Here, we showed that CD4+ T cells carrying a mutant gp130 that transduces the SHP2/Gab/MAPK pathway but not the STAT3-mediated one failed to develop into Th17, while CD4+ T cells whose mutant gp130 transduces the STAT3 signal only generated Th17, indicating that IL-6 acts directly on T cells through the tyrosine residues of gp130 required for STAT3 activation to promote the development of Th17. Moreover, we found that gp130-STAT3 pathway is essential for Th17 development and for the expression of ROR gamma t by using T cells specifically lacking gp130 and STAT3. Noteworthy is that the regulatory T cell (Treg) percentages and numbers were comparable between all mutant mice we tested in vivo, although we showed that IL-6-gp130-STAT3 pathway suppressed Treg development in vitro. Thus, we conclude that IL-6 acts directly to promote the development of Th17 by activating the T cell gp130-STAT3 pathway but has a minimum effect on Treg development at least in the steady state in vivo. Therefore, blockade of IL-6-gp130-STAT3 pathway in CD4+ T cells could be a good target for controlling unwanted T(h)17-mediated immune responses including autoimmune diseases.
• IL-6-gp130-STAT3 in T cells directs the development of IL-17+T-h with a minimum effect on that of Treg in the steady state

  Mika Nishihara, Hideki Ogura, Naoko Ueda, Mineko Tsuruoka, Chika Kitabayashi, Fumio Tsuji, Hiroyuki Aono, Katsuhiko Ishihara, Eric Huseby, Ulrich A. K. Betz, Masaaki Murakami, Toshio Hirano  INTERNATIONAL IMMUNOLOGY  19-  (6)  695  -702  2007/06  [Not refereed][Not invited]
   

  IL-17-producing T-h (T(h)17) comprise a distinct lineage of pro-inflammatory T-h that are major contributors to autoimmune diseases. Treatment with IL-6 and transforming growth factor beta (TGF beta) induces naive CD4(+) T cells to generate Th17, which also requires expression of the IL-6/TGF[3 target ROR gamma t. We reported that IL-6 transduces two signaling pathways via tyrosine redidues of the signal transducer gp130: one depends on signal transducers and activators of transcription (STAT)-3 activation and the other on Src homology region 2 domain-containing phosphatase 2 (SHP2)/Grb2 associated binder (Gab)/mitogen-activated protein kinase (MAPK) activation. Here, we showed that CD4+ T cells carrying a mutant gp130 that transduces the SHP2/Gab/MAPK pathway but not the STAT3-mediated one failed to develop into Th17, while CD4+ T cells whose mutant gp130 transduces the STAT3 signal only generated Th17, indicating that IL-6 acts directly on T cells through the tyrosine residues of gp130 required for STAT3 activation to promote the development of Th17. Moreover, we found that gp130-STAT3 pathway is essential for Th17 development and for the expression of ROR gamma t by using T cells specifically lacking gp130 and STAT3. Noteworthy is that the regulatory T cell (Treg) percentages and numbers were comparable between all mutant mice we tested in vivo, although we showed that IL-6-gp130-STAT3 pathway suppressed Treg development in vitro. Thus, we conclude that IL-6 acts directly to promote the development of Th17 by activating the T cell gp130-STAT3 pathway but has a minimum effect on Treg development at least in the steady state in vivo. Therefore, blockade of IL-6-gp130-STAT3 pathway in CD4+ T cells could be a good target for controlling unwanted T(h)17-mediated immune responses including autoimmune diseases.
• Regulation of MHC class 2 by IL-6

  北村 秀光, 村上 正晃  Clinical immunology & allergology  47-  (2)  157  -163  2007/02
• Verella-Garcia, J. Kappler, P. Marrack, and T. Hirano. Homeostatic proliferating CD4 T cells are involved in the pathogenesis of an Omenn syndrome murine model.

  J. Clin. Invest.  17: 1270-1281-  2007  [Not refereed][Not invited]
  
• Guihua Jin, Masaaki Murakami, and Toshio Hirano

  2007  [Not refereed][Not invited]
  
• IL-6 inhibits PLCg activation in TCR-mediated signaling

  2007  [Not refereed][Not invited]
  
• Guihua Jin, Masaaki Murakami, and Toshio Hirano

  2007  [Not refereed][Not invited]
  
• Verella-Garcia, J. Kappler, P. Marrack, and T. Hirano. Homeostatic proliferating CD4 T cells are involved in the pathogenesis of an Omenn syndrome murine model.

  J. Clin. Invest.  17: 1270-1281-  2007  [Not refereed][Not invited]
  
• Antigen independent induction of a CD4+ T cell-dependent arthritis.

  2007  [Not refereed][Not invited]
  
• IL-6 inhibits PLCg activation in TCR-mediated signaling

  2007  [Not refereed][Not invited]
  
• Antigen independent induction of a CD4+ T cell-dependent arthritis.

  2007  [Not refereed][Not invited]
  
• Liver acute-phase reactions convert innate immune signaling into adaptive immune responses

  2007  [Not refereed][Not invited]
  
• Liver acute-phase reactions convert innate immune signaling into adaptive immune responses

  2007  [Not refereed][Not invited]
  
• The point mutation of tyrosine 759 of the IL-6 family cytokine receptor gp130 augments collagen-induced arthritis in DBA/1J mice.

  Fumio Tsuji, Hiroyuki Aono, Miwa Yoshimi, Osamu Katsuta, Miwa Takai, Masaaki Murakami, Toshio Hirano, Katsuhiko Ishihara  ARTHRITIS AND RHEUMATISM  54-  (9)  S174  -S174  2006/09  [Not refereed][Not invited]
  
• Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function

  Hidemitsu Kitamura, Hideyuki Morikawa, Hokuto Kamon, Megumi Iguchi, Shintaro Hojyo, Toshiyuki Fukada, Susumu Yamashita, Tsuneyasu Kaisho, Shizuo Akira, Masaaki Murakami, Toshio Hirano  NATURE IMMUNOLOGY  7-  (9)  971  -977  2006/09  [Not refereed][Not invited]
   

  Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.
• Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function

  Hidemitsu Kitamura, Hideyuki Morikawa, Hokuto Kamon, Megumi Iguchi, Shintaro Hojyo, Toshiyuki Fukada, Susumu Yamashita, Tsuneyasu Kaisho, Shizuo Akira, Masaaki Murakami, Toshio Hirano  NATURE IMMUNOLOGY  7-  (9)  971  -977  2006/09  [Not refereed][Not invited]
   

  Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.
• CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8(+) T cells in a manner dependent on IL-4

  Naoko Ueda, Hiroko Kuki, Daisuke Kamimura, Shinichiro Sawa, Kenichiro Seino, Takuya Tashiro, Ken-ichi Fushuku, Masaru Taniguchi, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  18-  (9)  1397  -1404  2006/09  [Not refereed][Not invited]
   

  CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GaICer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GaICer enhanced homeostatic proliferation of CD8(+) but not CD4(+) T cells and this effect of alpha-GaICer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8(+) T cell homeostatic proliferation. Importantly, the expression of IL-4R alpha and STAT6 in CD8(+) T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8(+) T cells. Consistent with this, the level of IL-4R alpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4(+) T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8(+) T cell homeostatic proliferation in vivo.
• CD1d-restricted NKT cell activation enhanced homeostatic proliferation of CD8(+) T cells in a manner dependent on IL-4

  Naoko Ueda, Hiroko Kuki, Daisuke Kamimura, Shinichiro Sawa, Kenichiro Seino, Takuya Tashiro, Ken-ichi Fushuku, Masaru Taniguchi, Toshio Hirano, Masaaki Murakami  INTERNATIONAL IMMUNOLOGY  18-  (9)  1397  -1404  2006/09  [Not refereed][Not invited]
   

  CD1d-restricted NKT cells are activated by TCR-mediated stimulation via CD1d plus lipid antigens such as alpha-galactosylceramide (alpha-GaICer). These cells suppressed autoimmunity and graft rejection, but sometimes enhanced resistance to infection and tumor immunity. This double-action phenomenon of NKT cells is partly explained by cytokines produced by NKT cells. Therefore, roles of cytokines from activated NKT cells have been extensively examined; however, their roles on T cell homeostatic proliferation in lymphopenic condition have not been investigated. Here, we showed that alpha-GaICer enhanced homeostatic proliferation of CD8(+) but not CD4(+) T cells and this effect of alpha-GaICer was required for NKT cells. IL-4 was essential and sufficient for this NKT cell action on CD8(+) T cell homeostatic proliferation. Importantly, the expression of IL-4R alpha and STAT6 in CD8(+) T cells was essential for the NKT activity, indicating a direct action of IL-4 on CD8(+) T cells. Consistent with this, the level of IL-4R alpha expression on memory phenotype CD8(+) T cells was higher than that on naive phenotype one and CD4(+) T cells. Thus, these results showed the 'involvement' of IL-4 that is produced from activated NKT cells for CD8(+) T cell homeostatic proliferation in vivo.
• IL-6-STAT3信号伝達系による樹状細胞の機能制御 (特集1 樹状細胞とT細胞の相互作用)

  北村 秀光, 村上 正晃  臨床免疫・アレルギー科  46-  (2)  141  -147  2006/08
• IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb

  D Kamimura, Y Sawa, M Sato, E Agung, T Hirano, M Murakami  JOURNAL OF IMMUNOLOGY  177-  (1)  306  -314  2006/07  [Not refereed][Not invited]
   

  IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 eDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8(+) T and NK1.1(high) cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44(high)CD8(+) population, and the increased population was maintained for > 300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.
• IL-2 in vivo activities and antitumor efficacy enhanced by an anti-IL-2 mAb

  D Kamimura, Y Sawa, M Sato, E Agung, T Hirano, M Murakami  JOURNAL OF IMMUNOLOGY  177-  (1)  306  -314  2006/07  [Not refereed][Not invited]
   

  IL-2 is a potent immunostimulant and has been tested for clinical use, including in immunotherapy for cancers and HIV infection. Here we show that a widely used neutralizing anti-murine IL-2 mAb (S4B6) exhibits unexpected activities that enhance the treatment effects of IL-2 in vivo. Coinjection of the anti-IL-2 mAb with a plasmid carrying murine IL-2 eDNA significantly increased the serum IL-2 levels and induced a substantial increase in the division of CD8(+) T and NK1.1(high) cells in vivo. Injection of the mAb premixed with recombinant murine IL-2 showed the same enhanced effect. A 5-day treatment with the anti-IL-2 mAb alone gradually increased the CD44(high)CD8(+) population, and the increased population was maintained for > 300 days, suggesting that the mAb can gradually maintain and potentially enhance the bioactivity of endogenous IL-2 for extended periods. Furthermore, combined treatment with the anti-IL-2 mAb plus the IL-2 plasmid markedly enhanced Ag-specific CTL activity in vivo and partially protected mice from tumor metastasis to the lungs, compared with the anti-IL-2 mAb or IL-2 plasmid alone. These results demonstrated IL-2-enhancing effects of the anti-IL-2 mAb in vivo and suggest that combining a neutralizing anti-IL-2 Ab with IL-2 gene delivery might be used effectively to enhance IL-2 functions in clinical applications.
• Autoimmune arthritis associated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/IL-7-dependent homeostatic proliferation of CD4(+) T cells

  S Sawa, D Kamimura, GH Jin, H Morikawa, H Kamon, M Nishihara, K Ishihara, M Murakami, T Hirano  JOURNAL OF EXPERIMENTAL MEDICINE  203-  (6)  1459  -1470  2006/06  [Not refereed][Not invited]
   

  Mice homozygous for the F759 mutation in the gp130 interleukin ( IL)-6 receptor subunit have enhanced gp130-mediated signal transducer and activator of transcription ( STAT)3 activation and spontaneously developed a lymphocyte-mediated rheumatoid arthritis-like joint disease. Here, we show that the development of the disease is dependent on both major histocompatibility complex ( MHC)II-restricted CD4(+) T cells and IL-6 family cytokines. In spite of the necessity for CD4(+) T cells, the gp130 mutation was only required in nonhemtopoietic cells for the disease. The gp130 mutation resulted in enhanced production of IL-7. Conditional knockout of STAT3 in nonlymphoid cells showed that the enhancement of IL-7 production was dependent on STAT3 activation by IL-6 family cytokines. Homeostatic proliferation of CD4(+) T cells was enhanced in gp130 mutant mice and acceleration of homeostatic proliferation enhanced the disease, whereas the inhibition of homeostatic proliferation suppressed the disease. Anti-IL-7 antibody treatment inhibited not only the enhanced homeostatic proliferation, but also the disease in gp130 mutant mice. Thus, our results show that autoimmune disease in gp130 mutant mice is caused by increased homeostatic proliferation of CD4(+) T cells, which is due to elevated production of IL-7 by nonhematopoietic cells as a result of IL-6 family cytokine-gp130-STAT3 signaling.
• Autoimmune arthritis associated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/IL-7-dependent homeostatic proliferation of CD4(+) T cells

  S Sawa, D Kamimura, GH Jin, H Morikawa, H Kamon, M Nishihara, K Ishihara, M Murakami, T Hirano  JOURNAL OF EXPERIMENTAL MEDICINE  203-  (6)  1459  -1470  2006/06  [Not refereed][Not invited]
   

  Mice homozygous for the F759 mutation in the gp130 interleukin ( IL)-6 receptor subunit have enhanced gp130-mediated signal transducer and activator of transcription ( STAT)3 activation and spontaneously developed a lymphocyte-mediated rheumatoid arthritis-like joint disease. Here, we show that the development of the disease is dependent on both major histocompatibility complex ( MHC)II-restricted CD4(+) T cells and IL-6 family cytokines. In spite of the necessity for CD4(+) T cells, the gp130 mutation was only required in nonhemtopoietic cells for the disease. The gp130 mutation resulted in enhanced production of IL-7. Conditional knockout of STAT3 in nonlymphoid cells showed that the enhancement of IL-7 production was dependent on STAT3 activation by IL-6 family cytokines. Homeostatic proliferation of CD4(+) T cells was enhanced in gp130 mutant mice and acceleration of homeostatic proliferation enhanced the disease, whereas the inhibition of homeostatic proliferation suppressed the disease. Anti-IL-7 antibody treatment inhibited not only the enhanced homeostatic proliferation, but also the disease in gp130 mutant mice. Thus, our results show that autoimmune disease in gp130 mutant mice is caused by increased homeostatic proliferation of CD4(+) T cells, which is due to elevated production of IL-7 by nonhematopoietic cells as a result of IL-6 family cytokine-gp130-STAT3 signaling.
• Grasp a pTyr-peptide by its SOCS

  T Hirano, M Murakami  DEVELOPMENTAL CELL  10-  (5)  542  -544  2006/05  [Not refereed][Not invited]
   

  Signaling via suppressors of cytokine signaling (SOCS) is an important negative feedback system for cytokine-mediated signal transduction. Recently in Molecular Cell, Babon et al. (2006) described the tertiary structure of SOCS3 in complex with a phosphotyrosine-containing peptide from the IL-6 receptor sub-unit gp130, and they identified the specific amino acids that are critical for binding.
• TRIF-GEFH1-RhoB cascade regulates surface expression of MHC class II in dendritic cells that is critical for CD4+ T cell activation.

  EMBO J.  25: 4108-4119-  2006  [Not refereed][Not invited]
  
• TRIF-GEFH1-RhoB cascade regulates surface expression of MHC class II in dendritic cells that is critical for CD4+ T cell activation.

  EMBO J.  25: 4108-4119-  2006  [Not refereed][Not invited]
  
• Grasp a pTyr-peptide by its SOCS.

  Dev. Cell  10:542-544-  2006  [Not refereed][Not invited]
  
• IL-6-STAT3 controls intracellular MHC class II alpha beta dimer level through cathepsin S activity in dendritic cells

  H Kitamura, H Kamon, SI Sawa, SJ Park, N Katunuma, K Ishihara, M Murakami, T Hirano  IMMUNITY  23-  (5)  491  -502  2005/11  [Not refereed][Not invited]
   

  We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alpha beta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alpha beta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alpha beta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alpha beta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alpha beta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alpha beta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.
• IL-6-STAT3 controls intracellular MHC class II alpha beta dimer level through cathepsin S activity in dendritic cells

  H Kitamura, H Kamon, SI Sawa, SJ Park, N Katunuma, K Ishihara, M Murakami, T Hirano  IMMUNITY  23-  (5)  491  -502  2005/11  [Not refereed][Not invited]
   

  We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alpha beta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alpha beta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alpha beta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alpha beta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alpha beta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alpha beta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.
• IL-6-STAT3 controls intracellular MHC class II alpha beta dimer level through cathepsin S activity in dendritic cells

  H Kitamura, H Kamon, SI Sawa, SJ Park, N Katunuma, K Ishihara, M Murakami, T Hirano  IMMUNITY  23-  (5)  491  -502  2005/11  [Not refereed][Not invited]
   

  We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alpha beta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alpha beta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alpha beta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alpha beta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alpha beta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alpha beta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.
• IL-6-STAT3 controls intracellular MHC class II alpha beta dimer level through cathepsin S activity in dendritic cells

  H Kitamura, H Kamon, SI Sawa, SJ Park, N Katunuma, K Ishihara, M Murakami, T Hirano  IMMUNITY  23-  (5)  491  -502  2005/11  [Not refereed][Not invited]
   

  We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alpha beta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alpha beta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alpha beta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alpha beta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alpha beta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alpha beta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.
• gp130シグナルの異常とリウマチ様関節炎

  澤新一郎, 村上正晃  実験医学(増刊)  23-  (11)  224  -229  2005  [Not refereed][Invited]
  
• IL-12関連サイトカインIL-27のシグナル伝達機構

  浅川 正幸, 森島 紀子, 大脇 敏之, 村上 正晃, 平野 俊夫, 水口 純一郎, 善本 隆之  日本免疫学会総会・学術集会記録  34-  126  -126  2004/11
• VEGFR-1 (Fit-1) tyrosine kinase signaling enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow hematopoietic stem cells, and promotes rheumatoid arthritis

  M Murakami, S Iwai, S Hirasuka, Y Iwakura, Y Maru, M Shibuya  BLOOD  104-  (11)  223A  -223A  2004/11  [Not refereed][Not invited]
  
• Evidence of a novel IL-2/15R beta-targeted cytokine involved in homeostatic proliferation of memory CD8(+) T cells

  D Kamimura, N Ueda, Y Sawa, S Hachida, T Atsumi, T Nakagawa, SI Sawa, GH Jin, H Suzuki, K Ishihara, M Murakami, T Hirano  JOURNAL OF IMMUNOLOGY  173-  (10)  6041  -6049  2004/11  [Not refereed][Not invited]
   

  The homeostasis of memory CD8(+) T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8(+) T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25(+)CD4(+) T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8(+) T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44(high)IL-2/15Rbeta(high) CD8(+) T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15Rbeta signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8(+) T cells, although the only cytokines known to act through IL-2/15Rbeta are IL-2 and IL-15; and 4) the expression of IL-2/15Rbeta molecules on memory CD8(+) T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8(+) T cell division by an IL-15-independent but by an IL-2/15Rbeta-dependent mechanism, suggesting the existence of a novel IL-2/15Rbeta-utilizing cytokine that acts directly on memory CD8(+) T cells to promote cell division.
• Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells

  T Mizutani, S Fukushi, M Murakami, T Hirano, M Saijo, Kurane, I, S Morikawa  FEBS LETTERS  577-  (1-2)  187  -192  2004/11  [Not refereed][Not invited]
   

  Severe acute respiratory syndrome (SARS) has become a global public health emergency. p38 mitogen-activated protein kinase (MAPK) and its downstream targets are activated in SARS coronavirus (SARS-CoV)-infected Vero E6 cells and activation of p38 MAPK enhances the cytopathic effects of SARS-CoV infection. In addition, weak activation of Akt cannot prevent SARS-CoV infection-induced apoptosis in Vero E6 cells. In the present study, we demonstrated that signal transducer and activator of transcription (STAT) 3, which is constitutively phosphorylated at tyrosine (Tyr)-705 and slightly phosphorylated at serine (Ser)-727 in Vero E6 cells, was dephosphorylated at Tyr-705 on SARS-CoV infection. In addition to phosphorylation of p38 MAPK in virus-infected cells, other MAPKs, i.e., extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were phosphorylated. Although inhibitors of ERK 1/2 and JNK (PD98059 and SP600125) bad no effect on phosphorylation status of STAT3, inhibitors of p38 MAPK (SB203580 and SB202190) partially inhibited dephosphorylation of STAT3 at Tyr-705. Tyr-705-phosphorylated STAT3 was localized mainly in the nucleus in mock infected cells, whereas STAT3 disappeared from the nucleus in virus-infected cells. As STAT3 acts as an activator of transcription in the nucleus, these results suggest that STAT3 lacks its activity on transcription in SARS-CoV-infected Vero E6 cells. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells

  T Mizutani, S Fukushi, M Murakami, T Hirano, M Saijo, Kurane, I, S Morikawa  FEBS LETTERS  577-  (1-2)  187  -192  2004/11  [Not refereed][Not invited]
   

  Severe acute respiratory syndrome (SARS) has become a global public health emergency. p38 mitogen-activated protein kinase (MAPK) and its downstream targets are activated in SARS coronavirus (SARS-CoV)-infected Vero E6 cells and activation of p38 MAPK enhances the cytopathic effects of SARS-CoV infection. In addition, weak activation of Akt cannot prevent SARS-CoV infection-induced apoptosis in Vero E6 cells. In the present study, we demonstrated that signal transducer and activator of transcription (STAT) 3, which is constitutively phosphorylated at tyrosine (Tyr)-705 and slightly phosphorylated at serine (Ser)-727 in Vero E6 cells, was dephosphorylated at Tyr-705 on SARS-CoV infection. In addition to phosphorylation of p38 MAPK in virus-infected cells, other MAPKs, i.e., extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were phosphorylated. Although inhibitors of ERK 1/2 and JNK (PD98059 and SP600125) bad no effect on phosphorylation status of STAT3, inhibitors of p38 MAPK (SB203580 and SB202190) partially inhibited dephosphorylation of STAT3 at Tyr-705. Tyr-705-phosphorylated STAT3 was localized mainly in the nucleus in mock infected cells, whereas STAT3 disappeared from the nucleus in virus-infected cells. As STAT3 acts as an activator of transcription in the nucleus, these results suggest that STAT3 lacks its activity on transcription in SARS-CoV-infected Vero E6 cells. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Evidence of a novel IL-2/15R beta-targeted cytokine involved in homeostatic proliferation of memory CD8(+) T cells

  D Kamimura, N Ueda, Y Sawa, S Hachida, T Atsumi, T Nakagawa, SI Sawa, GH Jin, H Suzuki, K Ishihara, M Murakami, T Hirano  JOURNAL OF IMMUNOLOGY  173-  (10)  6041  -6049  2004/11  [Not refereed][Not invited]
   

  The homeostasis of memory CD8(+) T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8(+) T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25(+)CD4(+) T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8(+) T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44(high)IL-2/15Rbeta(high) CD8(+) T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15Rbeta signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8(+) T cells, although the only cytokines known to act through IL-2/15Rbeta are IL-2 and IL-15; and 4) the expression of IL-2/15Rbeta molecules on memory CD8(+) T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8(+) T cell division by an IL-15-independent but by an IL-2/15Rbeta-dependent mechanism, suggesting the existence of a novel IL-2/15Rbeta-utilizing cytokine that acts directly on memory CD8(+) T cells to promote cell division.
• IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

  SJ Park, T Nakagawa, H Kitamura, T Atsumi, H Kamon, S Sawa, D Kamimura, N Ueda, Y Iwakura, K Ishihara, M Murakami, T Hirano  JOURNAL OF IMMUNOLOGY  173-  (6)  3844  -3854  2004/09  [Not refereed][Not invited]
   

  Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130 (F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.
• IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

  SJ Park, T Nakagawa, H Kitamura, T Atsumi, H Kamon, S Sawa, D Kamimura, N Ueda, Y Iwakura, K Ishihara, M Murakami, T Hirano  JOURNAL OF IMMUNOLOGY  173-  (6)  3844  -3854  2004/09  [Not refereed][Not invited]
   

  Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130 (F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.
• New IL-6 (gp130) family cytokine members, CLC/NNT1/BSF3 and IL-27.

  Growth Factors.  22, 75-7.-  2004  [Not refereed][Not invited]
  
• New IL-6 (gp130) family cytokine members, CLC/NNT1/BSF3 and IL-27.

  Growth Factors.  22, 75-7./,-  2004  [Not refereed][Not invited]
  
• Development of Atherosclerosis in Osteopontin Transgenic Mice

  Chiba Satoru, Okamoto Hiroshi, Akino Masatoshi, Jia Nan, Sugawara Takeshi, Shimizu Toshihiro, Matsui Yutaka, Kitabatake Akira, Murakami Masaaki, Uede Toshimitsu  Circulation journal : official journal of the Japanese Circulation Society  66-  144  -144  2002/03/31
• Single molecule imaging of H-Ras activation in living cells

  M Murakami, T Kobayashi, C Ohshima, H Murakoshi, M Sakuma, Y Takeda, R Iino, S Minoguchi, A Yoshimura, A Kusumi  MOLECULAR BIOLOGY OF THE CELL  12-  402A  -402A  2001/11  [Not refereed][Not invited]
  
• (2)トランスジェニックマウスを用いたデキサメサゾン誘導胸腺細胞アポトーシスにおけるDIG1遺伝子の機能解析(第20回日本胸腺研究会)

  土佐 紀子, 岩田 誠, 横山 峯介, 岩渕 和也, 猪部 学, 村上 正晃, 上出 利光  日本呼吸器外科学会雑誌  15-  (5)  1a  -2a  2001/07/15
• SF7a-1 CD40Ig及びCTLA4Ig遺伝子組込みアデノウイルスベクターを併用した新しい遺伝子治療によるラット移植心の長期生着

  山下 健一郎, 柳田 尚之, 竹原 めぐみ, 小林 篤寿, 越前谷 勇人, 野村 克, 砂原 正男, 北河 徳彦, 崎浜 秀康, 村上 正晃, 古川 博之, 上出 利光, 藤堂 省  日本外科学会雑誌  102-  (0)  92  -92  2001/03/10  [Not refereed][Not invited]
  
• Functional analysis of HVEM, a member of TNFR family, by using a transgenic mice expressing soluble form of HVEM

  M Inobe, T Uenishi, M Murakami, T Uede  FASEB JOURNAL  15-  (4)  A352  -A352  2001/03  [Not refereed][Not invited]
  
• The functional analysis of DIG1 molecule, a putative G protein-coupled receptor, in dexamethasone-induced thymocyte apoptosis.

  N Tosa, M Murakami, M Iwata, WY Jia, M Yokoyama, K Iwabuchi, M Inobe, T Uede  FASEB JOURNAL  15-  (4)  A315  -A315  2001/03  [Not refereed][Not invited]
  
• Homeostasis of alpha beta TCR+ T cells

  P Marrack, J Bender, D Hildeman, M Jordan, T Mitchell, M Murakami, A Sakamoto, BC Schaefer, B Swanson, J Kappler  NATURE IMMUNOLOGY  1-  (2)  107  -111  2000/08  [Not refereed][Not invited]
   

  Cytokines contribute to T cell homeostasis at all stages of T cell existence. However, the particular cytokine involved varies as T cells progress from a naive through an activated to a memory state. In many cases the important cytokines are members of the interleukin 2 subfamily of the short-chain type I cytokines. A case is made for the idea that the evolutionary divergence of the short-chain family allowed for concurrent divergence in leukocytes.
• CTLA4IgG gene delivery prevents autoantibody production and lupus nephritis in MRL/lpr mice

  M Takiguchi, M Murakami, Nakagawa, I, A Hashimoto, T Uede  FASEB JOURNAL  14-  (6)  A1207  -A1207  2000/04  [Not refereed][Not invited]
  
• Maintenance of memory CD8+T cells is dependent upon a balance of IL-2 family cytokines.

  M Murakami, CC Ku, A Sakamoto, J Kappler, P Marrack  FASEB JOURNAL  14-  (6)  A989  -A989  2000/04  [Not refereed][Not invited]
  
• Combination therapy of CTLA4IG and FTY720 completely prevent the development of obliterative bronchiolitis and respiratory epitherial injury in a murine airway model.

  K Konishi, M Murakami, M Inobe, A Yamada, H Echizenya, K Yamashita, M Nomura, T Omura, S Todo, T Uede  TRANSPLANTATION  69-  (8)  S301  -S301  2000/04  [Not refereed][Not invited]
  
• Long term liver graft acceptance with an novel adenovirus mediated CD40IgG gene transfer on rat.

  M Nomura, K Yamashita, M Murakami, M Takehara, K Konishi, H Echizenya, N Yanagida, Z Xu-Dong, T Omura, A Kishida, H Furukawa, T Uede, S Todo  TRANSPLANTATION  69-  (8)  S279  -S279  2000/04  [Not refereed][Not invited]
  
• JAK 2 is associated with a box 1 like motif and phospholylates a critical tyrosine residue in the cytoplasmic region of CTLA-4.

  S Chikuma, M Murakami, M Inobe, T Uede  FASEB JOURNAL  14-  (6)  A1178  -A1178  2000/04  [Not refereed][Not invited]
  
• Immunotherapy of Osteosarcoma Using B7-1-transfected Tumor Vaccine in Rat Orthotopic Implantation Model

  KAWAGUCHI T.  日本整形外科學會雜誌  74-  (3)  S680  2000/03/25
• SF7a-4 CTLA4Ig及びFTY720の慢性拒絶反応の抑制効果

  小西 勝人, 村上 正晃, 野村 克, 山下 健一郎, 柳田 尚之, 越前谷 勇人, 竹原 めぐみ, 大村 孝志, 古川 博之, 岸田 明博, 上出 利光, 藤堂 省  日本外科学会雑誌  101-  (0)  122  -122  2000/03/10  [Not refereed][Not invited]
  
• Blocking the CD28-B7 T-cell costimulatory pathway abrogates the development of obliterative bronchiolitis in a murine heterotopic airway model

  A Yamada, K Konishi, GLE Cruz, M Takehara, M Morikawa, Nakagawa, I, M Murakami, T Abe, S Todo, T Uede  TRANSPLANTATION  69-  (5)  743  -749  2000/03  [Not refereed][Not invited]
   

  Background. CTLA4IgG that binds to B7 effectively inhibits the signaling of CD28/B7 pathway and induces antigen specific T-cell unresponsiveness in vitro and in vivo. We examined whether the development of obliterative bronchiolitis in a murine heterotopic airway transplantation model is T cell dependent and whether CTLA4IgG abrogates the development of obliterative bronchiolitis. Methods. Tracheae with main bronchi from C3H/He (H2(k)), BALB/C (H2(d)), or C57BL/6 (H2(b)) mice were transplanted heterotopic:ally into subcutaneous pockets on the backs of BALB/C or BALB/C nu/nu mice on day 0. Recipient mice were untreated or intraperitoneally treated with either CTLA4IgG or human IgG with different time and dose schedules. Results. The development of obliterative bronchiolitis, which leads to luminal obliteration by fibrous tissue in a murine heterotopic airway transplantation model, was T cell dependent and the development of obliterative bronchiolitis was significantly abrogated by the CTLA4IgG treatment. However, the normal ciliated columnar respiratory epithelial cells in allografts were lost and replaced by flattened attenuated epithelial cells even after the CTLA4IgG treatment. We further demonstrated that CTLA4IgG treatment did not result in the induction of donor-specific unresponsiveness. Conclusions. We demonstrated that the development of obliterative bronchiolitis in a murine heterotopic airway model involves both CD28/B7-dependent and -independent processes. The luminal obliteration by fibrous tissue is clearly CD28/B7 dependent and can be inhibited by CTLA4IgQ. The luminal obliteration of allografted trachea by fibrous tissues and the loss of ciliated columnar respiratory epithelial cells represent distinct disease processes.
• 好中球はオステオポンチン産生能を有する

  上野 博史, 村上 正晃, 奥村 正裕, 廉沢 剛, 上出 利光, 藤永 徹  日本獣医学会学術集会講演要旨集  129回-  140  -140  2000/03  [Not refereed][Not invited]
  
• Adenovirus vectorを用いたオステオポンチン発現系の樹立

  RASHID M M, 猪部学, 今重之, 村上正晃, 上出利光  日本免疫学会総会・学術集会記録  30-  2000
• 肝炎遷延化におけるTh1/Th2レギュレーター,オステオポンチンの役割

  猪部学, 村上正晃, 今重之, 上出利光  日本免疫学会総会・学術集会記録  30-  2000
• Control of homeostasis of CD8+ memory T cells by opposing cytokines

  KU CC  Science  288-  (5466)  675  -678  2000  [Not refereed][Not invited]
  
• Osteopontin exacerbates atherosclerotic lesions in transgenic mice

  S Chiba, H Okamoto, Y Matsui, T Shimizu, K Kumamoto, M Watanabe, M Murakami, T Uede, A Kitabatake  CIRCULATION  100-  (18)  696  -696  1999/11  [Not refereed][Not invited]
  
• CTLA4IgG発現アデノウイルスベクターを用いたループス腎炎への遺伝子治療の試み

  TAKIGUCHI MITSUYOSHI, MURAKAMI MASAAKI, NAKAGAWA IZUMI, SAITO IZUMI, HASHIMOTO AKIRA, KAMIDE TOSHIMITSU  日本獣医学会学術集会講演要旨集  128th-  20  -20  1999/09/01  [Not refereed][Not invited]
  
• Combination of CTLA41GG and FTY720 suppresses development of obliterative bronchiolitis and respiratory epithelial injury in a murine heterotopic airway model

  K Konishi, M Murakami, A Yamada, M Takiguchi, M Morikawa, T Omura, S Todo, T Uede  TRANSPLANTATION  67-  (9)  S605  -S605  1999/05  [Not refereed][Not invited]
  
• MRL/lprマウス肺病変形成におけるCD28/CTLA4‐B7補助シグナルの役割

  TAKIGUCHI MITSUNOBU, MURAKAMI MASAAKI, NAKAGAWA IZUMI, YASUDA JUN, HASHIMOTO AKIRA, KAMIDE TOSHIMITSU  日本獣医学会学術集会講演要旨集  127th-  68  -68  1999/03/01  [Not refereed][Not invited]
  
• P782 オステオポンチントランスジェニックマウスにおける動脈硬化の促進

  千葉 知, 岡本 洋, 松井 裕, 清水 紀宏, 熊本 秀樹, 中川 泉, 渡邊 正司, 米谷 圭史, 小野塚 久夫, 三神 大世, 北畠 顕, 村上 正晃, 上出 利光  Japanese circulation journal  63-  (1)  646  -646  1999/03/01  [Not refereed][Not invited]
  
• O-483 CTLA4IgG発現アデノウィルスベクターのCre/loxPによる発現制御

  竹原 めぐみ, 村上 正晃, 中川 泉, 小西 勝人, 野村 克, 柳田 尚之, 大村 孝志, 古川 博之, 岸田 明博, 鐘ケ江 裕美, 斉藤 泉, 上出 利光, 藤堂 省  日本外科学会雑誌  100-  228  -228  1999/02/10
• Effects of a novel immunosuppressant, FTY720, on heart and liver transplantations in rats

  K. Yamashita, M. Nomura, T. Omura, M. Takehara, T. Suzuki, T. Shimamura, A. Kishida, H. Furukawa, M. Murakami, T. Uede, S. Todo  Transplantation Proceedings  31-  (1-2)  1178  -1179  1999/02  [Not refereed][Not invited]
  
• オステオポンチンの過剰発現による肉芽腫性肺病変の出現

  千葉知, 村上正晃, RASHID M M, 白岩宙丈, 今重之, 猪部学, 岡本洋, 北畠顕, 上出利光  日本免疫学会総会・学術集会記録  29-  1999
• Persistent and secondary gene expression on using Adex1CACTLA4IgG

  MURAKAMI Masaaki, NAKAGAWA Izumi, ISHIKURA Hiroshi, KANEGAE Yumi, SAITO Izumi, UEDE Toshimitsu  日本分子生物学会年会プログラム・講演要旨集  21-  590  -590  1998/12/01
• Control of CTLA4IgG expression from adenovirus vector using Cre/loxP system

  TAKEHARA Megumi, MURAKAMI Masaaki, NAKAGAWA Izumi, KANEGAE Yumi, SAITO Izumi, TODO Satoru, UEDE Toshimitsu  日本分子生物学会年会プログラム・講演要旨集  21-  591  -591  1998/12/01
• 1prマウス肺病変形成におけるCD28/CTLA4-B7補助シグナルの役割

  滝口 満喜, 村上 正晃, 中川 泉, 橋本 晃, 上出 利光  日本臨床免疫学会会誌  (26回抄録集)  376  -376  1998/10
• CTLA4Ig overexpression enables secondary adenovirus administration and prolongs adenovirus-mediated gene transfer

  Nakagawa, I, H Okamoto, S Chiba, H Kumamato, K Yoneya, M Watanabe, M Murakami, T Uede  CIRCULATION  98-  (17)  796  -796  1998/10  [Not refereed][Not invited]
  
• Adenovirus-mediated gene transfer of CTLA4Ig is highly efficient and prolongs cardiac allograft survival

  Nakagawa, I, H Okamoto, S Chiba, H Kumamato, K Yoneya, M Watanabe, M Murakami, T Uede  CIRCULATION  98-  (17)  399  -399  1998/10  [Not refereed][Not invited]
  
• lprマウス肺病変形成におけるCD28/CTLA4‐B7補助シグナルの役割

  TAKIGUCHI MITSUYOSHI, MURAKAMI MASAAKI, NAKAGAWA IZUMI, HASHIMOTO AKIRA, UEDE TOSHIMITSU  日本免疫学会総会・学術集会記録  28-  376  1998/10  [Not refereed][Not invited]
  
• Persistent and secondary adenovirus-mediated hepatic gene expression using adenovirus vector containing CTLA4IgG

  Izumi Nakagawa, Masaaki Murakami, Kenichi Ijima, Shunsuke Chikuma, Izumu Saito, Yumi Kanegae, Hiroshi Ishikura, Takashi Yoshiki, Hiroshi Okamoto, Akira Kitabatake, Toshimitsu Uede  Human Gene Therapy  9-  (12)  1739  -1745  1998/08/10  [Not refereed][Not invited]
   

  Adenovirus vectors can transfer recombinant genes efficiently into a wide variety of cells in vivo, but have serious limitations: gene expression is transient and secondary gene transfer is inefficient or impossible because of cellular and humoral immune responses against adenovirus-transduced cells. To solve these limitations, we have constructed an adenovirus vector, Adex1CACTLA4IgG, that expresses CTLA4IgG molecules. After in vivo administration of Adex1CACTLA4IgG (9.0 x 109 PFU), the peak level of serum CTLA4IgG was 29.8 mg/ml on day 4. The serum CTLA4IgG concentration gradually fell but was still 5.7 mg/ml on day 90, However, the serum concentration of CTLA4IgG was elevated after a second administration of Adex1CACTLA4IgG. The production of antibody against adenovirus was completely prevented after treatment with Adex1CACTLA4IgG. In addition, coadministration of Adex1CALacZ with Adex1CACTLA4IgC induced persistent hepatic expression of β-Gal molecules, while administration of Adex1CALacZ alone induced transient expression of β-Gal molecules. More importantly, on day 160 a secondary challenge with Adex1CALacZ was possible in mice treated with Adex1CALacZ plus Adex1CACTLA4IgG. Thus, we have demonstrated that (1) gene expression of a recombinant adenovirus, Adex1CACTLA4IgG, is persistent in liver and secondary administration of this adenovirus is possible, (2) coadministration of Adex1CACTLA4IgG virus with another adenovirus, AdexCALacZ, prolongs AdexCALacZ-mediated gene expression, and (3) Adex1CACTLA4IgG is useful for secondary challenge with Adex1CALacZ.
• Concern of CD28/CTLA4-B7 support signal in pathogeny of MRL/lpr mouse.

  TAKIGUCHI MITSUYOSHI, MURAKAMI MASAAKI, NAKAGAWA IZUMI, YASUDA JUN, HASHIMOTO AKIRA, KAMIIDE TOSHIMITSU  日本獣医学会学術集会講演要旨集  126th-  22  -22  1998/08  [Not refereed][Not invited]
  
• Blockade of CD28/CTLA4 pathway prevented almost diseases and lymphadenopathy but not lung disease in MRL/lpr mice.

  M Takiguchi, M Murakami, Nakagawa, I, A Yamada, S Chikuma, T Uede  FASEB JOURNAL  12-  (5)  A1093  -A1093  1998/03  [Not refereed][Not invited]
  
• イヌの脳性ナトリウム利尿ペプチド遺伝子のクローニング

  浅野 和之, 村上 正晃, 遠藤 大二, 木村 享史, 奥村 正裕, 廉澤 剛, 藤永 徹  日本獣医学会学術集会講演要旨集  125回-  267  -267  1998/03  [Not refereed][Not invited]
  
• キチン・キトサンのマウス常在腹腔マクロファージに対する作用

  森 崇, 村上 正晃, 上出 利光, 蒲 直樹, 田中 一郎, 奥村 正裕, 廉澤 剛, 藤永 徹  日本獣医学会学術集会講演要旨集  125回-  275  -275  1998/03  [Not refereed][Not invited]
  
• The functional role of B7 molecules on the induction of thymocyte activation and apoptosis.

  Naoko Aoki, Manabu Inobe, Masaaki Murakami, Ryo Abe, Hajime Iizuka  Collected papers from the Institute of Immunological Science Hokkaido University  21-  281  -291  1998
• Expression and costimulatory function of B7-2 on murine CD4+ T cells.

  Rie Hakamada-Taguchi, Takuma Kato, Hiroshi Ushijima, Masaaki Murakami, Toshimitsu Uede, Hideo Nariuchi  Collected papers from the Institute of Immunological Science Hokkaido University  21-  301  -309  1998
• The mechanism by which proteolysis enhances the ligand binding activity of guinea pig typeII Fc receptor for IgG (FcγRIIB).

  Yasuhiro Isashi, Toshiyuki Yamashita, Shigeharu Nagasawa, Kumiko Tanaka, Masaaki Murakami, Toshimitsu Uede  Collected papers from the Institute of Immunological Science Hokkaido University  21-  292  -300  1998
• Selective antigen presenting activity of cortical thymic epithelial cells against CD4+ T cells associated with both lack of costimulatory molecules and inefficient presentation of MHC-peptide ligands

  Rika Kaneda, Kazuya Iwabuchi, Michiyuki Kasai, Masaaki Murakami, Toshimitu Uede, Kazunori Onoé  Cellular Immunology  181-  (2)  163  -171  1997/11/01  [Not refereed][Not invited]
   

  Selective activation among several effector functions of a T cell clone, DB14, specific for pigeon cytochrome c 43-58 (p43-58) and restricted to I- A(b/d) was induced by antigen (Ag) presentation with nonprofessional Ag- presenting cells (APC), cortical thymic epithelial cells (c-TEC) (B7-1- CD40-), whereas full activation of the DB14 was induced with another nonprofessional APC, I-Ab L cell (B7-1+CD40+). In the present study, to elucidate the mechanism underlying the selective activation of DB14 cells by c-TEC, we established c-TEC transfected with human CD40 alone (huCD40-c-TEC) or both human CD40 and murine B7-1 (huCD40/mB7-1-c-TEC), and compared the APC functions with those of the original c-TEC and I-Ab L cell. FN-γ production but not the proliferative response of DB14 was elevated by Ag presentation with huCD40-c-TEC as compared with unmanipulated c-TEC. On the other hand, upon stimulation with Ag plus huCD40/mB7-1-c-TEC both a significant proliferative response and IFN-γ production were induced in DB14. However, the level of these responses did not reach that induced in the presence of I- Ab L cell. A similar pattern of APC functions was demonstrated with the other B7-independent T cell clone, PAB3, or T cell hybridomas (DBhy22 and BD7-5) which are basically independent of costimulation for the activation. The present finding along with our previous report that several structural differences of I-Ab molecules are present between c-TEC and I-Ab L cell suggests that the distinct APC activity of c-TEC is attributable not only to a lack of B7-1 and CD40 but also to inefficient presentation of MHC-peptide complex on the c-TEC.
• CD80 combination activity shown by a phage library clone to combine with the anti-CTLA4 antibody.

  鳥越直彦, 福元隆浩, 川畑慎一, 米田明広, 村上正晃, 上出利光, 西徹, 伊東祐二, 杉村和久  日本免疫学会総会・学術集会記録  27-  199  1997/10  [Not refereed][Not invited]
  
• Immunoregulation activity of phage library clone combining with anti-CTLA4 antibody.

  福元隆浩, 鳥越直彦, 川畑慎一, 村上正晃, 上出利光, 西徹, 伊東祐二, 杉村和久  日本免疫学会総会・学術集会記録  27-  139  1997/10  [Not refereed][Not invited]
  
• Analysis of a peptide motif to combine with the anti-CD86 antibody using the phage library.

  米田明広, 鳥越直彦, 福元隆浩, 川畑慎一, 村上正晃, 上出利光, 西徹, 伊東祐二, 杉村和久  日本免疫学会総会・学術集会記録  27-  199  1997/10  [Not refereed][Not invited]
  
• Anti Tumor Immunity of B7-1 or B7-2 Induced Mouse Osteosarcoma Cells

  NAGAMORI M.  日本整形外科學會雜誌 = The Journal of the Japanese Orthopaedic Association  71-  (8)  S1368  1997/08/25
• B細胞上のCD28/CTLA-4結合分子ACBM (リンパ球表面分子と免疫調節)

  村上 正晃, 高橋 陽子, 今 重之  免疫  (1997)  106  -111  1997/08
• CD28,CTLA4/B7-1,B7-2とclonal anergy (特集 寛容導入をめざす移植免疫学)

  村上 正晃, 山田 陽, 上出 利光  モレキュラ-メディシン  34-  (7)  842  -853  1997/07
• Recent Progress on Costimulatory Molecules. New CTLA-4 binding molecule, ACBM expressed on resting B lymphocytes.

  今重之, 村上正晃  臨床免疫  29-  (8)  1997
• Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein?

  T Uede, Y Katagiri, J Iizuka, M Murakami  MICROBIOLOGY AND IMMUNOLOGY  41-  (9)  641  -648  1997  [Not refereed][Not invited]
  
• Identification of an alternative CTLA4 ligand on mouse B cells.

  M Murakami, Y Takahashi, Y Isashi, N Aoki, S Kon, WY Jia, M Inobe, R Abe  JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY  99-  (1)  886  -886  1997/01  [Not refereed][Not invited]
  
• Limb Allografts in Rats Enhance the Survival of Skin Allografts

  TSUCHIDA Y.  日本整形外科學會雜誌 = The Journal of the Japanese Orthopaedic Association  70-  (8)  S1539  1996/08/25
• 炎症部位における好中球Fc_γRの活性化機構

  山下 俊之, 井指 康裕, 長澤 滋治, 村上 正晃, 上出 利光  日本分子生物学会年会プログラム・講演要旨集  19-  20  -20  1996/08
• 静止期のB細胞に発現する新しいCD28, CTLA4結合分子, ACBMの同定とその機能

  村上 正晃, 上出 利光  日本分子生物学会年会プログラム・講演要旨集  19-  20  -20  1996/08
• Activation of JNK and ERK2 by CD40-mediated signals in WEHI231 cells.

  T Hara, M Murakami, M Hibi, T Uede  FASEB JOURNAL  10-  (6)  395  -395  1996/04  [Not refereed][Not invited]
  
• The role of B7-1a molecule, an alternatively spliced form of murine B7-1(CD80), on T cell activation.

  Manabu Inobe, Naoko Aoki, Peter S. Linsley, Jeffrey A. Ledbetter, Ryo Abe, Masaaki Murakami, Toshimitsu Uede  Collected papers from the Institute of Immunological Science Hokkaido University  19-  292  -298  1996
• Identification and characterization of an alternative cytotoxic Tlymphocyte-associated protein 4 binding molecule on B cells.

  Masaaki Murakami, Yohko Takahashi, Yasuhiro Isashi, Shigeyuki Kon, Wen-Yi Gia, Manabu Inobe, Ryo Abe, Toshimitsu Uede  Collected papers from the Institute of Immunological Science Hokkaido University  19-  287  -291  1996
• Vaccination of tumor cells transfected with B7-1 (CD80) gene Induces the'anti-metastatic effect and tumor immunity in mice.

  Hideki Fujii, Manabu Inobe, Fuminari Kimura, Jun Murata, Masaaki Murakami, Yasuharu Onishi, Ichiro Azuma, Toshimitsu Uede, Ikuo Saiki.  Collected papers from the Institute of Immunological Science Hokkaido University  19-  160  -165  1996
• Long-term acceptance of major histocompatibility complex- mismached cardiac allograft induced by a low does of CTLA4IgM plus FK506.

  Akira Yamada, Masaaki Murakami, Kenichi Ijima, Hideo Yagita, Yagita, Kou Okumura, Sakuzo Komatsu, Toshimitsu Uede  Collected papers from the Institute of Immunological Science Hokkaido University  19-  281  -286  1996
• Analysis of appearance of ACBM, a new CTLA4 combination molecule to appear in a B cell, in a mouse in vivo.

  高橋陽子, 青木直子, 今重之, 村上正晃, 上出利光  日本免疫学会総会・学術集会記録  26-  1996
• Analysis of combination region on CTLA-4 for ACBM, a new CTLA-4 combining molecule.

  今重之, 高橋陽子, 青木直子, 村上正晃, 上出利光  日本免疫学会総会・学術集会記録  26-  1996
• Isolation of DIG-1 gene induced dexamethasone dependently and thymus gland specifically.

  こ文い, 今重之, 村上正晃, 上出利光  日本免疫学会総会・学術集会記録  26-  1996
• Analysis of the CD40 signal transfer mechanism.

  原豊道, 今重之, 村上正晃, 上出利光  日本免疫学会総会・学術集会記録  26-  1996
• Identification of ACBM, a new combined molecule of CD28 and CTLA4 to appear in a B cell, and its function.

  村上正晃, 高橋陽子, 井指康裕, 今重之, か文い, 猪部学, 阿部良, 上出利光  日本免疫学会総会・学術集会記録  26-  1996
• ANGIOTENSIN-CONVERTING ENZYME GENE POLYMORPHISM IN JAPANESE PATIENTS WITH HYPERTROPHIC CARDIOMYOPATHY

  K YONEYA, H OKAMOTO, M MACHIDA, H ONOZUKA, M NOGUCHI, T MIKAMI, H KAWAGUCHI, M MURAKAMI, T UEDE, A KITABATAKE  AMERICAN HEART JOURNAL  130-  (5)  1089  -1093  1995/11  [Not refereed][Not invited]
   

  To examine the contribution of the angiotensin-converting enzyme (ACE) gene to hypertrophic cardiomyopathy (HCM), we determined the ACE insertion/deletion (I/D) polymorphism in 80 patients with HCM and 88 of their unaffected siblings and children. Patients were divided into familiar or solitary HCM (FHCM or SHCM) groups with or without affected family members. Genotypes were identified by the polymerase chain reaction (PCR) with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA prepared from peripheral leukocytes. D-allele frequencies were 0.38 in all subjects, 0.42 in patients with HCM, and 0.35 in relatives (p < 0.05). The probability ratios were 1.98, 1.46, and 2.97 in patients with HCM, FHCM, and SHCM, respectively. The D allele frequency was higher in SHCM than in FHCM (p < 0.05). The findings suggest that HCM, especially in solitary cases, is partially determined by genetic disposition. Findings imply that the ACE D allele is one of the genetic contributing factors associated with cardiac hypertrophy in HCM.
• Anti adhesion therapy〔邦文〕

  山田 陽, 村上 正晃, 上出 利光  日本臨床  53-  (3)  p761  -770  1995/03
• Functional analysis of the osteopontin molecule

  Katagiri Yohko, Mori Kiyoshi, Hara Toyomichi, Tanaka Kumiko, Murakami Masaaki, Uede Toshimitsu  Collected papers from the Institute of Immunological Science Hokkaido University  18-  317  -320  1995
• IDENTIFICATION OF ALTERNATIVELY SPLICED FORM OF THE MURINE B7 HAVING ALTERED RECEPTOR-BINDING AND FUNCTIONAL-PROPERTIES

  M INOBE, M MURAKAMI, Y NAGAI, PS LINSLEY, JA LEDBETTER, T UEDE  FASEB JOURNAL  8-  (5)  A1014  -A1014  1994/03  [Not refereed][Not invited]
  
• THE ROLL OF COSTIMULATORY SIGNALS IN PROLIFERATION OF DOUBLE-NEGATIVE (DN) T-CELLS OF LPR MICE

  Y NAGAI, M INOBE, M MURAKAMI, T UEDE  FASEB JOURNAL  8-  (5)  A1002  -A1002  1994/03  [Not refereed][Not invited]
  

Research Grants & Projects

• 副腎皮質機能亢進症関連肝障害による肝細胞癌発症機序の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/04 -2026/03 

  Author : 滝口 満喜, 村上 正晃, 佐々木 東, 新坊 弦也, 横山 望
• Deep phenotyping toward the precision medicine for chronic airway diseases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 今野 哲, 村上 正晃, 鈴木 雅, 杉森 博行, 清水 薫子, 木村 孔一
• 迷走神経刺激によるぶどう膜炎抑制治療

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/04 -2025/03 

  Author : 南場 研一, 村上 正晃, 北市 伸義
• Ocular gateway reflex, which is mediated by sensory nerve, and relapsing control by neurotransmitter

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 柳井 亮二, 村上 正晃, 園田 康平, 湧田 真紀子, 木村 和博, 寺西 慎一郎

   

  難治性ぶどう膜炎は炎症再燃を繰り返すことにより視機能を喪失させる。ステロイドは炎症再燃を抑制できず、先行研究はケモカインや免疫細胞による急性炎症を対象に病態解明に取り組んでいるが、炎症再燃に関しては決め手がない。私たちは免疫細胞を制限し眼内炎症を抑制する血液眼関門が炎症により妨げられていることに着目して研究をすすめてきた。本研究は、知覚神経による血液眼関門の制御，特に神経伝達物質サブスタンスP による免疫細胞ゲートの形成・制御を精査するとともに、私たちが見出したサブスタンP 由来のFGLM-NH2ペプチドを投与し，より効果的に炎症再燃を抑制することを目的とする。中枢神経系で明らかになった免疫細胞ゲートによる炎症回路：ゲートウェイ反射の観点から眼局所におけるゲートウェイ反射機構を解明し、炎症再燃の本質的理解と解決を目指す画期的な研究である。本研究の目的は、神経伝達物質により部位特異的な免疫病態が誘導され、眼内の炎症再燃が調節されていることをin vivo 系を用いた臨床的な観点から検証する。本年度は、神経伝達物質サブスタンスP による免疫細胞ゲートの形成と局在への影響を検討するため、三叉神経切除によるサブスタンスP 欠乏動物モデルおよび網膜光凝固によるサブスタンスP 過剰発現動物モデルを作成し，各々に自己免疫性ぶどう膜炎を誘導して、各々の群における眼内炎症の評価を行う。免疫細胞ゲート形成の評価は回収した眼球から組織切片を作成し、HE および免疫染色（CD4＋, CD8＋, CD11c+）により自己反応性CD4+T 細胞や抗原提示細胞、マクロファージの細胞数をカウントすることで免疫細胞ゲート形成へのサブスタンスP の影響を確認する。
• The effect of steroid for calcineurin inhibitors nephrotoxicity in kidney transplant recipients

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 田邉 起, 村上 正晃, 篠原 信雄, 堀田 記世彦

   

  腎移植後の長期成績の向上にはカルシニューリン阻害薬（CNI）による慢性腎毒性の克服が必要である。これまでのCNI腎毒性の概念は腎細動脈の中膜平滑筋の硝子様変化による血管毒性とされており、これにより腎の間質線維化や尿細管委縮が始まり移植腎機能低下を起こすと考えられていた。この動脈硝子化に代表される血管内皮細胞変化が慢性腎毒性のマーカーとされてきたが、尿細管や間質の上皮細胞系に注目したマーカーが必要である。 近年CNIによる炎症性サイトカインの産生の誘導が指摘されており、慢性炎症のメカニズムのひとつであるNF-κB経路とSTAT3経路の活性化が相乗的に起こる炎症増幅回路の活性化が関与している可能性がある。一方で、免疫抑制剤の一つであるステロイドには抗炎症作用があり、CNIよる炎症性サイトカイン産生誘導を抑制している可能性がある。よって本研究の目的は尿細管間質においてCNI腎毒性をステロイドが軽減しているかを検索し、さらに新規バイオマーカーの開発に取り組むことである。 まずステロイドがCNIによる腎毒性に良い影響を及ぼすかを臨床データおよび経時的な移植腎組織より解析した。当院の腎移植患者でステロイドが継続されている群と、ステロイドを早期に中止した群で、移植腎サンプルの見直しを行った。Periodic acid-Schiff stain（PAS）染色を用いて、これらの細動脈の硝子化を国際基準であるBanff分類のAlternate quantitative scoring for Hyaline Arteriolar Thickening（aahスコア）で3段階に分けて評価した。現段階でステロイド中止群においてCNI毒性が有意に高い結果がでている。この結果はステロイドが慢性腎毒性を抑制する可能性を示す重要な知見である。
• Development of immunomonitoring system that predicts chronic antibody-mediated rejection in kidney transplant recipients

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 堀田 記世彦, 村上 正晃, 田邉 起, 篠原 信雄

   

  腎移植の短期成績は飛躍的に向上しているが、長期的にはドナー特異的抗体による慢性抗体関連型拒絶反応(CAAMR)により移植腎機能廃絶となる症例が多く、この克服が長期成績の向上に必要不可欠である。しかし、CAAMRに対する有効な治療法は未だ確立していない。当研究の目的は、CAAMRとなりうる患者を早期に発見する診断法を開発することである。当研究は３つからなり令和3年度の研究実績は以下の通りである。 1. パラフィン移植腎生検標本を用いたCAAMR関連遺伝子の検索：腎機能が正常である患者において移植後1年目、2年目に行ったプロトコール移植腎生検を用いて遺伝子解析した。結果炎症に関連する数種類の遺伝子の発現を組み合わせることにより5年目にCAAMRに至る症例と至らない症例との区別がつく可能性を見いだせた。 ２．CAAMR早期診断のための尿中バイオマーカーの検索：過去に報告した尿中ORM1とSYT17の他に数種類の遺伝子候補について解析中である。 ３．末梢血リンパ球反応による早期診断法の開発： 200症例につきCFSE/MLR assayを行った結果、腎機能が正常で病理学的にも問題ない患者においてはドナーに対するCD8陽性T細胞の反応は認めないが、CAAMRの患者ではドナーに対するCD8陽性T細胞の強い反応が認めた。また、CD4に関しても同様の所見を認めたが、その中でもTh1，Th17細胞の反応が強く見られた。また、抗ドナー抗体が陽性で腎生検で拒絶を認めない、前CAMR状態の患者においてもCAMRの患者と同様の反応を認めた。CFSE/MLR assayによりCAAMRが未然に診断できる可能性を見いだせた。
• 破骨細胞に発現する免疫チェックポイント分子シグレック-15の機能と治療応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 高畑 雅彦, 村上 正晃, 津田 真寿美

   

  初年度は主に破骨細胞膜上のシグレック-15によるT細胞活性抑制効果の検証を中心におこなった。野生型マウス由来骨髄マクロファージから分化させたシグレック１５を高発現する破骨細胞またはシグレック-15遺伝子欠損マウス由来骨髄マクロファージからII型コラーゲンコートディッシュ上で培養したシグレック１５発現のない破骨細胞を用いてT-cellとの相互作用実験を行った。マウスシグレック-15発現破骨細胞または非発現破骨細胞と細胞透過性色素CFDA-SEで蛍光染色したCD4+T (Th1, Th17)、CD8+T細胞を共培養したところ、T細胞の細胞増殖性には明らかな差は生じなかったものの、シグレック-15発現破骨細胞はT-cellからのサイトカイン（IFNγ, IL-17, IL-22）分泌量を減少させることがわかった。 ヒト末梢血単球由来破骨細胞共培養系とヒトT-cellについても同様の実験を行い、破骨細胞由来のシグレック15はT細胞増殖性には明らかな変化を与えないが、T-cellからのサイトカイン分泌量を減少させ活性を抑制することを確認した。ヒト由来破骨細胞ではシグレック15遺伝子をノックダウンした細胞（単核破骨細胞）を用いた。 ２年目の目標であるin vivoでの検証の準備として、転移性骨がんのモデルはマウス乳癌細胞株E0771およびルイス肺がん由来細胞株を免疫不全マウス、野生型マウスおよびシグレック１５遺伝子欠損マウスに尾動脈経由で移植するモデルを作成中であり、免疫不全マウス以外ではモデルの最適化を行なっているところである。また、野生型マウスとシグレック１５遺伝子欠損マウスを用いて、アジュバント誘導関節炎をモデルを作成中である。
• Multi-scale Imaging of Water Molecules using MRI and Isotope Microscope

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 工藤 與亮, 村上 正晃, 小畠 隆行, 小牧 裕司, 杉森 博行, 坂本 直哉, 亀田 浩之, 安井 正人

   

  ①MRI撮像法開発：O-17標識水の存在によるT2値の短縮を定量的に計測してO-17濃度を定量解析するため、プリパルスを利用した高速T2 mapping法を開発して最適化を行った。異なる濃度のO-17標識水を含有した濃度ファントムを作成し、高速T2 mapping法と従来のFSE法によるT2 mapping法の精度を比較した。従来法と比較して高精度のT2値測定が可能となった。 ②正常動物・疾患モデル動物でのMRI撮像：正常マウスやラットにてO-17標識水の静脈内投与法や髄腔内投与法、頸動脈内投与法、腹腔内投与法などを確立した。静脈内投与や頸動脈内投与によって脳内の有意なMRI信号変化を確認した。水中毒モデルラットにO-17標識水を腹腔内投与してMRI撮像を行い、AQP4欠損ラットとの比較を行った。AQP4欠損によって脳内の水貯留が増加することが明らかになった。ALSモデルマウス・ラットにてO-17標識水を静脈内投与してMRI撮像を行った。野生型と比較して錐体路での水漏出が増加していることが明らかとなった。 ③同位体顕微鏡による水分子イメージング：新たに導入した多機能コーティング装置を用いて凍結下での標本作成から同位体顕微鏡によるイメージングまでの解析手順を確立した。ラット脳にO-18標識水を直接注入し、注入部位でのO-18濃度の上昇を確認した。摘出したラット肝の門脈内にO-18標識水を注入し、血管内や類洞内のO-18濃度の上昇を確認した。 ④ヒトでのMRI撮像：認知症患者を対象にしたO-17標識水の髄腔内投与研究にて、特発性正常圧水頭症患者とアルツハイマー型認知症患者で、髄腔内の水吸収速度に差があることを見出した。
• ストレスゲートウェイ反射により「病は気から」の分子機構を解き明かす

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2024/03 

  Author : 村上 正晃, 田中 勇希, 山崎 理絵

   

  ストレスゲートウェイ(SG)反射では、それ自体では病気を起こさない軽度のストレスが特定の神経回路を活性化し、脳特定血管の変容から当該血管周囲へ自己 反応性T細胞の浸潤を誘導する。その後、さらにその部位の血管周囲に分布する神経回路がATP依存性に活性化し、消化管障害、心機能不全などから突然死が起こる。 R3年度は複数の関節リウマチモデルを用いることで、炎症性疾患で遠隔部位に左右対称な炎症病変を生じさせる分子機構の解明に関して大きな進捗があった。 関節リウマチ、間質性肺炎、乾癬などの炎症性疾患では、遠隔部位に左右対称な炎症病変が形成され、神経系の関連が示唆されていましたが、その実態は長らく全く不明でした。村上らは、新規のゲートウェイ反射が形成に関与していると考え、関節リウマチモデルマウスを用いて検証し、新規の分子機構である「遠隔炎症ゲートウェイ反射」を発見しました。 遠隔炎症ゲートウェイ反射では、関節リウマチモデルマウスの片側の足関節の炎症で生じるATPにより、まず感覚神経、続いて脊髄のプロエンケファリン陽性介在神経の順で活性化し、その後、それに伴って反対側の足関節に分布する感覚神経が活性化しました。その結果、活性化した感覚神経から逆行性にATPが放出され、反対側の血管内皮細胞、線維芽細胞などでIL-6アンプが活性化されることで炎症が誘導されました。すなわち、遠隔炎症ゲートウェイ反射では、ATPが神経伝達物質かつ炎症増悪因子として作用することがわかりました。これらの結果から、遠隔炎症ゲートウェイ反射の神経回路とATPは、関節リウマチ、間質性肺炎、乾癬など遠隔炎症を引き起こす炎症性疾患の治療標的となることが期待されます。
• 精神心理状態の変容による病態修飾の分子機序の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/07 -2023/03 

  Author : 村上 正晃

   

  精神、心理状態の変化が身体に影響を及ぼすことは経験的にも知られているが、その分子機構は解明されておらず、それを客観的に評価できる バイオマーカーは存在しない。さらに、好ましくない環境や逆に楽しい環境などさまざまな心理状態が脳内でどのような神経回路を形成し健康や病気に影響するのかもほとんど理解されていない。これらの解明は現象が複雑であるがゆえ非常に挑戦的な研究課題であるが、精神、心理状態の変容と病気の関係を捉えられるためにゲートウェイ反射という申請者の独自の発見である観点に絞ってアプローチする。 R3年度は、すでにストレスゲートウェイ反射発見時に実施したものを基本に、テレメトリー、エコーを含む生理学的検証、行動試験を含む神経生理学的検証にて表現型を解析、病理学的検証を実施、さらに、分子生物学的手法のほか、透明化技術、マススペクトロメトリー技術、神経科学技術も採用し進めている。具体的には、透明化法・連続切片と免疫染色法等の組み合わせ、全身連続切片とイメージングMSの組み合わせ、ソート、レーザーマイクロダイセクション法、リボゾームトラップ後の一細胞解析を含むRNAseq、LCMS、ChIPseqなどのRNA発現、分子、DNA修飾の網羅的検討、さらに、オプトジェネティクス、ケモジェネティクスによる特定神経回路機能の検証を実施してストレスゲートウェイ反射の詳細を分子レベルでの解明を遂行中である。 また得られた知見を元に、北大病理学教室、法医学教室と共同研究にて特に加齢突然死、自己免疫疾患などのヒト検体での検証を同時に行なっている。
• 副腎－肝臓連関によるイヌ肝細胞癌発症メカニズムの解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2023/03 

  Author : 滝口 満喜, 村上 正晃, 山崎 淳平, 池中 良徳

   

  犬の肝細胞癌における、ステロイドホルモン産生過剰を起点とする発がん仮説の検証を目指し、前年度に引き続いた検討を行った。2年目となる本年度は肝細胞癌・副腎皮質機能亢進症罹患犬における血中ステロイドホルモン・代謝産物のプロファイリング、ならびに肝臓組織でのエピゲノム変異の解析を遂行した。 ステロイドプロファイリングでは前年度に確立した測定系を発展拡大させ、新たに6種の副腎皮質ホルモンと4種の代謝産物を合わせた、合計19種類のステロイドプロファイル解析が可能となった。この新たな測定系を用いて、肝細胞癌36症例、副腎皮質機能亢進症15症例、併発11症例、コントロール19症例を対象として、血液中のステロイドプロファイルを解析した。しかし、血液中のステロイドプロファイルに疾患特異性、もしくは疾患群間での差が認められなかった。 DNAメチル化の解析では、実験的ステロイドホルモン誘発性脂肪肝、副腎皮質腫機能亢進症併発肝細胞癌および肝細胞癌単独症例の腫瘍組織と腫瘍近傍組織の、合計3種類の肝臓組織を用い、多段階発癌仮説の検証を試みた。その結果、脂肪肝・近傍組織・腫瘍組織と段階的なメチル化変化が存在し、腫瘍においてメチル化レベルの高い遺伝子が6、低い遺伝子が12、抽出できた。 現時点では血中ステロイドプロファイルに肝細胞癌と副腎皮質機能亢進症との関連を示唆する血中ステロイドプロファイリングは見つかっていない。一方で、ステロイド肝から腫瘍への段階的なDNAメチル化レベルの変化が存在することが明らかになった。犬の肝細胞癌においてステロイドが発症に役割を果たしていることは十分に予想され、本年度の成果を複合的かつ詳細に解析することで、多段階発がん説の検証が進む。
• Analysis of epidermal growth factor receptor dependent inflammation amplifier activation in uveitis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2023/03 

  Author : 南場 研一, 村上 正晃, 北市 伸義

   

  初年度には、ぶどう膜炎群（サルコイドーシス、フォークト-小柳-原田病、ベーチェット病、HLA―B２７関連急性前部ぶどう膜炎）およびコントロール群として健常人について、血清中の上皮増殖因子受容体およびそのリガンドであるエピレグリン等の濃度をマルチプレックス法にて測定し、ぶどう膜炎群ではエピレグリンならびにその他の上皮増殖因子（EGF）受容体のリガンド（アンフィレグリン、ベタセルリン、TGF-アルファ、HB-EGF）がコントロール群と比べ有意に上昇していることを報告した（マンホイットニーのU検定：P＜０.０５）。これらの結果から、他疾患で報告されているようにぶどう膜炎患者においても上皮増殖因子の炎症増幅への関与が示唆された。昨年度はF759遺伝子変異マウスにおいて実験的自己免疫性ぶどう膜網膜炎（ EAU ）が増強されるかどうかを検討する予定であった。F759 遺伝子変異マウスではネガティブシグナルが阻害されてIL-6 産生が亢進し、上皮増殖因子が関与する炎症増幅経路はさらに増強されるため、EAUが強く惹起される可能性が示唆された。F759遺伝子変異マウスを当方の北海道大学医歯学共同研究動物施設に受入する手続きを進めているところで新型コロナウイルス感染拡大のため研究停止となり、現在まで受入が進んでいない。そこで、研究室に凍結保管していたぶどう膜炎患者血清を用いてEGF受容体およびそのリガンドについて測定し、症例数を増やし結果を得た。本年度は、マウスEAUの網脈絡膜を摘出し、網脈絡膜組織のmRNAからcDNAを作成し、マイクロアレイ法にて関連する分子の発現の評価を行った。EGF受容体のリガンドであるエピレグリン、アンフィレグリン、EGFが上昇していること、逆にEGF受容体は発現が低下していることが示され、マウスEAUにおいてEGF受容体を介するシグナル伝達が病態形成に関わっていることが示唆された。
• Analysis of molecules activating the inflammation amplification loop in fibromatosis of the palmar fascia

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : Matsui Yuichiro

   

  Dupuytren's contracture is a pathological fibrosis of the palmar aponeurosis that causes irreversible flexion contracture of the fingers and pain in the palmar region. Although several risk factors are known, the detailed pathogenesis of the disease remains unclear. This study focused on the presence of chronic inflammation in Dupuytren's contracture and the previously reported disease-associated genes. The results revealed that the IL-6 amplifier, a mechanism for amplification of chronic inflammation by simultaneous activation of IL-6 and NF-κB in non-immune cells, is activated in this disease. Furthermore, SFRP4, one of the disease-associated genes, was found to contribute to the activation of IL-6 amplifiers.
• Establishment of stress immunology

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/06 -2021/03 

  Author : MURAKAMI Masaaki

   

  We have previously discovered the “stress gateway reflex,” a neuro-immune interaction that lead to fatal gastrointestinal and heart failure by induction of brain micro-inflammation triggered by chronic stress. To find other stress related inflammatory disorders, we firstly focused on light stimulation. Exposure to photopic light down-regulated the ocular inflammation by suppressing beaching of the blood-retina barrier. This discovery also indicates that gateway reflex can be suppressed by environmental stimuli. Moreover, we focused on chronic kidney allograft rejection, a kind of stress associated disease. Pathological analysis revealed that patients’ tubular cells expressed STY17 and ORM1 as well as inflammatory transcription factors. Since these molecules were highly detected in urine, these molecules can be non-invasive candidate biomarkers for stress-mediated inflammatory diseases and will contribute to establish stress immunology in the future.
• Development of an immunological monitoring system to predict chronic rejection in kidney transplant recipients.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Hotta Kiyohiko

   

  Chronic active antibody-mediated rejection (CAAMR) is the main cause of graft loss in the long-term. Pathological changes during CAAMR occur gradually even without clinical manifestations, followed by dysregulation of the function of the kidney transplant. Therefore, a reliable immunoresponse monitoring system to identify recipients who will develop CAAMR in the future is required. A mixed lymphocyte reaction (MLR) assay is a method to measure T cells alloreactivity via direct pathway (DP). Recent studies indicate that DP also contribute the pathogenesis of chronic injury. However, there are no reports of MLR assay to monitor CAAMR. Therefore, we tried to develop a novel MLR monitoring system that predicts CAAMR in kidney transplant recipients. To this end, we measured the anti-donor response using our novel MLR in 150 kidney recipients whose graft status were evaluated by graft biopsy and analyzed the relationship between the anti-donor response and pathological findings of allograft.
• 心理免疫学の創成：精神・心理に関連するゲートウェイ反射の同定と解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2018/04 -2021/03 

  Author : 村上 正晃
• Creation of Space Immunology by space and ground experiments

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/06 -2019/03 

  Author : Murakami Masaaki, Kamimura Daisuke, Arima Yasunobu, Shirakawa Masaki, Shiba Dai, Yumoto Akane, Ueda Hiroki, Tainaka Kazuki

   

  We have reported that gravity stimulation induces regional neural activations to create an immune cell gateway to the CNS via alteration of specific vessels, a phenomenon called the gateway reflex. In this study, we aimed to study a relation between gravity and inflammatory diseases by a combination of ground and space experiments, and develop a research field of the space immunology. We have performed feasibility studies including the establishment of pathogenic T cell clones, overcome of time lag till sampling, and effects of over-gravity and impact during launch, with successful results. Although the space experiment was originally scheduled within the research term, it was postponed, which will be conducted in 2019.
• Analysis on the mechanism of fibrosis of the palmar fascia in Dupuytren's contracture

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2019/03 

  Author : Matsui Yuichiro

   

  Activation of STAT3 and NF-κB p65 was identified in the affected palmar fascial tissues from the patients with Dupuytren’s contracture. In addition, in the SNP analysis, genetic loci of risk were observed with uneven distribution in the patients with Dupuytren’s contracture. These included a SNP present between the genes SFRP4 and EPDR1. The activation of STAT3 and NF-κBp65 in the palmar fascial tissues from the patients with Dupuytren’s contracture suggests that an inflammatory circuit is activated in this disease. The relationship between the genes and SNPs with uneven distribution and the inflammatory circuit will be functionally analyzed in a knock-down experiment.
• The mechanisms of chronic inflammation by Gateway Reflex and inflammation amplifier

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : Murakami Masaaki

   

  A correlation between pain intensity and the pathogenesis of inflammation is poorly established and still unclear. In this application, we elucidated that a pain evoked relapse of multiple sclerosis. Moreover, we revealed that brain micro-inflammation around specific vessels caused by chronic-stress could be switch to activate new neural pathway to regulate organ homeostasis. In addition, we identified positive-regulator of inflammation amplifier by genome wide screening. Among them, we focused on BCR and Rbm10, and clarified the mechanisms of inflammation amplifier.
• Why does sleep disorder cause diseases?

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2017/03 

  Author : Murakami Masaaki

   

  The central nervous system (CNS) equips the blood-brain barrier (BBB) to limit exchanges of substances. However, immune-cell infiltration occurs in CNS. We have identified the dorsal vessels of the fifth lumbar (L5) spinal cord as a gateway for immune cells to CNS, which is critical for neuro-inflammation development. In this proposal, how stress-mediated neural activations affect the gateway was examined. I found that specific nerve activations by sleep disorder completely change the gateway site from L5 to the brain, followed by sudden death by gastrointestinal bleeding. This “stress-gateway reflex” explains a classical proverb “Illness starts in the mind.”, which we generally experience but the mechanisms are not well known. By continuing the gateway reflex research, it can be expected that manipulations of local neural activations will be a new therapeutic approach for certain diseases.
• 炎症の増幅回路の交感神経による活性化機構の解析と病態形成への関与

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2014/04 -2017/03 

  Author : 村上 正晃, STOFKOVA ANDREA, STOFKOVA Andrea

   

  我々は、線維芽細胞や血管内皮細胞などの非免疫細胞に存在するケモカイン・IL-6の過剰産生機構である「炎症回路」を発見して解析を続け、最近では炎症回路が局所の感覚神経-交感神経の興奮によって過剰に活性化されることを証明した。また、定常状態のマウスでは重力に伴う神経活性化によって、第５腰髄背側血管での炎症回路が過剰に働くことが分かり、さらに、血中に中枢神経系抗原を認識する自己反応生T細胞が存在する場合には、この部位から中枢神経系に侵入して炎症が慢性化し、多発性硬化症に似た病態を形成することが判明した。この局所感覚神経-交感神経の活性化による血管機能変化をゲートウェイ反射と呼んでいる。本研究では、研究が進んでいる多発性硬化症モデル(EAE)に加え、ベーチェット病モデルである実験的自己免疫性ぶどう膜炎（EAU）を新たに利用してこれらの知見をさらに詳細に解析し、自己免疫疾患発症時における神経刺激の役割について明らかにすることを目的とした。EAUでは免疫後10日目からCD4 T細胞を初めとして免疫細胞の網膜への浸潤が認められた。この時期にマウスを暗所から明所へ飼育場所を移動させると、炎症性メディエーターであるインターロイキン6やケモカインの発現や網膜病態の臨床スコアも含め、網膜炎症の状態が変化することを見出した。このことは光の強度が網膜炎症に影響を及ぼすことを示唆しており、光ゲートウェイ反射の存在を示唆している。また、網膜炎症に対する新たな治療法に結びつく可能性が考えられる。これらの結果は学会等で発表し、また論文作製中である。
• The novel therapeutic strategy for pulmonary hypertension targeting inflammation amplification loop mediated by IL-6

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2015/03 

  Author : NAKAOKA YOSHIKAZU, SHIRAI Mikiyasu, MURAKAMI Masaaki, KOMURO Issei

   

  The molecular mechanisms of interleukin-6(IL-6)-mediated pathogenesis of PAH have been elusive. Here, we identified IL-21 as a novel downstream target of IL-6-signaling in PAH. First, we found that IL-6 blockade by the monoclonal anti-IL-6 receptor antibody, MR16-1, ameliorated hypoxia-induced pulmonary hypertension (HPH) and prevented the hypoxia-induced accumulation of Th17 cells and M2 macrophages in the lungs. Consistently, the expression levels of IL-17 and IL-21 genes, one of the signature genes for Th17 cells, were significantly upregulated after hypoxia exposure in the lungs of mice treated with control antibody, but not in those treated with MR16-1. Whereas IL-17 blockade with an anti-IL-17A neutralizing antibody had no effect on HPH, IL-21 receptor-deficient mice were resistant to HPH and exhibited no significant accumulation of M2 macrophages in the lungs. These findings suggest that IL-6/IL-21-signaling is critical in the pathogenesis of pulmonary arterial hypertension.
• Proposal of a disease model by optogenetics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : MASAAKI Murakami

   

  We have stimulated nerves in sympathetic ganglia around the fifth lumbar cord by using light stimulation in Thy1 channel rhodopsin transgenic mice. This stimulation induced the increment of chemokine expression in dorsal vessels in the fifth lumbar cord. Furthermore, an intensity of chemokine expression was correlated with intensity and times of light stimulations. And we could induce the development of EAE by using light stimulations for sympathetic ganglia in MOG reactive T cell transferred mice. This result indicated that regional neural activation could induce the development of EAE and put forward a new model to analysis of autoimmune diseases including multiple sclerosis.
• Formation of blood-brain barrier and activation of Interleukin-6 amplifier

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : MURAKAMI Masaaki

   

  Activation of NFkB and STAT induces massive production of chemokines in non-immune cells, which leads to local inflammation. We have been studying this inflammation amplifying mechanism, termed inflammation amplifier. In this study, we aimed to characterize mechanisms by which immune cells infiltrate into the central nervous system (CNS), and regulation of blood-brain barrier. In a murine model of multiple sclerosis, EAE, we found that high-performance MRI revealed that inflammation with edema occurred at fifth lumbar region where immune cells firstly enter the CNS. In addition, this edema was associated with ischemia/reperfusion injury that deteriorated the CNS inflammation. We also discovered that various stresses modulated the entry sites of immune cells into the CNS and altered the development of EAE in mice.
• Characterization of intracellular Zn signaling and its relationship to diseases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2015/03 

  Author : HIRANO Toshio, MURAKAMI Masaaki

   

  Zinc is one of the essential metal elements, which is indispensable for the functions of various molecules in the body. However, it is not well understood how zinc levels inside and/or outside cells are regulated. In the course of research about interleukin-6 and its signal transduction, we found that zinc acts as a second messenger in a cell, and established a scientific field of “zinc signaling”. In this study, we revealed molecular mechanisms and physiological functions of zinc signaling using various knock-out mice related to zinc signaling such as zinc-binding molecules and zinc transporters.
• 急性期蛋白による自己免疫疾患・感染症の制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2010 -2011 

  Author : 村上 正晃

   

  本研究はT細胞の生体内での反応性を肝臓などの非免疫臓器からのサイトカイン発現を起点に検討することを目的としている。この目的のために以下の3つの実験を行った。 1. IL-7を肝臓特異的に欠損させたマウスでの自己免疫疾患の発症 IL-7floxマウスを京都大学の生田先生からいただきアルブミンCreマウスとかけ合わせて肝臓特異的にIL-7が欠損した変異マウスを作製した。多発性硬化症モテルの実験的脳脊髄炎(EAE)を誘導したところ有意にその発症か抑制された。この結果は研究代表者が2009年にImmunlty誌に発表したハイドロダイナミック法とshRNAを用いた方法を同様のものであった。すでにIL-7を肝細胞から誘導する1型IFN受容体の欠損でも同様な表現型を得ることができた。 2. 脊髄の血管内皮細胞に存在する病原T細胞の中枢神経系への侵入口 第5腰椎の背側の血管の内皮細胞にサイトカインIL-6と神経刺激によるノルアドレナリンの刺激にてSTAT3とNFkBが活性化してケモカインの過剰発現系IL-6アンプが活性化し免疫細胞の中枢神経系への入り口となっていることが判った。血液中に中枢神経系の抗原に対する自己反応性T細胞が存在すればこの部位から侵入して病気を発症した(Cell 2012)。 3. 血管内皮細胞でのケモカイン発現機構IL-6アンプの制御潰伝子のゲノムワイドな解析 実験2にて解析したIL-6アンプの活性化制御遺伝子を同定する目的でshRNAを搭載したレンチウイルスを用いたゲノムワイドな解析を行った。その結果、IL-6アンプの活性化は1000個以上の遺伝子にて制御されていることが判った。これらの遺伝子にはこれまで遺伝学的に病気、病態との関運か証明されたのもが10%以上存在して疾患関連遺伝子が濃縮されていることが判った(under revlsion)。
• Controlling of chronic inflammatory diseases via regulation of inflammation stroma cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : MURAKAMI Masaaki, KAMIMURA Daisuke

   

  We defined the inflammation stroma cells as cells having excessive chenokine production after NFkB and STAT3 stimulation. The inflammation stroma cells have type 1 collagen molecules and include endothelial cells, fibroblasts, astrocytes, and epithelial cells. The main founding in this project is that excessive activation of the he inflammation stroma cells by an excessive neural activation particularly via sympathetic neuron' s creates a gateway for immune cells in blood vessels including autoreactive CD4+ T cells followed by the development of an autoimmune disease.
• Activation of Zinc signal and its biological significance

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2010 

  Author : HIRANO Toshio, MURAKAMI Masaaki, KAMIMURA Daisuke, FUKUSHIMA Toru, FUKADA Toshiyuki

   

  We here attempt to investigate intracellular Zn signaling, which is important for cell fate and/or cell activation status determination followed by various diseases and disorders. We mainly have three results, which are obtained by this grant. (I) Investigation of intracellular Zn signals, (II) Molecular basis of Zn signal to analyze Zn-binding sites in the target molecules and their mechanism, and (III) in vivo significances of Zn signal using various Zn-related molecule deficient mice and several disease/disorder models.
• 肝臓からのサイトカインの発現とT細胞反応性の制御

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2008 -2009 

  Author : 村上 正晃

   

  私たちは、線維芽細胞にてIL-6信号によって生体内のIL-7の発現量が制御されていることを示すことができた。最近、"LPSが肝細胞からIL-7を免疫細胞由来のTRIF=>IFNβを介して誘導してT細胞の反応性を亢進させる"ことを発見し、論文に発表した(Immunity 2009)。今年度は以下の2つの点に関して実験を行った。 (i) LPSあるいは他のTLRリガンドの刺激にて肝臓から発現されてT細胞の反応性に大きな影響を与えるIL-7以外の分子を同定することを目的にDNAアレーから自己免疫誘導時に肝臓にて発現する可溶性分子を同定した。これまでに約20個の分子を肝臓にて強制発現させてT細胞の分裂能を指標に解析した。その結果、2つの分子を強制発現させた場合にT細胞の分裂が促進した。現在、それらの分子に関して肝臓特異的にノックダウンさせたときのT細胞依存性自己免疫疾患の発症を解析している。 (ii) マウス生体へのTLRリガンド刺激にて肝臓からのIL-7発現は上昇したが、脾臓およびリンパ節からのIL-7の発現は減少した。脾臓あるいはリンパ節では線維芽細胞が主にIL-7を産生している事が知られている。肝臓と線維芽細胞でのIL-7発現制御メカニズムを比較解析して『なぜ、脾臓およびリンパ節にてTLRリガンド刺激後にIL-7の発現が低下する必要が有るのか?』を解析している。各種ノックアウトマウスをLPS刺激してリンパ節からのIL-7発現を解析した。現在までに25種類のノックアウトマウスにて解析を行ったが、その発現がLPS刺激前後に変化しない変異マウスは同定できていない。そのため、現在、SOCS3を線維芽細胞特異的にノックアウトしたマウスを作製してIL-7発現を解析している。
• Regulation of Immune responses and autoimmune diseases by cytokines

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2007 

  Author : HIRANO Toshio, MURAKAMI Masaaki, YAMASHITA Susumu

   

  Our aim of this study was to know how Y759F mutation in gp130, an IL-6 signal transducer, induces an autoimmune arthritis disease in vivo and to investigate a role of IL-6-mediated signaling in the normal immune responses. We obtained two important concepts showing how tissue specific autoimmune diseases are induced as described below. (1) Excess IL-7 expression induced by activation of non-hematopoietic cells via an excess IL-6 signaling triggers an excess homeostatic proliferation of CD4+ T cells followed by the development of autoimmune arthritis in F759 mice. We demonstrated how a dysregulated interaction between non-hematopoietic cells and hematopoietic cells, CD4+ T cells, induces a tissue specific autoimmune diseases in vivo. (2) There is an IL-17-mediated IL-6 positive feedback loop in non-hematopoietic cells even in normal condition in vivo. Dysregulation of the IL-6 loop by a genetic abnormality such as Y759F mutation in F759 mice induces an excess production of IL-6 via the loop followed by the development of autoimmune arthritis. Moreover, we proved the existence of intracellular Zinc signaling, which is also important for regulation of immune cell functions.
• Acquisition of Signal Information for Immune Surveillance and Determination of Immune Responses

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2006 

  Author : SAITO Takashi

   

  We aimed to clarify the mechanism on the acquisition of the information of immune recognition and regulation of immune responses. As the central mechanism of immune surveillance, dendritic cells (DC) first recognize pathogens upon infection, and then innate immunity is activated, which in turn induces antigen-specific recognition and activation of T cells. In this project, we have clarified that IRAK-4, a central regulatory serin/threonine kinase in innate immune signaling, plays also a critical role in T cell activation as the central response in the adaptive immunity. In IRAK-4-deficient mice, proliferation and cytotoxic function of CD8+ T cells were impaired upon LCMV infection. We analyzed whether the impaired response was attributed to the defects in innate response by DCs or T cells by using T cell transfer experiments, and found that T cell activation was impaired in both MHC class I and II-restricted responses. Indeed, we found that not only in vivo T cell responses but also in vitro responses including allogenic responses, super-antigen responses, and responses upon anti-TCR stimulation. By analyzing the defective signaling pathways, particularly NF-AT or NF-κB activation pathways, we found that NF-κB activation was suppressed and impaired phosphorylation of PKCθ appeared to be responsible for the defective NF-κB activation. These results indicate that IRAK-4 plays a critical role in TCR activation signals by directing towards NF-κB activation. We further analyzed the mechanism of IRAK-4-mediated NF-κB specific activation, and found that IRAK-4 associates with ZAP-70 in the over-expression system. IRAK-4 appears to be recruited together with ZAP-70 to the vicinity of TCR upon stimulation, and is involved in activation regulation. Our result that IRAK-4 is important for NF-KB activation in both innate and acquired immunities suggests that NF-κB activation by IRAK-4 has been conserved through phylogenic development.
• IL-2依存性樹状細胞によるメモリーCD8+T細胞の維持機構

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2003 

  Author : 村上 正晃
• Identification of a chromosomal region that is responsible for memory T cell homeostasis in a mutant mouse.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2003 

  Author : MURAKAMI Masaaki

   

  It is well known that memory T cells proliferate faster and react to the pathogens stronger compared to naive T cells. I found a female mutant mouse carrying excess amount of memory T cells of both CD4+ and CD8+ in a SPF colony in National Jewish Medical Center, Denver, CO. The phenotype is inherited as an autosomal recessive manor. Thymus size is small (about 10% of controls) and the numbers of double positive cells reduces dramatically. T cell differentiation is partially but dominantly blocked at a step where preTCR signaling is necessary named DN3. Platelets significantly increased in serum and this result is consistent with excess amount of megakaryocytes in spleen. In addition, the number of immature B cells decreased significantly and differentiation of B cells is blocked at large preB stage where preBCR signaling is important. I performed bone marrow transplantation experiment to analyze if the mutation in lymphoid progenitor is or if it in microenvironment is necessary for the phenotype of T and B cells in the mutant mice. The mutation in lymphoid progenitor cells themselves is necessary to see the phenotype. In order to identify a DNA fragment carrying a responsible gene that induces the phenotype in the mutant mice, we employed SSPL method. We crossed B10#4 mice with NZB mice to get F2 mice and got over 100 phenotype + and -offspring. We identified 1cM DNA fragment in chromosome #2 that is responsible for the mutant and the fragment has about 40 genes according to NCBI database. Result of RT-PCR experiment for a gene in the fragment was fascinating, since splicing pattern of the gene is different between the B10#4 and control mice. I analyzed sequence of the gene and found that there were two mutations in the introns of the gene.
• 過剰のメモリーT細胞を持ち膵島炎を発症する突然変異マウスからの責任遺伝子単離

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2002 

  Author : 村上 正晃

   

  1)申請者はデンバーのマラック教授の教室に留学中に正常C57BL10コロニーから末梢血中に過剰のメモリー表現型T細胞を持ち、肝臓および膵島にリンパ球浸潤が認められる突然変異マウスBL10#4を見つけることができた。その後の研究から、この形質は常染色体劣勢遺伝し、1遺伝子で規定されること、T細胞およびB細胞分化の両方に異常が認められ、それぞれプレTおよびプレB細胞受容体信号が必要な時期(プレT細胞から未熟T細胞及びプレB細胞から未熟B細胞)で重度な分化障害が有ること、巨核球数が増加して血小板が正常個体の10倍以上にまで増加していること、中程度の貧血が認められ、抗赤血球抗体の検出できる個体が有ること、骨髄移植の実験から変異表現型は環境因子では無く、骨髄細胞自体に由来することが判明した。ヒトの1型糖尿病を含めた自己免疫疾患は多因子疾患であるが、それらの発症に関連する遺伝的要因の一つに、このマウスの責任遺伝子が成りうる可能性がある(日本免疫学会、東京、2002年、発表)。 2)申請者は特定領域研究、ゲノム医科学に、本研究課題にて本年度から参加させていただきました。本年度、本予算を用いてBL10#4とNZBマウスをかけ合わせて、100匹以上の形質陽性および陰性F2マウスを得ることができた。それらのF2マウスの肝臓からDNAを抽出し、BL10とNZBで長さの異なるMITマーカーを用いてSSLP法にてBL10#4の形質の責任遺伝子の存在を解析した。幸運なことに責任遺伝子の存在する1cM程度の領域を2番染色体に同定することができた。セレラ社のデータベースで解析してみるとその領域には約45個の遺伝子が存在していることが判明した。
• Analysis of regulation of host defense response by osteopopontin and its applocation to diagnosis and therapy strategy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -2000 

  Author : UEDE Toshimitsu, MAEDA Masahiro

   

  1. Highly metastatic adenocarcinoma cells tend to secrete osteopontin(OPN). OPN binds to β1 integrin-containing receptors in an autocrine fashion and induce motility of tumor cells. The association of β1 integrins with CD44v on tumor cell surface is essential for OPN-induced tumor cell motility. 2. The new-vessel formation as defined by CD34 positive endothels was significantly increased in OPN and VEGF positive stage I lung adenocarcinoma tissues and corelated very well with poor prognosis of patients. 3. Upon high fat diet feeding, OPN transgenic mice developed considerably severe atherosclerosis as compared to control mice. In OPN transgenic mice, macrophages within atherosclerotic lesions expressed significant levels of OPN. 4. Upon intravenous injection of zymosan, mice develoved liver granuloma, the development of granuloma was significantly inhibited by simultaneous injection of anti-OPN antibody, indicating the critical role of OPN in the granuloma development.
• 新しいCTLA4結合分子、ACBMのcDNA単離とその機能解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1997 -1997 

  Author : 村上 正晃, 上出 利光

   

  T細胞の活性化はT細胞受容体からの信号と接着分子からの補助信号が必須である。我々は補助信号分子としてCD28/CTLA4を研究してきた。CD28/CTLA4は共通のリガンドであるB7-1/B7-2を持つ、T細胞上の分子である。CD28は静止期のT細胞から発現していて活性化のための副信号を伝達する。一方、CTLA4は活性化T細胞に発現誘導され、T細胞の不活性化を誘導する。本研究では新たに発見したB細胞上のCTLA4結合分子ACBMの機能解析とcDNAの単離を目的としている。新しいCTLA4結合蛋白を同定するために既知のリガンド、B7-1/B7-2と結合できない可溶性変異型CTLA4の作製を行った。この分子を用いて細胞株を検索し、結合できる細胞が新しいCTLA4結合蛋白を発現していると考えた。CD28/CTLA4の細胞外領域には動物間で高度に保存されたMYPPPYモチーフが存在したのでこのMYPPPYモチーフに点突然変異を加えていった。その結果、最後のPYをAに置換したPYAA変異体がB7-1/B7-2と結合できなかった。このPYAA分子の細胞外領域をIgG1のFc領域と結合させたPYAAIgGを作製し、細胞株を検索してマウス未熟B細胞株、WEHI231細胞に結合を認めた。WEHI231からACBM分子を免疫沈降すると130kDのホモダイマーであることが判明した。ACBMのcDNAを単離するためにCTLA4IgG/PYAAIgGを用いたアフィニティーカラムを作製した。しかし、CTLA4IgG/PYAAIgGの単体への共有結合後、ACBMとの結合が弱まり、目的よりかなり少量のACBMを精製できたのみであった。そのため、精製を続けるためには大量のCTLA4IgG/PYAAIgG分子が必要となり、アデノウイルスを用いた発現系を作製した。この発現系を用いてmgオーダーのCTLA4IgG/PYAAIgGを得ることが可能となり、ACBMの精製とあわせて、CD28/CTLA4-B7/ACBM信号の生体内での機能解析が可能となった。

Educational Activities

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Position History

• 2016年4月1日 

  - 

  2018年3月31日 

  遺伝子病制御研究所長
• 2018年4月1日 

  - 

  2020年3月31日 

  遺伝子病制御研究所長
• 2022年4月1日 

  - 

  2024年3月31日 

  遺伝子病制御研究所長
• 2020年4月1日 

  - 

  2022年3月31日 

  遺伝子病制御研究所副所長
• 2020年4月1日 

  - 

  2022年3月31日 

  遺伝子病制御研究所附属感染癌研究センター長
• 2016年4月1日 

  - 

  2018年3月31日 

  教育研究評議会評議員
• 2018年4月1日 

  - 

  2020年3月31日 

  教育研究評議会評議員
• 2022年4月1日 

  - 

  2024年3月31日 

  教育研究評議会評議員