Rheumatoid Arthritis: Uzuki M

Sawai T, Uzuki M. · Division of Leading Pathophysiology, Department of Pathology, School of Medicine, Iwate Medical University. · Clin Calcium. · Pubmed #19252242 No free full text.

Abstract: Histopahological features of rheumatoid arthritis, beginning from synovitis through deteriorating cartilage and bone to joint destruction has basically unchanged since the old days. On the other hand many inflammatory factors initiating, sustaining and/or activating inflammation such as cytokines and proteolytic enzymes, were successively detected, and followed by genetic analysis using animal models such as transgenic and knockout methods. Newly developed therapies by biological products remarkably have influenced the inflammatory these factors and genes, and seemed to modify the histopathological features. This article refers the histopathlogical features of RA in topics such as places involved in early stage, and the cellular origin, especially about the fibroblast like cells (FLS) which have been paid attention recently as key cells presenting immunological, histiocytic and fibroblastic properties, furthermore, participating the bone destruction in part as well as osteoclast in RA. We also introduce the several animal models of RA applied by many researchers for therapeutic and genetic analyses in RA.

Uzuki M, Sawai T, Sasaki Y. · Iwate Medical University, School of Medicine, Department of Pathology. · Clin Calcium. · Pubmed #17404475 No free full text.

Abstract: Recent technologies proceed the remarkable development of genetical and protein analysis. However, we are apt to lose opportunities to observe about the disease as a whole feature. In this article, we describe the inflammatory process from synovial inflammation to cartilage and bone destruction. We notice that there are many problems to be solved in rheumatoid arthritis (RA) from the point of inflammatory process. It is often needed for us to stand still and look over the whole features of disease.

Matsushita I, Uzuki M, Matsuno H, Sugiyama E, Kimura T. · Department of Orthopaedic Surgery, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. · Mod Rheumatol. · Pubmed #17165004 No free full text.

Abstract: We report a 62-year-old man with rheumatoid arthritis (RA) who developed nodulosis after methotrexate (MTX) treatment. The epithelioid cells of nodules were positive for matrix metalloproteinases (MMP)-2, MMP-3, MMP-9, and Ki67. The synovial tissues obtained from the same patient were negative for MMP-3, MMP-9, and Ki67. This study demonstrated that MTX-induced nodules are different from synovial tissues in terms of MMP expression, suggesting the presence of different pathologic mechanisms and differential MTX susceptibility.

Ogawa Y, Ohtsuki M, Uzuki M, Sawai T, Onozawa Y, Nakayama J, Yonemura A, Kimura T, Matsuno H. · Sankyo Co. Ltd., Tokyo, Japan. · Arthritis Rheum. · Pubmed #14673986 links to  free full text

Abstract: OBJECTIVE: To examine the suppressive effect of anti-human Fas monoclonal antibody (mAb) on osteoclastogenesis in rheumatoid arthritis (RA) both in vitro and in vivo. METHODS: For in vitro analysis, activated CD4+ T cells derived from peripheral blood mononuclear cells were left untreated or were treated with humanized anti-human Fas mAb (R-125224) and cocultured with human monocytes. On day 12, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was counted. For in vivo analysis, tissue derived from human RA pannus was implanted with a slice of dentin subcutaneously in the backs of SCID mice (SCID-HuRAg-pit model). R-125224 was administered intravenously once a week for 3 weeks. The implanted tissue and dentin slice were removed, and the pits formed on the dentin slice were analyzed. RESULTS: In vitro, coculture of activated CD4+ T cells and peripheral monocytes induced osteoclastogenesis. The number of TRAP-positive multinucleated cells was reduced when activated CD4+ T cells were treated with R-125224. We established a new animal model for monitoring osteoclastogenesis, SCID-HuRAg-pit. We found that with R-125224 treatment, the number of pits formed on the implanted dentin slices was significantly reduced and the number of lymphocytes in the implanted RA synovial tissue was dramatically reduced in this model. CONCLUSION: This is the first study to demonstrate the suppressive effect of anti-human Fas mAb on osteoclastogenesis in RA synovial tissues through the induction of T cell apoptosis. Induction of apoptosis of infiltrated lymphocytes could be a useful therapeutic strategy for RA, in terms of suppressing both inflammation and bone destruction.

Ishizuka M, Hatori M, Suzuki T, Miki Y, Darnel AD, Tazawa C, Sawai T, Uzuki M, Tanaka Y, Kokubun S, Sasano H. · Department of Pathology, Tohoku University School of Medicine, Sendai, Japan. · Clin Sci (Lond). · Pubmed #14570589 No free full text.

Abstract: Rheumatoid arthritis (RA) is a disease characterized primarily by chronic inflammatory synovitis and is well-known to be associated with significant sex differences in its prevalence and clinical features. Sex steroids have been proposed to be involved in the pathogenesis of RA, but details pertaining to the expression of sex steroid receptors in RA synovial tissue have yet to be fully characterized. In the present study, we examined oestrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR) and androgen receptor (AR) mRNA expression using real-time reverse transcriptase-PCR (RT-PCR) in eight female RA synovial tissues and six female synovial tissues without inflammation, and determined immunolocalization of ERalpha, ERbeta, PR-A, PR-B and AR using immunohistochemistry in synovial tissues obtained from 22 RA patients. Real-time RT-PCR analysis demonstrated the expression of ER, PR and AR mRNAs in both RA and non-inflamed synovial tissues. Relative abundance of ER mRNAs was significantly higher in RA synovial tissue than non-inflamed synovial tissue (P<0.05). In addition, the relative ERalpha/ERbeta mRNA expression ratio was significantly lower in RA than non-inflamed synovial tissue (RA, 2.34 +/- 1.60; and non-inflamed, 20.7 +/- 19.1; P<0.05). There were no significant differences in relative abundance of PR mRNA. Relative abundance of AR mRNA was significantly lower in RA (P<0.05). Immunoreactivity for ERalpha, ERbeta, PR-B and AR was detected in the lining cells, inflammatory cells and fibroblasts in all the patients examined. The labelling indices for ERbeta and PR-B were more abundant in both lining cells (ERbeta, 54.2 +/- 12.2%; PR-B, 73.6 +/- 18.9%) and inflammatory cells (ERbeta, 74.6 +/- 16.2%; PR-B, 75.9 +/- 16.1%) than in fibroblasts (ERbeta, 36.5 +/- 15.6%; PR-B, 49.4 +/- 18.0%). Labelling indices for ERalpha and AR were significantly higher in lining cells (ERalpha, 14.4 +/- 8.6%; AR, 31.2 +/- 11.3%) and fibroblasts (ERalpha, 12.1 +/- 7.5%; AR, 20.1 +/- 9.6%) than those in inflammatory cells (ERalpha, 5.7 +/- 3.3%; AR, 9.2 +/- 4.4%). There were significant differences (P<0.05) in the labelling indices for ERalpha, ERbeta and PR-B between men and women under 50 years of age in fibroblasts of RA synovial tissues. These results indicate that sex steroid receptors are present in RA and non-inflamed synovial tissues, including inflammatory cells in RA, and suggest that sex steroids may play important roles in the regulation of inflammation of RA synovial tissue.

Matsuno H, Yudoh K, Nakazawa F, Sawai T, Uzuki M, Nishioka K, Yonehara S, Nakayama J, Ohtsuki M, Kimura T. · Department of Orthopaedic Surgery, Toyama Medical and Pharmaceutical University, Japan. · J Rheumatol. · Pubmed #12180717 No free full text.

Abstract: OBJECTIVE: Anti-Fas monoclonal antibodies (Mab) are considered to be a potential therapeutic agent for rheumatoid arthritis (RA). However, Fas mediated liver and chondrocyte damage is a serious problem in its clinical application. m-HFE7A, a novel anti-Fas Mab, selectively induces apoptosis in inflammatory cells. We succeeded in humanizing m-HFE7A to obtain h-HFE7A. We investigated the therapeutic effects of h-HFE7A Mab in RA. METHODS: We investigated the apoptosis-inducing activities of h-HFE7A on human Fas ligand transfected cells and cultured human activated lymphocytes (human peripheral blood mononuclear cells and isolated human RA synovial lymphocytes), synoviocytes, and chondrocytes. We then examined the effects of h-HFE7A Mab in vivo using SCID-HuRAg mice implanted with human RA tissue. RESULTS: Administration of h-HFE7A Mab alone did not induce apoptosis in cultured human Fas ligand transfected cells and activated lymphocytes. However, apoptosis-inducing activities were noted by this Mab crosslinking with a secondary antibody or Fcgamma receptor positive cells. In contrast, no apoptosis induction by h-HFE7A was observed on cultured synoviocytes and chondrocytes with or without crosslinking. Thus the crosslinking with Fcgamma receptor positive cells is essential for the efficacy of this Mab in vivo. In the implanted tissue of the SCID-HuRAg mice, the number of inflammatory cells was significantly decreased in the h-HFE7A Mab treated group compared to the IgG treated control group. Moreover, there were only negligible effects in synoviocytes and chondrocytes with the h-HFE7A Mab. CONCLUSION: Administration of this novel humanized anti-Fas Mab may provide a new treatment for RA by inducing Fas mediated apoptosis in inflammatory cells.

Matsuno H, Yudoh K, Uzuki M, Nakazawa F, Sawai T, Yamaguchi N, Olsen BR, Kimura T. · Department of Orthopaedic Surgery, Toyama Medical and Pharmaceutical University, Japan. · J Rheumatol. · Pubmed #12022345 No free full text.

Abstract: OBJECTIVE: An endostatin that inhibits angiogenesis dependent tumor growth is being tested as an antitumor agent. The neoangiogenesis condition of cancer is essentially identical to that of rheumatoid arthritis (RA). Thus antiangiogenic treatment has potential for treatment of RA. We investigated the effects of human recombinant endostatin on human RA synovial tissue by use of a novel model of RA, in which human RA tissue is grafted into SCID mice (SCID-HuRAg). METHODS: Ten or 50 mg/kg of human recombinant endostatin was administered by percutaneous direct intrasynovial injection in each of 7 SCID-HuRAg mice. We examined the volume of the grafted tissue mass and the histological changes 7 days after endostatin administration. Six control mice received phosphate buffered saline in the same manner. RESULTS: The grafted synovial volume of SCID-HuRAg mice was significantly decreased by endostatin administration. The number of inflammatory cells (macrophages and lymphocytes) was also significantly reduced in a dose dependent manner. The number of vessels that were counted by von Willebrand factor VIII and type IV collagen positive cells was decreased, although apoptotic cells were increased in RA synovia. CONCLUSION: The results suggest that antiangiogenesis treatment using endostatin represents a potential new therapeutic strategy for RA.

Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M, Sawai T, Yonezawa T, Saeki Y, Panayi GS, Pitzalis C, Kimura T. · Department of Orthopedic Surgery, Toyama Medical and Pharmaceutical University, Toyama, UK. · Rheumatology (Oxford). · Pubmed #11934972 links to  free full text

Abstract: OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.

Itoh T, Uzuki M, Shimamura T, Sawai T. · Department of 1st Pathology, Department of Orthopedic Surgery, School of Medicine, Iwate Medical University, Morioka-city. · Ryumachi. · Pubmed #11925908 No free full text.

Abstract: OBJECTIVE: Matrix metalloproteinase (MMP) is a novel proteolytic enzyme that plays an important role in joint destruction in rheumatoid arthritis (RA). To elucidate the dynamics of MMPs in serum and synovial fluid, we measured the concentration and activity of MMP-1, -9, -13 in serum and synovial fluid of RA patients. Among them especially we focused on newly defined MMP-13 and compared with MMP-1 and MMP-9. METHODS: Serum, synovial fluid and synovial, and pannus tissues used in this study were obtained from RA patients. To compare the dynamics of each enzymic protein, we performed the following procedures: Firstly, we measured concentration of MMP-1, -9, -13 by using ELISA kit. Secondly, the activity of MMP-1, -9, -13 were also measured by using the MMP activity assay system. Then we obtained the activity ratio of each MMP from calculation of activity/concentration. We also examined the expression of MMP-13 in synovial tissues by immunohistochemical and in situ hybridization studies. RESULT: Concentration and activity levels of MMP-1, -9, -13 were significantly higher in RA serum and synovial fluid than in OA. Activity ratio of MMP-1, MMP-9 MMP-13 were 3.60 +/- 1.56, 1.03 +/- 1.75, 35.30 +/- 24.28 (ODA450/ng) in RA serum and 1.60 +/- 2.02, 3.97 +/- 14.83, 14.25 +/- 15.04 (ODA450/ng) in synovial fluid. In synovial and pannus tissues. MMP-13 positive cells were diffusely demonstrated by immunohistochemical and in situ hybridization studies. They were synovial lining cells, endothelial cells, fibroblasts, monocytes, osteoblasts, and chondrocytes. CONCLUSION: MMP-13 positive cells were diffusely presented in joint regions including synovial and pannus tissues. Although the concentration of MMP-13 was not so high, its activity ratio was elevated in serum and synovial fluid in the patients with rheumatoid arthritis.

Nakazawa F, Matsuno H, Yudoh K, Katayama R, Sawai T, Uzuki M, Kimura T. · Department of Orthopedic Surgery, Toyama Medical and Pharmaceutical University, Japan. · J Rheumatol. · Pubmed #11508582 No free full text.

Abstract: OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.

Munakata T, Uzuki M, Shimamura T, Sawai T. · Department of Orthopaedic Surgery, School of Medicine, Iwate Medical University, Morioka-city. · Ryumachi. · Pubmed #11505512 No free full text.

Abstract: OBJECTIVES: IL-18 is a novel cytokine that plays an important role in the Th1 response. The aim of this study is to investigate the dynamics of IL-18 in serum, synovial fluid and synovial membrane in the patients with rheumatoid arthritis. MATERIALS AND METHODS: The serum, synovial fluid and synovial membrane were obtained from RA patients at operation. The levels of IL-18 in the serum and synovial fluid were measured by ELISA. We then examined the expression of IL-18 in synovial tissues using anti-human IL-18 monoclonal antibody in immunohistochemical study. RESULTS: The levels of IL-18 in serum and synovial fluid in RA patients were 193.7 +/- 109.7 pg/ml and 258.8 +/- 238.0 pg/ml, respectively. Compared with OA patients and normal volunteers, the level of IL-18 in RA patients was higher in both serum and synovial fluid. (P < 0.05) In synovial membrane, the cells positive for anti IL-18 antibody were confirmed not only in RA (n = 26) but also in OA (n = 7) patients. The positive cells were the synovial lining cells, macrophages, fibroblasts and endothelial cells. However, a large number of positive cells were demonstrated in synovial tissues in RA compared with OA patients.

Uzuki M, Sasano H, Muramatsu Y, Totsune K, Takahashi K, Oki Y, Iino K, Sawai T. · The First Department of Pathology, Iwate Medical University, School of Medicine, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan. · Clin Sci (Lond). · Pubmed #11352772 No free full text.

Abstract: Urocortin is a newly identified member of the corticotropin-releasing factor (CRF) neuropeptide family, and is known to be involved in the modulation of the inflammatory process. We examined the expression of urocortin, CRF and their receptors (CRF receptor; CRF-R) in the synovial tissue of patients with rheumatoid arthritis (RA) in order to study the possible biological roles of urocortin. Synovial tissues/fluids were obtained from 38 patients with RA, nine patients with osteoarthritis and four with trauma. We studied the concentration of urocortin in the synovial fluid using RIA, and the expression of urocortin in synovial tissue using immunohistochemistry, mRNA in situ hybridization and reverse transcriptase-PCR (RT-PCR). In addition, we examined the immunolocalization of CRF and the expression of CRF-R1, -R2-alpha and -R2-beta mRNAs utilizing RT-PCR in these synovial tissues. Urocortin concentrations in synovial fluid were higher in RA patients (79.8+/-154 pg/ml) than in control patients (12.3+/-4.8 pg/ml; P< or =0.05). Urocortin immunoreactivity and mRNA signals were both detected in synovial cells, lymphocytes, fibroblasts and macrophages. The number of urocortin-positive cells in the synovium was significantly higher in RA (73.1+/-32.1 cells per high-power field) than in control (18.4+/-10.4 cells per high-power field) patients. In addition, both urocortin immunoreactivity and mRNA signals in the synovium reached maximum levels in the active stage of RA inflammation. Moreover, the number of immunoreactive urocortin-positive cells was significantly correlated with the urocortin concentration in synovial fluid (r=0.705; P<0.001) and with histologically defined local inflammatory activity (r=0.641; P<0.001). The distribution and number of immunoreactive CRF-positive cells in synovial tissue were similar to those of urocortin-positive cells (r=0.701; P<0.001). Urocortin, CRF-R1 and CRF-R2-alpha mRNAs detected by RT-PCR were expressed in in the synovium of 10/10, 10/10 and 2/10 RA patients respectively, but CRF-R2-beta was not expressed. Urocortin was actively synthesized in the synovium of RA patients. The present study suggests that urocortin may play an important role as an autocrine and/or paracrine regulator of synovial inflammation in RA.

Hishinuma T, Nakamura H, Sawai T, Uzuki M, Itabash Y, Mizugaki M. · Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan. · Prostaglandins Other Lipid Mediat. · Pubmed #10560620 No free full text.

Abstract: We devised an effective purification for the microdetermination of prostaglandin E2 (PGE2) in human joint fluid using gas chromatography/selected ion monitoring and determined PGE2 in the joint fluid in rheumatoid arthritis (RA) patients using this method. The methyl estermethoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 566.4 for PGE2 and at m/z 570.4 for the internal standard (PGE2-d4). A good linear response over the range of 10 pg to 50 ng was demonstrated. We detected PGE2 to a level of about 46 pg/ml in the joint fluid of RA patients. The level of PGE2 in RA patients was significantly higher than that in osteoarthritis patients used as controls. Moreover, we measured inflammatory cytokine (IL-1beta, TNFalpha, IL-6 receptor) levels in joint fluid by using enzyme-linked immunosorbent assay. A relationships between the PGE2 level in joint fluid and these cytokines or biochemical data as the indicator of RA disease was not observed. We found that the PGE2 level in each patient was influenced by therapeutic drugs. The PGE2 level in RA patients with non-steroidal anti-inflammatory drugs was lower than with steroids.