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OP0035 The production of cxcr3 agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis

Background IFN-g-producing Th1 or Th0 cells predominate in the synovium of rheumatoid arthritis (RA). It is now known that the accumulation of Th1 cells is mediated by Th1 cell expression of the chemokine receptors CCR5 and CXCR3 and the local expression of their agonistic chemokines. Accordingly, T cell infiltrates in RA synoivum have been shown to mostly express CCR5 and CXCR3. Objectives Since synovial fibroblasts are known as an active participant in joint destruction and chronic inflammation, we investigated the expression of CXCR3 agonists such as IP-10, Mig, and I-TAC in synovial fibroblasts, as well as synovial tissues from RA patients. In addition, the importance of these fibroblast-derived chemokines in the chemotactic response of RA blood CD4+ T cells was determined. Methods Synovial fibroblast cell lines, prepared from RA synovial tissues, were incubated with or without IFN-g, IL-1, TNF-a, IL-17, or various combinations. Levels of protein and mRNA for IP-10, Mig, and I-TAC were measured by ELISA and RT-PCR, respectively. The distribution of chemokine-producing cells in RA synovial tissue was determined by immunohitochemistry. Fibroblasts were cultured overnight on chamber slides and their chemokine synthesis was detected by intracellular staining and a confocal laser scanning microscopy. The chemotactic response of RA blood CD4+ T cells to fibroblast culture supernatants with or without antibodies against IP-10, Mig, and I-TAC was determined by an in vitro chemotaxis microchamber technique. Results Concentrations of CXCR3 agonistic chemokines in RA synovial fluid were much higher than those in RA peripheral blood or osteoarthritis (OA) synovial fluid. Immunohistochemical analysis of RA synovial tissue showed that these chemokines were expressed mostly on large cells and vascular endothelial cells in the sublining layer, as well as in the lining layer. The staining was prominent around the lymphocyte aggregate. Synovial tissues form RA more strongly expressed mRNA for all three CXCR3 agonists than OA tissues, and spontaneously released large amounts of their proteins. Synovial fibroblast cell lines isolated from RA, when stimulated with IFN-g, were able to produce these chemokines at the mRNA and protein levels, and this chemokine production was significantly augmented by IL-1, TNF-a, and IL-17. Culture supernatants of activated fibroblasts contained the chemotactic activity for blood CD4+ T cells from RA, and this activity was partially but significantly inhibited by antibodies against these CXCR3 agonists. Conclusion These results indicate that synovial fibroblasts may contribute to the continued Th1 response in RA in part by secreting CXCR3 agonists. In addition, inflammatory cytokines derived from Th1 cells and macrophages, IFN-g, IL-17, IL-1, and TNF-a, are important inducers of these chemokines in the joint.