THU0094 Role of sphingosine-1-phosphate receptor 3 signallingin collagen-induced arthritis

Background Sphingosine-1-phosphate (S1P) is a biologically active phospholipid, which is derived from membrane lipid. It binds to the receptors, named S1P1–5, and regulates several signalling pathways involved in inflammation, cell survival, angiogenesis and cell migration. Concentration of S1P and expression of S1P receptors can vary according to local tissue conditions. RA is a chronic inflammatory disorder of joints and the concentration of S1P in synovial fluid is higher in RA patient than in OA patient. In vitro, S1P3 expression in RA synoviocyte is upregulated by TNFa treatment. On the other hand, it is not clarified whether S1P/S1P3 signalling pathway contributes to arthritis in RA. Objectives The objective of this study is to investigate the role of S1P/S1P3 signalling in inflammatory arthritis. Methods Collagen-induced arthritis (CIA) was induced by subcutaneous injection of bovine type II collagen emulsified in complete Freund’s adjuvant in wild-type (WT) or S1P3-knock-out (S1P3-KO) 7–9 week-old DBA/1J mice. Arthritis severity were evaluated by visual scoring and histological analysis. The severity was assessed over time by using the arthritis score, in which each paw was scored on a scale of 0–4 and the scores of all four paws were cumulated, resulting in a maximum possible score of 16 per mouse. For histopathological examination, mice were sacrificed on the 42nd day and the hindlimbs were removed and fixed in 4% buffered formaldehyde. Paraffin embedded sections of the knee joints stained with hematoxylin and eosin were systematically scanned in a microscope and scored based on cell infiltration, cartilage destruction and bone erosion parameters. S1P3 mRNA expression was examined by realtime PCR method with total RNA extracted from knee joint capsules of CIA or normal WT mice. Murine primary fibroblast like synoviocytes (FLS) were obtained from CIA mice. We examined S1P3 expression after TNFa treatment and measured cytokine produced after S1P treatment with or without TNFa pretreatment in FLS. Results S1P3 deficiency resulted in modest symptoms of arthritis and a significant reduction in synovial inflammation and bone erosions in histological analysis. S1P3 mRNA expression in knee joint capsule in CIA mice was about five times as high as that in normal mice. TNFa treatment upregulated S1P3 expression and S1P treatment enhanced IL-6 production in WT-FLS significantly. TNFa-priming enhanced S1P-induced IL-6 production, which is significantly higher in WT-FLS than in KO-FLS. This effect was not observed in MCP-1 production of WT-FLS. Conclusions S1P3-KO reduced severity of arthritis, inflammation and bone erosions in CIA. S1P3 mRNA was upregulated in inflamed joint capsule. S1P induces IL-6 production via S1P3 upregulation by TNFa in CIA-FLS. S1P3 inhibition could be a good target of the therapy for arthritis. References [1] Blaho VA and Hla T. An update on the biology of sphingosine 1-phosphate receptors. J Lipid Res55:1596–608. [2] Zhao C, Fernandes MJ, and Bourgoin SG, et al. Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-alpha. J Lipid Res49: 2323–37. Disclosure of Interest None declared