| Summary: | Rheumatoid arthritis (RA) is an autoimmune disease characterised by joint inflammation. The resulting joint destruction is mediated by enhanced secretion of degradative enzymes and pro-inflammatory cytokines. Joint inflammation is accompanied by elevated levels of glutamate in the synovial fluid. Work in this thesis investigates the hypothesis that the increased glutamate concentration in RA synovial fluid can induce pathological changes associated with synovial joint destruction. To determine whether cells of the synovial joint can respond to glutamate RNA was purified from various cells and tissues of the rat knee. RT-PCR revealed that metabotropic (mGluR4) and ionotropic glutamate receptors (NMDA NR1, KA1, AMPAGluR2, AMPAGluR3) and glutamate transporters (EAATsl to 3) are expressed in the synovial joint. Differences in expression between RA and normal fibroblast-like synoviocytes (FLS) were observed. The effect of modulating these receptors and transporters in normal and RA FLS was investigated. RA and normal FLS were treated with a range of glutamate concentrations and inhibitors of glutamate transporters and receptors. Markers of inflammation and matrix degradation were measured. Pro-MMP2, TEMPI, TEMP2 and IL-6 levels were modulated with changes in extracellular glutamate. Elevated production of IL-6, pro- MMP2, TEMPI and TIMP2 by RA FLS was observed in the presence of glutamate transporter inhibitors. Inhibition of kainate receptors decreased IL-6 production by RA FLS and inhibition of NMDA receptors increased pro-MMP2 in these cells. Glutamate receptor and transporter inhibition caused different responses in normal FLS. The effect of TNFa and IL-6 on expression of the glutamate transporter EAAT1 was also determined. RT-PCR, immunohistochemistry (IHC) and Western blotting revealed that expression of EAAT1 mRNA and protein was increased in RA FLS in response to IL-6. This was not observed in normal FLS. IHC also demonstrated that TNFa increased EAAT1 protein expression in RA FLS but not in normal FLS. To investigate the function of glutamate receptors, RA FLS were pre-loaded with the calcium indicator fluo-4 and stimulated with glutamate, NMDA or kainate. Upon stimulation, scanning confocal microscopy revealed increases in fluorescence generated by intracellular free calcium indicating functional NMDA and AMPA/kainate receptors in human RA FLS. Using a 14C-glutamate uptake assay it has also been demonstrated that glutamate transporters are functional in RA FLS. This data demonstrates that glutamate receptors and transporters are expressed in the synovial joint in vivo and are functional in human FLS. Furthermore, modulation of glutamate receptors and transporters influences release of IL-6, TIMPs and pro-MMP2 by FLS. Expression and responses differ between RA and normal FLS but the high levels of glutamate found in RA synovial fluid may activate glutamate receptors to induce a pro inflammatory and degradative phenotype in FLS. Tins may be influenced through IL-6 and TNFa induced increases in EAAT1 expression, indicating a possible feedback mechanism. It is proposed that the kainate receptor pathway that increases IL-6 release in response to glutamate warrants further investigation as a therapeutic target for RA.
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