73 Ionotropic glutamate receptors are functional in human fibroblast-like synoviocytes and modulate IL-6 and MMP-2 expression

Abstract

Rheumatoid arthritis (RA) affects 350,000 people in the UK. Glutamate levels increase 54 fold in the synovial fluid of RA patients and correlate with RANTES, IL-8 and MIPa levels. We have detected mRNA expression of various glutamate receptor subunits (NMDAR1, KA1, AMPAGluR2, AMPA GluR3, mGluR4) in tissues of the rat knee. All receptor subunits mRNAs were expressed in rat patella. The fibrocartilagenous meniscus and articular cartilage chondrocytes expressed mGluR4 and both AMPA receptor subunits. Human synoviocytes express NMDAR1 and KA1 mRNA. To investigate receptor function, synoviocytes were preloaded with a fluorescent indicator of intracellular free calcium (iCa), stimulated with glutamate, NMDA or kainate (500 AM), and fluorescence recorded by real time confocal microscopy. Glutamate stimulated release of iCa in 25% of synoviocytes whereas NMDA and Kainate each stimulated 15% of cells. NMDA responses increased to 57% in the absence of Mg. To determine whether glutamatergic signalling influences human synoviocyte inflammatory/degradative responses, we cultured synoviocytes at various external glutamate concentrations (50 AM–2 mM) in the presence of glutamate receptor antagonists and measured release of IL-6 and MMP2 and 9. In 2 of 4 RA patients tested, glutamate stimulation increased synoviocyte pro MMP-2 release. This was not inhibited by ionotropic glutamate receptor antagonists (MK801 100 AM, DAP5 10 AM, CFM2 10 AM, NBQX 150 AM). TIMP1 and TIMP2 release were not affected by glutamate stimulation or co-treatment with receptor antagonists. IL-6 expression varied greatly in human synoviocytes derived from different RA patients (0–120 pg/ml media). Consistent with this, the effect of glutamate stimulation on IL-6 release varied. However, the AMPA/KA receptor antagonist NBQX significantly reduced IL-6 release at all glutamate concentrations. Since this inhibition was much greater than that by CFM2, we propose that activation of kainate receptors in human synoviocytes may lead to IL-6 release. Since, KA receptors are functional in human synoviocytes and regulate release of IL-6, the role of glutamatergic signalling in RA pathology warrants further investigation.