The studies proposed will examine three aspects of cytokine interactions in rheumatoid arthritis (RA): 1) the role of cytokine antagonism and synergy in the pathogenesis of RA; 2) the regulation of GM-CSF production in the synovium as well as the role of GM-CSF and its inhibitor(s) in synovial inflammation and in stimulating proliferation of pluripotential cells; and 3) the location and environment of cytokine producing cells in the rheumatoid synovium. For the first project, three indices of synoviocyte activation (collagenase production, proliferation, and HLA-DR expression) will be used to test combinations of cytokines for synergy and antagonism. In preliminary experiments TNF-alpha and IFN-gamma antagonized each other's activating effects on synoviocytes. This and other negative interactions might be important homeostatic control mechanisms in the synovium. The mechanism of mutual antagonism between IFN-gamma and TNF-alpha will be investigated by determining the effect of TNF-alpha and IFN-gamma on surface receptor binding, expression, and internalization and measuring cytokine mediated changes in the rate of HLA-DR gene transcription and mRNA degradation. The second set of experiments will focus on the role of GM-CSF and GM-CSF inhibitors in RA. Ribonuclease protection assays will be used to study GM-CSF mRNA regulation in cultured synoviocytes and other synovial tissue cells while immuno- and bioassays will be used to measure secreted protein. A modified version of a PBM GM-CSF bioassay developed in our laboratory will be used to detect pluripotential cells in the synovium. The role of GM-CSF in synovial hyperplasia will be investigated by measuring the ability of CSFs to expand and differentiate pluripotential cells that reside in the tissue. Finally, in situ hybridization of RA synovial tissue frozen sections will be used to identify and localize cytokine producing cells in the synovium.
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