(Taken from the application): Previous studies from the Principal Investigator's laboratory support the hypothesis that IgM-RF- bearing immune complexes are involved in initiating or perpetuating the chronic inflammatory state in juvenile rheumatoid arthritis (JRA). In addition to activating complement, with the formation of phlogistic C3a and C5a molecules, immune complexes isolated from JRA synovial fluid have been shown to induce the secretion of interleukin 1beta (IL-1beta) from monocytic U937 cells. Furthermore, model BSA-anti-BSA immune complexes induce other cytokines in addition to IL-1B, from human peripheral blood mononuclear cells (PBMC's), including two believed to be involved in the pathophysiology of rheumatoid disease: GM-CSF and IL-8. Preliminary data from the P.I.'s laboratory also suggest that the ability of immune complexes to induce leukocyte cytokine expression may be dependent upon their size and composition. This proposal will test that hypothesis and examine in further detail the cellular mechanisms responsible for the induction of cytokines by JRA serum and synovial fluid immune complexes. In the first set of experiments, immune complexes purified from JRA serum and synovial fluids will be characterized in detail (Specific Aim 1). Specifically, Western blotting to identify immunoglobulin and complement content, quantitative ELISA assays for bound complement components, and qualitative ELISA assays for the presence of IgA, IgM, and IgG-rheumatoid factors (RF's) within the complexes will be undertaken. The effects of IgM-RF on complement binding to the immune complexes will also be examined. In the next set of experiments (Specific Aim 2), JRA immune complexes will be incubated with PBMC's to determine whether these complexes or some subset of them can induce 4 cytokines believed to be critical in the pathophysiology of synovial inflammation: IL-1 beta, IL-6, IL-8, and GM-CSF. Cellular/molecular mechanisms involved in cytokine expression in these experiments will also be examined. Finally, the roles of immune complex size and composition in determining their pathologic potential will be examined in an in vitro model (Specific Aim 3). In these experiments, model immune complexes of specific size and composition will be constructed in the laboratory. The capacity of these complexes to induce IL-1 beta from monocytic U937 cells and the cellular signalling mechanisms associated with that expression (specifically, tyrosine kinase activation and intracellular calcium release), will then be examined in detail.
