It is likely that both in RA and JRA two macrophage/fibroblast derived cytokines (TNF-alpha and IL-1) are major contributors to the induction of the chronic inflammatory state and to the development of severe tissue destruction. This study will be focused on one of them; TNF-alpha. To demonstrate that increased levels of TNF-alpha expression in synovium may be an important determinant of joint damage in JRA, we propose: 1) to estimate levels of TNF-alpha activity and TNF receptor expression in inflamed synovium, 2) to define cell sources and tissue distribution of TNF secretion as well as its potential cell targets and 3) to determine clinical correlates of TNF-alpha expression. Given the very small volume of synovial fluid obtainable in this pediatric population, semiquantitative PCR methodology was chosen as the best initial approach. In preliminary studies, elevated levels of TNF- alpha transcripts were demonstrated in a majority of JRA patients, and TNF-alpha appeared to be the most abundant cytokine of those tested. Immunoassays will be used to confirm that elevated levels of TNF-alpha transcripts in synovial fluid reflect increased amounts of cytokine protein. Next, a combination of in situ hybridization and immunohistochemistry will be used to detect cells secreting TNF-alpha as well as cells expressing TNF receptors. Identification of cell types expressing TNF receptors and, therefore, capable of responding to TNF- alpha will also identify the main mechanisms through which pathogenic effects of the TNF are carried out in JRA synovium. Further confirmation of major role of TNF-alpha in development of joint destruction could come from the demonstration of positive correlations between levels of TNF-alpha and/or its receptors in synovium and some clinical features of the disease reflecting the degree of joint damage. In the long term, these studies will lead to understanding of the relative contributions of components of the immune system to the disease as well as identifying the optimal targets for therapeutic intervention.
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