Destruction of articular cartilage is a common feature of the final pathway in arthritides such as rheumatoid arthritis (RA) and osteoarthritis (OA). This process is thought to be controlled largely by cytokines released from the synovium or the cartilage matrix itself under the influence of various noxious stimuli. Interleukin-1(IL-1) produced by joint tissues is a major inducer of matrix metalloprotease (MMP) gene expression in chondrocytes and synoviocytes. The binding of IL-1 to its receptor (IL-IR) is an obligatory step resulting in elicitation of signal transduction and to MMP up-regulation. This developmental and feasibility project will test the hypothesis that free forms of interleukin-1 receptor (IL-IR) obtained from chondrocytes or a receptor fragment obtained by enzymatic cleavage may be useful as reagents to antagonize IL-1 binding to IL-IR. Two approaches to produce receptor will be used in these studies: 1.) chondrocytes will be grown in culture, membranes solubilized with detergent, and the soluble receptor (dsIL-IR) purified by affinity chromatography on immobilized antibodies to human T-lymphocytic cell-line IL-IR (type 1). Further purification will be achieved by chromatography on immobilized conconavalin-A. Purified dsil-IR will be identified by SDS- polyacrylamide gel electrophoresis followed by transfer to membranes and probing with 125I-labeled IL-1beta. 2) A second approach will aim to produce a recombinant IL-IR) by expression cloning of the extracellular portion of chondrocyte IL-IR. Proteolytic fragments of receptor (IL- IRFs) will be generated by enzymatic digestion with various proteolytic enzymes and purified by High Liquid Chromatography (HPLC). IL-IR and IL- IRFs will be tested in vitro for ability to block IL-1 binding to its receptor. Rabbit-derived IL-IR and fragments thereof will be tested in the rabbit partial meniscectomy OA model to assess the efficacy of the preparations in vivo.
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