The central focus of this proposal is to determine the functional role(s) of IL-6 and its receptors in synoviocytes found in normal and rheumatoid arthritis (RA) joints. Interleukin-6 is the most prominently expressed cytokine in synovial fluids from patients with RA. Recruited inflammatory cells (monocytes, neutrophils and T and B lymphocytes) as well as fibroblasts may contribute to the high levels of IL-6. For cells to respond to IL-6 they must possess a cognant IL-6 receptor subunit (IL- 6R) and its signal transducing subunit (gp130). It is not currently known what cells in the RA lesion possess the IL-6 receptors nor if expression of the receptor is altered in the synoviocytes as part of the etiology of this disorder. Not all cell types express IL-6R however, the signal transducing subunit (gp130) is found in nearly all cell types. Induction of IL-6R during an arthritic crisis could make quiescent cells responsive to the cytokine. Experiments are planned using sensitive ribonuclease protection assays (RPA) and reverse transcriptase polymerase chain reaction (RT-PCR) methodologies to quantitate IL-6R and gp 130 mRNAs in synoviocytes from normal and induced arthritic joints of an animal model. Additionally, the IL-6R mRNA and protein from human synoviocyte cell lines will be measured. Of considerable interest is the finding that soluble forms of the two subunits of the IL-6 receptor complex have been identified both in serum and urine. Unlike soluble forms of receptors from other cytokines, sIL-6R can bind with IL-6 and this complex activate membrane bound gp130. This feature makes the sIL- 6R an agonist, and renders cells IL-6 responsive, which by themselves cannot bind IL-6. Another experimental aim of this project will be determined the concentration of sIL-6R in synovial fluid and from conditioned medium of synoviocytes exposed to factors known to be present in the arthritic synovial fluids. sIL-6R is expressed by being proteolytically cleaved by a yet to be identified cell surface protease. Experiments are described in which this protease will be identified using a genetically constructed form of the IL-6R for a functional assay system. It has recently been reported that when IL-6 and sIL-6R are added to a co-culture of osteoblasts and bone marrow stromal cells a dramatic increase in the number of osteoclast-like cells are formed. Since bone dissolution is a part of the pathology of rheumatoid arthritis an understanding of how these cells are formed from the IL-6 signal will be investigated.
Our specific aims will be to identify the cells responsible for producing sIL-R, determine what factors alter the expression of the receptor, identify the protease that cleaves the receptor from the cell surface and to determine some of the events that lead to osteoclast formation. We believe results from this investigation will provide new information on the cellular and molecular events that take place in the RA lesion.
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