Rheumatoid arthritis (RA) is a chronic, progressive, destructive joint disease which causes immense morbidity and excess mortality. Histological assessment of RA synovium reveals prominent infiltrates of blood-derived leukocytes; activation of these cells sustains the inflammatory response which leads to joint injury in RA. Leukocyte accumulation in inflammatory foci is a multi-faceted process that begins with cell adhesion to microvascular endothelium. This crucial process results from interactions between endothelial and leukocyte surface molecules, including members of the Selectin gene family. Recent work from our laboratory has identified the ligand for E-Selectin as a carbohydrate, the sialylated form of the Lewis x blood group related molecule (sLex). The underlying hypotheses of this proposal are that: (1) leukocyte accumulation in RA joints is induced by selective expression of Selectins, and (2) inhibition of Selectin-ligand binding will suppress leukocyte accumulation and prevent joint damage. To test these hypotheses, synovium from patients with RA and from two well-characterized animal models of RA (ovalbumin-induced arthritis in the rabbit knee and streptococcal cell wall arthritis in the rat) will be used. An essential prerequisite for the research program will be to generate cloned cDNA's, recombinant proteins, and neutralizing antibodies to Selectins to perform studies in rabbit, rat, and human tissue. The first experimental goal will be to prepare and validate the activity of these reagents, by assessing Selectin expression in in vitro adhesion models using cells derived from each test species. A well-characterized in vitro assay of leukocyte adhesion to endothelium will be used to test the inhibitory activity of the neutralizing antibodies, sLex and other carbohydrate analogs, and the recombinant Selectin molecules. The next experimental goal will be to localize Selectin protein expression in the arthritic joint, by immunohistochemistry, and to assess the correspondence of the sites of specific leukocyte cell-type accumulation with local expression of Selectins and their ligands. The final component of this project will determine whether administration of soluble forms of Selectin ligands, neutralizing antibodies, or recombinant Selectins inhibits the development of arthritis. These studies will delineate the contribution of Selectin molecules to the development of the arthritic lesion, and they may provide the scientific foundation for novel therapeutic interventions for RA, based on these molecular interactions.
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