A key event in the pathogenesis of rheumatoid arthritis (RA) is the recruitment or homing of activated T cells and monocytes to synovium. Recruitment of activated T cells involves a complex series of events including T cell rolling, chemokine receptor activation, increased T cell adhesiveness, and eventually transendothelial migration up a chemokine gradient. A central regulatory step wherein the decision is made for a rolling leukocyte to leave the circulation and enter a site of inflammation is at the stage of all adhesion or chemokine-mediated increase in cell adhesiveness, and one of the hypotheses of this proposal is that signal transduction through chemokine receptors on activated T cells may also function to regulate the adhesiveness and molecular interactions of the CD44 adhesion molecule. CD44 binds the chemokine MIP-1beta and can potentially create a gradient to guide leukocytes into the synovium. We also hypothesize that the binding and presentation of chemokines by CD44 may be regulated by conformational changes in CD44. An emerging concept is that chemokines and their receptors can target lymphoid cells to specific anatomical sites. Little is currently known about which chemokines or chemokine receptors regulate the influx of T cells into the synovium. The overall goal of this project is to better define the molecular mechanisms by which chemokine regulate the influx of T lymphocytes into the RA joint. We will accomplish these objectives by first defining the chemokine milieu of the inflamed synovium in RA and identifying the chemokine receptors that are functional on synovium-infiltrating lymphocytes using RNAse protection assays, RT-PCR, chemokine ELISA, and measuring Ca++ flux and competitive ligand binding in chemokine receptor- transfected RBL-2H3 cells. We will study in detail the mechanisms by which chemokines may regulate cell adhesiveness through CD44 and its variant isoforms including studies to test CD44 phosphorylation, cytoskeletal association and function in binding to hyaluronan. We will characterize the molecular interactions between MIP-1beta and CD44 using a variety of CD44 fusion proteins and MIP-1beta mutants, and determine whether structural changes in CD44 alters MIP-1beta binding and presentation.
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