The general goal of this project is to study the cellular and molecular determinants of leukocyte interactions with components of the rheumatoid synovial microenvironment. In the past four years, we have shown that the transmembrane hyaluronate receptor (CD44) is a central, multifunctional proinflammatory molecule in RA, and is the same as the Hermes class of lymphocyte homing receptors that mediates mononuclear cell adhesion to synovial endothelial venules. Recent data have shown that CD44 represents a family of molecules with isoforms. In this project, we propose to define to define the proinflammatory functions of each of the isoforms of the CD44 with the goal of devising novel strategies of inhibition of CD44 proinflammatory function as potential new therapies for RA. Individual specific aims in this project are: 10 We will develop oligonucleotide probes that are specific for known isoforms of CD44. We will develop mabs that specifically react with the human CD44R1 (CD44E) form of CD44, and profile the expression of CD44 isoforms in normal and RA PB T cells, PMNs and monocytes, and in normal, OA and RA synovial T cells, macrophages and fibroblasts. 2) We will characterize the size of CD44 isoforms on purified purified synovial macrophages, T cells and fibroblasts, and compare with T cells, B cells, monocytes and PMNs and various cell lines. 3) We will We will study the origin of soluble CD44 in serum and synovial fluid. 4) recombinant (r) CD44-immunoglobulin (Ig) fusion molecules (rCD44Rg-2), 2), rCD44, rCD44R1 (CD44E), affinity purified transmembrane CD44, and purified soluble CD44S from synovial fluid and serum, we will determine the determine the immunoregulatory effects of various CD44 isoforms on in vitro monocyte function. 5) Using purified proteins, we will determine the the ability of regions of CD44 to bind to cytokines (IL3, GM-CSF, TGF-beta, FGF) and to bind to components of the extracellular matrix. Using cytoskeletal components, we will determine the ability of various forms of purified CD44 to associate with the cytoskeleton, and, using deletion mutants of CD44 cytoplasmic domain, define functional sequelae of CD44- cytoskeletal interactions. 6) To map functional domains of CD44, we will prepare a series of deletion mutants of CD44 extracellular or cytoplasmic regions. 7) Using a novel system of synovial tissue grown in vivo in severe combined immune deficient (SCID) mice, we will study the ability of autologous and allogeneic T cells to migrate to human synovial xenografts. Taken together, these studies should provide critical new knowledge necessary for the design of new therapies to interrupt multiple stages of inflammation in RA.
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