Recruitment and retention of chronic inflammatory leukocytes (e.g.,T cell subsets and monocytes) is a pivotal step in initiation and progression of chronic inflammatory diseases including atherosclerosis and autoimmune diseases. In this revised application, we propose a detailed plan to study the role of CD47 and its ligands, SIRPa and SIRPy, and thrombospondin-1 and -2 (TSPs), at a molecular level in mononuclear leukocyte recruitment using in vitro and in vivo models.
In SPECIFIC AIM 1, the role ofthe CD47 - SIRP and CD47 - TSPs adhesive interactions in recruitment of mononuclear leukocytes will be studied by multiple strategies using in vivo and in vitro approaches. The contributions of this pathway to mononuclear leukocyte transendothelial cell migration will be dissected in a live-cell imaging model of transmigration under defined shear flow conditions in vitro. We will make use of function blocking mAbs, shRNA knockdown, and leukocytes and cultured endothelial cells from CD47'''and TSP-1/2''"""""""" mice. These analyses will be corroborated by direct visualization of leukocyte interactions with the microvessel wall using an intravital microscopy approach. In addition, the ability of CD47 to cluster around actively transmigrating leukocytes and to trigger intracellular signaling events required for leukocyte transendothelial cell migration will also be addressed.
In Specific Aim 2, studies will investigate CD47 mediated regulation of leukocyte VLA-4 and LFA-1 integrin function. CD47 associates with avp3 and VLA-4 integrins and has been shown to regulate integrin function by """"""""in cis"""""""" interactions that signal through a heterotrimeric Gi-coupled complex. Based on biochemical evidence other studies suggest CD47 regulation of integrin ligand binding does not work via its signaling and instead requires direct physical interaction between its extracellular Ig domain and Ov integrins. We hypothesize that through direct association with VLA-4 integrins, CD47 regulates integrin diffusivity in the plasma membrane, and that this has functional consequences for integrin activation. In support of our hypothesis, our preliminary data working in T cells indicate VLA-4 integrin diffused three-fold greater in CD47 null cells and that CD47 null cells exhibit a marked reduction in binding to VCAM-1 and ICAM-1 under shear flow conditions. We propose to investigate the mechanism of CD47 regulation of VLA-4 and LFA-1 integrin dependent functions in mononuclear leukocytes by cell biological, biophysical and biochemical assays.
In SPECIFIC AIM 3, we will determine the role of CD47 in mononuclear leukocyte recruitment in an experimental model of immunemediated disease using CD47""""""""'"""""""" mice. Only a limited number of studies have evaluated the role of CD47 in chronic immune or inflammatory processes in vivo. Our experimental approach will be to determine the role of CD47 in the recruitment of pathogenic T cells in a murine model of experimental autoimmune encephalomyelitis (EAE). The proposed studies in these three Aims will provide new insights for CD47 dependent regulation of chronic inflammatory leukocyte function and recruitment and may provide new avenues for anti-inflammatory therapies.
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