The Role of CD47 in Mononuclear Leukocyte Recruitment

慢性炎性白细胞的募集和保留（例如，T细胞亚群和单核细胞）是一种 慢性炎性疾病包括动脉粥样硬化的起始和进展的关键步骤， 自身免疫性疾病在这个修改后的申请中，我们提出了一个详细的计划，研究CD 47的作用及其在细胞中的作用。 SIRP α和SIRP γ配体以及血小板反应蛋白-1和-2（TSP）在单核细胞中的分子水平 使用体外和体内模型进行白细胞募集。在特异性AIM 1中，CD 47- SIRP和 将通过多项研究来研究单核白细胞募集中的CD 47-TSP粘附相互作用。 使用体内和体外方法的策略。该通路对单核白细胞的作用 将在定义的条件下，在迁移的活细胞成像模型中解剖跨内皮细胞迁移。 体外剪切流动条件。我们将利用功能阻断单克隆抗体，shRNA敲低，白细胞 以及来自CD 47和TSP-1/2小鼠的培养的内皮细胞。这些分析将得到直接的证实。 使用活体显微镜方法可视化白细胞与微血管壁的相互作用。在 此外，CD 47聚集在主动迁移的白细胞周围并触发细胞内 还将讨论白细胞跨内皮细胞迁移所需的信号传导事件。在特定 目的2：研究CD 47对白细胞VLA-4和LFA-1整合素功能的调节作用。 CD 47与α ν β 3和VLA-4整联蛋白相关，并已显示通过“顺式”调节整联蛋白功能。 通过异源三聚体GI偶联复合物进行信号传导的相互作用。根据生化证据其他 研究表明，整合素配体结合的CD 47调节不通过其信号传导起作用，而是需要 其细胞外IG结构域和Ov整联蛋白之间的直接物理相互作用。我们假设通过 CD 47与VLA-4整联蛋白直接结合，调节整联蛋白在质膜中的扩散性， 这对整联蛋白活化具有功能性影响。为了支持我们的假设，我们的初步数据 在T细胞中的工作表明，VLA-4整联蛋白在CD 47缺失细胞中的扩散比在CD 47缺失细胞中大三倍， 在剪切流条件下与VCAM-1和ICAM-1的结合显著降低。我们建议 研究CD 47调节VLA-4和LFA-1整合素依赖性功能的机制， 通过细胞生物学、生物物理学和生物化学测定法测定单核白细胞。具体目标3： 确定CD 47在免疫介导的实验模型中单核白细胞募集中的作用， 使用CD 47+小鼠的疾病。只有有限数量的研究评估了CD 47在 体内慢性免疫或炎症过程。我们的实验方法将是确定 CD 47在实验性自身免疫性小鼠模型中致病性T细胞募集中的作用 脑脊髓炎（EAE）。这三个目标中的拟议研究将为CD 47提供新的见解 慢性炎症性白细胞功能和募集的依赖性调节，并可能提供新的 抗炎治疗的途径。

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.