The Role of CD47 in Mononuclear Leukocyte Recruitment

慢性炎性白细胞(如T细胞亚群和单核细胞)的募集和保留是一种 动脉粥样硬化和慢性炎症性疾病的发生和发展的关键步骤 自身免疫性疾病。在这个修订的申请中，我们提出了一个详细的计划来研究CD47及其 SIRPA和SIRPy，以及凝血酶敏感蛋白-1和-2(TSPs)在分子水平上的单核细胞 利用体外和体内模型进行白细胞募集。在特定目标1中，CD47-SIRP和CD47-SIRP的作用 CD47-TSPs黏附相互作用在单个核白细胞募集中的研究 使用体内和体外方法的策略。这一途径对单核白细胞的贡献 在定义的跨内皮细胞迁移的活细胞成像模型中，将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抗、shRNA敲除和白细胞 以及培养的CD47‘’和TSP-1/2‘“小鼠的内皮细胞。这些分析将得到直接的 使用活体显微镜观察白细胞与微血管壁的相互作用。在……里面 此外，CD47聚集在主动迁移的白细胞周围并触发细胞内的能力 还将讨论白细胞跨内皮细胞迁移所需的信号事件。具体而言 目的研究CD47对白细胞VLA-4和LFA-1整合素功能的调节作用。 CD47与avp3和vla-4整合素相关，已被证明通过“in cis”来调节整合素的功能。 通过异三聚体Gi偶联复合体发出信号的相互作用。基于其他生化证据 研究表明，CD47对整合素配体结合的调节不是通过其信号转导起作用的，而是需要 其胞外Ig结构域与卵子整合素之间的直接物理相互作用。我们假设通过 CD47直接与VLA-4整合素结合，调节整合素在质膜上的扩散，并且 这对整合素的激活有功能上的影响。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整合素在CD47缺失细胞中的扩散是CD47缺失细胞的三倍 在剪切流动条件下，与VCAM-1和ICAM-1的结合显著减少。我们建议 探讨CD47对VLA-4和LFA-1整合素依赖功能的调节机制 通过细胞生物学、生物物理和生化分析检测单个核白细胞。在具体的目标3中，我们将 在免疫调节的实验模型中确定CD47在单核细胞募集中的作用 疾病使用CD47“‘”小鼠。只有有限数量的研究评估了CD47在 体内的慢性免疫或炎症过程。我们的实验方法将是确定 CD47在实验性自身免疫小鼠模型致病T细胞募集中的作用 脑脊髓炎(EAE)。这三个目标的拟议研究将为CD47提供新的见解 慢性炎性白细胞功能和募集的依赖性调节，并可能提供新的 消炎治疗的途径。

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.