The Role of CD47 in Mononuclear Leukocyte Recruitment

CD47 在单核白细胞募集中的作用

• 批准号: 7753041
• 负责人: FRANCIS W. LUSCINSKAS
• 金额: $ 61.36万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7753041
• 关键词: Actins; Address; Adhesions; Adhesives; Affinity; Agonist; Air; Anti-Inflammatory Agents; Anti-inflammatory; Apical; Atherosclerosis; Autoimmune Diseases; Avidity; Binding; Biochemical; Biological; Biological Assay; CD47 Antigen; CD47 gene; Cell Adhesion; Cell membrane; Cells; Chronic; Clinical; Complex; Cytoskeleton; Data; Diffuse; Disease; Encephalomyelitis; Endothelial Cells; Endothelium; Event; Exhibits; Experimental Autoimmune Encephalomyelitis; Experimental Models; Functional disorder; Genetic; Imagery; Immune; Immune System Diseases; In Vitro; Inflammation; Inflammatory; Integrin alpha4beta1; Integrins; Intercellular Junctions; Intercellular adhesion molecule 1; Knockout Mice; Lateral; Leukocytes; Life; Ligand Binding; Ligands; Mediating; Microcirculation; Microscopy; Modeling; Molecular; Mononuclear Leukocytes; Mus; Names; Null Lymphocytes; PTPNS1 gene; Pathogenesis; Pathway interactions; Pertussis Toxin; Physiological; Play; Principal Investigator; Process; Proteins; Regulation; Role; SHPS-1 protein; Signal Transduction; Skin; Small Interfering RNA; Symptoms; T-Lymphocyte; T-Lymphocyte Subsets; Thrombospondin 1; Thrombospondins; Tyrosine Phosphorylation; Vascular Cell Adhesion Molecule-1; Work; base; cadherin 5; cell motility; cell type; cellular imaging; extracellular; in vivo; in vivo Model; insight; intravital microscopy; migration; monocyte; particle; programs; small hairpin RNA; thrombospondin 2; Actins; Address; Adhesions; Adhesives; Affinity; Agonist; Air; Anti-Inflammatory Agents; Anti-inflammatory; Apical; Atherosclerosis; Autoimmune Diseases; Avidity; Binding; Biochemical; Biological; Biological Assay; CD47 Antigen; CD47 gene; Cell Adhesion; Cell membrane; Cells; Chronic; Clinical; Complex; Cytoskeleton; Data; Diffuse; Disease; Encephalomyelitis; Endothelial Cells; Endothelium; Event; Exhibits; Experimental Autoimmune Encephalomyelitis; Experimental Models; Functional disorder; Genetic; Imagery; Immune; Immune System Diseases; In Vitro; Inflammation; Inflammatory; Integrin alpha4beta1; Integrins; Intercellular Junctions; Intercellular adhesion molecule 1; Knockout Mice; Lateral; Leukocytes; Life; Ligand Binding; Ligands; Mediating; Microcirculation; Microscopy; Modeling; Molecular; Mononuclear Leukocytes; Mus; Names; Null Lymphocytes; PTPNS1 gene; Pathogenesis; Pathway interactions; Pertussis Toxin; Physiological; Play; Principal Investigator; Process; Proteins; Regulation; Role; SHPS-1 protein; Signal Transduction; Skin; Small Interfering RNA; Symptoms; T-Lymphocyte; T-Lymphocyte Subsets; Thrombospondin 1; Thrombospondins; Tyrosine Phosphorylation; Vascular Cell Adhesion Molecule-1; Work; base; cadherin 5; cell motility; cell type; cellular imaging; extracellular; in vivo; in vivo Model; insight; intravital microscopy; migration; monocyte; particle; programs; small hairpin RNA; thrombospondin 2

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.

慢性炎性白细胞的募集和保留（例如，T细胞子集和单核细胞）是 慢性炎症性疾病（包括动脉粥样硬化和 自身免疫性疾病。在此修订的应用中，我们提出了一个详细的计划，以研究CD47及其的作用 配体，sirpa和sirpy，以及在单核中的分子水平 使用体外和体内模型的白细胞募集。在特定目标1中，CD47 -SIRP和 CD47-在募集单核白细胞中的TSP粘合剂相互作用将由多个研究 使用体内和体外方法的策略。该途径对单核白细胞的贡献 跨内皮细胞迁移将在定义的迁移的活细胞成像模型中阐述 体外剪切流条件。我们将利用功能阻塞mAb，shRNA敲低和白细胞 来自CD47'''和TSP-1/2'“小鼠的培养的内皮细胞。这些分析将通过直接来证实 使用插入显微镜方法与微血管壁的可视化白细胞相互作用。在 此外，CD47围绕主动迁移白细胞聚集并触发细胞内的能力 还将解决白细胞跨内皮细胞迁移所需的信号事件。具体 AIM 2，研究将研究CD47介导的白细胞VLA-4和LFA-1整联蛋白功能的调节。 CD47与AVP3和VLA-4整联蛋白相关，并已证明可以通过“ CIS”调节整联蛋白功能 通过异三聚体GI耦合复合物发出信号的相互作用。基于生化证据其他 研究表明，CD47整联蛋白配体结合的调节不起作用，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。我们通过 与VLA-4整联蛋白的直接关联，CD47调节质膜中整合素的扩散率，并且 这对整合素激活具有功能后果。为了支持我们的假设，我们的初步数据 在T细胞中工作表明VLA-4整合素在CD47无效细胞中扩散了三倍，并且CD47无效细胞 在剪切流条件下，与VCAM-1和ICAM-1的结合显示出明显的降低。我们建议 研究VLA-4和LFA-1整联蛋白依赖性功能的CD47调节的机理 细胞生物学，生物物理和生化测定的单核白细胞。在特定的目标3中，我们将 确定CD47在单核白细胞募集中的作用 使用CD47“”小鼠疾病。只有有限的研究评估了CD47在 体内慢性免疫或炎症过程。我们的实验方法是确定 CD47在实验自身免疫的鼠模型中募集致病性T细胞的CD47 脑脊髓炎（EAE）。这三个目标中提出的研究将为CD47提供新的见解 慢性炎症白细胞功能和招募的依赖调节，并可能提供新的 抗炎疗法的途径。