MOLECULAR MECHANISMS OF MONOCYTE CHEMOTAXIS

单核细胞趋化性的分子机制

• 批准号: 2232518
• 负责人: MARGARET R GYETKO
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2232518
• 关键词: 3T3 cells; CD antigens; Cryptococcus neoformans; cellular immunity; chemotaxis; complement receptor; cryptococcosis; cytokine; flow cytometry; genetically modified animals; glycosylation; immunoelectron microscopy; inflammation; laboratory mouse; lectin; leukocyte adhesion molecules; mannose; microorganism immunology; monocyte; phospholipase D; plasminogen activator; thin layer chromatography; transfection

Mononuclear phagocytes are recruited to sites of pulmonary inflammation. These sites can be associated with infectious, fibrosing and granulomatous lung diseases. Recruitment is accomplished by directed migration (chemotaxis). CD87 (the urokinase plasminogen activator (uPA) receptor) is required for mononuclear phagocyte chemotaxis. The central hypothesis of this proposal is that the essential role CD87 plays in mononuclear phagocyte chemotaxis is dependent upon its expression, its localization to specific plasma membrane micro-domains called caveolae, and its sequential interaction with signaling proteins and other cell surface receptors. The overall objective of this proposal is to delineate the mechanism by which CD87 mediates mononuclear phagocytes chemotaxis. Our specific objectives are: l) Determine a) whether aggregation of CD87 within caveolae is required, and b) determine whether caveolin, the protein lining caveolae, participates in signal transduction in during chemotaxis. 2) Determine whether CD87 associates with complement receptor 3 (CR3) via carbohydrate- lectin interactions to effect chemotaxis. 3) Determine if a glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) mediated mechanism contributes importantly to CD87 shedding. 4) Using transgenic uPA deficient mice, determine if binding of uPA participates importantly in CD87 function during chemotaxis in vitro, and cellular recruitment in response to pulmonary inflammation induced by C. neoformans infection in vivo. l) Visual localization of CD87 to caveolae and colocalization of CD87 and caveolin will be accomplished by immunolabeling and electron microscopy and immunofluorescent quantitative confocal microscopy. Physical binding of CD87 and caveolin will be assessed by immunoprecipitation and Western blotting. Signal transduction via caveolin will be assessed by immunoprecipitation and Western blotting with anti- phosphorotyrosine antibodies. 2) Carbohydrate-lectin interactions between CD87 and CR3 will be assessed by colocalization studies in the presence and absence of specific saccharides. Studies evaluating chemotaxis will be done in parallel. Direct interaction between CR3 and CD87 and the specific relevant domains of CD87 will be assessed using murine cells transfected with CR3 and soluble human recombinant CD87. 3) PMA-and cytokine-induced CD87 shedding will quantitated by ELISA and the mechanism determined by analysis of the molecular weight of shed CD87 compared with known CD87 variants. Specific inhibitors of candidate mechanisms will be used to assess the relative contribution of each to agonist-specific CD87 shedding. 4) Using uPA deficient mice, we will determine whether uPA plays a role in mononuclear phagocytes chemotaxis in vitro, or in leukocyte recruitment in response to pulmonary C. neoformans infection in vivo. These studies will elucidate the molecular mechanism of CD87 dependent mononuclear phagocyte chemotaxis. Through this knowledge, specific therapeutic interventions may be developed to control and modulate inflammatory responses.

单核细胞吞噬细胞被募集到肺部炎症部位。 这些部位可伴有感染性、纤维化和肉芽肿性 肺部疾病招聘是通过定向迁移完成的 （趋化性）。CD 87（尿激酶纤溶酶原激活物（uPA）受体）是 单核吞噬细胞趋化性所必需的。的中心假设 这一建议是，CD 87在单核细胞中起着重要作用， 吞噬细胞的趋化性依赖于其表达、其定位， 特定的质膜微域称为小窝，其顺序 与信号蛋白和其他细胞表面受体的相互作用。的 本建议的总体目标是界定一种机制， CD 87介导单核吞噬细胞趋化性。我们的具体目标 1）确定a）小窝内CD 87的聚集是否是 B）确定小窝蛋白，即小窝衬里蛋白， 参与趋化过程中的信号转导。2)确定 CD 87是否通过碳水化合物与补体受体3（CR 3）结合， 凝集素相互作用以影响趋化性。3)确定是否 糖基磷脂酰肌醇特异性磷脂酶D（GPI-PLD）介导 这一机制对CD 87脱落有重要作用。4)利用转基因 uPA缺陷小鼠，确定uPA的结合是否重要地参与 在体外趋化过程中CD 87的功能， 对C.新生儿感染 vivo. l）CD 87对小窝的视觉定位和CD 87的共定位。 CD 87和小窝蛋白将通过免疫标记和电子显微镜来实现。 显微镜和免疫荧光定量共聚焦显微镜。 将通过以下方法评估CD 87和小窝蛋白的物理结合： 免疫沉淀和Western印迹。小窝蛋白介导的信号转导 将通过免疫沉淀和免疫印迹法进行评估， 磷酸酪氨酸抗体。2)糖-凝集素相互作用 将通过共定位研究评估CD 87和CR 3， 和缺乏特异性抗体。评估趋化性的研究将是 并行进行。CR 3和CD 87之间的直接相互作用和特异性的 将使用转染的鼠细胞评估CD 87的相关结构域 CR 3和可溶性人重组CD 87。3)PMA和苦参碱诱导 通过ELISA定量CD 87脱落，并通过ELISA确定机制。 脱落CD 87与已知CD 87相比的分子量分析 变体。候选机制的特异性抑制剂将用于 评估每种对激动剂特异性CD 87的相对贡献 脱落4)使用uPA缺陷的小鼠，我们将确定uPA是否发挥作用， 在单核吞噬细胞体外趋化性中的作用，或在白细胞中的作用 肺循环C反应的募集。体内新生菌感染。 这些研究将有助于阐明CD 87依赖性细胞凋亡的分子机制。 单核吞噬细胞趋化性 通过这些知识，具体 可以开发治疗干预以控制和调节 炎症反应。