MOLECULAR MECHANISMS OF MONOCYTE CHEMOTAXIS

单核细胞趋化性的分子机制

• 批准号: 2392766
• 负责人: MARGARET R GYETKO
• 金额: $ 21.07万
• 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/2392766
• 关键词: 3T3 cells; CD antigens; Cryptococcus neoformans; cellular immunity; chemotaxis; complement receptor; cryptococcosis; cytokine; flow cytometry; genetically modified animals; glycosylation; glycosylphosphatidylinositols; 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.

单核巨噬细胞被招募到肺部炎症部位。 这些部位可能与感染性、纤维化性和肉芽肿有关。 肺部疾病。招聘是通过定向迁移完成的 (趋化性)。CD87(尿激酶型纤溶酶原激活物(UPA)受体) 单核巨噬细胞趋化所必需的。的中心假说 这一建议是CD87在单核细胞中所起的关键作用 吞噬细胞的趋化作用依赖于其表达，其定位于 称为小凹的特定质膜微区及其序列 与信号蛋白和其他细胞表面受体的相互作用。这个 这项提案的总体目标是勾勒出 CD87介导单核巨噬细胞趋化作用。我们的具体目标 L)确定a)CD87在小窝内的聚集是否 必需的，以及b)确定小窝，小窝的蛋白质衬里， 参与趋化过程中的信号转导。2)确定 CD87是否通过碳水化合物与补体受体3(CR3)结合 凝集素相互作用影响趋化作用。3)确定是否存在 糖基磷脂酰肌醇特异性磷脂酶D(GPI-PLD)介导 机制对CD87的脱落起重要作用。4)使用转基因技术 UPA缺陷小鼠，确定uPA结合是否重要参与 CD87在体外趋化和细胞募集中的作用 肺炎克雷伯氏菌感染引起的肺部炎症反应 活着。L)CD87在小窝的可视定位和共定位 CD87和小窝蛋白将通过免疫标记和电子技术完成 显微镜和免疫荧光定量共聚焦显微镜。 CD87和小窝的物理结合将通过以下方式进行评估 免疫沉淀和免疫印迹。小窝蛋白介导的信号转导 将通过免疫沉淀和Western blotting与抗 磷酸酪氨酸抗体。2)碳水化合物-凝集素相互作用 CD87和CR3将在现场通过共址研究进行评估 以及缺乏特定的糖类。评估趋化性的研究将是 并行不悖。CR3和CD87的直接相互作用与特异性 CD87的相关结构域将通过转基因的小鼠细胞进行评估 用CR3和可溶性人重组CD87表达。3)PMA和细胞因子诱导 用酶联免疫吸附试验定量检测CD87的脱落及其作用机制 CD87与已知CD87分子量的比较分析 变种。候选机制的特定抑制剂将用于 评估每种受体对激动剂特异性CD87的相对贡献 正在脱落。4)利用uPA缺陷小鼠，我们将确定uPA是否发挥作用 在单核巨噬细胞体外趋化或白细胞趋化中的作用 在体内对肺部新生芽孢杆菌感染的反应。 这些研究将阐明CD87依赖的分子机制。 单核巨噬细胞趋化作用。通过这种知识，具体地 可以开发治疗性干预措施来控制和调节 炎症反应。