ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

CD45 在吞噬细胞运动和信号转导中的作用

• 批准号: 5200846
• 负责人: L HARVATH
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200846
• 关键词: CD antigens; antibody receptor; biological signal transduction; calcium flux; cell motility; chemoattractants; crosslink; flow cytometry; fluorescent dye /probe; human tissue; immunoprecipitation; monoclonal antibody; neutrophil; phagocytosis; phosphorylation

The leukocyte common antigen (CD45) family is a group of high molecular weight glycoproteins that are expressed on the plasma membranes of all leukocytes. We have previously shown that epitopes of CD45 are involved in neutrophil chemotaxis to the chemoattractants, leukotriene B4 and recombinant human C5a (rHuC5a). The purpose of this project is to better understand the role of CD45 in phagocyte signal transduction during exposure to chemoattractant or phagocytic stimuli. The physical proximity of FcgammaRII (CD32), the low affinity receptor for IgG, and CD45 on the surface of human neutrophils (PMN) was investigated with fluorescence resonance energy transfer (FRET) and co-immuno- precipitation. CD45 epitopes were labeled with mAb conjugated to a donor fluorochrome, FlTC, and CD32 epitopes were labeled with mAb conjugated to an acceptor fluorochrome, tetramethylrhodamine (Rh). FRET between PMN-bound FITC-CD45 mAb and Rh-CD32 mAb was quantified by FACS analysis. The FRET efficiency was 16-40% at 4 degrees C and 14-45% at 37 degrees C. Physical association of CD45 and CD32 was examined by immunoprecipitation from lysates of cell surface biotinylated PMN. CD45 mAb co-immunoprecipitated CD32 (40 kDa) and CD32 mAb co-immunoprecipitated CD45 (180 kDa). FACS analysis of intracellular calcium mobilization was performed in Fluo-3 AM-loaded PMN when CD32 mAb was bound to PMN and either crosslinked to itself or co-crosslinked to bound CD45 mAb with a secondary F(ab)'2 antibody. CD32 crosslinked to itself mobilized intracellular calcium and stimulated protein tyrosine phosphorylation, whereas, co-crosslinking CD32 with CD45 decreased the calcium response and protein tyrosine phosphorylation. These results indicate that extracellular domains of CD32 and CD45 are in close physical proximity on isolated human PMN. The perturbation of these proteins by co-crosslinking modulates FcgRII-mediated mobilization of intracellular calcium and protein tyrosine phosphorylation.

白细胞共同抗原（CD45）家族是一组高分子抗原 所有细胞质膜上表达的重量糖蛋白 白细胞。 我们之前已经证明 CD45 的表位参与其中 中性粒细胞对化学引诱剂白三烯 B4 和 重组人 C5a (rHuC5a)。 该项目的目的是为了更好 了解 CD45 在吞噬细胞信号转导中的作用 暴露于化学引诱剂或吞噬细胞刺激。 低亲和力受体 FcgammaRII (CD32) 的物理接近性 IgG 和人中性粒细胞 (PMN) 表面的 CD45 为 通过荧光共振能量转移（FRET）进行研究 共免疫沉淀。 CD45 表位用单克隆抗体标记 供体荧光染料、FlTC 和 CD32 表位均用 mAb 标记 与受体荧光染料四甲基罗丹明 (Rh) 缀合。 烦恼 通过 FACS 对 PMN 结合的 FITC-CD45 mAb 和 Rh-CD32 mAb 之间的差异进行定量 分析。 FRET 效率在 4°C 时为 16-40%，在 4°C 时为 14-45% 37°C。CD45和CD32的物理结合通过以下方法检查 细胞表面生物素化 PMN 裂解物的免疫沉淀。 CD45 mAb 免疫共沉淀 CD32 (40 kDa) 和 CD32 mAb 免疫共沉淀 CD45 (180 kDa)。 细胞内FACS分析 当 CD32 mAb 时，在装载 Fluo-3 AM 的 PMN 中进行钙动员 与 PMN 结合并与其自身交联或与 PMN 共交联 将 CD45 mAb 与 F(ab)'2 二抗结合。 CD32 交联至 本身动员细胞内钙并刺激蛋白质酪氨酸 磷酸化，而 CD32 与 CD45 共交联则降低了 钙反应和蛋白质酪氨酸磷酸化。 这些结果 表明 CD32 和 CD45 的胞外域紧密相连 孤立的人类 PMN 上的物理接近度。 这些的扰动 蛋白质通过共交联调节 FcgRII 介导的动员 细胞内钙和蛋白质酪氨酸磷酸化。