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: 2569051
• 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/2569051
• 关键词: CD antigens; antibody receptor; calcium flux; cell motility; chemoattractants; chemotaxis; flow cytometry; human tissue; immunoglobulin G; immunoprecipitation; intermolecular interaction; leukotrienes; neutrophil; phosphoprotein phosphatase; western blottings

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 FcgRII (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 monoclonal antibodies (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 flow cytometric analysis. The FRET efficiency for different CD45 mAb was 7-35% at 37 degrees C and 8-33% at 4 degrees C. Physical association of CD45 and CD32 was examined by immunoprecipitation of PMN lysates. Western blotting with CD45 mAb detected CD45 (180 kDa) in CD45 and CD44 immunoprecipitates but not in CD32, normal mouse serum or protein G immunoprecipitates. Flow cytometric 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（CD 45）家族是一组高分子量的 重量的糖蛋白，表达在质膜上的所有 白细胞 我们以前已经表明，CD 45的表位参与了 在中性粒细胞趋化性的化学引诱剂，白三烯B4和 重组人C5 a（rHuC 5a）。 这个项目的目的是为了更好地 了解CD 45在吞噬细胞信号转导中的作用， 暴露于化学引诱物或吞噬刺激物。 物理 IgG的低亲和力受体FcgRII（CD 32）和CD 45的接近 研究了人中性粒细胞（PMN）表面的 荧光共振能量转移（FRET）和共免疫- 降水 用单克隆抗体标记CD 45表位 (mAb)与供体荧光染料、FlTC和CD 32表位缀合， 用与受体荧光染料缀合的mAb标记， 四甲基罗丹明（Rh）。 PMN结合的FITC-CD 45 mAb与 Rh-CD 32 mAb通过流式细胞术分析定量。 的FRET 对于不同的CD 45 mAb，在37 ℃下的效率为7-35%，而在37 ℃下的效率为8-33%。 4摄氏度。 CD 45和CD 32的物理结合通过 PMN裂解物的免疫沉淀。 Western blotting with CD45 mAb 在CD 45和CD 44免疫沉淀物中检测到CD 45（180 kDa），但在 CD 32、正常小鼠血清或蛋白G免疫沉淀。 流 进行细胞内钙动员的细胞计数分析 在Fluo-3 AM负载的PMN中，当CD 32 mAb与PMN结合时， 与自身交联或与结合的CD 45 mAb共交联， 二抗F（ab）2。 与自身交联的CD 32动员 细胞内钙和刺激的蛋白酪氨酸磷酸化， 而CD 32与CD 45共交联降低了钙反应， 和蛋白质酪氨酸磷酸化。 这些结果表明 CD 32和CD 45的细胞外结构域在物理上非常接近 分离的人类中性粒细胞 这些蛋白质的扰动， 共交联调节FcgRII介导的细胞内 钙和蛋白质酪氨酸磷酸化。