ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

白细胞共同抗原(CD45)家族是一组高分子的 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的表位与 在中性粒细胞对趋化剂白三烯B4和 重组人C5a(RHuC5a)。这个项目的目的是为了更好地 了解CD45在吞噬细胞信号转导中的作用 暴露在化学诱导剂或吞噬细胞刺激下。体能 低亲和力免疫球蛋白受体FcgRII(CD32)与CD45的亲和性 对人中性粒细胞(PMN)表面进行了研究。 荧光共振能量转移(FRET)和联合免疫 降水。用单抗标记CD45表位 (MAb)与供体荧光色素、FltC和CD32表位偶联 用标记到受体荧光色素上的单抗标记， 四甲基罗丹明(Rh)。PMN结合的FITC-CD45单抗和FRET之间的FRET 用流式细胞仪对rh-CD32单抗进行定量。烦躁不安 不同CD45单抗的效价在37℃时为7-35%，在37℃时为8-33% 4℃。CD45和CD32的物理结合通过 中性粒细胞裂解物的免疫沉淀。CD45单抗的Western blotting 在CD45和CD44免疫沉淀物中检测到CD45(180 KDa)，而在 CD32、正常小鼠血清或G蛋白免疫沉淀物。流动 进行细胞内钙离子动员的细胞学分析 当CD32单抗与PMN结合时，在Fluo-3 AM负载的PMN中 与自身交联或与结合的CD45单抗共交联用 二抗F(Ab)2。CD32自身交联体动员 细胞内钙和被刺激的蛋白质酪氨酸磷酸化， 然而，CD32和CD45的共同交联会降低钙反应 和蛋白质酪氨酸磷酸化。这些结果表明， CD32和CD45的胞外结构域在物理上非常接近 在分离的人中性粒细胞上。这些蛋白质受到的干扰 共交联调节FcgRII介导的细胞内动员 钙和蛋白质酪氨酸磷酸化。

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.