ROLE OF CD45 IN PHAGOCYTE MOTILITY AND SIGNAL TRANSDUCTION

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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.