The CD45 antigen 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 chemo-attractants, 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. Two different cell systems are used in these studies: human neutrophils from normal donors and a transformed murine macrophage cell line, RAW264. During the year, flow cytometric studies were undertaken in which rhodamine-labeled C5a and fluorescein-labeled CD45 monoclonal antibodies (mAb) were exposed to phagocytes and subsequently evaluated for colocalization on the cell membrane with a fluorescence resonance energy transfer technique. Preliminary data suggest that the C5a receptor and CD45 are closely associated on human neutrophils. Energy transfer studies are in progress with RAW264 cells and mutagenized variant clones of RAW264 that lack CD45 expression. A flow cytometric assay for intracellular calcium was set up using the fluorescent probe fluo-3 and intracellular calcium changes were monitored in human neutrophils in which Fc receptors were crosslinked with mAbs or stimulated with chemoattractants. Crosslinking Fc receptors results in an increase in intracellular calcium. When the Fc receptors are crosslinked with CD45, the intracellular calcium signals are reduced. Since the association of CD45 with Fc receptors appears to alter the calcium responses in neutrophils, similar studies will be conducted in RAW264 cells and the CD45- mutants to determine whether CD45 normally functions in regulating the Fc receptor mediated calcium response.
