Activation of human polymorphonuclear leukocytes (PMN) plays a key role in chronic and acute inflammatory processes including infectious disease, septic shock, ischemia-reperfusion injury, arthritis, nephritis, and perhaps resistance to cancer. This program's overall goal is to understand the mechanism of PMN activation. We have discovered that certain cell surface proteins physically interact with complement receptor type 3 (CR3), an inflammation-associated integrin. To capitalize on this finding, we will address the following specific aims using PMNs and/or transfectants expressing native or engineered receptors. We will test the hypothesis that certain glycophospholipid-linked membrane proteins, such as urokinase receptors (uPAR), reversibly interact with CR3. Experimental methods sensitive to receptor-receptor proximity and lateral mobility will be used to characterize these interactions, which will be linked with signal transduction and cell migration. We will examine CR4-to uPAR interactions on PMNs and provide experimental tests of the phase-locked signal transduction hypothesis and its cellular origin. The mechanism of uPAR-integrin interactions and their physiological relevance will be examined. To analyze the molecular mechanism of FcRIII-CR3 interaction, allotypic variants of FcRIIIB in PMNs and transfectants expressing FcRIII with selected deletion of N-linked consensus sequences will be studied. We will test the hypothesis that FcRII interacts with CR3 using transfectants that express these native proteins of defective forms (FcRII- or CR3-) that lack their cytoplasmic domains and internalization ability. Using a reverse genetic complementation approach, we will test the ability of wild-type CR3 or FcRII to rescue internalization mediated by ligation of FcRII- or CR3-, respectively. Physical association will be studied using biophysical and immunoprecipitation techniques whereas functional associations will be monitored using transmembrane signaling and phagocytosis assays. Our studies will determine the nature and mechanisms of inter-receptor interactions, which may lead to the development of new anti-inflammatory agents.
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