The purpose of the studies described in this application is tocharacterize the ATP receptor(s) on cytokine-treated and untreated macrophages using monoclonal antibodies (MoAb) that are capable of blocking ATP-mediated lysis. With these MoAb, a cDNA library made from interferon (IFN) gamma-treated macrophages will be screened to identify, and ultimately sequence, a putative ATP receptor protein. Next, with the MoAb and the cDNA clones which identify the ATP receptor on macrophages, the biological role of this receptor on other cells will be explored by expression cloning techniques, flow cytometry, and Northern blotting analysis. To determine the basis for the differential effect of ATP on IFNgamma- and GM-CSF-treated macrophages, the intracellular signaling pathways which mediate the ATP lysis of these cells will be examined. The role(s) of Ca++, calmodulin-linked enzymes, phosphoinositol metabolism, protein kinases, and apoptosis will be probed so as to identify the mechanism of lysis of cytokine-treated macrophages. The relevance of macrophage differentiation on their sensitivity to ATP will be assessed using monocytic tumor cell lines, the maturation status of which is well-characterized.
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