REGULATION OF MACROPHAGE FUNCTION BY PROTEIN KINASES

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Macrophages can be stimulated to states of heightened microbicidal and tumoricidal activity that are functionally defined as activated. The intracellular events by which macrophage activation is regulated have not yet been defined. We propose to systematically define the rapid alterations in protein phosphorylation that are associated with stimulation of macrophages and identify the protein kinases involved in these processes. Two different stimuli will be used in these studies; colony stimulating factor (CSF-1) and bacterial lipopolysaccharide (LPS). LPS and CSF-1 induce many common functional changes in macrophages, but each also induces unique functions. For example, only LPS induces tumoricidal activity. In order to determine how CSF-1 and LPS alter macrophage activity both a pharmacologic and biochemical approach will be employed. The pharmacologic approach will utilize agents that may mimic or inhibit activities of LPS or CSF-1 to help identify the second messenger/protein kinase systems that might mediate the actions of CSF-1 and LPS. The biochemical approach will determine the rapid alterations in protein phosphorylation induced by each stimuli. After initial pharmacologic and biochemical studies have given an indication of possible protein kinases mediating specific actions, the protein kinases involved will be identified. This will be accomplished by comparing the in vivo phosphorylation pattern with the phosphorylation pattern obtained by stimulating specific protein kinases in cell preparations in vitro. The protein kinases that will be stimulated will be chosen based on the results of the pharmacologic and phosphorylation studies. Comparisons will be made between the in vitro and in vivo phosphorylations to determine if the phosphorylations are on the same phosphoprotein and on the same site of that phosphoprotein. These results will allow us to determine if the phosphorylation reaction is mediated by the specific protein kinase studied. It is hoped that this will help define the mechanism(s) by which macrophage function is regulated.