ACTIVATION OF THE HUMAN NEUTROPHIL

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Generation of superoxide anion and degranulation are critical to the microbicidal function of neutrophils, but may also contribute to tissue damage during inflammation. Therefore an understanding of the function. These studies are designed to investigate the activation of human neutrophils by phagocytic and non-phagocytic stimuli, with particular reference to the role of calcium movements, membrane lipids, and protein kinases as transducers or modulators of stimulus-response coupling. Calcium and protein kinase C have been implicated in both triggering and down regulating neutrophil functions. Ligands such as the chemotactic peptide f-Met-Leu-Phe, activate a phospholipase C which cleaves intracellular Ca, and diayclglycerol, a cofactor for the activation of protein kinase C. Other sources of diglyceride will be evaluated including phosphatidyl inositol, glycosyl-phosphatidyl inositol, phosphatidyl choline and de novo localization and molecular species of the diglyceride, both critical considerations in determining cofactor activity. Diacylglycerol is an important signalling element in neutrophil activation and its removal may serve as an important control for cell activation. Localization and activity of the enzymes critical to the regulation of diacyl glycerol, such as diacylglycerol kinase, will be determined. Multiple immunoreactive species of protein kinase C, the alpha-, beta and gamma,n-isozymes, have been demonstrated in the neutrophil. It is hypothesized that different isotypes of protein kinase C play selective roles in neutrophil functions. The beta-protein kinase C, that is translocated from cytosol to membrane in response to elevated Ca or to phorbol myristate acetate, as well as a novel non-translocated alpha- protein kinase C will be purified. The differential cofactor requirements and substrate specificities of these different isotypes of protein kinase C will be determined as a key to understanding the selective roles of alpha- PKC and beta PKC in neutrophil functions. A novel glycosyl-phosphatidyl inositol glycosyl-Pl) has been demonstrated in neutrophils, which may play backbone by phospholipase C, and selectively inhibits superoxide but not degranulation. Thus the polar head group of glycosyl-Pl is a unique and physiologic probe for examining the signalling for superoxide generation. An understanding of the generation of positive and negative signals, and of their regulation is essential in devising appropriate strategies for enhancing those functions necessary for host defense and selectively down regulating functions responsible for tissue damage.