The neutrophil carries out its primary function of destroying foreign cells by a complex sequence that begins with binding of a stimulus to membrane receptors and proceeds to membrane and cytoplasmic events that culminate in the activation of contractile elements oxidative metabolism and degranulation. Microbicidal activity is achieved by the formation of reduced oxygen derivatives, secretion of granule enzymes and accumulation of these toxic agents in the phagocytic vacuole. We propose a detailed investigation of the process of activation of the metabolic burst and degranulation in human neutrophils and monocytes. We shall emphasize early events at the membrane level, kinetic analyses of the cellular responses, isolation and characterization of specific cell components involved in the responses, and studies of cells from patients with defined neutrophil dysfunction syndromes such as chronic granulomatous disease. Membrane depolarization will be monitored fluorometrically using lipophilic cationic cyanine dyes. Kinetics of the respiratory burst (02 consumption, formation of superoxide anion and H202) will be compared with those of depolarization and degranulation. We will examine specific cell components participating in the respiratory burst, including cytochrome b, quinones, flavins and oxidases acting on NADPH or NADH. These components will be studied in intact cells and in myeloid precursors as they mature in vitro, localized and isolated in fractionated cells, examined for changes in activity and location with stimulation of cells and recombined in mixtures of cell fractions. Special effort will be made to isolate and characterize cytochrome b and prepare an antibody to it for use as a probe of cell function. Methods will be developed for measurement of pH and pCa in phagocytic vacuoles since these parameters may be determinants of microbicidal function. The early kinetics of degranulation will be emphasized using a new continuous fluorometric assay for myeloperoxidase release. Comparisons will be made of azurophil and specific granules and of the kinetics of enzyme secretion, 02 metabolism and depolarization. The role in these events of fusion of granules with the plasma membrane and phagocytic vacuole will be evaluated. The proposed studies will provide valuable new data on stimulus-response coupling in phagocytic cells, thereby advancing our understanding of host defense mechanisms and of important clinical syndromes of phagocyte dysfunction.
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