Modulation of Neutrophil-Reduced Pyridine Nucleotide Content Following Stimulation With Phorbol Myristate Acetate and Chemotactic Factors

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Abstract
Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 μg/ml of PMA results in a time-dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7–15 min of stimulation with PMA. Simultaneous measurement of PMA-stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all-or-none response while H2O2 production showed a graded response. These data suggest that with PMA stimulation, a threshold exists beyond which constitutive NAD(P)+ reduction is suppressed and complete oxidation of NAD(P)H occurs, while H2O2 production is proportional to the concentration of PMA. PMA-stimulated oxidation of NAD(P)H was reversible, and fluorescence returned to the initial level or higher after 40–60 min. Oxidation of NAD(P)H also occurred when cytochalasin B-treated PMN were stimulated with 25 nM C5a or 100 nM formyl-methionyl-leucyl-phenylalanine (f-MLP), but occurred more rapidly, peaking at 1 to 3 min. Fluorescence also returned by 5–6 min. This response to C5a and f-MLP was graded and proportional to the concentration of chemotactic factor used. Comparative studies showed that the cytochalasin-B treatment was essential for measurement of NAD(P)H oxidation, in response to C5a and F-MLP.

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