Hydrogen Peroxide Production in Chronic Granulomatous Disease: A CYTOCHEMICAL STUDY OF REDUCED PYRIDINE NUCLEOTIDE OXIDASES

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Abstract

The ability of polymorphonuclear leukocytes (PMN) to produce H2O2 in response to phagocytic stimulation was examined cytochemically using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Normal PMN oxidized diaminobenzidine within the phagocytic vacuole by a reaction dependent upon endogenous H2O2 and myeloperoxidase. CGD PMN failed to oxidize diaminobenzidine, which is consistent with the biochemical data showing a lack of H2O2-generating capacity. A plasma membrane enzyme (oxidase) activated by phagocytosis is capable of H2O2 production in PMN. The localization of this oxidase activity was explored in CGD PMN using a cytochemical technique specific for H2O2. The enzyme activity is stimulated by exogenous NADH, but not NADPH. Reaction product formation, indicative of activity of the oxidase, is dependent upon precipitation of cerium ions by the enzymatically generated H2O2. The advantage of this approach is that enzyme activity of individual cells can be assessed, allowing determination of numbers of reactive cells in the population and their relative degrees of reactivity. NADH oxidase was found to be active both on the plasma membrane and within the phagocytic vacuoles of control PMN, whereas those cells from three CGD patients showed greatly reduced activity in both these sites. Assessment of the reactivity of individual cells showed the number of cells with oxidase activity in CGD to be significantly reduced when compared to control values. Additionally, of those cells that do react, a higher percentage of them are only weakly reactive. Omission of NADH from the incubation medium reduced the percentage of control cells showing enzyme activity but had no effect on CGD PMN, implying that the enzyme is not saturated with substrate in control cells, but in CGD the diminished enzyme is fully saturated. The defect may lie in the fact that in CGD patients there are fewer cells capable of peroxide generation, and a majority of these reactive cells produce only reduced amounts of this bactericidal agent.

Authors

Richard T. Briggs, Manfred L. Karnovsky, Morris J. Karnovsky