MODIFICATION OF NEUTROPHIL OXIDANT PRODUCTION WITH DIPHENYLENEIODONIUM AND ITS EFFECT ON BACTERIAL KILLING

Diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase, has been used to distinguish between oxidative and nonoxidative killing of Sta phylococcus aureus and Escherichia coli by neutrophils. The rate of ki lling of S. aureus was inhibited by 77% in the presence of 10 mu M DPI , compared to 81% measured under anaerobic conditions. DPI represents a convenient and accessible alternative to an anaerobic environment or using neutrophils from patients with chronic granulomatous disease, f or eliminating oxidative killing. The killing of E. coli was also inhi bited by DPI. The effect was more apparent at 30 min than at 10 min, s uggesting that E. coli can be killed rapidly by nonoxidative mechanism s that become less efficient at later times. DPI was used at concentra tions less than 10 mu M to determine how this affected production of t he three major neutrophil oxidants, superoxide, hydrogen peroxide, and hypochlorous acid, and to determine the effect of partial inhibition of oxidant production on the killing of S. aureus. Unexpectedly, lower concentrations of DPI (0.1-2 mu M) inhibited hydrogen peroxide and hy pochlorous acid production 10-30% more than they inhibited superoxide production. Correlation of hydrogen peroxide or hypochlorous acid prod uction with the killing of S. aureus showed that up to 30% inhibition had no effect on the rate of killing, implying that agents that impair neutrophil oxidant production less than this will not compromise bact erial killing. Higher inhibition of oxidant production led to a linear decline in the rate of killing.