INVOLVEMENT OF SUPEROXIDE AND MYELOPEROXIDASE IN OXYGEN-DEPENDENT KILLING OF STAPHYLOCOCCUS-AUREUS BY NEUTROPHILS

We have used a quantitative assay that measures independent rate const ants for phagocytosis and killing of Staphylococcus aureus to investig ate the involvement of superoxide and myeloperoxidase in bacterial kil ling by human neutrophils, To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conj ugate was attached to the surface of S. aureus via protein A in its ce ll wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase -deficient neutrophils were used. Adding the NADPH oxidase inhibitor d iphenyleneiodonium, to prevent superoxide production, decreased the ki lling rate to 25%, indicating that oxidative killing mechanisms predom inate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria lin ked to inactivated enzyme. Superoxide dismutase had no effect in the p resence of diphenyleneiodonium, The rate of killing was decreased to 3 3% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils, Superoxide dismutase had no effect in the presence of azi de. On the assumption that the oxidative and nonoxidative components o f killing can be considered separately, the oxidative rate was decreas ed by almost half by superoxide dismutase and was about six times lowe r when myeloperoxidase was inactive, We conclude that myeloperoxidase- dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathw ay.