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Serum-opsonized bacteria are efficiently ingested and killed by neutrophils within the phagocytic vacuole, where they are exposed to an array of reactive oxygen metabolites and toxic lysosomal components. Although bacteria may be destroyed by oxygen-independent mechanisms alone, many types of bacteria are not killed effectively unless they are attacked by oxygen metabolites. However, the apparent inability of extracellular scavengers, or inhibitors, of oxygen metabolites to gain access to the phagocytic vacuole makes this system difficult to evaluate. Therefore, we investigated the ability of neutrophils triggered with phorbol myristate acetate to destroy unopsonized E. coli in a serum-free model system. Neutrophils incubated with phorbol myristate acetate at a cell-to-bacteria ratio of 1:4 caused a greater than 95% reduction in colony-forming units (CFU) of E. coli in 60 min at 37 degrees C. Destruction of E. coli by the stimulated neutrophils was dependent on neutrophil number, stimuli concentration, and the incubation period. The neutrophil-mediated bactericidal effect was stimulated by superoxide dismutase, but was inhibited by catalase, azide, or compounds known to scavenge hypochlorous acid. Although stimulated neutrophils can generate long-lived endogenous N- chloroamines , these compounds did not play a direct role in destruction of E. coli in our model system. However, in the presence of exogenous iodide, endogenous N- chloroamines exerted a powerful bactericidal effect. Finally, neutrophils triggered with opsonized zymosan could also mediate E. coli destruction by a qualitatively similar process. Thus, we have demonstrated that neutrophils have the potential to utilize the myeloperoxidase system to generate bactericidal quantities of a species with characteristics similar to, if not identical with, hypochlorous acid.

Human neutophils stimulated with phorbol myristate acetate were able to rapidly destroy autologous red blood cell targets. Neutrophil-mediated cytotoxicity was related to phorbol myristate acetate concentration and neutrophil number. The ability of stimulated neutrophils to lyse red blood cell targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for O2.- and H2O2 in the cytotoxic event, a variety of OH. and 1O2 did not effect cytolysis. The myeloperoxidase inhibitor cyanide did not reduce red blood destruction, while azide consistently impaired cytolysis. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase-H2O2-halide system. We propose that neutrophils, stimulated with phorbol myristate acetate, generate O2.- and H2O2, which play an integral role in a novel cytotoxic mechanism.

Human monocytes incubated with phorbol myristate acetate (PMA) or opsonized zymosan particles can chlorinate the beta-amino acid taurine to its monochloramine derivative. Taurine monochloramine can then be quantitated by its ability to oxidize 5-thio-2-nitrobenzoic acid to its disulfide or by its characteristic absorption peak at 252 nm. Stimulated, but not resting, monocytes chlorinated taurine by a process dependent on time, cell concentration, and pH. The formation of taurine chloramine by stimulated monocytes could be inhibited by catalase, azide, or cyanide, was unaffected by superoxide dismutase, and was stimulated by exogenous myeloperoxidase. Thus, taurine chloramine generation by human monocytes appeared dependent on both H2O2 and myeloperoxidase. Compared to human neutrophils, the monocyte could generate similar amounts of chloramine when stimulated with phorbol myristate acetate, but far less if opsonized zymosan particles were used as the trigger. Based on the known ability of the H2O2- myeloperoxidase-Cl- system to generate free HOCl, it would seem that this oxidant is the most likely species responsible for the monocyte- mediated chlorination reactions. Thus, we have used a simple quantitative assay to demonstrate the ability of the human monocyte to generate large quantities of a highly reactive and toxic oxygen metabolite.

Hydroxyl radical generation was demonstrated during heme-catalyzed decomposition of 15-hydroperoxy arachidonic acid. The hydroperoxide- mediated inactivation of prostacyclin synthetase seems to be related to the generation of this highly reactive species. A relationship between hydroperoxides and the hydroxyl radical may be important in the modulation of prostacyclin synthesis in diseased states.