Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.

Significance: When neutrophils kill microorganisms, they ingest them into phagosomes and bombard them with a burst of reactive oxygen species. Recent Advances: This review focuses on what oxidants are produced and how they kill. The neutrophil NADPH oxidase is activated and shuttles electrons from NADPH in the cytoplasm to oxygen in the phagosomal lumen. Superoxide is generated in the narrow space between the ingested organism and the phagosomal membrane and kinetic modeling indicates that it reaches a concentration of around 20 μ M . Degranulation leads to a very high protein concentration with up to millimolar myeloperoxidase (MPO). MPO has many substrates, but its main phagosomal reactions should be to dismutate superoxide and, provided adequate chloride, catalyze efficient conversion of hydrogen peroxide to hypochlorous acid (HOCl). Studies with specific probes have shown that HOCl is produced in the phagosome and reacts with ingested bacteria. The amount generated should be high enough to kill. However, much of the HOCl reacts with phagosomal proteins. Generation of chloramines may contribute to killing, but the full consequences of this are not yet clear. Critical Issues: Isolated neutrophils kill most of the ingested microorganisms rapidly by an MPO-dependent mechanism that is almost certainly due to HOCl. However, individuals with MPO deficiency rarely have problems with infection. A possible explanation is that HOCl provides a frontline response that kills most of the microorganisms, with survivors killed by nonoxidative processes. The latter may deal adequately with low-level infection but with high exposure, more efficient HOCl-dependent killing is required. Future Directions: Better quantification of HOCl and other oxidants in the phagosome should clarify their roles in antimicrobial action. Antioxid. Redox Signal. 18, 642–660.