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.

Prxs (peroxiredoxins) are a family of proteins that are extremely effective at scavenging peroxides. The Prxs exhibit a number of intriguing properties that distinguish them from conventional antioxidants, including a susceptibility to inactivation by hyperoxidation in the presence of excess peroxide and the ability to form complex oligomeric structures. These properties, combined with a high cellular abundance and reactivity with hydrogen peroxide, have led to speculation that the Prxs function as redox sensors that transmit signals as part of the cellular response to oxidative stress. Multicellular organisms express several different Prxs that can be categorized by their subcellular distribution. In mammals, Prx 3 and Prx 5 are targeted to the mitochondrial matrix. Mitochondria are a major source of hydrogen peroxide, and this oxidant is implicated in the damage associated with aging and a number of pathologies. Hydrogen peroxide can also act as a second messenger, and is linked with signalling events in mitochondria, including the induction of apoptosis. A simple kinetic competition analysis estimates that Prx 3 will be the target for up to 90% of hydrogen peroxide generated in the matrix. Therefore, mitochondrial Prxs have the potential to play a major role in mitochondrial redox signalling, but the extent of this role and the mechanisms involved are currently unclear.

Neutrophil NET formation induced by PMA, bacteria, and ionomycin has different requirements for NADPH oxidase activity and myeloperoxidase. Release of NETs by neutrophils is linked with immune protection and host damage. A variety of stimuli promotes NET formation. However, findings from different laboratories often vary, and it is possible that more than one mechanism of NET formation exists. NET formation induced by PMA has been shown to require NADPH oxidase activity, and there is evidence that the granule enzyme MPO is also involved. However, requirements for NADPH oxidase or MPO with other stimuli are less well established. We investigated the role of oxidants in NET formation by human neutrophils induced with PMA, several bacterial genera, and the calcium ionophore ionomycin. With the use of inhibitors of the NADPH oxidase and MPO, oxidant scavengers, and cells from a MPO-deficient individual, we observed that requirements for oxidant generation depend on the stimulus. NADPH oxidase activity was required with PMA and bacterial stimulation but not with ionomycin. Whereas MPO was required for efficient NET formation with PMA, incubation with bacteria induced NETs independently of MPO activity. Although the specific mechanisms whereby oxidants participate in NET formation remain to be clarified, it is possible that other stimuli that mobilize calcium act like ionomycin via an oxidant-independent mechanism, and it cannot be inferred from results with PMA that MPO is required with more physiological stimuli.