Myeloperoxidase: a front-line defender against phagocytosed microorganisms

Abstract

Successful immune defense requires integration of multiple effector systems to match the diverse virulence properties that members of the microbial world might express as they initiate and promote infection. Human neutrophils--the first cellular responders to invading microbes--exert most of their antimicrobial activity in phagosomes, specialized membrane-bound intracellular compartments formed by ingestion of microorganisms. The toxins generated de novo by the phagocyte NADPH oxidase and delivered by fusion of neutrophil granules with nascent phagosomes create conditions that kill and degrade ingested microbes. Antimicrobial activity reflects multiple and complex synergies among the phagosomal contents, and optimal action relies on oxidants generated in the presence of MPO. The absence of life-threatening infectious complications in individuals with MPO deficiency is frequently offered as evidence that the MPO oxidant system is ancillary rather than essential for neutrophil-mediated antimicrobial activity. However, that argument fails to consider observations from humans and KO mice that demonstrate that microbial killing by MPO-deficient cells is less efficient than that of normal neutrophils. We present evidence in support of MPO as a major arm of oxidative killing by neutrophils and propose that the essential contribution of MPO to normal innate host defense is manifest only when exposure to pathogens overwhelms the capacity of other host defense mechanisms.

Figure 1.. Phagocytosis of bacteria by a neutrophil.

A thin section electron micrograph of a single neutrophil with three Escherichia coli (A), visible within two phagocytic vacuoles (B). Reprinted from ref. [1].

Figure 2.. Reactions catalyzed by MPO inside the neutrophil phagosome.

The catalytic activities of MPO that will occur in the phagosome include production of HOCl (blue) and dismutation of superoxide (red). Superoxide should also recycle Compound II formed by reduction of Compound I (orange) and maintain the dominant activities of MPO [28].

Figure 3.. Reactions of HOCl that have been used to demonstrate that MPO is active inside neutrophil phagosomes.

Reactions with tyrosine and methionine are shown for residues on proteins (−P) [45, 62, 72, 73].

Figure 4.. Impaired bacterial killing by MPO-deficient human neutrophils.

(A) Staphylocidal activity of leukocytes from normal individuals and those with MPO deficiency (MPO-def) or CGD [134]. (B) Bacterial killing, measured as the loss of viable organisms at 30 min, by normal (open bars), CGD (light gray bars), and MPO-deficient (dark gray bars) neutrophils using an optimized method for disruption of bacteria [135]. S. aureus, Staphylococcus aureus; C. albicans, Candida albicans. (C) Rate constants for killing of S. aureus by neutrophils from an individual with MPO deficiency or those from normal donors in the absence or presence of diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase, or azide and 2-TX, which inhibit MPO [136, 137]. Data have been replotted from the original references.

Figure 5.. Susceptibility of MPO-deficient mice to infections.

(A) Pulmonary infection with C. albicans in WT (○) and homozygous mutant mice (●). Mice were injected intratracheally with 4.3 × 10⁶ CFU C. albicans. At the indicated times after the challenge, whole lungs were homogenized, and aliquots of the homogenates were plated. Five mice or more were used in each group. Results represent mean ± sd log10 CFU/organ [140]. (B) Mortality was monitored in WT (○) and littermate MPO-deficient mice (●) after inducing sepsis by ligating and puncturing the blind-ended cecum to release intestinal microflora into the abdominal cavity [38]. (C) Mortality rates in MPO-deficient and WT mice in response to i.p. challenge with K. pneumoniae [141]. Data have been replotted from the original references.

Figure 6.. The MPO-mediated antimicrobial system: properties of MPO are well-suited to conditions of the neutrophil phagosome (blue) in the production of microbicidal concentrations of HOCl.

Contact with an ingestible particle triggers assembly from cytoplasmic and intrinsic membrane precursors of a NADPH oxidase (NOX2), which transfers electrons from cytoplasmic (gray) NADPH to dissolved molecular oxygen in the phagosome. A voltage-gated proton channel concurrently allows for transfer of H⁺ to balance charge and to provide protons for subsequent reactions. Granules (green) containing MPO fuse with the phagosome and release their contents. The superoxide formed by NADPH oxidase is expected to react predominantly with the ferric and Compound III forms of MPO. The net result is efficient dismutation that generates from superoxide and protons, molecular oxygen, and H₂O₂ in equal amounts. Chloride is available to the phagosome from a combination of pinocytosis during phagocytosis and transport through one or more chloride channels, including the CFTR that is defective in CF patients. MPO then catalyzes the H₂O₂-mediated oxidation of chloride to form HOCl, which reacts with the bacterium (yellow) or neutrophil proteins to produce cytotoxic chloramines [monochloramine (NH₂Cl) and protein chloramine (PNHCl)]. See text for a review of evidence that microbicidal amounts of HOCl can be generated in phagosomes of human PMN.