Phagocyte and extra-phagocyte myeloperoxidase-mediated microbicidal action

Myeloperoxidase (MPO) is produced by neutrophil leukocytes and monocytes. These leukocytes are the phagocyte effectors of the acute inflammatory response and provide innate protection against infecting pathogens. In that regard MPO is the enzymatic effector of phagocyte microbicidal action. MPO is unique in its ability to catalyze the hydrogen peroxide (H2O2) dependent oxidation of chloride (Cl) to hypochlorite (OCl), i.e., the microbicidal component of bleach. Hypochlorite directly reacts with a second H2O2 to produce singlet molecular oxygen (O2*), an electronically excited state of oxygen with potent electrophilic reactivity. As a metastable excited state, O2* has a microsecond half-life that restricts microbicidal reactivity to within a radius of about 0.2 micron from its point of generation. In unstimulated phagocytes, MPO is stored in the azurophilic granules. Following the phagocytosis of a microbe, azurophilic granules fuse with the microbe-containing phagosome to form a phagolysosome. In the process of phagocytosis NADPH oxidase is activated and incorporated into the membrane of the phagosome, and ultimately, the membrane of the phagolysosome. NADPH oxidase catalyzes the one-equivalent reduction of oxygen (O2) producing hydrodioxylic acid (HO2; aka, hydroperoxyl radical) with an acid dissociation constant (pKa) of 4.8. As such, production of HO2 drives the pH of the phagolysosomal space toward 4.8. At neutral pH, HO2 dissociates yielding a proton (H) and its conjugate base, the superoxide anion (O2). Although anionic charge repulsion prevents direct disproportionation of O2 in neutral to alkanine conditions, in an acid milieu HO2 readily reacts with O2 yielding the H2O2 that drives MPO production of the OCl and O2* necessary for effective microbicidal action. Healthy human adults release about 10 neutrophils containing about 0.4 mg MPO into the circulating blood each day; i.e., about 4 femtograms MPO/neutrophil. The production of neutrophils and the quantity of MPO per neutrophil increase in inflammatory states and with G-CSF treatment. After a relatively short circulating lifetime, neutrophils leave the blood and migrate into body spaces including the mouth, urinary tract, vagina and gastrointestinal tract. Greater than 10 neutrophils can be routinely lavaged from the mouth of healthy humans, and in inflammatory conditions, the quantity of neutrophils lavaged increases in proportion to the blood neutrophil count. The oropharyngeal and vaginal spaces are normally colonized with lactic acid bacteria (LAB), such as viridans streptococci and lactobacilli. These LAB lack cytochromes and produce lactic acid and H2O2 as end products of metabolism. Consequently, the neutrophil leukocytes that migrate into such spaces can release MPO into an acidic milieu containing H2O2 adequate to facilitate extra-phagocyte microbicidal action. MPO selectively binds to microbes, showing relatively low binding to LAB and high binding to other bacteria. The short half-life of O2* demands proximity to target for effective killing, and as such, when MPO is limiting, selective binding results in selective killing. The fundamental elements required for phagolysomal and extracellular microbicidal action are MPO, Cl and H2O2 in an acidic milieu. As such, combining purified MPO and a H2O2-generating oxidase with oxidase substrate results in a potent microbicidal system. In that regard, effective and broad spectrum MPO microbicidal action has been demonstrated using glucose oxidase (GO) with glucose as substrate. This GO-MPO system demonstrates in vitro microbicidal action against all bacteria and yeast tested. The efficacy of applying this GO-MPO microbicidal formulation for the prevention of surgical site and wound infections has also been demonstrated in animal test models.