Stimulation of neutrophilic leukocytes results in secretion of toxic proteins and the enzyme myeloperoxidase (MPO) from cytoplasmic granules and activates neutrophil oxygen (O2) metabolism, which produces toxic oxidants. Neutrophil toxins combat infection by killing invading microorganisms but also contribute to inflammatory tissue destruction. Long-range goals are to characterize the oxidants, determine their role in antimicrobial activity and damage to host cells, and elucidate mechanisms that regulate their potency and selectivity. The working hypothesis is that hydrogen peroxide (H202) and products of the MPO/H202/chloride (C1-) system are the major oxidative toxins. Superoxide (02-) is the first product of leukocyte 02 metabolism, but the principal role of 02- is not as a toxin. Instead, 02- is an intermediate in H202 production and an oxidant for extracellular reductants such as ascorbate (vitamin C) and sulfhydryl compounds. Oxidation of these reductants by 02- increases H202 production and consumes the reductants so that they can't detoxify H202 and products of the MPO system. Toxicity of the MPO system is also regulated by ammonia (NH4+), and lysine-rich peptides from the cytoplasmic granules. MPO catalyzed the oxidation of C1- by H202 to yield hypochlorous acid (HOC1), which reacts with NH4+ to yield the bactericidal agent monochloramine (NH2C1). However, NH2C1 can also suppress toxicity by inhibiting neutrophil 02 metabolism, and NH4+ or NH2C1 may inhibit MPO secretion. The reaction of HOC1 with the lysine-peptides yields relatively non-toxic though long-lived peptide-chloramines. The proposed studies will characterize the interaction of 02-, H202, and products of the MPO system with ascorbate and other extracellular reductants, and examine effects of the reductants on antibacterial and cytotoxic activities of isolated neutrophils. The ability of manganese (Mn2+) to promote oxidation of reductants by MPO will be studied, to determine whether MPO could act as an oxidase and produce toxic oxidants in neutrophils with defective 02 metabolism. The effect of NH4+ on neutrophil 02 metabolism, MPO secretion, and antibacterial activity will be measured, and the interaction of lysine-peptides with the MPO system will be studied using purified peptides and MPO. These studies will provide new insights into the role of oxidants and reductants in neutrophil function and may lead to improved methods for combating infection while suppressing damage to host tissues.
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