Human mononuclear phagocytes : Production of reactive oxygen species generated by the NADPH oxidase

Abstract: Human professional phagocytes constitute the first line of defence against invading microorganisms. With a membrane-bound enzyme system, the NADPH oxidase, these cells generate bactericidal metabolites. Activation of NADPH oxidase results in a generation of reactive oxygen species, e.g. superoxide anion and hydrogen peroxide. These oxygen species constitute an important part of the host defence against invading microbes. The subcellular localization of the NADPH oxidase is of great importance, since activation of plasma-membrane-localized oxidase system results in cellular release of potentially hannful oxygen species in the vicinity of the phagocyte. A regulated intracellular store of NADPH oxidase could focus the bactericidal arsenal on the phagocytosed prey and thereby reduce the risk of extracellular release and tissue damage in the host A new technique was developed that allowed measurement of the release, as well as the intracellular generation, of reactive oxygen species. The technique was used to compare the response induced in different types of phagocytic leukocytes, polymorphonuclear neutrophils, monocytes and macrophages. Stimulating neutrophils with the chemoattraclant peptide fMet-Leu-Phe induce an association of the ligand-receptor complex to the cell cytoskeleton. An event that has been suggested to terminate the activation of NADPH oxidase. Termination of the NADPH oxidase actiVity in monocytes occurs without any association of the complex to the cytoskeleton. Obviously, different mechanisms to terminate the fMet-Leu-Phe-induced oxidase response opemte in monocytes and neutrophils. No qualitative difference was observed between neutrophils and monocytes, with respect to the localization of the respiratory burst. However, macrophages lack the intracellular phase of the respiratory burst, and the molecular background of this difference could be identified by using indirect immunofluorescence and subcellular fractionation to localize the membrane component of the oxidase (cytochrome bsss). In neutrophils, most of the cytochrome b558 as well as most of the donnant oxidase activity, was recovered in the specific granules, whereas only a minor part was recovered in the plasma membrane and secretory vesicles. Considering disintegrated macrophages, NADPH oxidase activity was found exclusively in a fraction enriched in plasma membranes. These results  indicate that macrophages are incapable of intracellular generation of reactive oxygen species due to the absence of a granule-localized pool of the NADPH oxidase. Despite large similarities between different professional phagocytes, fundamental differences are apparent when comparing structural and regulatory processes related to the NADPH oxidase.