Adam P Levine | bioRxiv Science

bioRxiv2015

The NADPH oxidase and microbial killing by neutrophils, with a particular emphasis on the proposed antimicrobial role of myeloperoxidase within the phagocytic vacuole

This review is devoted to a consideration of the way in which the NADPH oxidase of neutrophils, NOX2, functions to enable the efficient killing of bacteria and fungi. It includes a critical examination of the current dogma that its primary purpose is the generation of hydrogen peroxide as substrate for myeloperoxide catalysed generation of hypochlorite. Instead it is demonstrated that NADPH oxidase functions to optimise the ionic and pH conditions within the vacuole for the solubilisation and optimal activity of the proteins released into this compartment from the cytoplasmic granules, which kill and digest the microbes. The general role of other NOX systems as electrochemical generators to alter the pH and ionic composition in compartments on either side of a membrane in plants and animals will also be examined.

Immunology↗

bioRxiv2014

The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH

Activation of the NADPH oxidase (NOX2) of the phagocytic vacuole of neutrophils is essential for innate immunity. Sustained activity of the oxidase requires that charge movements across the membrane are balanced. A role for the proton channel, HVCN1, has been proposed but not proven. Using the ratiometric pH indicator SNARF, introduced into the cytosol and separately into the vacuole coupled to Candida, we used confocal microscopy to measure changes in pH in these two compartments in human and mouse neutrophils. Shortly after phagocytosis by human cells, the vacuolar pH rose to ~9, at which it was maintained for ~20 minutes, while the cytosol showed a small acidification of ~0.25 pH unit. Alkalinisation has important consequences for the microbicidal and digestive functions of vacuolar enzymes. In HVCN1 knock out mouse neutrophils, the phagocytosis induced respiratory burst was halved to ~3 fmols per Candida, the vacuolar pH rose to >11 and the cytosol acidified excessively to pH ~6.75. These changes were prevented by the protonophore CCCP. The rate of extrusion of protons into the extracellular medium following phagocytosis was not significantly different from wild type neutrophils suggesting that cytoplasmic acidification resulted from the loss of the proton sink into the vacuole. HVCN1 phagocytic vacuoles showed considerable swelling, and this was blocked by CCCP and decreased by valinomycin. Stoichiometric considerations indicated that the HVCN1 channel compensates 90-95% of the oxidase-induced charge in normal cells, and in its absence, charge is carried by ions other than protons, including K+.

Cell Biology↗