Hv1 proton channels differentially regulate the pH of neutrophil and macrophage phagosomes by sustaining the production of phagosomal ROS that inhibit the delivery of vacuolar ATPases (original) (raw)
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2014
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 toCandida, 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 perCandida, the vacuolar pH rose to >11 and the cytosol acidified excessively to pH ~6...
Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis
Proceedings of the National Academy of Sciences, 2009
Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH i in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na ؉ /H ؉ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH i decreased sharply, and recovery required both Na ؉ /H ؉ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn 2؉ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn 2؉ . Both the rate and extent of acidification in HVCN1deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis. innate immunity ͉ ion channels ͉ phagocyte ͉ respiratory burst ͉ SNARF Author contributions: D.M. and T.E.D. designed research; D.M., B.M., and V.V.C. performed research; M.C., E.R., and M.J.S.D. contributed new reagents/analytic tools; D.M., B.M.,
Journal of Experimental Biology, 1992
V-ATPases in phagocytic cells are known to mediate the acidification of most intracellular organelles. Proton-pump-mediated acidification of these organellar compartments is vital to numerous cell processes, including receptor recycling, protein processing and sorting and microbial degradation. Recent studies have suggested a role for V-ATPases in cytoplasmic pH homeostasis. The present discussion will review the current knowledge regarding the presence of V-ATPases in both the plasmalemmal and organellar membranes of phagocytic cells, the regulation of proton pump activity in these locations and the functional significance of pump-mediated proton translocation.
The Journal of Cell Biology, 1996
In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H ÷ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H ÷ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H + exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutro-phils. Since phagocytosis involves Fc~/(Fc~R) and complement receptors such as CR3 (a 132 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against Fc~/Rs (Fc-,/RII > Fc-yRIII) but not 132 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-132 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and Fc~Rs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcvR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-1 activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca 2÷ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc~R cross-linking. In summary, Fc~/Rs and 132 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation. N EUTROPmLS react to invading microorganisms and mediators present within an inflammatory milieu with a variety of rapid and coordinated responses which include movement of cells out of the vascular space along a gradient of chemotactic molecules followed by phagocytosis and killing of the microorganisms. This bactericidal function is effected by complex processes involving secretion of proteolytic enzymes and reactive oxygen intermediates (produced by the NADPH oxidase) into the phagolysosome (for review see . During these processes, dynamic alterations occur in leu-
Neutrophils kill microorganisms by inducing exocytosis of granules with antibacterial properties. Four isoforms of the "a" subunit of V-ATPase-a1V, a2V, a3V, and a4V-have been identified. a2V is expressed in white blood cells, that is, on the surface of monocytes or activated lymphocytes. Neutrophil associated-a2V was found on membranes of primary (azurophilic) granules and less often on secondary (specific) granules, tertiary (gelatinase granules), and secretory vesicles. However, it was not found on the surface of resting neutrophils. Following stimulation of neutrophils, primary granules containing a2V as well as CD63 translocated to the surface of the cell because of exocytosis. a2V was also found on the cell surface when the neutrophils were incubated in ammonium chloride buffer (pH 7.4) a weak base. The intracellular pH (cytosol) became alkaline within 5 min after stimulation, and the pH increased from 7.2 to 7.8; this pH change correlated with intragranular acidification of the neutrophil granules. Upon translocation and exocytosis, a2V on the membrane of primary granules remained on the cell surface, but myeloperoxidase was secreted. V-ATPase may have a role in the fusion of the granule membrane with the cell surface membrane before exocytosis. These findings suggest that the granule-associated a2V isoform has a role in maintaining a pH gradient within the cell between the cytosol and granules in neutrophils and also in fusion between the surface and the granules before exocytosis. Because a2V is not found on the surface of resting neutrophils, surface a2V may be useful as a biomarker for activated neutrophils.
Free radical research, 2014
Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼ 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additiona...
Journal of leukocyte biology, 2002
The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described. The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca(2)(+)/H(+) voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.