Determinants of the Phagosomal pH in Neutrophils

Phagosomes formed by neutrophils are much less acidic than those of other phagocytic cells. The defective acidification seen in neutrophils has been attributed to consumption of protons during the dismutation of superoxide, because a large, sustained acidification is unmasked when the cells are treated with inhibitors of the NADPH oxidase. Consumption of protons transported into the phagosome by dismutation would tightly couple the activities of the NADPH oxidase and the vacuolar type H+-pump (or V-ATPase). We tested the existence of the predicted coupling using microfluorimetry and digital imaging and found that the rate of superoxide generation was independent of the activity of the H+-pump. Moreover, we failed to detect the alkalinization predicted to develop through dismutation when the pump was inhibited. Instead, two other mechanisms were found to contribute to the inability of neutrophil phagosomes to acidify. First, the insertion of V-ATPases into the phagosomal membrane was found to be reduced when the oxidase is active. Second, the passive proton (equivalent) permeability of the phagosomal membrane increased when the oxidase was activated. The increased permeability cannot be entirely attributed to the conductive H+ channels associated with the oxidase, since it is not eliminated by Zn2+. We conclude that the NADPH oxidase controls the phagosomal pH by multiple mechanisms that include reduced proton delivery to the lumen, increased luminal proton consumption, and enhanced backflux (leak) into the cytosol. Phagosomes formed by neutrophils are much less acidic than those of other phagocytic cells. The defective acidification seen in neutrophils has been attributed to consumption of protons during the dismutation of superoxide, because a large, sustained acidification is unmasked when the cells are treated with inhibitors of the NADPH oxidase. Consumption of protons transported into the phagosome by dismutation would tightly couple the activities of the NADPH oxidase and the vacuolar type H+-pump (or V-ATPase). We tested the existence of the predicted coupling using microfluorimetry and digital imaging and found that the rate of superoxide generation was independent of the activity of the H+-pump. Moreover, we failed to detect the alkalinization predicted to develop through dismutation when the pump was inhibited. Instead, two other mechanisms were found to contribute to the inability of neutrophil phagosomes to acidify. First, the insertion of V-ATPases into the phagosomal membrane was found to be reduced when the oxidase is active. Second, the passive proton (equivalent) permeability of the phagosomal membrane increased when the oxidase was activated. The increased permeability cannot be entirely attributed to the conductive H+ channels associated with the oxidase, since it is not eliminated by Zn2+. We conclude that the NADPH oxidase controls the phagosomal pH by multiple mechanisms that include reduced proton delivery to the lumen, increased luminal proton consumption, and enhanced backflux (leak) into the cytosol. A complex and highly organized series of events takes place at sites of tissue injury to limit the extent of microbial infection. Neutrophils are crucial contributors to this host response, as they are the first line of defense against invading microorganisms. When activated by bacterial or inflammatory chemoattractants, circulating neutrophils traverse capillary walls by diapedesis and migrate to the sites of infection, where they recognize and eliminate foreign organisms (1Elsbach P. Weiss J. Immunol. Lett. 1985; 11: 159-163Crossref PubMed Scopus (33) Google Scholar, 2Cohen M.S. Clin. Infect. Dis. 1994; 18 Suppl. 2: 170-179Crossref Scopus (76) Google Scholar). Phagocytosis, the process of internalization of the microorganisms into a membrane-bound vacuole, is central to the microbicidal response (1Elsbach P. Weiss J. Immunol. Lett. 1985; 11: 159-163Crossref PubMed Scopus (33) Google Scholar, 2Cohen M.S. Clin. Infect. Dis. 1994; 18 Suppl. 2: 170-179Crossref Scopus (76) Google Scholar). The membrane of newly formed phagosomes displays a composition similar to that of the plasma membrane, while the phagosomal contents are initially similar to the extracellular milieu. However, shortly after sealing, the phagosome undergoes drastic remodeling by fusion with endomembrane compartments, while maintaining its approximate size through coordinated fission events. Such remodeling involves delivery into the phagosomal lumen of a variety of microbicidal agents, including lytic enzymes and cationic peptides, as well as insertion into the phagosomal membrane of proteins that also contribute to the killing and disposal of microorganisms (3Levy O. Blood. 2000; 96: 2664-2672Crossref PubMed Google Scholar, 4Tjelle T.E. Lovdal T. Berg T. Bioessays. 2000; 22: 255-263Crossref PubMed Scopus (138) Google Scholar). The proteins acquired by the phagosomal membrane during maturation include the NADPH oxidase and the H+-pumping vacuolar ATPase (V-ATPase). 1V-ATPaseH+-pumping vacuolar ATPaseDPIdiphenylene iodoniumpHpphagosomal pHMES4-morpholineethanesulfonic acidPBSphosphate-buffered saline The NADPH oxidase, an enzymatic complex consisting of both membrane-bound and cytosolic subunits, assembles at the phagocytic membrane, where it facilitates the transfer of one electron from cytosolic NADPH to molecular oxygen. The resulting superoxide anion and the reactive oxygen metabolites generated therefrom, which include hypochlorous acid and hydroxyl radicals, are potent microbicidal agents (5Miller R.A. Britigan B.E. Clin. Microbiol. Rev. 1997; 10: 1-18Crossref PubMed Google Scholar). The V-ATPase catalyzes the vectorial transport of H+ into the phagosomal lumen (6Hackam D.J. Rotstein O.D. Zhang W.J. Demaurex N. Woodside M. Tsai O. Grinstein S. J. Biol. Chem. 1997; 272: 29810-29820Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). H+-pumping vacuolar ATPase diphenylene iodonium phagosomal pH 4-morpholineethanesulfonic acid phosphate-buffered saline Like neutrophils, macrophages also utilize phagocytosis to contribute to the innate immune response (2Cohen M.S. Clin. Infect. Dis. 1994; 18 Suppl. 2: 170-179Crossref Scopus (76) Google Scholar, 7Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2092) Google Scholar). In these cells acidification of the phagosomal lumen, which has been reported to reach pH 5.5 (6Hackam D.J. Rotstein O.D. Zhang W.J. Demaurex N. Woodside M. Tsai O. Grinstein S. J. Biol. Chem. 1997; 272: 29810-29820Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 8Lukacs G.L. Rotstein O.D. Grinstein S. J. Biol. Chem. 1990; 265: 21099-21107Abstract Full Text PDF PubMed Google Scholar), appears to be stringently required for effective bacterial killing (9Sturgill-Koszycki S. Schlesinger P.H. Chakraborty P. Haddix P.L. Collins H.L. Fok A.K. Allen R.D. Gluck S.L. Heuser J. Russell D.G. Science. 1994; 263: 678-681Crossref PubMed Scopus (1071) Google Scholar). Two lines of evidence indicate that the V-ATPase is responsible for the luminal acidification: (a) the accumulation of H+ requires cytosolic ATP (8Lukacs G.L. Rotstein O.D. Grinstein S. J. Biol. Chem. 1990; 265: 21099-21107Abstract Full Text PDF PubMed Google Scholar), and (b) acidification is inhibited by specific V-ATPase inhibitors like bafilomycin and concanamycin (6Hackam D.J. Rotstein O.D. Zhang W.J. Demaurex N. Woodside M. Tsai O. Grinstein S. J. Biol. Chem. 1997; 272: 29810-29820Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 8Lukacs G.L. Rotstein O.D. Grinstein S. J. Biol. Chem. 1990; 265: 21099-21107Abstract Full Text PDF PubMed Google Scholar). As V-ATPases are also inserted into the phagosomes of neutrophils, a comparable phagosomal acidification would be expected in these cells. Remarkably, however, phagosomal pH (pHp) in neutrophils has been reported to undergo a biphasic change; an alkalinization occurs during the first few minutes, which is then slowly followed by a modest secondary acidification to pH ∼6.5. It is unclear whether this apparent discrepancy resulted from differences in the methodology used or whether phagosomal pH is truly regulated differently in these two cell types. Segal et al. (10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar) proposed a mechanism to account for the biphasic pHp changes reported in neutrophils. The initial alkalosis was attributed to consumption of H+ during the process of dismutation of superoxide to H2O2, while the secondary acidification was proposed to reflect the ongoing activity of the V-ATPase, which becomes unmasked when the respiratory burst subsides. Consistent with this notion, in neutrophils from chronic granulomatous disease patients, which lack an active NADPH oxidase, pHp was found to acidify at rates comparable with those seen in macrophages (6Hackam D.J. Rotstein O.D. Zhang W.J. Demaurex N. Woodside M. Tsai O. Grinstein S. J. Biol. Chem. 1997; 272: 29810-29820Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar). Since the original hypothesis of Segal et al. (10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar), it has become apparent that additional H+ transport systems operate in phagocytes, which are activated in parallel with the respiratory burst. In particular, a H+ conductive pathway or “channel” was found to exist in both neutrophils and macrophages. While the molecular identity of the channel is still the subject of debate, it is generally agreed that large H+-selective currents can be mediated by this entity, which can impact on both the cytosolic and phagosomal pH. Passage of H+ through these channels could account for the inability of phagosomes to acidify rapidly and fully in neutrophils, as they do in macrophages. The objective of the present experiments was to reanalyze the determinants of the phagosomal pH of human neutrophils, with particular attention to the possible contribution of passive H+transport pathways. To this end, we used fluorescence ratio imaging to select and study single phagosomes in adherent neutrophils. This approach overcomes some of the limitations inherent in earlier studies, which may have affected their interpretation (see “Discussion”). We report that the activation of the NADPH oxidase alters pHpnot only by promoting the consumption of intraphagosomal H+(equivalents) but also by altering the permeability and fusogenic properties of the phagosomal membrane. Nigericin, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, Oregon Green 514 succinimidyl ester, SNAFL1 succinimidyl ester, SNAFL2 succinimidyl ester, dihydrorhodamine, valinomycin, and zymosan were purchased from Molecular Probes, Inc. (Eugene, OR). Concanamycin A was obtained from Kamiya Biochemical Company (Thousand Oaks, CA). Polyclonal antibody to the 39-kDa subunit of the V-ATPase was prepared as described (11Schapiro F. Sparkowski J. Adduci A. Suprynowicz F. Schlegel R. Grinstein S. J. Cell Biol. 2000; 148: 305-315Crossref PubMed Scopus (94) Google Scholar). Monoclonal antibody against CD66b was obtained from Serotec Ltd. (Oxford, England). Fibronectin was obtained from Roche Molecular Biochemicals. Diisopropyl fluorophosphate and diphenylene iodonium (DPI) were from Toronto Research Chemicals Inc. (Toronto, Canada). Human IgG, protease inhibitor mixture for mammalian cell extracts, superoxide and were purchased from of pH The and to pH at The and to pH at The pH the but was with and pH was to The and or or acid or In the was to with the to pH at Neutrophils were from obtained by using and as described A. J. Clin. Suppl. Google Scholar). neutrophils were at in and at used than zymosan was in followed by to and then and in at a of and the succinimidyl of Oregon Green or were in at The was by of the to of zymosan The mixture was then to for at with was by and the was in of with of at pH and to the This was two or while the was The was in and in The properties of the were using a fluorescence using while the were by with followed by two with Neutrophils were these which were in a with and on the of a fluorescence with a A and the in of a To and to the a was used to the of the and was to was and and to the cells by a fluorescence was through a and with a was by on an The was at by a in of the an additional in the the was to a of cell by Two independent of were the of the a of fluorescence ratio pH was obtained in by with to pH and were using a of in for to the zymosan were by the extracellular to be to the phagosomal pH these against the fluorescence were obtained using both Neutrophils were treated with or concanamycin for at to was then by the cells cells in with zymosan was When by reactive oxygen the is into the of the cell was then the and fluorescence was and using the described was using the as described Blood. 1981; PubMed Google Scholar). To phagosomes from human neutrophils, we used a of the of and M. J. Cell Biol. 1994; PubMed Scopus Google Scholar). Neutrophils in were initially treated with fluorophosphate a potent protease for at the of inhibitor by the cells were at in To were by at for with human followed by a and in of the were in a a which of the neutrophil were with an of the The were to to and the the cells and The was and to the and neutrophils. The was then in pH at with protease inhibitors and a of a mixture of protease inhibitors for mammalian cell The cells from in parallel were using a for was slowly to of and of the neutrophils. A of the was the to that cell The was at for to cells. The the was with an of pH at a of This was then on of a and with and The was then to at for at using a of the the the and the phagosomes was The was into of and at at for The the phagosomes was in and used for the and were using using the and transfer membrane were then for in a in The were with the for at The antibody were and CD66b The were then with in were with of or for followed by were using the enhanced and using of To the pHp in we zymosan that were with a of two and Oregon Green We of the a of these two and to the of of pH as in The zymosan were with human to internalization by neutrophils that been to to A digital imaging that fluorescence ratio imaging with was used to cells that with to adherent and and to the pH of the resulting in that were not in A and indicate that the of experiments both the at and the fluorescence ratio for at to the the fluorescence ratio was highly to the pH of the in the pH as predicted from the of The changes in fluorescence at and in the ratio that into phagosomes are by in A in the and in the ratio was to develop during the after In of the of the fluorescence to this would be as a in However, that the pH was In it that the pH of the been reduced after internalization the of the fluorescence ratio when pH was and was a of that seen in extracellular (see This was not to of the phagosomal pH with the extracellular during since were obtained with and of with It was reported earlier that similar to the used to pH were to by of phagocytic cells Green S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and Green S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) that the highly reactive hypochlorous which the of it was found that the fluorescence of these is This the that the pH to zymosan phagosomes generation of reactive oxygen for their This was tested using a potent inhibitor of activity Blood. PubMed Google Scholar). As in the of the fluorescence in in Moreover, the of the ratio as a of pH was also indicate that of the can during phagosomal in neutrophils and that of is for of pH by of superoxide and failed to inability to of these enzymes in the phagosomal lumen to not experiments were in with is also an inhibitor of the respiratory neutrophils have few and entirely on for their J. Clin. PubMed Scopus Google Scholar). their to and to pump H+ was by (see where of the was we found that the phagosomal pH of neutrophils for a of at Two indicate that the were fully (a) the of to the extracellular a to of and (b) the were to It is Segal et al. (10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar), we were to detect an alkalinization shortly after This could be attributed to the modest of the pH used in the since the a of is and that of Oregon is To of a possible the pH was by using acidic during However, initial alkalosis was whether the extracellular pH at the of phagocytosis was or In we tested of and The a of these are and to study pHp in the phagosomal alkalinization could be using these indicate that in single adherent neutrophils, the phagosomal pH not the pH of the extracellular that is with the We to the contribution of the NADPH oxidase to the of This was using a inhibitor of the oxidase J. J. 1999; PubMed Scopus Google Scholar). When neutrophils were to in the of a and acidification of the phagosome was In pHp at and at this for at The acidification was fully by that it is generated by The proposed earlier by Segal and (10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar) that H+ by the V-ATPase the for superoxide generation and the for its dismutation and that the two are This that of the V-ATPase would in the generation of a membrane the of a pH. The of an would in the activity of the oxidase. were tested The of concanamycin on pHp was tested in cells where the oxidase was active. to the described the V-ATPase inhibitor not an alkalinization Second, we the rate of generation of reactive oxygen using This is to the To that phagosomal to oxidase activity was we experiments where phagosomes were by and only the were by digital fluorescence imaging and We then the rate of generation in cells in the and of As in was in the activity of the oxidase when the pump was active In the fluorescence after was and in the and of indicate that the V-ATPase is not an of for the NADPH oxidase. It is that the of to is by H2O2, that similar of the were in phagosomes with and This would that the H+ by the V-ATPase is not the and not the of H+ for the dismutation The of a phagosomal acidification of the NADPH oxidase was initially to that consumption by superoxide or its proton accumulation in the phagosomal To this we at of As in the inhibitor the oxidase when after the has the of or after of As a and acidification was when the oxidase was inhibited from the and in pHp was in its However, the of after resulted in a much and While in cells pHp for (see the of with were since consumption of H+ by the oxidase was to account for the of the phagosome to acidify. We that other contribute to the inability of the phagosome to at mechanisms can be for the of of the phagosomes to become First, the activity of the proton may This in could be to of the phagosomal V-ATPases or to a in their Second, the activity of the which are M. Annu. Rev. Cell Biol. 1997; PubMed Scopus Google Scholar), may become by a in the permeability of the phagosomal membrane to the passive permeability to H+ the may have were The plasma membrane of neutrophils, which to the is of Instead, the V-ATPases are to the phagosome by fusion with are that the activity of the oxidase may the of including some in with chronic granulomatous disease (10Segal A.W. Geisow M. Garcia R. Harper A. Miller R. Nature. 1981; 290: 406-409Crossref PubMed Scopus (278) Google Scholar). It was possible that the generation of oxygen in some the insertion of V-ATPases into the phagosomal membrane. This was tested by the of V-ATPase into the membrane of phagocytosis in the and of the neutrophils were and the phagosomes were by The V-ATPase of the phagosomal was using and with to the 39-kDa subunit of the As V-ATPases were in with the original cell however, the accumulation of was in phagosomes from cells treated with the NADPH oxidase inhibitor and of from experiments that the of V-ATPases was in phagosomes from cells This is to be to of the V-ATPase and is to reflect fusion with endomembrane This is by the that also increased the of CD66b in phagosomes and CD66b is a of the membrane of neutrophil that one or of the NADPH oxidase the of fusion with the As a the of V-ATPases on the phagosomal membrane is in for the of the phagosome to acidify. in V-ATPases are still present in the membrane of these failed to acidify (see This that other contribute to the inability of these phagosomes to acidify. A mechanism could a in the required to the of the that the of may be by of the NADPH oxidase. To the of phagocytosis was to in that this would be the of the phagosomal This not the activity of the which a large and acidification in cells with As in phagosomes formed by cells with an active oxidase failed to acidify. To whether this was to the cells were then treated with valinomycin, a conductive the phagosomal membrane to this to the lumen, the of to H+ We earlier that in neutrophils, since the rapidly the when to cells A. Grinstein S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). As in the of not the acidification of that the process was not by the into the of H+ by the V-ATPase can acidification of the This backflux or is an of the pH of J. S. M.S. T.E. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). It is possible that of the NADPH oxidase may have this to the of the pH To the we first the possible and of the passive H+ (equivalent) in phagosomes from cells. As in the to a pH phagosomes have a which can be unmasked when the V-ATPase is inhibited by the plasma membrane of a and J. PubMed Scopus Google Scholar), we whether a similar pathway could contribute to the To this end, neutrophils were to zymosan in a as well as As in the of reduced the rate of of pH the of This that a pathway to the backflux of H+ from the phagosomal lumen to the cytosol. We then whether an of this pathway is responsible for the inability of phagosomes to acidify when the oxidase is were to phagosomes in the of as but was from the the of the phagosomes were to acidify when the oxidase was active We that increased H+ to the inability of these phagosomes to the are to Zn2+. To the that other of develop as a of oxidase we the passive permeability of phagosomes in cells that were treated with or Neutrophils were to develop phagosomes and were To similar the V-ATPases were inhibited with and the phagosomal pH was by the cells in of pH a pH was the phagosomal membrane by rapidly the cytosolic pH to This was by the pH to and to the the rate of alkalinization of the phagosomal lumen, we could the passive H+ (equivalent) permeability of cells with active or oxidase. The in that the rate of pHp was in than in cells. In the rate was at in than in cells. This that the activity of the oxidase increased the passive permeability of the phagosomal membrane to In we used an imaging to the determinants of pHp in neutrophils. a used in (a) It neutrophils while adherent to which the much than the neutrophils used in the (b) We to the of the used for pH may have in earlier studies, the interpretation of the imaging single we that only and not those to the neutrophil were We used after This pHp than in parallel we that the phagosomes of neutrophils from those of macrophages in that their pH is As the can be attributed to the activity of the NADPH oxidase, which is much in neutrophils. However, indicate that the of pH is not to the consumption of H+ by the dismutation of Instead, we that two to the acidification: (a) of the delivery of V-ATPases to the phagosomal membrane and (b) of a passive H+ on the phagosomal membrane. The is associated with a reduced fusion of with the phagosomal membrane. The mechanism this process is at but it may be to of the phagosomal membrane, resulting from an of the The of the pathway also It is not to and from the H+ reported to exist in we cannot the that reactive oxygen in the the V-ATPase is to that are to M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and be of the the NADPH oxidase. to the V-ATPase activity of phagosomes from and cells. the of the enzymatic activity was for whether using or We for the contribution to the phagosomal superoxide

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