Alkalinity of Neutrophil Phagocytic Vacuoles Is Modulated by HVCN1 and Has Consequences for ...

20 Pages·2015·3.48 MB·English

RESEARCHARTICLE
Alkalinity of Neutrophil Phagocytic Vacuoles
Is Modulated by HVCN1 and Has
Consequences for Myeloperoxidase Activity
AdamP.Levine1,MichaelR.Duchen2,SimondeVilliers3,PeterR.Rich3,Anthony
W.Segal1*
1 DivisionofMedicine,UniversityCollegeLondon,London,UnitedKingdom,2 DepartmentofCelland
DevelopmentalBiology,UniversityCollegeLondon,London,UnitedKingdom,3 GlynnLaboratoryof
Bioenergetics,DepartmentofBiology,UniversityCollegeLondon,London,UnitedKingdom
* [email protected]
Abstract
TheNADPHoxidaseofneutrophils,essentialforinnateimmunity,passeselectronsacross
thephagocyticmembranetoformsuperoxideinthephagocyticvacuole.Activityoftheoxi-
OPENACCESS
daserequiresthatchargemovementsacrossthevacuolarmembranearebalanced.Using
Citation:LevineAP,DuchenMR,deVilliersS,Rich thepHindicatorSNARF,wemeasuredchangesinpHinthephagocyticvacuoleandcyto-
PR,SegalAW(2015)AlkalinityofNeutrophil
solofneutrophils.Inhumancells,thevacuolarpHroseto~9,andthecytosolacidified
PhagocyticVacuolesIsModulatedbyHVCN1and
slightly.Bycontrast,inHvcn1knockoutmouseneutrophils,thevacuolarpHroseabove11,
HasConsequencesforMyeloperoxidaseActivity.
PLoSONE10(4):e0125906.doi:10.1371/journal. vacuolesswelled,andthecytosolacidifiedexcessively,demonstratingthatordinarilythis
pone.0125906 channelplaysanimportantroleinchargecompensation.Protonextrusionwasnotdimin-
AcademicEditor:AmitGaggar,Universityof ishedinHvcn1-/-mouseneutrophilsarguingagainstitsroleinmaintainingpHhomeostasis
Alabama-Birmingham,UNITEDSTATES acrosstheplasmamembrane.Conditionsinthevacuoleareoptimalforbacterialkillingby
Received:December30,2014 theneutralproteases,cathepsinGandelastase,andnotbymyeloperoxidase,activityof
whichwasunphysiologicallylowatalkalinepH.
Accepted:March21,2015
Published:April17,2015
Copyright:©2015Levineetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany Introduction
medium,providedtheoriginalauthorandsourceare
Neutrophilsthatencounterabacteriumorfungusengulfitintoaphagocyticvacuoleofinvagi-
credited.
natedplasmamembrane,intowhichcytoplasmicgranulesreleasetheircontentsofpotentially
DataAvailabilityStatement:Allrelevantdataare
lethalenzymes(Fig1).Theseprocessesareassociatedwithaburstofnon-mitochondrialrespi-
withinthepaperanditsSupportingInformationfiles.
rationinwhichelectronsarepassedacrossthemembraneofthevacuolebyanNADPHoxi-
Funding:ThisworkwassupportedbytheWellcome
dase,NOX2,thatgeneratessuperoxide[1].Thiselectrontransportisessentialforefficient
TrustGrantWT081695,theBiotechnologyand
killingofthemicrobesasevidencedbythesevereimmunodeficiencysyndromeofChronic
BiologicalSciencesResearchCouncil,andtheIrwin
GranulomatousDisease(CGD)inwhichthefunctionofNOX2isabsentorcompromised[2].
JoffeMemorialFellowship(APL).Thefundershadno
roleinstudydesign,datacollectionandanalysis, Thetransportofelectronsintothephagocyticvacuoleiselectrogenic,causingalarge,rapid,
decisiontopublish,orpreparationofthemanuscript. membranedepolarisationwhichwillitselfcurtailfurtherelectrontransportunlessthereis
compensatoryionmovement[3]bythepassageofcationsintothevacuoleand/oranionsin
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. theoppositedirection(Fig1).Thenatureoftheionsthatcompensatethechargemayhavea
PLOSONE|DOI:10.1371/journal.pone.0125906 April17,2015 1/20
NeutrophilPhagocyticVacuolarpH
Fig1.SchematicrepresentationoftheneutrophilphagocyticvacuoleshowingtheconsequencesofelectrontransportbyNOX2ontooxygen.The
proposedionfluxesthatmightberequiredtocompensatethemovementofchargeacrossthephagocyticmembranetogetherwithmodulatorsofionfluxes
areshown.
doi:10.1371/journal.pone.0125906.g001
directeffectonthepHwithinthevacuoleandthecytosol.Attemptstocharacterisemecha-
nismsofchargecompensationhaveconcentratedontheroleofprotonchannels[4],character-
isedusingdivalentcationssuchasZn2+andCd2+asinhibitors[3,5,6].Theseionsare
reasonablyselectiveforprotonchannelsinthelowmicromolarrange[4,7],buthavemultiple
othertargetswhenusedatmillimolarconcentrations[8–12].Cloningofthegeneforthepro-
tonchannelHvcn1[13,14],andthesubsequentgenerationofHvcn1knockoutmice[15]hasal-
lowedamoreprecisedefinitionofitsroleinneutrophilbiology.Contrarytopredictionsfrom
previousstudiesusinghighconcentrationsofZn2+[8],completeeradicationoftheHVCN1
channelonlyreducedoxidaseactivitybyabout50%[15,16],andhadasurprisinglysmalleffect
onmicrobialkilling[15].InhibitionordeletionoftheHVCN1channelhasbeenshowntore-
sultinexaggeratedacidificationofthecytosolafterphagocytosisofzymosan[17]orstimula-
tionoftheoxidasewithphorbolmyristateacetate(PMA)[18]whichledtothesuggestionthat
thischannelmightbeimportantfortheexpulsionofprotonsfromtheneutrophilcytosol
[17,18]althoughthiswasnotmeasureddirectlyineitherofthesestudies.Thoseobservations
raisethepossibilitythatthedepressanteffectofthelossoftheHVCN1channelonthe
NADPHoxidasemightbeduetothedevelopmentofanexcessivelyacidiccytosol,whichin-
hibitstheoxidase[19],ratherthanasaconsequenceofimpairedchargecompensation.
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NeutrophilPhagocyticVacuolarpH
InasubsequentstudyofHvcn1-/-neutrophilsphagocytosingzymosanparticles[20],averag-
ingthepHoftheentirepopulationofneutrophilphagosomesdidnotyieldasignificantdiffer-
encebetweenwildtypeandHvcn1-deficientcells.However,thepHofHvcn1-/-phagosomes
werefoundtobeheterogeneous,withathirdbeingalkalineand14%acidic.
InordertodefinetheroleoftheHVCN1channelinneutrophilbiology,wehaveusedthe
ratiometricfluorescentpHindicator,SNARF,tomeasurethepHdirectlyinindividualphago-
cyticvacuolesandinthecytosolofhuman,andwildtype(WT)andHvcn1-/-mouseneutro-
philsduringphagocytosis.ThedataconfirmthattheHVCN1channelplaysamajorrolein
chargecompensationacrossthephagocyticvacuolemembrane,andinitsabsencenon-proton
fluxespredominateinchargecompensation.
Therearetwoopposingviewsastothemechanismsbywhichtheoxidasecontributestomi-
crobialkilling.Ontheonehanditisbelievedthatmyeloperoxidase(MPO)promoteskilling
throughtheperoxidaticgenerationofhypochlorousacidfromH O producedbytheoxidase
2 2
[21].Analternativeviewisthatkillingismediatedthroughthedigestiveactionofneutralpro-
teasesthatareactivatedbytheelevatedpH[22]andinfluxofK+[23]generatedbytheoxidase.
Todeterminewhichofthesemechanismsisthemorefeasiblewithintheenvironmentofthe
phagocyticvacuole,wemeasuredthepHdependenceoftheactivityofMPOandthatofthe
twomajorneutralproteases,cathepsinGandelastase,andrelatedthesetotheobservedphago-
cyticvacuolarpHinnormalneutrophils.
Results
TherespiratoryburstisimpairedinHvcn1-/-neutrophilswhilst
extracellularacidreleaseisnormal
InWTneutrophils,oxygenconsumptionwasincreasedtenfoldbyPMAandtoasimilarlevel
bytheadditionofopsonisedCandida.PMAstimulatedoxygenconsumptionwasreducedto
73%ofnormalincellsisolatedfromHvcn1-/-mice(p=0.035),consistentwithpreviousobser-
vations[15,16,18]wasabolishedbytheoxidaseinhibitorDPI(p<0.001),andwasabsentin
neutrophilslackinggp91phox(p<0.001)(Fig2A).OxygenconsumptionbyHvcn1-/-cells
phagocytosingopsonisedCandidawas50%ofthatbyWTmouseneutrophils(p<0.001),and
significantlylowerthaninthesamecellsafterPMAstimulation(p<0.001)buthigherthanthe
20%measuredbyothers[20].UsingAmplexRedtomeasureH O generatedbyPMAstimu-
2 2
latedcells,oxidaseactivitywasdecreasedtosimilarlevelsinHvcn1-/-at58.4%±18.1(SD)
(n=5)ofthatbyWTcells.
ActivationoftheoxidaseusingPMAorCandidaincreasedtheextracellularacidification
rate(ECAR),tosimilarlevelsinHvcn1-/-andWTmouseneutrophilsandlevelsachievedwere
equivalentwithPMAorwithCandida(p=nsforallcomparisons).Theratewassignificantly
reducedinthepresenceofDPI(p=0.035)andremainedatbaselinelevelsinneutrophilslack-
inggp91phox(p<0.01)(Fig2B).Therelativelyhighextracellularacidificationobservedwhen
oxygenconsumptionbyPMAstimulatedcellsisblockedbyDPIcanbeexplainedbytheinhib-
itoryeffectofDPIonmitochondrialelectrontransport[24]aswellasthatoftheneutrophilox-
idase.Inhibitionofmitochondrialelectrontransportleadstoanaerobicglycolysiswiththe
productionoflactateandthereleaseofmoreacid[25].Nodifferencewasobservedbetween
theextracellularacidificationbetweenWTandHvcn1-/-cellsinthepresenceofPMAandDPI
(datanotshown).
OxygenconsumptionbyhumanneutrophilsphagocytosingCandida,asmeasuredwitha
Clarkoxygenelectrode,wasdeterminedas5.8fmols±0.4(SD)(n=5)perCandidaphagocy-
tosedandtherespiratoryburstwasbrief,withapeakratelastingabouttwominutes[26].In
neutrophilsfromtheHvcn1-/-micetheequivalentmeasurementwas3.2fmolscorresponding
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Fig2.Oxygenconsumptionandextracellularacidificationratebyneutrophils.(A)Oxygenconsumptionrate(OCR)and(B)extracellularacidification
rate(ECAR)byneutrophilsfromWT,Hvcn1-/-andgp91phox-/-miceinresponsetostimulationwithPMA(withandwithoutDPI)oropsonisedCandida.The
numbersofindependentexperimentsisshownoverthetotalnumberofmeasurements.Statisticalsignificance:***p<0.001,**p<0.01and*p<0.05.
DifferencesbetweenPMAstimulatedWTandgp91phox-/-werep<0.001in(A)andp<0.05in(B).Therewerenosignificantdifferencesin(B)betweenPMA
andCandidaorbetweenWTandHvcn1-/-cells.Median,quartilesand95%centilesareshown.
doi:10.1371/journal.pone.0125906.g002
tothe~50%reductionobserved(Fig2A).Giventhe4:1ratioofelectronspassingacrossthe
vacuolarmembranetooxygenconsumed(Fig1),theconsumptionof~6fmolsofoxygenper
Candidawouldrequire~24fmolsofcompensatingchargeinnormalneutrophilsand~12
fmolsinHvcn1-/-cells.
ThephagocyticvacuoleofhumanandWTmouseneutrophilsreaches
pH8.5–9whilstinHvcn1-/-cellsitexceeds11
ThefluorescenceratioofSNARFcoupledtoheatkilledCandidashowedasigmoidalrelation-
shipwithpHandwasapproximatelylinearwithpH7–10(Fig3D).WetrackedchangesinpH
inthephagocyticvacuolesofindividualneutrophilsfromthetimethatlabelledCandidawere
engulfed.Shortlyfollowingengulfment,thevacuoleunderwentasignificantalkalinisation
(Fig3).Inhumanneutrophils,thisstartedalmostimmediately(Fig3EandS1Video).The
meanmaximumpHreachedpost-phagocytosiswas9.0(SD8.3–10.2)(Fig3E)andthiselevat-
edpHwasmaintainedfor20–30minutes.WhentheNADPHoxidasewasinhibitedbyDPI,
thevacuoleacidifiedtoabout6.3(SD6.1–6.56)(Fig3Band3E),althoughSNARFdetermina-
tionsarenotveryaccurateatthispH,theseresultsaresimilartopreviousfindings[27]and
whenaddedfollowingphagocytosis,DPIrapidlyreducedthevacuolarpH(Fig4CandS2
Video)[20].TheseelevationsinvacuolarpHoccurreddespitethebufferingcapacityofthe
heat-killedCandida(S1Fig).ThemaximumdifferenceinthevacuolarpHinhumanneutro-
philsinthepresenceorabsenceofDPI(~6.5and~9,respectively(Fig3E))required1.9fmols
ofOH-perCandida.AssumingthispHrisewasentirelyduetonon-protoncharge
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Fig3.TimecoursesofchangesinpHinthevacuoleandcytoplasmofphagocytosingneutrophils.RepresentativeimagesofCandidaphagocytosed
byhumanneutrophils(A),withDPI(B),andbyHvcn1-/-neutrophils(C).StandardcurvesfortherelationshipbetweenSNARFratioandpHoforganismsand
cytoplasmareshownin(D).Candidaalonewereaddedtotwodifferentbuffersystems,labelledTrisorBarbital,andintracellularorganismswereexposedto
theBarbitalbuffersafterpermeabilisationofneutrophilswithsaponin.PanelsE-LshowtimecoursesofthepHchangesofphagocytosedCandidaand
cytoplasmofhuman(E,I),mouseWT(F,J)andHvcn1-/-(G,K)neutrophilssynchronisedtothetimeofparticleuptake(0minutes).InE-GandI-K,each
individualblacklinerepresentsserialmeasurementsoftheSNARFratioofasinglephagocytosedCandidaorneutrophilcytoplasm,respectively.Mean±SD
(shadedareas)areshown.InthecompositepanelsHandLthemeandatahavebeensmoothed.DataareplottedaccordingtoSNARFratiowiththe
approximatecorrespondingpHshownontherighty-axis.Thenumberofindependentexperimentsoverthetotalnumberofindividualcellsexaminedis
shown.TheeffectofDPIonvacuolarpHinhumanneutrophilsisshowninE(pinkanddashedlines,12cells).
doi:10.1371/journal.pone.0125906.g003
compensation,itwouldamountto~8%ofthetotal~24fmolcompensatingcharge,closeto
the~5%previouslyestimated[23].
WTmouseneutrophilsshowedasimilar,althoughlessextreme,alkalinisationofthevacu-
olereachingameanmaximumpHof~8.5(SD8.0–9.1)post-phagocytosis(Fig3F).Theinitial
risewasveryrapidandwasfollowedbyabrieffall,possiblyasaresultofdegranulationofacid
granulecontents,beforeresumingitsupwardcourseafteradelayofaboutoneminute.Bycon-
trast,thepHinthevacuolesofHvcn1-/-neutrophilswasrapidlyandgrosslyelevatedtoamean
valueof~11.1(SD9.7–12.5)(Fig3GandS3Video).Thesechangeswereobservedinalmostall
vacuoles.ThedifferencebetweentheareasunderthecurvesoftheWTandHvcn1-/-cellswas
highlysignificant(p<0.001).GiventhestrongbufferingcapacityofCandidainthealkaline
range,ariseinpHfrom~6.5inDPItreatedcellsto~11.1inHvcn1-/-required~9.2fmolsof
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Fig4.TheeffectofDPI,azide,4ABH,KCN,zinc,andCCCPonvacuolarpH.(A)Theeffectof300μMZn2+,DPIand60μMCCCPonvacuolarpHinWT
andHvcn1-/-neutrophilsat~30minutesfollowingtheadditionofCandidawithoutsynchronisationtoparticleuptake.(B)Theeffectof5μMDPI,5mMsodium
azide,50μM4ABHand1mMKCN,onvacuolarpHinhumanneutrophilsat~30minutesfollowingtheadditionofCandidawithoutsynchronisationtoparticle
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NeutrophilPhagocyticVacuolarpH
uptake.Theeffectoftheadditionofazide(C)orDPI(D)onvacuolarpHinhumanneutrophilssynchronisedtotimeofaddition(redarrows).Thenumbersof
independentexperimentsisshownoverthetotalnumberofmeasurements.Median,quartilesand95%centilesareshown.Statisticalsignificance:***
p<0.001.
doi:10.1371/journal.pone.0125906.g004
OH-perorganism(S1Fig),similartothe~12fmolsoftotalcompensatingcharge.Inasmall
numberofHvcn1-/-cells(3.3±3.8SDof8experimentsand810cells)thevacuolescollapsed
(e.g.S3VideoandS4Video),whichcoincidedwithsuddenalkalinisationofthecytoplasm.
Theseextremeconditionscouldimpairtheoxidaseinphagocytosingneutrophils.
ThealkalinisationinHvcn1-/-vacuoleswasalmostcompletelypreventedbytheadditionof
theprotonophorecarbonylcyanide-m-chlorophenylhydrazone(CCCP),whichwillproducea
protonshunt,whereasZn2+,increasedalkalinisationofvacuolesinWTmouseneutrophilsata
concentrationof100μM(Fig4A).Sodiumazidehasbeenused[20,28]toinhibittheactionof
MPOwhichwasthoughttoquenchthefluorescenceoffluorescein,theflurophorepreviously
utilisedtodeterminevacuolarpH.Wethereforedeterminedtheeffectoftheadditionofazide
onSNARFfluorescence.Inendpointassays,azidesignificantlyreducedthepHinthevacuoles
at~30minutesinhumanneutrophils(p<0.001,Fig4B)oralmostimmediatelywhenadded
duringatimecourse(Fig4D).ThiseffectofazidewasnotduetoitsinhibitoryeffectonMPO
becauseneither4-aminobenzoicacidhydrazide(4ABH),aselectiveinhibitorofMPO,at
50μM,morethanthreetimestheIC50[29](Fig4B),nor1mMKCN,depressedvacuolarpH.
KCNshowedcompletesaturationofbindingtoMPOinintactneutrophilsatthisconcentra-
tion(S2Fig)atwhichitinhibitsitsperoxidaticactivity[30].WhenSNARF-labelledCandida
wereaddedtobuffersatvaryingpHintheabsenceorpresenceofazide,noeffectofazidewas
seenontheSNARFratio(S3Fig)oftheCandidathemselves.
CytoplasmicpHfallsafterphagocytosis,andthisisexaggeratedin
Hvcn1-/-cells
InhumanorWTmouseneutrophilstherespiratoryburstwasassociatedwithamodestacidifi-
cationofthecytoplasm,fromapHof7.56(SD7.49–7.62)(S4DFig)toameannadirpHof
7.30(SD7.09–7.49)(Fig3IandS1Video),withhomeostasisbeingachievedovertheensuing
20–30minutesasdescribedpreviously[31–33].NeutrophilsfromWTmicebehavedsimilarly
(Fig3J).InHvcn1-/-neutrophilstheacidificationwasmuchmorepronounced(p<0.001,Fig
3KandS3Video),reachingaminimummeanpHof~6.72(SD6.08–7.12)at2.0minutesafter
phagocytosis,beforereturningtonormalafterabout20–30minutes.Therateofrecoveryofcy-
toplasmicpHafteracidificationwith20mMNH Clforfiveand20minuteswasnodifferent
4
betweenWTandHvcn1-/-neutrophils(S4AandS4BFig,respectively).
Thebufferingcapacityoftheneutrophilcytosolhaspreviouslybeendeterminedatbetween
28[34]and50[32]mMunit-1.Givenanapproximateneutrophilvolumeof~300μm3[35]
andthe0.2unitfallincytoplasmicpHinhumanneutrophils(from7.5to7.3)thisequatesto
theaccumulationof~2.3fmols(39mMunit-1×0.2unit×300μm3)ofprotonsleftinthecyto-
solwhenthechargeiscompensatedbyalternativeions(9.6%of~24fmol).InHvcn1-/-cellsthe
fallincytoplasmicpHto6.6equatesto~10.5fmolofprotons(calculatedasabove).
Hvcn1-/-vacuolesarelargerthannormal
ThevacuolesofHvcn1-/-neutrophilscontainingCandidaunderwentaprofoundswelling,
whichwasmuchmoreobviousthaninWTmouseorhumancells(S1VideoandS3Video).To
determinetheextentofthisswellingandtodistinguishitfromthatinducedbytheosmoticef-
fectsoftheproductsofdigestionoftheCandida,wemeasuredthecross-sectionalareasof
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Fig5.VacuolarsizeinWTandHvcn1-/-neutrophilscontainingasinglelatexparticle.Thecross-sectionalareaofalatexparticleis~7μm2.
RepresentativeimagesofaWT(A)andHvcn1-/-neutrophilcontainingasinglelatexparticleareshownin(A)and(B),respectively.(C)Quantitationof
vacuolarswellinginHvcn1-/-neutrophilscomparedwithWT,andtheeffectsof5μMDPI,60μMCCCPand3μMvalinomycin.Thenumbersofindependent
experimentsisshownoverthetotalnumberofmeasurements.Median,quartilesand95%centilesareshown.Statisticalsignificance:***p<0.001and**
p<0.01.
doi:10.1371/journal.pone.0125906.g005
vacuolescontainingasingleindigestiblelatexparticlewithadiameterof3μm(across-section-
alareaof7.1μm2),similartothatofCandida.InneutrophilsfromHvcn1-/-mice,thevacuoles
swelledtoamediancross-sectionalareaof11.2μm2(quartiles8.5–16.6)comparedwith
7.9μm2(6.8–9.1)inneutrophilsfromWTmice(p<0.001)orHvcn1-/-treatedwith5μMDPI
(7.9μm2)(6.8–8.8)orCCCP(7.9μm2)(6.9–9.0)(Fig5C)after30minutes.Theseresultsindi-
catethatosmoticallyactiveionsaredrivenintothevacuolebytheoxidaseintheabsenceofthe
protonchannel.Thisswellingwasreducedsignificantly(p<0.001)by3μMvalinomycintoa
medianareaof9.9μm2(7.1–12.8).Theestimatedvolumesutilisingtheradiuscalculatedfrom
themediancross-sectionalareaandassumingsphericalvacuoleswere:latexparticles
(14.2μm3);WTvacuoles(16.7μm3)andHvcn1-/-(28.2μm3)withoutand(23.4μm3)with
valinomycin,respectively.
IfthechargecompensationinHvcn1-/-cells(~12fmols)werepurelyaccomplishedbythe
influxofcations,e.g.K+,toaconcentrationthatbalancedthecytosolicosmoticpressureof
~300mOsm,takenasclosetothatofextracellularfluid[36],itwouldproduceavacuolarvol-
umeof~40μm3(12fmols/300mM)greaterthanthatofthelatexparticlesalone,considerably
largerthanthatobserved.Thissuggeststhatnon-osmoticallyactivecations,suchasprotons,
passintothevacuole,orthatanionssuchasCl-,passout,oracombinationofthesetwoevents.
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Fig6.EffectsofpHonenzymaticactivity.TheeffectofvariationsinpHonperoxidaticandchlorinatingactivitiesofMPOandontheproteaseactivitiesof
cathepsinGandofelastaseareshown.Resultsshownarethemean+SDofatleastthreeseparateassaysandareexpressedasapercentageofthe
maximalobservedactivity.
doi:10.1371/journal.pone.0125906.g006
PhysiologicalpHinhumanneutrophilvacuolesisoptimalforactivityof
granuleenzymescathepsinGandneutrophilelastasebutincompatible
witheffectiveperoxidaticandchlorinatingfunctionofMPO
TheinfluenceofpHontheperoxidaticandchlorinatingactivitiesofMPOandontheprotease
activitiesofcathepsinGandelastaseareshowninFig6.Peroxidaseandchlorinatingactivities
ofMPOweremaximalatacidpH,withkcatforTMBoxidationandMCDchlorinations-1at
pH5.0being36and19,respectively.BothactivitiesfelloffsubstantiallywithelevatedpHuntil
virtuallynothingofeithercouldbedetectedatpH9or10.Bycontrast,activityofbothcathep-
sinGandelastasewaslowatpH5,andthatforcathepsinGpeakedatbetween7and9whereas
elastasewasmostactivebetween8and10.
TheSODactivityofMPOshowedasimilarrelationshiptopHasdiditsperoxidaseactivity
whereasthecatalaticactivitywaslowestatneutralpHandincreasedatpH5and6,and10at
whichkcat=3O generateds-1(S5Fig).
2
Discussion
Theoriginaldescription[22]ofanalkalinisationoftheneutrophilphagocyticvacuolebythe
NADPHoxidasetoapHof~7.5–8hasbeenreplicatedanumberoftimes[27,37,38].Inthose
studies,fluoresceinwasemployedaspHindicator.FluoresceinsaturatesatapHof~8and
above,wasnotgenerallyusedinaratiometricmanner.Inaddition,ithasbeenthoughtby
sometobecomebleachedbytheactionofMPOinthephagocyticvacuole[39,40].
SNARFismuchbettersuitedtothedeterminationofthevacuolarpH.Ithasadynamic
rangeofbetweenpH6and11withapproximatelinearitybetween7and10(Fig3D),isratio-
metric,andcanalsobeusedtosimultaneouslymeasurethepHinthevacuoleandcytoplasm.
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OutsideofpHinterval,SNARF-inferredpHmeasurementsarelessprecise.Wehaveshownin
severalwaysthatitsfluorescentpropertiesarenotalteredbytheconditionsinthephagocytic
vacuole.Theextentanddurationofalkalinisationweobservedinthehumanneutrophilswas
muchgreaterthanthatpreviouslydescribedbyus[22]andothers,inpartbecauseSNARFis
moresuitedtomeasurementsinthealkalinerange.Inaddition,weonlymeasuredthepHof
Candidainsidevacuoles,sothatthefluorescencesignalwasnotinfluencedbythatofthenon-
phagocytosedorganismsthatarebufferedbytheextracellularmedium.Synchronisingthe
changesinfluorescencetothetimeofparticleuptake,gaveamoreaccurateindicationofthe
changesinpHovertime.
BothstudiesthatwereunabletodetectanelevationofneutrophilvacuolarpHaboveneutral
[20,28]includedsodiumazideinallthesolutionstocounteractbleachingofthefluoresceinby
MPO[28].WefoundherethatazidesignificantlyreducedthevacuolarpH(Fig4Band4D)by
amechanismthatdidnotinvolvetheinhibitionofMPO;4ABH,whichisamorespecificMPO
inhibitor,hadnosucheffectataconcentrationofoverthreetimestheIC50[29]andtheaddi-
tionofazideproducedarapiddropinthepHofpreviouslyalkalinevacuoles(Fig4D).KCN,
whichinhibitsMPO[41],alsohadnoeffectonvacuolarpHatconcentrationsatwhichwe
demonstratedittobindtoMPOtosaturationinintactneutrophils.ElChemalyandcolleagues
[20]observedthreedifferentpopulationsofphagocyticvacuoleinHvcn1-/-neutrophils,athird
reachingapHofover8.3,surprisinglyhighwhenusingfluoresceinasindicator[27],and14%
wereacidic.TheycorrectlyconcludedthatthealkalinevacuolesindicatedthattheHVCN1
channeltransportedprotonsintothevacuole.Theysuggestedthattheoxidaserepelled
V-ATPasesfromassociationwiththevacuoles,andthattheacidicvacuoleswerecausedby
weakoxidaserepulsionoftheseV-ATPaseswhichthenpumpedprotonsintothevacuole.We
didnotobservethesameheterogeneitywithourcellswhichwerepurifiedfromperipheral
blood(Fig3E).Wealsoobservedaveryalkalineandamoreacidicpopulationofvacuoles
whenweperformedexperimentsonbonemarrow(datanotshown).Itispossiblethatsome
immatureneutrophilsinthebonemarrowhaveanabnormallylowrespiratoryburst.
TheaccuratemeasurementofvacuolarpHinnormalhumanneutrophilsisabsolutelycen-
traltounderstandingthemechanismsbywhichthesecellskillbacteriaandfungi.Astrong
bodyofopinionsupportstheconceptthatMPOis“afrontlinedefenderagainstphagocytosed
microorganisms”[21]andthatitkillsmicrobeswithinthiscompartmentthroughthegenera-
tionofHOCl.ThevirtualabsenceofperoxidaseandchlorinatingactivityofMPOatpHofbe-
tween8.5and9.5pertaininginthevacuole,andthedemonstrationthattheantimicrobial
activityofMPOisonlyobservedunderacidconditions[41],arguesstronglyagainstthispossi-
bility.However,asshownhere,thesepHprovideanoptimalmilieuforthemicrobicidalanddi-
gestivefunctionsofthemajorgranuleproteases,elastaseandcathepsinGwhichareactivated
bythiselevatedpHandtheinfluxofK+intothevacuole[23,42].Theseresultssupportthe
findingthatknock-outmicelackingcathepsinGandelastase,whilstretainingnormaloxidase
andMPOactivities,arerenderedundulysusceptibletoinfectionbyStaphylococcusaureus,
CandidaalbicansandAspergillusfumigatus[23,43,44].ItispossiblethatMPOhastwodiffer-
entfunctionsinvivo,dependinguponthelocalenvironment.ThepHof~9inthevacuolefa-
vourscatalaseactivityratherthatofaperoxidase,whereas,whentheneutrophilencountersan
organismthatitisunabletofullyengulf,suchasafungalmycelium,thesituationisdifferent
[45].TheconcentrationofMPOandH O willbemuchlower,thepH,whichislowininflam-
2 2
matoryfociat~6[46]isoptimalforperoxidaticactivity,andthesupplyofchlorideislimitless.
ThegrossdifferenceinthevacuolarpHbetweenHvcn1-/-neutrophilsandthosefromWT
miceandhumansmustindicatethatfewerprotonspassintothevacuoleintheHvcn1-/-cells,
providingfirmevidencethatunderphysiologicalconditionstheHVCN1channelcompensates
theoxidaseinducedchargebyconductingprotonsacrossthevacuolarmembrane.The
PLOSONE|DOI:10.1371/journal.pone.0125906 April17,2015 10/20