CorrespondencehahnedrfzdeContributedequallyDepartmentofRheumatologyandClinicalImmunologyCharit ID: 380742
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RESEARCHARTICLEOpenAccessHumanmonocytesandmacrophagesdifferintheirmechanismsofadaptationtohypoxiaMoniqueFangradt,MartinHahne,TimoGaber,CindyStrehl,RomanRauch,PaulaHoffMaxLöhning,Gerd-RüdigerBurmesterandFrankButtgereitAbstractIntroduction:Inflammatoryarthritisisaprogressivediseasewithchronicinflammationofjoints,whichismainly *Correspondence:hahne@drfz.deContributedequallyDepartmentofRheumatologyandClinicalImmunology,CharitéUniversityHospital,Charitéplatz1,Berlin,10117GermanyFulllistofauthorinformationisavailableattheendofthearticleetalArthritisResearch&Therapyhttp://arthritis-research.com/content/14/4/R181 ©2012Fangradtetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. ATPproduction,becausethisprocessdoesnotrequireoxygen[6,7].Hypoxiaoccursinjointinflammationsuchasduringthepathogenesisofrheumatoidarthritis[1,5],fracturehematomas[8],andmalignanttumors[9].Ngetal.demonstratedinrecentinvivostudiesthataninversecorrelationexistsbetweensynovialoxygenpartialpres-sureandinflammatoryactivityinpatientswitharthritis:thestrongertheinflammation,themorepronouncedthelocalhypoxia.Kennedyetal.showedanti-TNF-therapy,widelyusedtotreatRA,increasestheoxygenpartialpressureinthejoint.Fortheinitialinflammatoryenvir-onmentinanearlyfracturehematoma,thespecificcyto-kinepatternandtypicalgenesignaturesinimmunecellsreflectasituationoflocalhypoxia[8].Inaddition,Vaupeletal.showedtheimportantroleofhypoxiaandhypoxia-induciblefactors(HIFs)intumorigenesis[9].Duringmonocytopoiesis,monocyteprecursorcellsinthebonemarrow,monocytesinthebloodandmacro-phagesinthetissuearesubjectedtoverydifferentoxy-genlevels.Forexample,anoxygenpartialpressure)of10mmHg(1.3%O)ispresentinthebonemarrowofmice[10].Incontrast,muchhigherpOvaluesof50to100mmHg(6to13%O)intheperiph-eralbloodandof20to50mmHg(approximately2.5to6.0%O)inhealthytissueweremeasured[11].Further-more,ininflamedareasmacrophagesfacepathophysio-logicalhypoxia.Intheseregions,theoxygencontentislowerthaninhealthytissuesbecauseofimbalancebetweenprovisionandconsumptionofoxygen.Dis-turbedbloodcirculationandinflammatoryswellingsresultinginalengtheningofthediffusiondistancedecreasetheoxygensupplywhereastheinfluxofmeta-bolicallyactiveimmunecellsstronglyincreasestheoxy-genconsumption[12].Forthesereasons,cellsareforcedtoadaptimmediatelytothereducedpOlevelswhenenteringtheselowoxygenatedareas.Themechan-ismofthisadaptationandthetemporalrelationshipofthisresponsetoactivationandmigrationarenotyetfullyunderstood.RapidadaptationofmonocytestohypoxiamayinvolveHIForotherfactors.Inothercells(forexample,T-cells),itisknownthatthetranscriptionfactorHIF-1underhypoxicconditionsistranslocatedintothenucleusandbindstopromoterregionsoftargetgenestoenablethenecessaryadaptationandmaintenanceofbasicfunctionslikemotion,activationandeffectorcellfunction[12,13].However,therearedivergentviewsontheexpressionandfunctionofHIFinprimaryhumanmonocytes.NeitherHIF-1,HIF-2norHIF-3werefoundbyElbarghatietal.inprimarymonocytesafterincubationunderhypoxiafor24h.Theauthorssuspectedthatthe-subunitofHIFisnotexpressed,becausetheperipheralbloodasaplaceofresidenceofcirculatingmonocytesischaracterizedphysiologicallybyahighpO[11,14].How-ever,itshouldbenoted,thatmonocyteshavetoadapttoloweroxygenlevelsimmediatelyoncetheystartthepro-cessofbeingattractedtothevesselswall,migratingintotheinflamedtissueandstartingtodifferentiateintomacrophages.CXCR4transcriptlevelshavebeenshowntoincreaseinmonocytesfacinghypoxia,whichsuggestsHIFiscruciallyinvolvedinregulatingthetrafficking[15].Furthermore,inmyeloidcelllineslikeTHP-1cellsincu-batedunderhypoxia,HIF-1wasdetectable[16].TherehavealsobeenreportsthatnuclearfactorofkappalightpolypeptidegeneenhancerinB-cells(NFB),atranscrip-tionfactoralsoregulatedbyhypoxia,isinvolvedintheadaptationofprimaryhumanmonocytestohypoxia[17].Herewehaveexaminedhowhumanmonocytesadapttohypoxicconditionsduringtheirdifferentiationintomacrophages.WefocusedontheanalysisofexpressionandfunctionofHIF-1,butalsoconsideredalternativepathwaysinvolvingNFMaterialsandmethodsAntibodiesandreagentsPMA,macrophagecolonystimulatingfactor1(hM-CSF),andGÖ6976werepurchasedfromSigmaAldrichChe-mieGmbH(Hamburg,Germany),ImmunoTools(Frie-soythe,Germany),MerckKgaA(Darmstadt,Germany).Fortolllikereceptor(TLR)stimulation,hTLRligandSetIIwasboughtfromApotech(Epalinges,Switzerland).Forimmunoblotting,mousemonoclonalanti-HIF-1bodywasboughtfromBDTransductionLaboratories(Heidelberg,Germany);mouseanti--actinantibodywaspurchasedfromSigma-Aldrich(Hamburg,Germany);goatpolyclonalanti-HIF-2,goatpolyclonalanti-LaminBandmouseanti-Jun-BantibodywereboughtfromSantaCruzBiotechnology(Heidelberg,Germany);mousemonoclonalanti-NFBp100/p52,anti-NFBp105/p50,Bp65,anti-c-Rel,anti-c-Fos,anti-c-Junanti-bodywereboughtfromCellSignaling(Frankfurt/Main,Germany);anti-mouseIgGHRPandanti-goatIgGHRPwereboughtfromPromega(Mannheim,Germany).Monocyteisolation,cultureandmacrophageHumanperipheralblood(buffycoats)wasobtainedfromhealthydonors(DRK-BlutspendedienstOstgemeinnützigeGmbH,Berlin/Brandenburg/Sachsen,Germany,withapprovalfromtheCharitéEthicsReviewBoard).PeripheralbloodmononuclearcellsfromthesebuffycoatswerethenimmediatelyisolatedbydensitygradientcentrifugationusingFicoll-PaquePlustechni-que(AmershamBioscienceAB,Freiburg,Germany).Toensureastableexperimentalsetupandcomparablestartingconditions,CD14monocyteswereenrichedupto99%purityand&#x-361;95%viability(datanotshown)byetalArthritisResearch&Therapyhttp://arthritis-research.com/content/14/4/R181Page2of12 MACSusinganti-humanCD14conjugatedmagneticbeads(MiltenyiBiotec,BergischGladbach,Germany)andthenimmediatelyusedfortheexperiments.Mono-cyteswereculturedat4×10cells/mlinRPMI1640supplementedwith10%volume/volumepercent(v/v)heat-inactivatedFCS(Sigma-Aldrich,Hamburg,Germany),100units/mlpenicillinG,100g/mlstrepto-mycin(bothPAALaboratories,Pasching,Austria),andM2-ME(Sigma-Aldrich,Hamburg,Germany).Monocyteswereincubatedwith25nMhM-CSFfor7daystodifferentiateintomonocytederivedmacro-phages(hMDM).CultureofcelllinesTHP-1,HL-60,andU937cells(AmericanTypeCultureCollection)wereculturedinRoswellParkMemorialInsti-tutemedia(RPMI)1640supplementedwith10%(v/v)heat-inactivatedFCS,100units/mlpenicillinG,100g/mlstreptomycin,and50M2-ME.Humanmicrovascularendothelialcells(HMEC-1)werepurchasedfromtheCen-terofDiseaseControl(Atlanta)andwerecultivatedinendothelialbasalmedium(PAALaboratoriesGmbH,Pasching,Austria)supplementedwith5%(v/v)heat-inacti-vatedFCS(Sigma-Aldrich,Hamburg,Germany),100units/mlpenicillinG,100g/mlstreptomycin(bothPAALaboratoriesGmbH,Pasching,Austria),1%(v/v)200mML-glutamine,0,01%(v/v)endothelialgrowthfactor(EGF)g/ml),0,2%(v/v)hydrocortisone(380M)(bothSigma)andgrownin0.2%gelatine-coated75cmflasksor24-wellplates,respectively.InductionofhypoxiaandstimulationCellswereincubatedinahumidifiedhypoxicchamber(Binder,Tuttlingen,Germany)at5%COlevelandlessthan1%ObalancedwithN.Normoxiccontrolswereincubatedat5%COinahumidifiedatmospherewith18%O.StimulationwasdonewithPMA10ng/ml(16nM)(SigmaAldrichChemieGmbH,Hamburg,Germany).Forkineticanalyzesunderhypoxia,monocyteswereincubatedinawater-jacketchambersealedwithaClark-typeoxygenelectrode(StrathkelvinInstruments,NorthLanarkshire,Scotland)asdescribedpreviously[12].RNAisolationandquantitativereal-timePCR(qPCR)ofselectedgenesAftercelllysis,totalRNAwasextracted(RNeasyMiniKit;Qiagen,Hilden,Germany)andthequalitywasassessedonaBioanalyzer(Agilent).ThecDNAwassynthesizedbyreversetranscriptionusingTaqManReverseTranscriptionReagents(AppliedBiosystems,Darmstadt,Germany).qPCRwascarriedoutusingtheFastStartDNAMasterSYBRGreenIKit(Roche,Mannheim,Germany).Datawerenormal-izedtotheexpressionof-actin(ACTB).AllprimersusedwereobtainedfromTIBMolbiol(Berlin,Germany):-actin(CACT,TgATCCACATCTgCTggAAggT;hypoxia-induci-blefactor1,alphasubunit(CCATTAgAAAgCAgTTCCgC,TgggTAggAgATggAgATgC;lactatedehy-drogenaseA(LDHACCCAAAATgCAAggAACACT;phosphoglyceratekinaseATggATgAggTggTgAAAgC,CAgTgCTCA-chemokine-receptor4(ggCATgACggACAAgTACAggCT,AAAgTACCAgTTTgC-CACggC(Table1).Celllysis:forwholecellextractsofmonocytes,10werelysedin20lLaemmlibuffer.Forthepreparationofnuclear/cytoplasmicfraction,4*10cellswerelysedusingtheNuclearExtractKitfromActiveMotif(LaHulpe,Belgium),accordingtothemanufacturersinstructions.Immunodetectionofproteins:20lofwholecellextractornuclear/cytoplasmicfractionwasseparatedbySDS-PAGEandblottedontopolyvinylidenedifluoridemembranes(Millipore,Darmstadt,Germany).Blottedproteinswereanalyzedasindicatedandvisualizedbyenzymaticchemiluminescence(AmershamBiosciences,FreiburgGermany).StatisticalanalyzesDatashownarereportedasthemean±SDofatleastsixexperiments.Differencesbetweennormallydistribu-tedgroupswerecomparedusingtheStudent-testandinnon-normallydistributedgroupswiththeMann-Whitney-testforindependentgroupsandwiththe-testfordependentsamples.ResultsisstabilisedunderhypoxiainhumanmonocytesbutremainsinthecytoplasmInordertoinvestigatefirstthestabilisationofHIF-1afunctionofpOvaluesanddurationofincubation,MACS-isolatedCD14monocyteswereincubatedinaClark-typeelectrodefor5h(Figure1A)withasubse-quentreoxygenationtimeof12minutes.ImmunoblotanalyzesrevealedthatmonocytestabilisationofHIF-1beginswhenhypoxia(pO2%)commences.Withincreasingdurationofhypoxia,theaccumulationofHIF-increased(Figure1B).Therewasmarkedaccumula-tionofHIF-1duringincubationunderhypoxia.Asexpected,reoxygenationcausedanimmediatedegrada-tionofHIF-1(Figure1B).Next,weanalyzedthecytosolicandnuclearfractionsafter5hincubation,inordertodefinetheexactloca-tionofHIF-1.HIF-1wasfoundexclusivelyinthecytoplasmandnotinthenuclearfractionofhypoxicmonocytes(Figure1C).etalArthritisResearch&Therapyhttp://arthritis-research.com/content/14/4/R181Page3of12 TLRstimulationdoesnotaffectHIF-1Followingtheseobservations,weinvestigatedwhetherconcurrentTLRstimulationofhumanhypoxicmonocytesisneededfortranslocationofHIF-1intothenucleus.Weincubatedthecellsfor5hunderhypoxia,withcon-currentstimulationofTLR1-9usingarangeofligands(Figure2A).TLRstimulationunderhypoxicconditionsdidnotleadtotranslocationofHIF-1intothenucleus,regardlessoftheligandandconcentrationused.Represen-tativeexperimentalresultsareshowninFigure2B-D,obtainedwithPam3HCl(toll-likereceptor(TLR)-1/2stimulation),lipopolysaccharideLPS(TLR4stimulation)andR-848(TLR7/8stimulation).Underallhypoxiccondi-tionstested,HIF-1wasdetectableexclusivelyinthecyto-solfractionofprimaryhumanmonocytes.isessentialforHIF-1WeexaminedwhetherstimulationwithPMAleadstotranslocationofHIF-1intothenucleus.PMAisusuallyappliedtodifferentiatemonocytesoverashorttimetoamacrophage-likephenotype.HIF-1cannotbefoundinunstimulatedmonocyteswhenincubatedundernor-moxia,asshownbyimmunoblotanalyzes(Figure3A).However,ifthecellswerestimulatedwithPMAfor5hundernormoxia,aweakHIF-1signalinthecytosolfractionwasdetectable.AlthoughHIF-1wasdetectableunderhypoxiainunstimulatedmonocytesexclusivelyinthecytoplasm,inhypoxicPMA-stimulatedmonocytesitwasdetectablenotonlyinthecytoplasm,butalsointhenucleus.ThesignalstrengthofHIF-1seeninhypoxicPMA-stimulatedcellswasstrongerthaninhypoxicunsti-mulatedmonocytes.SincePMAisknowntobeaPKCactivator,weincubatedmonocytesfor5hunderhypoxiastimulatedwithPMA,withconcurrentadditionofthePKC--inhibitor,Gö6976,atincreasingconcentrations.Figure3Bshowsthattheinhibitorataconcentrationof50nMreducedtheaccumulationofHIF-1inthenucleus.WithaGö6976concentrationof100nM,HIF-1wasnolongerdetectableinthenucleus.ThesedatademonstratethatPKC-essentialforthetransportofHIF-1fromthecytoplasmintothenucleus.MonocytedifferentiationtomacrophagesleadstoHIF-1Weconsideredwhetherdifferentiationofhumanmono-cytesintomacrophages(hMDMs)usinghM-CSFalsocausedhypoxia-inducedtranslocationofHIF-1intothenucleus.ThemacrophagenuclearfractionwasidentifiedusingthelocationofLaminB;thelocationofactinwasnotusedasthismaybefoundinthenuclearfractionofmacrophagesduetoalterationsinthecytoskeletonofmacrophagesafterstimulation,asdescribedbyHartwigandJanmey[18].Incubationofmonocyteswith25nMhM-CSFover7daysledtodifferentiationintohMDM.AfterincubationofmonocytesandhMDMsfor5hunderhypoxia,HIF-1themonocytescouldonlybedetectedinthecytosoliccompartment,whileinhMDMsHIF-1wasseentoresideinthenuclearextract(Figure4A).Wealsoconsideredwhetheranypossibleprioradhesionofhumanmonocytestoendothelialcells,asaninitialstepofmigration,couldinitiatethetranslocationofHIF-1intothenucleus.Humanmonocyteswereincubatedfor5hinplateswithwellscoatedwithhumanmicrovascularendothelialcells(HMEC-1),underhypoxiaandundernor-moxia.ThecellularlocalizationofHIF-1wasinvesti-gated.Weobservedthatco-culturewithendothelialcellsofthistypedidnotinduceaccumulationofHIF-1inthenucleusofthemonocytes.HIF-1wasfoundexclusivelyinthecytosolfractionofmonocytes,asassessedbyimmu-noblot(Figure4B);HIF-1wasnotdetectableundernor-moxia.Takentogether,thesefindingssuggestthatadhesionofmonocytetothevascularwallisnotsufficientfortranslocationofHIF-1tothenucleus.Hypoxia-inducedgeneexpressionofhumanmonocytesversushMDMsNextwecomparedtheexpressionofselectedhypoxia-inducedgenesinhumanmonocytesandhMDMs.We Table1PrimersetsusedGenesymbolGenenameGenefunctionStructuralhousekeepergACAggATgCAgAAggAgATCACTTgATCCACATCTgCTggAAggThypoxia-induciblefactor1,alphasubunitTranscriptionfactorCCATTAgAAAgCAgTTCCgCTgggTAggAgATggAgATgClactatedehydrogenaseAACCCAgTTTCCACCATgATTCCCAAAATgCAAggAACACTphosphoglyceratekinase1ATggATgAggTggTgAAAgCCAgTgCTCACATggCTgACTchemokine-receptor4chemokine-receptorggCATgACggACAAgTACAggCTAAAgTACCAgTTTgCCACggCetalArthritisResearch&Therapyhttp://arthritis-research.com/content/14/4/R181Page4of12 examinedgenesthathavebeenidentifiedastypicalHIF- 1targetgenessuchastheglycolysisenzymes LDHA and PGK1 ,andthechemokinereceptor CXCR4 . Inmonocytesincubatedunderhypoxicconditions-in contrasttonormoxia-genesfor LDHA and CXCR4 weresignificantly( P 0.05)upregulated,althoughHIF- 1 a isnotpresentinthenucleus;thegenefor PGK1 showedincreasedexpressionbutthisdidnotreachsta- tisticalsignificance(Figure5A-C). HIF1A asatarget geneofHIF-1itselfisregulatedattheproteinleveland Figure1 Hypoxia-induciblefactoralpha(HIF-1 a )isstabilizedunderhypoxiabutremainsinthecytoplasm .( A )Schemeofsample acquisitionforkineticanalyzesofmonocytesincubatedinawater-jacketchambersealedwithaClark-typeoxygenelectrode,whichfacilitates theconstantmonitoringofoxygensaturationduringtheexperimentalsetup(sampleacquisitionisindicatedbyarrows).( B )DetectionofHIF-1 a and,fornormalizationpurposes, b -actininmonocytewholecellproteinsamplesacquiredasshownin(A)byimmunoblot.Underhypoxia, monocytesstabilizeHIF-1 a inatime-dependentmanner.( C )DetectionofHIF-1 a , b -actinandfornormalizationpurposes,thenuclearprotein LaminB,inmonocytenuclearfraction(NF+)andcytosolic(NF-)cellfractionsusingimmunoblot.HIF-1 a wasexclusivelydetectedinthe cytoplasminunstimulatedmonocytesincubatedunderhypoxicconditions( n =6). Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page5of12 thereforeshowednomeasurableinduction(Figure5D). Macrophagesundernormoxiashowedhigherexpression ofthegenes LDHA (1.7-fold)and PGK1 (2.3-fold)than monocytes(Figure5Aand5B).Therewasnosignificant differenceintheexpressionofthesegenesunder hypoxiaversusnormoxia,presumablybecausemetabo- lismhadalreadybeenswitchedfromoxidativephos- phorylation/respirationtoa naerobicglycolysisduring differentiation. Similarfindingswereobservedfor CXCR4 (Figure5C), wherehypoxicincubationdidnotleadtoasignificant increaseof CXCR4 expressioninhMDMs.Alsoin hMDMs,hypoxiadidnotinduceanymeasurableupre- gulationof HIF1A (Figure5D). Incubationofmonocytesunderhypoxialeadsto translocationoftranscriptionfactorNF B1intothe nucleus AstheexpressionofHIFtargetgenes(forexample, LDHA , CXCR4) inmonocyteswasinducedbyhypoxia althoughHIF-1 a wasnotpresentinthenucleus,we consideredothertranscriptionfactorsthatcouldbe involved. Weexaminedthecellularlocalizationoftranscription factorsNF Bp100/p52,c-Rel,andc-Jun(Figure6A), NF Bp105/p50andc-Fos(Figure6B),andJunBand NF Bp65(Figure6C)inmonocytesthatwereincu- batedfor5hunderhypoxia.Althoughothertranscrip- tionfactors(forexample,c-FosandNF Bp65)didnot changetheirlocalizationwhenincubatedunderhypoxic versusnormoxicconditions,NF Bp105(theinactive formofNF B1)remainedinthecytoplasm,whilethe activeformNF Bp50wastranslocatedintothenucleus. TheactiveformofNF B2(NF Bp52)andc-Juncould beseeninthenucleusundernormoxic,butnotunder hypoxicconditions. THP-1,HL-60,andU937cellsexpressHIF-1 a inthecell nucleusunderhypoxicandnormoxicconditions Myeloidcelllinesareoft enusedasanexperimental modelforprimaryhumanmonocytes.Weconsideredin whichcellularcompartmentHIF-1 a couldbefoundin Figure2 Toll-likereceptor(TLR)stimulationdoesnotaffecthypoxia-induciblefactoralpha(HIF-1 a )localization .( A )TableofTLRsand theircorrespondingligandsusedinourexperimentswithinthegivenconcentrationrange.( B - D )DetectionofHIF-1 a ,LaminBand b -actinin monocytenuclear(NF+)andcytosolic(NF-)cellfractionsusingimmunoblot.MonocyteproteinlysateswereacquiredafterTLRstimulationand incubationfor5hunderhypoxiausing( B )TLR1/2stimulationbyPam 3 CSK 4 3HCl,( C )TLR4stimulationbyLPSor( D )TLR7/8stimulationbyR- 848. Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page6of12 unstimulatedandPMA-stimulatedmyeloidcelllines (THP-1,HL-60,andU937)underhypoxicconditions. WeidentifiedHIF-1 a inthenucleusbothundernor- moxicandhypoxicconditionswithorwithoutPMAsti- mulation(SeeFigureS1A-CinAdditionalfile1).We concludethatinthisregard,thecelllinesclearlydiffer fromprimaryhumanmonocytesandbehavelike hMDMs.Thisisofconcernasthesecelllines,butnot humanmonocytes,areroutinelyusedforresearchon bioenergeticissues(forexample,adaptationtohypoxia). Discussion Circulatingbloodmonocytesfaceoxygenconcentrations ofmorethan40mmHg,whichfueloxidativephosphory- lation.However,uponmigrationtoinflamedjoints, monocytesencounterhypoxicconditionsandmustadapt immediatelytothereducedpO 2 .Forseveraldifferentcell types,ithasbeenshownthatthetranscriptionfactor HIF-1underhypoxicconditionsistranslocatedintothe nucleuswhereitbindstopromoterregionsoftarget genes.Thisenablescellstoadaptandmaintaintheir basicandspecificfunctions[19,20].Elbarghati etal. reportedrecentlythatprima ryhumanmacrophagesbut notmonocytesrapidlyup-regulateHIF-1 a andHIF-2 a proteinsuponexposuretohypoxia,withtranslocationof theseproteinsintothenucleus[14]. WedemonstrateherethatthetranscriptionfactorHIF- 1 a alsoaccumulatesinquiescenthumanmonocytes underhypoxia,butispresentsolelyinthecytosol.For thisreason,wepostulatethatitcannotberesponsiblefor thetranscriptionalinductio noftypicalhypoxiatarget genesinthenucleus.Itisnotclearwhymonocytesdiffer inthisregardfrommanyothercelltypes,whereHIF-1 a underhypoxiaistranslocatedintothenucleus[21-25]. OnepossibilityisthattheHIF-inducedadaptation mechanisminmonocytesisnotnecessarybecauseofthe plentifuloxygensupplypresentinperipheralblood.The stabilisationofHIF-1 a inthecytosolunderhypoxiccon- ditionsmay,therefore,reflectapre-activestatethat becomesactivewhenthecellsstarttheirmigrationinto lowoxygentissueareas.However,itshouldbenotedthat wealsostudiedquiescentandPHA-stimulatedperipheral Figure3 Proteinkinase(PKC)- a / b 1 isessentialforhypoxia-induciblefactoralpha(HIF-1 a )translocation .( A+B )DetectionofHIF-1 a , LaminBand b -actininnuclear(NF+)andcytosolic(NF-)cellfractionsofprimaryhumanmonocytesusingimmunoblot.(A)Monocyteprotein lysateswereacquiredafterPMAstimulationandincubationfor5hunderhypoxiaandundernormoxiaasindicated.(B)Monocyteprotein lysateswereacquiredafterPMAstimulationfollowingPKC- a / b 1 inhibitionusingGö6976asindicatedandsubsequentincubationfor5hunder hypoxia.( A , B )PMAstimulationfor5hunderhypoxialeadstonucleartranslocationofHIF-1 a ,andspecificinhibitionofPKC- a / b 1 byGö6976 preventstranslocationofHIF-1 a . Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page7of12 humanbloodCD3 + CD4 + T-cells,whichalsocirculatein oxygen-richblood.Incontrasttomonocytes,hypoxic conditionsinducedHIF-1 a inthesecells,withtransloca- tionintothenucleusasshownbyimmunoblotting[12]. Fromthisobservation,wesuggestthattheHIF-1 a regu- lationmechanismmaybeafeatureoftheevolutionary youngercellsoftheadaptiveimmunesystem,butnotof evolutionaryoldercellsoftheinnateimmunesystem, suchasmonocytes. TheapparentlackofinvolvementofHIF-1 a inregu- latingexpressionofhypo xia-inducedgenesinmono- cytessuggeststhatothertranscriptionfactorsmediate thiseffect.Intheliterature,ithasbeenreportedthat adaptiveresponsestohypoxiaareregulatedbyseveral transcriptionfactors,incl udingHIF-1,HIF-2,ETS-1, cAMPresponseelementbindingprotein,activatorpro- tein-1andnuclearfactor- B[26-33].Hence,weexam- inedvariouspossibletranscriptionfactorsandfound thattheactiveformofNF B1,NF Bp50,istranslocated intothenucleusofthehumanmonocytesasareaction towardsapO 2 of2%.Ingoodagreementwiththis observation,Battaglia etal. havepreviouslyshownDNA bindingofNF Bp50underhypoxiainprimaryhuman monocytesbymeansofasupershiftanalysis[17]. Furthermore,Oliver etal. havedescribedtheselective activationofthecanonicalNF- Bpathwayviap65by intermittentandsustainedhypoxiainHeLacells[34]. Thenon-canonicalNF- Bpathwayviap52isnot impactedbyhypoxia.Ourresultsareconsistentwith thesefindingsasweshow,toourknowledgeforthe firsttimeinprimarymonocytes,thatp50ispartofthe canonicalpathwayinducedbysustainedhypoxia. WethereforesuggestthatNF B1servesasakeyreg- ulatorenablingtheimmediateadaptationofmonocytes tohypoxiaduringmigrationfrombloodintothetissue environment.Wesuggestthat,duringthedifferentiation Figure4 Hypoxia-induciblefactoralpha(HIF-1 a )translocationafterdifferentiationofmonocytestomacrophagesandco-culturewith endothelialcells .( A , B )DetectionofHIF-1 a ,LaminBand b -actininmonocyteorhMDMnuclear(NF+)andcytosolic(NF-)cellfractionsusing immunoblot.( A )ProteinlysateswereacquiredafterincubationofhumanmonocytesandhMDMfor5hunderhypoxia.Differentiationof monocytestomacrophagesleadstoHIF-1 a translocationintothenucleus.( B )Monocyteproteinlysateswereacquiredafterincubationof humanmonocytesonhumanendothelialcells(HMEC-1,humanmicrovascularendothelialcells)coatedwellplatesfor5hundernormoxiaand underhypoxia.Co-cultureofmonocyteswithendothelialcellsisnotsufficientforthetranslocationofHIF-1 a . Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page8of12 processofhumanmonocytesintomacrophages,the morepotentandpossiblym orerobustHIF-1systemis activated.TheHIF-1systemmaybeneededforthe rapidadaptationtovaryingoxygenconcentrations, whichisofessentialfunctionalimportanceforlong-liv- ingtissuemacrophages.Indeed,wedemonstratehere thatthestimulationofthemonocyteswithPMA(which althoughnon-physiologicalisusuallyappliedtodiffer- entiatemonocytesoverashorttimetoamacrophage- likephenotype)andthemorephysiologicalinductionof monocytedifferentiationbymeansofM-CSFcausethe translocationofHIF-1 a intothenucleusoflong-living tissuemacrophages.ThepresenceofHIF-1 a inthe nucleusofmacrophagesorhMDMsunderhypoxiahas alreadybeenverifiedbyothergroups[14,35-37].HIF-1 a wasalsodetectableinthenucleusofdifferentmyeloid celllines(THP-1,HL-60,andU937)underhypoxiccon- ditions.Althoughoftenusedasexperimentalmodelsof monocytes,thesecelllinecel lsarehighlyproliferative andmalignantcellswithnu merousdifferencesfrom macrophages,hMDMs,andmonocytes.Withregardto theHIF-1pathways,thesecelllinesbehavelikemacro- phagesorhMDMs,butnotlikemonocytes.Thisshould beconsideredwhenusingthesecelllinesasmodelsto analyzethebioenergeticfunctionsofmonocytesand/or macrophagesininflammatoryarthritis. OurobservationthatbothPMAstimulationandM- CSF-induceddifferentiationofmonocytesintomacro- phagescausesthetranslocationofHIF-1 a intothe nucleuspromptedasearchfo rpotentialregulators. Figure5 Monocytes,incontrasttohumanmonocytederivedmacrophages(hMDMs),showhypoxia-inducedgeneexpression .Real- timePCRanalysisofmRNAexpressionlevelsoftheglycolyticenzymes LDHA ( A )and PGK1 ( B ),thehypoxiainduciblefactor(HIF)-1targetgene CXCR4 ( C )andthetranscriptionfactor HIF1A ( D )inmonocytesandhMDMthatwereincubatedundernormoxia(whitebars)andunderhypoxia (blackbars),respectively( n =6).ThemRNAexpressionlevelofß-actin(ACTB)wasusedfornormalization. Ct=Ct (GOI) -Ct (ACTB) .Valuesare means±SD. + P 0.1;* P 0.05;(Wilcoxon t -test). Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page9of12 SincePKC- a / b 1 isstronglyactivatedbyPMAstimula- tion,wehypothesizedthatthisproteinkinaseenzyme couldplayakeyrole.Indeed,ourexperimentsdemon- stratedthattheinhibitionofPKCbyGö6976leadsto abrogationofHIF-1 a translocationintothenucleus. Ourobservationisinagreementwithdataprovidedby ChangandBeezholdwhohaveprovedtheexistenceof bothprevailingPKC-isoforms a and b inprimary humanmonocytes[38].Lin etal. recentlyshowed Gö6976-mediatedinhibitionofPKC- a andmembrane translocationduringdifferentiationintoMDMs,and consequentlydiminisheddifferentiationofMDMs[39]. Itshouldbenoted,however, thattheexactmechanism ofthePKC-mediatedtransportofHIF-1 a intothe nucleusiscurrentlystillunclear. ItwasinterestingtorealizethatHIF-1 a isnotshifted intothenucleusifhumanmonocytesareincubatedunder hypoxiawithconcurrentTLRstimulation.Furthermore, contactofmonocyteswithendo thelialcells(thatis,.the initialmigrationstepandthereforethebeginningofthe differentiation)isnotsufficienttoinduceHIF-transloca- tion.Takentogether,thesedataclearlydemonstratethat neitherthecontactofmonocyteswithantigeninthe hypoxicareasnorthecontactofmonocyteswithendothe- lialcellscausesthetranslocationofHIF-1 a intothe nucleus;rather,itappearstobethedifferentiationprocess perse thatactivatestheHIF-1system. InagreementwithBosco etal. [40],weshowedthat hypoxiastrengthensthegeneticexpressionoftheglyco- lysisenzyme LDHA .HIF-1 a isnotpresentinthe nucleusundertheseconditionssoweinferthiseffectto bemediatedbyNF Bp50.However,macrophages demonstratesignificantlyhigherexpressionsofthegene LDHA (andanotherenzymeinvolvedinglycolysis, PGK1 )undernormoxiathanmonocytes.Inaddition,the expressionofthesegenesisnotincreasedbyincubating macrophagesunderhypoxicconditions( LDHAP =0.39, PGK P =0.26).AconstitutionalPKCover-expression constantlyinducingtheHIF-1systemmaybeapossible explanationoftheseobservations[41]. Interestingly,theobservationsmadefortheglycolysis genesdifferfromthoseforthechemokinereceptor CXCR4 .Itshouldbenotedthattheexpressionofthis chemokinereceptorisoxygen-dependent.Therefore,the chemotacticbehaviourofmonocytescanbeadaptedto variableoxygenconditions.Bosco etal. describeda geneticinductionof CXCR4 inatranscriptomecharac- terisationofmonocytesincubatedunderhypoxia[40]. Wealsofoundthe CXCR4 geneinmonocytestobesig- nificantlyinducedunderhypoxia.However,incontrast toglycolysisgenes,the CXCR4 geneexpressionunder normoxiainhMDMsisnotmorepronouncedthanin monocytes.Although CXCR4 expressionincreasedin hMDMswithhypoxia,thisincreasewasnotsignificant ( P =0.17).Asignificantincre aseinthegeneticexpres- sionfor CXCR4 underhypoxiainhMDMshasbeen describedbyFang etal. [42].Schioppa etal. showed thatinhumanmonocytesandhumanMDMs,hypoxia inducedexpressionof CXCR4 attheproteinlevel[15]. Theauthorsinterpretthenavigationprocesshypoxia/ HIF-1/CXCR4asanimportantmechanismfortheregu- lationofcellmigrationintohypoxictissuesorforthe retentionofcellsinhypoxictissues.Thisisinlinewith ourfindingofhypoxia/HIF-1/CXCR4forhMDMsbut, duetotheabsenceofHIF-1 a inthenucleusofmono- cytes,ashypoxia/NF Bp50/CXCR4formonocytes. Figure6 Incubationofmonocytesunderhypoxialeadstotranslocationoftranscriptionfactor,nuclearfactorofkappalight polypeptidegeneenhancerinB-cells(NF B1),intothenucleus .Detectionofhypoxia-induciblefactoralpha ( HIF-1 a ),LaminBand b -actin innuclear(NF+)andcytosolic(NF-)cellfractionsofprimaryhumanmonocytesusingimmunoblot.( A - C )Monocyteproteinlysateswereacquired afterincubationfor5hunderhypoxiaandundernormoxiaasindicated.ThecellularlocalizationoftranscriptionfactorsNF Bp100/p52,c-Rel, andc-Jun(Figure6A),NF Bp105/p50andc-Fos(Figure6B),andJunBandNF Bp65(Figure6C)weredeterminedbyimmunoblot.NF Bp105 (theinactiveformofNF B1)remainsinthecytoplasm,whiletheactiveformNF Bp50istranslocatedintothenucleusunderhypoxicconditions (Figure6B).Otherfactorsshowedeithernochangeintheirlocalizationor,asforc-JunandNF Bp52,werenolongertranslocatedintothe nucleusunderhypoxicincubation. Fangradt etal . ArthritisResearch&Therapy 2012, 14 :R181 http://arthritis-research.com/content/14/4/R181 Page10of12 ConclusionsInsummary,thelocalizationofthetranscriptionfactorisshiftedduringthedifferentiationprocessfromthecytosol(inmonocytes)tothenucleus(inmacro-phages),apparentlyasaPKC--mediatedadaptationtoalowoxygenenvironment.Inmonocytes,itisNFkB1,andnotHIF-1,thatisofcentralimportancefortheexpressionofhypoxia-adjustedgenes.Thesedatademonstratethat(i)duringdifferentiationcrucialcellu-laradaptationmechanismsaredecisivelychangedand(ii)bioenergeticaspectsareofcrucialimportancefortheunderstandingofunderlyingpathophysiologicalpro-cessesininflammatoryarthritis.AdditionalmaterialAdditionalfile1:FigureS1A-C.Myeloidcelllines(THP-1,HL-60,andU937)expressHIF-1inthecellnucleus.THP-1cells(),HL-60cells()andU937cells()expressHIF-1inthenucleusundernormoxiaandhypoxia,withorwithoutPMAstimulation(5h).THP-1andU937showsimilarexpressionofHIF-1undernormoxiaandhypoxia(),whereasHL-60cellsdemonstrateincreasedexpressionofhypoxia-induciblefactor(HIF)-1underhypoxia().AllcelllinesshowahigherexpressionofHIF-1inthepresenceofPMA().DetectionofHIF-1LaminBand-actininnuclear(NF+)andcytosolic(NF-)cellfractionsofmyeloidcelllinesusingimmunoblot.()Proteinlysateswereacquiredafterincubationfor5hunderhypoxiaandundernormoxiaasindicated.ACTB:ß-actin;ATP:adenosinetriphosphate;c-AMP:cyclicadenosinemonophosphate;CD:clusterofdifferentiation;cDN:complementarydeoxyribonucleicacid;CO:carbondioxide;c:thresholdcycle;CXCR:C-X-Cmotifchemokinereceptor;DC:dendriticcell;DRK:DeutschesRotesKreuz(GermanRedCross);EGF:endothelialgrowthfactor;FCS:fetalcalfserum;GÖ6976:12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole;HeLa:HenriettaLackscellline;HIF:hypoxia-induciblefactor;HL-60:humanpromyelocyticleukemiacells;hM-CSF:humanmacrophagecolony-stimulatingfactor;hMDM:humanmonocytederivedmacrophages;HMEC-1:humanmicrovascularendothelialcellline;HRP:horseradishperoxidase;IgG:immunoglobulinG;LDHA:lactatedehydrogenaseA;LPS:lipopolysaccharide;MACS:magneticactivatedcellsorting;2-ME:ß-mercaptoethanol;MHC:majorhistocompatibilitycomplex;mmHg:millimeterofmercury;N:molecularnitrogen;NF:nuclearfraction;B:nuclearfactorofkappalightpolypeptidegeneenhancerinB-cells;Omolecularoxygen;OxPhos:oxidativephosphorylation;PamPam3Cys-Ser-(Lys)4-Trihydrochloride;PCR:polymerasechainreaction;PGK1:phosphoglyceratekinase1;PHA:phytohemagglutinin;PKC:proteinkinaseC;PMA:phorbol12-myristate13-acetate;pO:oxygenpartialpressure;qPCR:quantitativepolymerasechainreaction;R-848:resiquimod;RA:rheumatoidarthritis;RNA:ribonucleicacid;RPMI1640:RoswellParkMemorialInstitutemedia1640;SD:standarddeviation;SDS-PAGE:sodiumdodecylsulfatepolyacrylamidegelelectrophoresis;THP-1:humanacutemonocyticleukemiacellline;TLR:tolllikereceptor;TNF:tumornecrosisfactor;U-937:humanhistiocyticleukemiacellline;v/v:volume/volumepercent.AcknowledgementsThisstudywassupported(inpart)byresearchfundingfromtheDFGthroughtheBerlin-BrandenburgSchoolforRegenerativeTherapies(GSC203)toMH.WewouldliketothankManuelaJakstadtfortechnicalassistance.AuthordetailsDepartmentofRheumatologyandClinicalImmunology,CharitéUniversityHospital,Charitéplatz1,Berlin,10117Germany.GermanRheumatismResearchCenter(DRFZ),Charitéplatz1,Berlin,10117Germany.Berlin-BrandenburgSchoolforRegenerativeTherapies(BSRT),FoehrerStraße15,Berlin,13353Germany.Berlin-BrandenburgCenterforRegenerativeTherapies(BCRT),FoehrerStraße15,Berlin,13353Germany.Allauthorshavemadesubstantialcontributionstoconceptionanddesignofthestudy,ortoacquisitionofdataandtheanalysisandinterpretation.Allauthorsreadandcommentedonthedraftversionsofthemanuscriptandhavegivenfinalapprovaloftheversiontobepublished.CompetinginterestsTheauthorsdeclarenocompetingfinancialornon-financialinterests.Received:29February2012Revised:30June2012Accepted:7August2012Published:7August20121.NgCT,BinieckaM,KennedyA,McCormickJ,FitzgeraldO,BresnihanB,BuggyD,TaylorCT,OSullivanJ,FearonU,VealeDJ:Synovialtissuehypoxiaandinflammationinvivo.AnnRheumDis2.BinieckaM,KennedyA,FearonU,NgCT,VealeDJ,OSullivanJN:damageinsynovialtissueisassociatedwithinvivohypoxicstatusinthearthriticjoint.AnnRheumDis3.BinieckaM,KennedyA,NgCT,ChangTC,BaloghE,FoxE,VealeDJ,FearonU,OSullivanJN:Successfultumournecrosisfactor(TNF)blockingtherapysuppressesoxidativestressandhypoxia-inducedmitochondrialmutagenesisininflammatoryarthritis.ArthritisResTher4.ButtgereitF,BurmesterGR,BrandMD:Bioenergeticsofimmunefunctions:fundamentalandtherapeuticaspects.ImmunolToday5.KennedyA,NgCT,ChangTC,BinieckaM,OSullivanJN,HeffernanE,FearonU,VealeDJ:Tumornecrosisfactorblockingtherapyaltersjointinflammationandhypoxia.ArthritisRheum6.StraubRH,CutoloM,ButtgereitF,PongratzG:Energyregulationandneuroendocrine-immunecontrolinchronicinflammatorydiseases.InternMed7.DziurlaR,GaberT,FangradtM,HahneM,TripmacherR,KolarP,SpiesCM,BurmesterGR,ButtgereitF:Effectsofhypoxiaand/orlackofglucoseoncellularenergymetabolismandcytokineproductioninstimulatedhumanCD4+Tlymphocytes.ImmunolLett8.KolarP,GaberT,PerkaC,DudaGN,ButtgereitF:Humanearlyfracturehematomaischaracterizedbyinflammationandhypoxia.ClinOrthopR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