DCSIGNIsaReceptorforHumanHerpesvirus8onDendriticCellsandMacrophagesGi - PDF document

DC-SIGNIsaReceptorforHumanHerpesvirus8onDendriticCellsandMacrophagesGiovannaRappocciolo,*FrankJ.Jenkins,*HeatherR.Hensler,*PaoloPiazza,*MarielJais,*LuannBorowski,*SimonC.Watkins,andCharlesR.Rinaldo,Jr.*Humanherpesvirus8(HHV-8)causesKaposi’ssarcomaandpleuraleffusionlymphoma.Inthisstudy,weshowthatdendriticcell-specicICAM-3grabbingnonintegrin(DC-SIGN;CD209)isareceptorforHHV-8infectionofmyeloidDCsandmacro-phages.DC-SIGNwasrequiredforvirusattachmenttothesecellsandDC-SIGN-expressingcelllines.HHV-8bindingandinfectionwereblockedbyanti-DC-SIGNmAbandsolubleDC-SIGN,andmannan,anaturalligandforDC-SIGN.Infectionof *DepartmentofInfectiousDiseasesandMicrobiology,GraduateSchoolofPublicHealth,andDepartmentofPathologyandDepartmentofCellBiologyandPhysiology,SchoolofMedicine,UniversityofPittsburgh,Pittsburgh,PA15261ReceivedforpublicationSeptember7,2005.AcceptedforpublicationNovember17,2005.Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpagecharges.Thisarticlemustthereforebeherebymarkedinaccordancewith18U.S.C.Section1734solelytoindicatethisfact.ThisworkwassupportedbytheNationalInstitutesofHealthGrantsR01CA82053 with10%heat-inactivatedFCSand-glutamineandwerenegativeformycoplasma(PCRELISA;Roche).Monocyte-derivedDCsandmacrophagesEnrichedCD14monocyteswereobtainedfromPBMCsandusedforgenerationofimmatureDCsaspreviouslydescribed(25).Forsomeex-periments,DCswerematuredwithtrimericCD40Lat1g/ml(Amgen).MacrophagesweregeneratedfromCD14monocytesbycultureinRPMI1640mediumwith5%heatinactivated,pooledhumanABserum,and1000U/mlrecombinanthumanGM-CSFfor7days,andtreatedovernightwith20ng/mlhumanIL-13(PeproTech).EndothelialcellsPrimaryhumanadultdermalmicrovascularendothelialcells(HMVEC-d;catalogCC2543;Cambrex)wereculturedaccordingtothesupplier’sin-structions.Cellswereremovedfromtheculturewellsforsurfacemarkerstainingandsubculturebytreatmentwith0.1mMEDTAinPBSwith0.25%BSA.ViruspuriÞcationandinfectionofcellsConcentratedsupernatantsandcell-freelysatesfrom2decanoylphorbol-13-acetate(TPA;Sigma-Aldrich)-inducedBCBL-1cellswereusedassourceofHHV-8andpuriedbyamethodmodiedfromCerimeleetal.(26).Briey,theHHV-8lyticcyclewasinducedinBCBL-1cellsbytreatmentwith20ng/mlTPAfor4days.Thecellswerethencentrifuged,andthesupernatantwasremovedandsaved.Thecellpelletwaslysedbythreefreeze-thawcycles,centrifuged,andpooledwiththeoriginalsupernatant.Theseviruspreparationswerethenputthroughamltertoeliminatecelldebrisandconcentratedovernightat4°Cwith7%polyethyleneglycol6000(EMBBiosciences).Theprecipitatewascentrifugedat15,000for1hat4°C,resuspendedinasmallamountofPBSwithCaandMgand0.1%BSA,andmicrocentrifugedat13,000rpm(BiofugeFresco).Theresultingsupernatantwasthencentri-fugedthrougha25%w/vsucrosecushionat40,000rpmat4°Cfor1hinaBeckmanSW41rotor.Thepelletwassoakedovernightat4°Cin1mlofPBScontaining0.1%BSAandthenlayeredovera5–45%sucrosegradientinPBS,andcentrifugedat40,000rpmat4°Cfor1hinaBeckmanSW41rotor.Thevisiblebandatthecenterofthegradientwascollected,dialyzedagainstPBSat4°Covernight,andthenfrozenat70°C.Insomeexperi-ments,pooledpelletsrecoveredfromthe25%sucrosecushionwerelay-eredoveradiscontinuousNycodenz(Sigma-Aldrich)gradientandcentri-fugedat24,000rpmat10°CinaBeckmanSW28rotor(27).Viruswascollectedatthegradientinterface,anddialyzedovernightagainststerilePBSandstoredasdescribedabove.Anaverageof1copiesofviralDNAwereusedtoinfect1cellsfor2hat37°C,washedandincubatedforupto5daysinAIM-Vmediumat37°Cin5%CO.Forblockingstudies,cellswerepretreatedwith20g/mlanti-human-DC-SIGNmAb(clone120507;R&DSystems,orcloneDCN46;BDBiosciences),anti-CD11amAb(BDBiosciences),mouseIgG(Sigma-Aldrich),or100g/mlmannan(Sigma-Aldrich),for1hat4°CbeforeexposuretoHHV-8.QuantitativePCRassayforHHV-8HHV-8DNAlevelsweremeasuredbyaquantitative,real-timeTaqManPCRassaywithaprimersetspecicfortheHHV-8alkalineexonucleasegene(ORF37)previouslyshowntobehighlyspecicforHHV-8DNA(28).StandardizationofHHV-8DNAquantitationwasdoneusingknownquantitiesofHHV-8DNA(AppliedBiosystems).TodetermineviralDNAlevelsininfectedcells,cellularDNAwasextractedusingtheQIAampBloodDNAMiniprepkit(Qiagen).TodetermineviralDNAlevelsfromencapsidatedvirusincellculturesupernatants,theexperimentalsampleswerepelletedbyultracentrifugationandtreatedwithDNaseI(InvitrogenLifeTechnologies)followedbyphenolchloroformextraction.Toverifythatthis

assaydetectedlyticvirusreplication,weanalyzedDNAsamplesfromBCBL-1cellscontainingthegeneunderthecontrolofatetracycline-responsivepromoter(giftfromJ.Jung,NewEnglandRegionalPrimateResearchCenter,Worcester,MA)(29).Weinducedviralreplica-tionbytheadditionof20ng/mldoxycyclineandcollectedsamplesat24,48,and72hforisolationofDNAandanalysisofHHV-8viralloadbyourreal-timeTaqManPCRassay.Ourresultsdemonstratedasignicantin-creaseinviralDNAintheinducedcellsrelativetotheuninducedcells(datanotshown).ImmunoßuorescenceassaysImmunouorescencewasdoneonHHV-8-infectedormock-infectedcellsusinganti-HHV-8ORF73ratmAbandmousemAbsdirectedagainstORF-K8.1A/BandORF59(AppliedBiosystems).Cellswerecountedandspottedonpoly--lysine-coatedslides,xedin4%paraformaldehydefor20min,andpermeabilizedwithbuffer(0.5%BSA,0.1%saponin,0.1%)for20minatroomtemperature.CellswereincubatedwiththeprimaryAbfor30minat4°C,washedextensivelyinbuffer,andthenstainedwithFITC-conjugatedgoatanti-mouseoranti-ratIgG(Sigma-Aldrich).ControlswereomissionoftheprimaryAbandstainingofunin-fectedcells.Insomeexperiments,cellswerealsostainedbydirectimmu-nouorescencewithFITC-conjugated,anti-DC-SIGNmAb(clone120507;R&DSystems),orwithK8.1A/BorORF59mAbsdirectlyconjugatedwithPEorTexasRedusingaZenonlabelingkit(MolecularProbes).ToavoidnonspecicbindingofIgG,ablockingstepwasaddedusingSuper-BlockBlockingBuffer(Pierce).Forconfocalmicroscopy,DCswereculturedasperourstandardpro-ceduresonglasscoverslips,xedwith2%paraformaldehyde,washedinPBS,blockedwithBSA/normalgoatserumfor40min,washedinPBS,stainedwithanti-K8.1A/BmAbfor1handwithgoatanti-mouseCY3-conjugatedF(ab(JacksonImmuno)for1h.Afterwashing,thecellsweretreatedwithanti-ORF73mAbandanti-DC-SIGN-FITCmAb(cloneDCN46;BDBiosciences)for1h,washed,andstainedwithCY5conjugatedgoatanti-ratserum(JacksonImmuno)for1h.ImagesweretakenwithanFluoViewBX61confocalmicroscope(OlympusAmerica).BlockingofHHV-8bindingForreceptor-blockingstudies,cellswereincubatedwithpuried,[midine-labeledHHV-8at4°CbythemethodofAkulaetal.(5,8).Insomeexperiments,cellswereincubatedwithanti-DC-SIGNmAb(20clone120507)ormannan(100g/ml)for1hat4°Cbeforeaddingtheradiolabeledvirus.Puried[H]thymidine-labeledHHV-8werealsoincubatedwith10g/mlsolubleDC-SIGN(agiftfromS.Butera,CentersforDiseaseControlandPrevention,Atlanta,GA)at37°Cfor1h,addedtothecellpellets,andmeasuredforradioactivityasdescribed(5).FlowcytometricanalysisofcellphenotypesExpressionofcellsurfacemoleculeswasexaminedbyowcytometry(BeckmanCoulterXL)usingFITC-conjugatedmAbspecicforMHCclassI(HLA-ABC)andMHCclassII(HLA-DR),CD80,CD86,CD83,andDC-SIGNfor30minat4°C,andthenxedwith1%paraformalde-hyde.Cellswerealsotreatedwithunconjugatedanti-integrinmAb(VLA-3;ChemiconInternational)andFITC-conjugatedgoatanti-mouseIgGantiserafor30mineachat4°C.CellswerestainedwithisotypeAborgoat-anti-mouseIgGFITCascontrols.FlowcytometricanalysisofendocytosisDCswereinfectedwithHHV-8for24or48h,orweremock-infected,andassessedforendocyticcapacityofFITC-dextran(,40,000)(1mg/ml;Sigma-Aldrich)at37°Cor4°Cfor30min,washedfourtimeswithcoldPBScontaining1%FCSand0.01%NaN,andanalyzed(BeckmanCoulterXL);cellsincubatedat4°Cwerethecontrol.ELISPOTassaySingle-cellIFN-productionbyCD8TcellswasmeasuredbyanELISPOTassayusingpeptidesrepresentingknownHLAA*0201inuenzaAvirusM158–66andEBVBMLF1280–288epitopes(25).CD8TcellswereobtainedfromCD14cell-depletedPBMCbypositive,immunomagneticbeadselection(MiltenyiBiotec),whichdoesnotinterferewithAgactivationoftheCD8Tcells(datanotshown).HHV-8infectsDCsWeexaminedwhetherDCsthatareofmyeloidoriginandexpressDC-SIGNcouldbeinfectedwithHHV-8.RepresentativedatafromeightindependentexperimentsusingarangeofvirusinputmultiplicitiesshowthatviralDNAcopiespercellremainedlowininfectedDCsduring72hofinfection,andtherewasnodetectablevirusfoundinthesupernatantsofinfectedcells(Fig.1).Al-thoughviralDNAreplicationwasverylowintheinfectedDCs,viralproteinscodedbyORF-K8.1andORF-59couldbedetectedupto48hpostinfection,whileORF73LNA-1expressionbecameapparentat48and72hofinfection(Fig.1anddatanotshown).ExpressionofbothK8.1andORF59diminishedgreatlyafter48h(Fig.1)andwasnotdetectedby72hpostinfection(datanotshown).ThepeaklevelofcellsexpressingtheseproteinsreachedDC-SIGNISARECEPTORFORHHV-8 50–75%by24–48h(Fig.1;TableI);LNA-1wasexpressedin72%(range60–100%)ofDCsat48h(Fig.1)and72h(datan

otThus,thereisgeneexpressionofK8.1andORF59proteinsduringtherst24–48hofinfectionofDCs,whichisreplacedbygeneexpressionby72hofinfection.Moreover,althoughcompleteviralreplicationwaslacking,therewasasmallbutsig-nicantamountofcelldeathassociatedwithinfection,astheper-centageofviableDCsintheHHV-8-infectedculturesat72hwas83(3.1)%,comparedwith92(4.7)%inuninfectedDCs(0.05;pairedtest).Finally,studiesdoneinparallelcon-rmedthatHHV-8infectionoftheDCswassimilartothatofvascularendothelialcells(30),whichrepresentmajortargetsofHHV-8infectioninvivo.Thus,thedatashowalackofasignif-icantincreaseinviralDNA,butproductionofviralproteins,overseveraldaysofHHV-8infectionofHMVEC-d(Fig.2).Anti-DC-SIGNmAbandmannaninhibitHHV-8infectionofWenextdeterminedthatHHV-8infectionofDCscouldbeinhib-itedbyblockingwithanti-DC-SIGNmAb(clone120507)asin-dicatedbya92%decreaseinK8.1A/Bexpressionat24h(Fig.3TableI)and48h(datanotshown).TreatmentoftheDCswithanti-CD11amAbhadnoeffectonHHV-8infection.Theseresultswereconrmedusingadifferentanti-DC-SIGNmAb(cloneDCN46)(datanotshown).DCsinfectedwithHHV-8for24hwerealsopositivefortheORF59viralprotein(TableI)andwerethesamecellsexpressingDC-SIGN(Fig.3).TreatmentoftheDCswithanti-DC-SIGNmAbbeforeHHV-8infectionblocked91%ofORF59expression(Fig.3;TableI)andalsoinhibitedthebindingoftheuorochromedye-labeledanti-DC-SIGNmAbtoDC-SIGN(Fig.3).SimilarlevelsofinhibitionofHHV-8infec-tionwerenotedafterpretreatmentoftheDCswithmannan,anaturalligandforDC-SIGN(datanotshown).DC-SIGN-transfectedcellsaresusceptibletoHHV8infectionK562cellsandB-LCLdonotconstitutivelyexpressDC-SIGNandwerenotsusceptibletoHHV-8infection(Fig.4;TableI).Incon-trast,K562cellsandB-LCLsstablytransfectedwithaplasmidthatoverexpressesDC-SIGN(K562DC-SIGNandB-LCL-DC-SIGN)weresusceptibletoHHV-8infectionasevidencedbyexpressionofHHV-8K8.1andORF59proteinsat24h(Fig.4;TableI).Inadditionalexperiments,weconrmedthatHHV-8infectedthesecellsviaDC-SIGNbyblockinginfectionwithanti-DC-SIGNmAb(TableI).HHV-8bindstoDC-SIGNandbindingisinhibitedbyanti-DC-SIGNmAb,solubleDC-SIGNandmannanToprovethatDC-SIGNwasactuallyasurfacereceptorforbind-ingofHHV-8,weassessedbindingofradioactivelylabeledvirustoDCsandB-LCL-DC-SIGNat4°Ctoallowbindingbutpreventinternalizationofvirus(8).BindingofradiolabeledHHV-8wasinhibitedby60%witheitheranti-DC-SIGNmAbormannan(Fig.5).Furthermore,pretreatmentofradiolabeledHHV-8withsolubleDC-SIGNgreatlyinhibitedthebindingofthevirustothesecells(Fig.5),conrmingthatHHV-8bindsDC-SIGN.Finally,anti-DC-SIGNmAb-mediatedinhibitionofvirusbindingtoDCswasdose-dependent(Fig.5HHV-8infectsmacrophagesthroughDC-SIGNMonocytesandmacrophagesareknowntobeinfectedwithHHV-8invivo(18)butarerefractorytoinfectioninvitrounlessactivated(31).DC-SIGNisup-regulatedbytreatmentofmono-cyte-derivedmacrophageswithIL-13invitro(9,15)andinin-ammatory,activatedmacrophagesinvivo(13,32).We,there-fore,determinedwhetherDC-SIGNwasareceptoronthesecellsforHHV-8.Non-IL-13-stimulated,monocyte-derivedmacro-phagesexpressedlowlevelsofDC-SIGNat24handsupportedlimitedHHV-8infection,i.e.,K8.1andORF59expression,me-dian,8%(TableI).Incontrast,IL-13-treatedmacrophagesex-pressedhighlevelsofDC-SIGNby24h(Fig.6).AlthoughtherewaslittleincreaseinviralDNAinthesecultures(datanotshown),themajorityofIL-13-treatedmacrophagesexpressedORF59(me-dian,88%)(Fig.6;TableI)andK8.1(median,63%)(Fig.6TableI)by24h.DC-SIGNwascoexpressedinHHV-8infectedmacrophagesasshowninFig.6.Finally,HHV-8infectionofthe FIGURE1.HHV-8infectsDCs.,QuantitativeDNAresultsfromreal-timePCRassayontotalDNAthatwasextractedfrominfected(inf)DCs(reddottedline)andthecorrespondingDNase-resistant,concentratedcellculturesupernatants(sup,pinkdottedline).Uninfectedcellsandsuperna-tantswerenegativeforHHV-8DNA(redsolidlineandpinksolidline,,ImmunouorescenceonDCsthatwereinfectedwithHHV-8andstainedwitheithermAbanti-K8.1A/B(cytoplasmicreduo-rescence),anti-ORF59(cytoplasmicandperinuclearreduorescence),oranti-LNA-1(ORF73,nucleargreenuorescence).UninfectedDCswerenegativeforeachofthethreeHHV-8proteins(Fig.1B,toprow).CellswerecounterstainedwithDAPI(blue)(600).Dataarefromoneexper-imentrepresentativeofeightindependentexperiments.TheJournalofImmunology non-IL-13-treatedmacrophageswascompletelyblockedbypre-treatmentofthecellswithanti-DC-SIGNmAb(TableI)orman-nan(datanotshown).Moreimportant

ly,thiseffectwasalsonotedinIL-13-treatedmacrophages,whereanti-DC-SIGNmAbde-creasedexpressionofORF59by100%(TableI)andK8.1by84%(Fig.6;TableI).integrinisnotessentialforHHV-8infectionofDC-SIGN-expressingcellsintegrinhasbeenidentiedasareceptorforHHV-8inhumanforeskinbroblastsandendothelialcells(8).Wefoundthat,incontrasttoDC-SIGN,thisintegrinwasnotexpressedontheDC-SIGNtransfectedB-LCLsandK562cellsthatweresus-ceptibletoHHV-8infection(TableI).Moreover,wasexpressedonnon-IL-13-activatedmacrophages,onlyafewofwhichexpressedDC-SIGNandsupportedHHV-8infection.HHV-8infectionresultsinadecreaseinDC-SIGNexpressionSurfaceexpressionofDC-SIGNisknowntodecreaseduetoin-ternalizationafterbindingtoHIV1oritsnaturalligandICAM-3(33).Inourstudies,DC-SIGNexpressionwasdown-regulatedonDCsafter24–72hofinfectionwithHHV-8(0.001deter-minedbytwo-tailedpairedStudent’stest)(Fig.7).ThelossofDC-SIGNexpressiononthesurfaceofinfectedDCswasmuchgreaterthanthatinducedbymaturationofDCsbytreatmentwithCD40L(e.g.,meanuorescenceintensity(MFI)forDC-SIGNonHHV-8-infectedDCsat72hpostinfection(MFI10),comparedwithuninfectedDCs(MFI245),andCD40L-stimulatedDCs205)(datanotshown).Wealsoobservedastrongdown-regulationofexpressionofDC-SIGNonthesurfaceofbothacti-vatedmacrophages(Fig.6)andB-LCL-DC-SIGN(datanotshown)afterinfectionwithHHV-8.Therewereinsignicantin-creasesinexpressionofDCmaturationmarkersHLA-DRandTableI.HHV-8infectionofprimarycellsandcelllinesexpressingDC-SIGN CelltypeK8.1ExpressionORF59ExpressiontreatedUntreatedDCs92(87–97)776(72–80)250(20–80)84(0–10)875(68–100)87(3–12)Macrophages5(2–10)471(40–75)48(6–10)40(0–3)48(0–16)40(0–3)IL-13Macrophages74(50–80)478(62–95)463(60–66)410(0–21)488(66–100)40(0–0)B-LCL0(0–0)51(0–2)30(0–0)50(0–0)30(0–0)50(0–0)B-LCL-DC-SIGN73(49–85)51(0–2)391(98–100)50(0–3.3)388(83–100)50(0–0)K5620(0–0)50(0–0)20(0–0)5ndndK562-DC-SIGN68(55–75)40(0–0)288(72–90)5ndndndDC-SIGNorintegrinexpressiononthesurfaceofcellsasmeasuredbyFACSandexpressedaspercentofpositiveornegativecellsforuorescenceafterstainingwithmAbspecicforDC-SIGNandascomparedtotheisotypecontrolormouseanti-IgGFITCalone.Numberofdeterminations.Dataareexpressedasmedian(range).PercentofcellsreactingpositivewithmAbagainstK8.1A/BorORF59at24hpostinfection.Dataareexpressedasmedian(range).Numberofdeterminations.nd,Notdone. FIGURE2.HHV-8infectionofendothelialcells.,QuantitativeDNAresultsfromreal-timePCRassayontotalDNAthatwasextractedfrominfected(inf)HMVEC-d(bluedottedline)andthecorrespondingDNase-resistantconcentratedcellculturesupernatants(sup,lightbluedottedline).UninfectedcellsandsupernatantswerenegativeforHHV-8DNA(bluesolidlineandlightbluesolidline,respectively).Dataarefromoneexper-imentrepresentativeofthreeindependentexperiments.,Immunouores-cenceresultsonHMVEC-dthatwereinfectedwithHHV-8andstainedwitheitheranti-K8.1A/BmAb(cytoplasmicreduorescence)oranti-LNA-1(ORF73)mAb(nucleargreenuorescence)at24and72h,respec-tively.UninfectedHMVEC-dwereusedascontrols.Cellswerecounter-stainedwithDAPI(blue)(600).Dataarefromoneexperimentrepresentativeofsixindependentexperiments. FIGURE3.InfectionofDCswithHHV-8isblockedbyantiDC-SIGN,ImmunouorescenceresultsonDCsthatwereleftuntreatedortreatedwitheitheranti-DC-SIGNmAb(clone120507)oranti-CD11amAb,infectedwithHHV-8,incubated,andstainedwithanti-K8.1A/BmAb(red)at24h.UninfectedDCswereusedascontrols.,Immunou-orescenceresultsonDCsthatwereeithertreatedwithanti-DC-SIGNmAb(clone120507)orleftuntreated,infected,andstainedat24hwithanti-DC-SIGNmAb(green)andanti-ORF59mAb(red).Theoverlayofcom-binedcolorsforanti-DC-SIGNandORF59isshown.Cellswerecoun-terstainedwithDAPI(blue)(600).Dataarefromoneexperimentrepresentativeofeightindependentexperiments.DC-SIGNISARECEPTORFORHHV-8 CD83by24h(Fig.7).ExpressionofCD83declinedtoback-groundlevelsby72h.HHV-8infectedDCsalsohadlowerex-pressionofHLAABCat48and72h.FurtheranalysisofHHV-8-infectedDCsstainedforDC-SIGNandORF59andexaminedbyconfocalmicroscopyat24and48hshowedthatDC-SIGNwasstronglydown-regulatedandlocalizedinthecytoplasmofinfectedDCs(Fig.7).ThissuggeststhatDC-SIGNmoleculeshavebeeninternalizedasaresultofviralinfection,oralternativelycouldhavechangedtheirstructure,andthereforecouldnotberevealedwiththemAbusedintheseassays.However,thereappearedtobenonewmoleculessynthesizedand/orbroughttothecellsurfaceduringthetimespanofth

eseexperiments(Fig.7HHV-8infectionofDCsimpairstheirfunctionHHV-8isknowntocodeforseveralproteinswithimmunomodu-latorypropertiesthatareconsideredimportantinitspathogenesis(34).WethereforedeterminedwhetherHHV-8infectioninducedimpairmentofAg-processingand-presentationfunctionsofDCs(35).WefoundastronginhibitionofendocytosisinHHV-8-in-fectedDCswhencomparedwithuninfected,immatureDCsorCD40L-maturedDCs(Fig.8).BecauseAguptakeispivotaltosubsequentAgpresentation,weexaminedwhetherHHV-8-in-fectedDCscouldstimulatevirusAg-specicmemoryCD8lymphocytes.HHV-8-infectedDCswerepoorAPCswhencom-paredwithuninfectedDCs,asshownbydecreasedproductionofbyautologousCD8TcellsinresponsetoimmunodominantinuenzaAvirusandEBVpeptides(Fig.8).Additionally,asmatureDCsaremoreefcientatpresentingAgtoTcells,wedeterminedwhetherHHV-8infectedmatureDCswereabletopresentAgmoreefcientlythaninfected,immatureDCs.Notably,maturationofDCsbytreatmentwithCD40LbeforeinfectionwasnotsufcienttoovercometheinhibitoryeffectofHHV-8infectiononDCAg-presentingfunction(Fig.8DC-SIGNisatypeIIC-typelectinthatservesasarecognitionreceptoronDCsforseveralvirusesandother,diversepatho-gens(16).BindingtoDC-SIGNandotherC-typelectinsleadstoAgprocessingandpresentationtoTcellsforactivationofadaptiveimmuneresponses.However,somepathogenscansub-vertthisprocessanduseDC-SIGNtoestablishinfectioninthehost.Inthisstudy,weshowthatDC-SIGNisareceptorforHHV-8onmyeloidDCsandmacrophages.EvidencethatDC-SIGNisareceptorforHHV-8isbasedonestablishedcriteria FIGURE4.DC-SIGNexpressionrendersresistantcellssusceptibletoHHV-8infection.,ImmunouorescenceresultsonK562andK562-DC-SIGNcellsthatwereinfectedwithHHV-8andstainedwithanti-K8.1A/BmAbat24h(red).,ImmunouorescenceresultsonB-LCLandB-LCLDC-SIGNthatwereinfectedwithHHV-8andstainedafter24hwithanti-K8.1A/BmAb(red).CellswerecounterstainedwithDAPI(600).Dataarefromoneexperimentrepresentativeofveindependentexperiments. FIGURE5.HHV-8bindstoDC-SIGN.,Inhibitionofbindingofradio-activelylabeledHHV-8bytreatmentoftargetcellswithanti-DC-SIGNmAb.DCsorB-LCL-DC-SIGNwerepretreatedwithanti-DC-SIGNmAb(clone120507)ormannan,orleftuntreated.Eachbarrepresentsthemeanpercentofbindinginhibition(SE)(comparedwithuntreatedcells)fromtwoduplicate,InhibitionofbindingofradioactivelylabeledHHV-8bytreatmentofviruswithsolubleDC-SIGN.Resultsarethemean(SE)per-centageofinhibitionofbindingofsolubleDC-SIGN-treatedHHV-8com-paredwithbindingofradiolabeleduntreatedvirustoeachcelltypefromtwo,DoseresponseofinhibitionofvirusbindingtoDCsbytreatmentwithanti-DC-SIGNmAb.Eachbarrepresentsthemeanofduplicatereactions(SE)fromduplicatedeterminations.Dataarefromoneexperimentrepresentativeofthreeindependentexperiments.TheJournalofImmunology (4),andincludes:1)DC-SIGNwaspresentonDCsandmac-rophagesthataresusceptibletoinfectionbythevirus,2)ex-pressionofDC-SIGNrenderednormallyresistantcelllinessus-ceptibletoHHV-8infection,and3)HHV-8infectionwasblockedbymAbsdirectedagainstDC-SIGN,andbysolubleDC-SIGNandanaturalligandofDC-SIGN(mannan).Immature,monocyte-derivedDCssupportedviralK8.1andORF59geneexpression,whichwasblockedbypretreatmentofthecellswithanti-DC-SIGNmAbormannan,orbypretreatmentoftheviruswithsolubleDC-SIGN.ExpressionofK8.1andORF59subsequentlydeclinedintheDCs,withpersistenceoflatent(LNA-1)viralgeneexpression.AnalysisofHHV-8DNAdemon-stratedverylowlevelsofviralDNAininfectedDCswithnoevidenceofvirusproduction.However,thevirallyticgenesandORF59andthelatencygenewereexpressedintheinfectedcells.TheexpressionofsomebutnotallviralgeneswithhighlyrestrictedDNAreplicationhasalsobeennotedforHHV-8infectionofhumanbroblastsandendothelialcells(30).Indeed,microarrayanalysishasshownthatHHV-8infectionofbroblastsandendothelialcellsdoesnotresultinaproductive,replicativecycle(30).Instead,thereisexpressionofasubsetoflyticcyclegenetranscripts(includingORF59andK8.1)whichquicklysub-sides,followedbypersistentexpressionoflatencygenetranscripts(ORF73).Moreover,onlylatent,andnotproductivelytic,HHV-8infectionhasbeennotedinvarioustypesofinfectedcells(8,36–38).InfectionofDCsbyHHV-8appearssimilartoinfectionoftheseothercelltypesinvitrointhatthereisaburstofgenetran-scriptionresultinginanonproductive,latentviralinfectionwithoutsignicantviralDNAreplication.Thislimitedlyticgeneexpres-sionwiththepersistenceoflatentgenesinvitroappearstobeuniqu

etothisherpesvirus(30),andrequiresfurtherstudy.OurstudyisthersttodemonstrateHHV-8infectionofmy-eloidlineageDCs.ThisissupportedbyarecentreportthatHHV-8caninfectCD34stemcellprecursorsofDCsinvitro(39).WeproposethatDCsthatexpressDC-SIGNinvivo,i.e.,maturemy-eloidDCsandimmatureplasmacytoidDCs(9,10,40),shouldbesusceptibletoinfectionbyHHV-8.Interestingly,DCshaveocca-sionallybeenfoundinKSlesions(18).Previously,however,therehavebeenconictingreportsonthepresenceofHHV-8inDCs(19–21).Notably,thesestudiesonlyexaminedDCsfornatural,invivoinfectionwithHHV-8inpersonswhowereeithernotas-sessed,orwerenegative,forHHV-8Abs.Furtherstudiesareon-goingtoexamineDCsforinfectionwithHHV-8inpersonswhoareHHV-8seropositiveorhaveKS.TheresultsshowthatmacrophageswerealsosusceptibletoHHV-8infectionviaDC-SIGN.LownumbersofunstimulatedmacrophagesexpressedDC-SIGNinassociationwiththeirinvitrotransformationfrommonocytes.TheseunstimulatedmacrophagesweresusceptibletoalowlevelofinfectionwithHHV-8,whichwasblockedbyanti-DC-SIGNmAbormannan.ActivationofmacrophageswithIL-13greatlyenhancedexpressionofDC-SIGN,conrmingpreviousreports(9,15),andrenderedthemhighlypermissiveforHHV-8infection.Moreover,HHV-8infec-tionwasblockedbypretreatmentoftheactivatedmacrophageswithanti-DC-SIGNmAbormannan.Previously,ithasbeenre-portedthatmonocyte-derivedmacrophagesfromsomenormaldo-norsthatarestimulatedinvitrowithallogeneicPBMCsaresus-ceptibletoHHV-8infection(31).Moreover,treatmentofbloodmonocytesfromKSpatientsinvitrowithproinammatorycyto-kinesresultsinpersistenceofHHV-8(41).IL-13isproducedbyTh2cells,aswellaseosinophils,mastcells,andbasophils.Inter-estingly,HHV-8encodesaviralmacrophageinhibitoryproteinIIthatisassociatedwithinltrationofTh2cellsandeosinophilsinKSlesions(42).ProductionofIL-13bytheseinltratingcellscouldpotentiallyenhanceHHV-8infectionofmonocytesandmacrophagesproximaltotheKSlesionbyup-regulatingexpres-sionofDC-SIGN.Integrins,particularlythedimer,havebeenshowntoserveasreceptorsforHHV-8infectionofendothelialcellsandbro-blasts(8).WehavefoundthatexpressionofDC-SIGNoncells FIGURE6.HHV-8infectionofIL-13-activatedmacrophagesisrelatedtoDC-SIGNexpression.,FlowcytometricanalysisshowingexpressionofDC-SIGNonHHV-8-infected(emptyhistogram,brokenline)orun-infected(emptyhistogram,solidline)IL-13-activatedmacrophages.Fullhistogram,isotypecontrols.,Im-munouorescenceresultsonIL-13-activatedmacro-phagesthatwereinfectedwithHHV-8for24handstainedforORF59(red)andDC-SIGN(green).Theoverlayofcombinedcolorsforanti-DC-SIGNandORF59isshown.,ImmunouorescenceresultsonIL-13-activatedmacrophagesthatwerepretreatedwithanti-DC-SIGNmAb(clone120507)ormouseIgG,infectedwithHHV-8for24handstainedforanti-K8.1mAb(red).CellswerecounterstainedwithDAPI(Dataarefromoneexperimentrepresentativeoffourin-dependentexperiments.DC-SIGNISARECEPTORFORHHV-8 normallyresistanttoHHV-8infection(B-LCLsandK562cells)renderedthemsusceptibletoHHV-8infection,althoughneitherofthesecellsexpressedontheirsurface.Nevertheless,herpesvirusescanhavemorethanonereceptorinthesamecell(4),ashasrecentlybeenreportedforepidermalgrowthfactorreceptorandintegrinforinfectionofbroblastsbyhumanCMV(43).orotherintegrinscouldservetogetherwithDC-SIGNasreceptorsforHHV-8incertaintypesofcells.Moreover,hepa-ransulfateisalsoknowntobindHHV-8onendothelialcellsandbroblasts(5–7).Indeed,theresiduallevelofbindingandinfec-tionofHHV-8incellspretreatedwithanti-DC-SIGNmAbinourstudies,whichissimilartothatreportedforothermicrobialagentsthatuseDC-SIGNasareceptor(44,45),suggeststhatthereareadditionalreceptorsforHHV-8onthesecells.StudiesdesignedtodistinguishtherolesofDC-SIGNandotherreceptorsinHHV-8infectionareinprogress.BindingofHHV-8toDC-SIGNinduceddown-regulationofthismoleculeonDCs,whichappearstobesequesteredinthecytoplasmofthecells.UptakeofDC-SIGNintolysosomalcom-partmentsisknowntooccurafterbindingofotherpathogenstothemolecule(46),andcouldbeinvolvedinprocessingofHHV-8Ags.WehavealsoshownthatHHV-8infectionofDCsinducedpro-foundperturbationsintwooftheirmostimportantfunctions,i.e.,adecreaseinendocytosis,orAguptake,andAgpresentationtoTcells.ThelattereffectcouldbedueinparttoadecreaseinexpressionofMHCclassImoleculesthatisawell-documentedfeatureofHHV-8infection(47,48).Indeed,wenotedadecreaseinMHCclassIexpressiononHHV-8-infectedDCs.Thesemod-ulatoryeffectsonthe

DCswereduetoHHV-8infection,asweusedpuriedHHV-8inthesestudiestoruleoutpossibleeffectsontheDCsofotherfactorsincrudeviruspreparations.Takento-gether,theseresultsindicatethatHHV-8infectionofDCsleadsto FIGURE7.EffectofHHV-8in-fectionofDCsonexpressionofDC-SIGNandcostimulatorymolecules.,DC-SIGNexpressiononunin-fectedorHHV-8infectedDCs.DataaremeanMFI(SE)fromsevenin-dependentexperiments.,Expres-sionofHLA-ABC,HLA-DR,CD83,andDC-SIGNoninfectedDCs.Bluehistogram,HHV-8-infectedDCs;yellowhistogram,uninfectedDCs;emptyhistogram,neline,andbro-kenlineisotypecontrolsfortheinfectedanduninfectedDCs,respec-tively.Dataarefromoneexperimentrepresentativeof10independentex-,ConfocalmicroscopyofHHV-8-infectedDCsstainedwithanti-DC-SIGN(green)andanti-ORF59(red)mAbsat24h(leftpanel)and48h(centerpanel)afterinfection.UninfectedDCsservedascontrolsrightpanel).Dataarefromoneex-perimentrepresentativeoftwoinde-pendentexperiments.TheJournalofImmunology defectsinAgprocessingandpresentationthatcouldbeimportantinimmuneevasionofthevirus.ThisDCdysfunctioncouldalsorelatetoourpreviousndingsofarelativelynonrobustCD8cellresponsetoHHV-8afterprimaryHHV-8infection(49).TheseresultssuggestthatamajorblockincellsresistanttoHHV-8infectionisthelackofexpressionofaproperreceptorsuchasDC-SIGN.Indeed,wehavefoundthatactivatedBlymphocytesexpressDC-SIGN,andmicrovascularendothelialcellsexpressaC-typelectinhomologofDC-SIGN,DC-SIGNR(DC-SIGNre-lated;CD209R),andthattheseserveasreceptorsforHHV-8onthesecells(ourunpublishedobservations).Finally,itshouldbenotedthatthecellularhostrangeofHHV-8infectioncanbeex-pandedarticiallytoincludecellsnormallyresistanttoHHV-8infectionbytreatmentofthesecellswithPolybrene(50),whichenhancesreceptor-independentinfection(51).WeconcludethatexpressionofthetypeIIC-typelectin,DC-SIGN,isnecessaryforreceptor-mediatedinfectionofmyeloidDCsandmacrophages.DC-SIGNcouldserveasaportalforHHV-8infectionofcellsthatareknowntoexpressDC-SIGNinvivo,i.e.,tissue-residentDCsandalveolarmacrophages(10,52),macrophagesactivatedbyproinammatorycytokines(9)andac-tivatedBlymphocytes(ourunpublishedresults).Thisnovelnd-ingontheroleofDC-SIGNintheinfectiousprocessofHHV-8couldhaveimportantimplicationsfordesigningeffectivestrate-giesforinhibitingHHV-8infectionandassociatedcancers.WegratefullyacknowledgehelpfuldiscussionswithP.Spear,P.Gupta,D.Rowe,P.Moore,Y.ChangandO.Flore,andtheassistanceofM.Tomaszewski,A.Pollard,andS.Alber.WethankAmgenforthegiftofCD40L(MTAn.200312724).Theauthorshavenonancialconictofinterest.1.Boshoff,C.,R.Weiss.2002.AIDS-relatedmalignancies.Nat.Rev.Cancer2.Bubman,D.,andE.Cesarman.2003.PathogenesisofKaposi’ssarcoma.tol.Oncol.Clin.NorthAm.17:717–745.3.Blasig,C.,C.Zietz,B.Haar,F.Neipel,S.Esser,N.H.Brockmeyer,E.Tschachler,S.Colombini,B.Ensoli,andM.Sturzl.1997.MonocytesinKa-posi’ssarcomalesionsareproductivelyinfectedbyhumanherpesvirus8.J.Virol.71:7963–7968.4.Spear,P.G.,andR.Longnecker.2003.Herpesvirusentry:anupdate.J.Virol.10179–10185.5.Akula,S.M.,N.P.Pramod,F.Z.Wang,andB.Chandran.2001.Humanher-pesvirus8envelope-associatedglycoproteinBinteractswithheparansulfate-like284:235–249.6.Birkmann,A.,K.Mahr,A.Ensser,S.Yaguboglu,F.Titgemeyer,B.Fleckenstein,andF.Neipel.2001.Cellsurfaceheparansulfateisareceptorforhumanherpesvirus8andinteractswithenvelopeglycoproteinK8.1.J.Virol.7.Wang,F.Z.,S.M.Akula,N.P.Pramod,L.Zeng,andB.Chandran.2001.Humanherpesvirus8envelopeglycoproteinK8.1Ainteractionwiththetargetcellsin-volvesheparansulfate.J.Virol.75:7517–7527.8.Akula,S.M.,N.P.Pramod,F.Z.Wang,andB.Chandran.2002.Integrin(CD49c/29)isacellularreceptorforKaposi’ssarcoma-associatedherpesvirus(KSHV/HHV-8)entryintothetargetcells.108:407–419. FIGURE8.EffectofHHV-8infectionofDCsonendocytosisandAg-specicactivationofCD8Tcells.,EndocytosisofFITC-dextranbyuninfectedDCs(leftpanel,openhistogram)orHHV-8infectedDCs(rightpanel,openhistogram).CD40L-maturedDCs(middlepanel,openhistogram)andcellsincubatedat4°C(shadedhistograms)servedascontrols.Dataarefromoneexperimentrepresentativeofveindependentexperiments.,TheabilityofHHV-8-infectedoruninfected,immature(iDC),andCD40L-maturedDCs(mDC)tostimulateAg-specicIFN-productioninautologousCD8Tcells.EachbaristhemeanSEoftriplicatewells.Dataarefromoneexperimentrepresentativeoftwoindependentexperiments.DC-SIGNISARECEPTORFORHHV-8 9.Soilleux,E.J.,L.S.Mo

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