Human

Human󰀆Secreted󰀆Stabilin-1−InteractingChitinase-like󰀆Protein󰀆Aggravates󰀆the

Inflammation󰀆Associated󰀆With󰀆RheumatoidArthritis󰀆and󰀆Is󰀆a󰀆Potential󰀆MacrophageInflammatory󰀆Regulator󰀆i...

ARTICLE󰀃󰀃in󰀃󰀃ARTHRITIS󰀃AND󰀃RHEUMATOLOGY󰀃·󰀃MAY󰀃2014

DOI:󰀃10.1002/art.38356

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11󰀉AUTHORS,󰀃INCLUDING:

Geng󰀃MengPurdue󰀃University

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Yanmei󰀃Zhao

Chinese󰀃Academy󰀃of󰀃Sciences

25󰀃PUBLICATIONS󰀃󰀃󰀃262󰀃CITATIONS󰀃󰀃󰀃

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Yuan󰀃Huihui

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Xiaofeng󰀃ZhengPeking󰀃University

77󰀃PUBLICATIONS󰀃󰀃󰀃747󰀃CITATIONS󰀃󰀃󰀃

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Available󰀃from:󰀃Xiaofeng󰀃ZhengRetrieved󰀃on:󰀃09󰀃March󰀃2016

ARTHRITIS&RHEUMATOLOGYVol.66,No.5,May2014,pp1141–1152DOI10.1002/art.38356

©2014,AmericanCollegeofRheumatology

HumanSecretedStabilin-1ϪInteractingChitinase-likeProtein

AggravatestheInflammationAssociatedWithRheumatoidArthritisandIsaPotentialMacrophage

InflammatoryRegulatorinRodents

WeichunXiao,1GengMeng,1YanmeiZhao,1HuihuiYuan,2TingtingLi,1YanyanPeng,1YongZhao,3MingLuo,4WenmingZhao,2ZhanguoLi,5andXiaofengZheng1Objective.Tostudytherelationshipbetweenthehumansecretedproteinstabilin-1؊interactingchitinase-likeprotein(SI-CLP)andrheumatoidarthri-tis(RA).

Methods.TheexpressionofSI-CLPinperipheralbloodmononuclearcells(PBMCs)andsynovialfluidfrompatientswithRAandtheeffectsofcytokinesonSI-CLPexpressionwereexaminedbyWesternblotting.Fluorescence-activatedcellsortinganalysiswasper-formedtoinvestigatethebindingbetweenSI-CLPandcells.Bonemarrow–derivedmacrophageswereisolatedfromwild-typeandSI-CLP؊/؊mice,andreal-timequantitativepolymerasechainreactionwasperformedtodetectthelevelsofmessengerRNAforcytokinesorSI-CLPinSI-CLP؊orcytokine-treatedmacrophages.Histologicstudieswereconductedtoevaluateinflamma-SupportedbytheNationalBasicResearchProgramofChina

(grants2010CB911800and2010CB529106),theNationalScienceFoundationofChina(grants30930020,30870487,and31170709),andtheInternationalCenterforGeneticEngineeringandBiotechnology(ICGEBgrantCRP/CHN09-01).

1WeichunXiao,PhD(currentaddress:CapitalMedicalUni-versity,Beijing,China),GengMeng,PhD,YanmeiZhao,PhD(cur-rentaddress:InstituteofBiophysics,ChineseAcademyofSciences,Beijing,China),TingtingLi,BSc,YanyanPeng,BSc,XiaofengZheng,PhD:StateKeyLabofProteinandPlantGeneResearch,SchoolofLifeSciences,PekingUniversity,Beijing,China;2HuihuiYuan,PhD,WenmingZhao,MD:CapitalMedicalUniversity,Beijing,China;3YongZhao,MD,PhD:PekingUniversityPeople’sHospital,Beijing,China;4MingLuo,PhD:UniversityofAlabamaatBirmingham;5ZhanguoLi,MD,PhD:ChineseAcademyofSciences,Beijing,China.

Drs.Xiao,Meng,andYanmeiZhaocontributedequallyto

thiswork.

AddresscorrespondencetoXiaofengZheng,PhD,School

ofLifeSciences,PekingUniversity,Beijing100871,China.E-mail:xiaofengz@pku.edu.cn.

SubmittedforpublicationApril17,2013;acceptedinrevised

formJanuary7,2014.

tionandtheexpressionofinterleukin-12(IL-12),IL-13,andSI-CLPinlesions.Enzyme-linkedimmunosorbentassayswereusedtodetectthecytokinelevelsinbonemarrow–derivedmacrophages.Ratsormicewithcollagen-inducedarthritis(CIA)andSI-CLP؊/؊micewereusedtostudythefunctionofSI-CLPinRA.

Results.SI-CLPexpressionwasincreasedinPBMCsanddetectableinsynovialfluidfrompatientswithRA.AdministrationofSI-CLPtoratswithCIAaggravatedarthritis-associatedinflammation.SI-CLPwasspecificallyattachedtothesurfaceproteinofmacro-phages,whichelevatedtheexpressionofIL-1␤,IL-6,IL-12,andIL-13inmacrophagesandmousebonemarrow–derivedmacrophages,up-regulatingERKphosphorylation.Moreover,SI-CLPwasup-regulatedbybothIL-12andIL-13throughJNKandJAK/STATsignaling,respectively.KnockoutofSI-CLPresultedinadecreaseintheexpressionofIL-1␤,IL-6,IL-12,andIL-13andlowersusceptibilitytoCIAcomparedwithwild-typemice.SI-CLPtreatmentalsoaggravatedarthritis-relatedinflammationinwild-typeandSI-CLP؊/؊mice.

Conclusion.SI-CLPfunctionsasaregulatoroftheinflammatoryresponsebymacrophages.Thede-creaseininflammation-associatedcytokinelevelsre-sultingfromSI-CLPknockoutmayexplainthelowersusceptibilitytoCIAinSI-CLP؊/؊mice.

Macrophagesprovideafirstlineofhostdefenseagainstpathogens.Macrophagesareactivatedbyinterferon-␥,interleukin-1(IL-1),andIL-12andper-formtheireffectorfunctionsimmediatelybyreleasingproinflammatorycytokinesandgrowthfactorsandthen

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1142initiatingadaptiveimmunity(1–4).MacrophagesarealsoactivatedbytheTh2cytokinesIL-4andIL-13andplayacrucialroleinprotectingthehostduringallergicinflammationandparasiticinfection(5,6).Overactiva-tionofmacrophagesleadstoinflammationandaseriesofclinicalandpathologicconsequencesandinducesthedevelopmentofautoimmunediseasessuchasarthritis.Therefore,activationofmacrophagesisessentialfortheimmunesystemtoinitiateinflammation,andinvestiga-tionsoftheseissuesareimportantforunderstandingthemechanismofautoimmunediseaseinducedbyoveracti-vationoftheimmunesystem.

Rheumatoidarthritis(RA)isachronicauto-immunediseasecharacterizedbymacrophageandlym-phocyteinfiltrationandinflammationofthesynovialjoints.Cytokinesarepivotalregulatorsofawidevarietyofimmuneandinflammatoryprocesses(7,8).Macro-phagesplayimportantrolesintheregulationofRA.MacrophagesareabundantinRAandarecloselyin-volvedinpathogenesisbypromotinginflammationandjointdestruction.Macrophagesinfiltrateintothein-flamedsynovialmembraneandareactivated,producingvariousproinflammatorycytokinesandchemokines(9–12).ThesecytokinespromotethedevelopmentofchronicinflammationandaggravatecartilageandbonedestructioninRAthroughmultiplemechanisms(13–16).

Stabilin-1isamarkerforalternativemacrophageactivation(17,18).Stabilin-1Ϫinteractingchitinase-likeprotein(SI-CLP),amemberofthechitinase-likeorGlyco_18family,isahumansecretedprotein.SI-CLPcontainsasignalpeptideandaGlyco_18domain(19),butthesequenceidentitybetweenSI-CLPandotherGlyco_18domainproteinsisϽ20%.SI-CLPinhumansandinmice/ratsshareshighidentity(ϳ90%)ontheaminoacidlevel,indicatingfunctionalconservationofSI-CLPinthesespecies.TheexpressionofSI-CLPisabundantinthebronchoalveolarlavagefluidofpatientswithchronicinflammatorydisordersoftherespiratorytractandinhumanperipheralbloodleukocytes(18),butthefunctionofSI-CLPremainslargelyunknown.

ToclearlyunderstandthecorrelationofSI-CLPwithRAandcytokines,weassessedtheregulationofSI-CLPoncytokinesaswellastheinfluenceofcytokinesonSI-CLP.WeusedanSI-CLPϪ/Ϫmousemodelofcollagen-inducedarthritis(CIA)toinvestigatetherolesofSI-CLPinRA.TheresultsofthisstudyindicatedthatSI-CLPfunctionsasanimmuneregulatorthroughitsassociationwithmacrophagesandbyup-regulatingtheexpressionofIL-1,IL-6,IL-12,andIL-13.Adeficiency

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ofSI-CLPinmicewithCIAhindersthedevelopmentofchronicinflammation.

MATERIALSANDMETHODS

Celllines,cellculture,andreagents.Thecelllines

weremaintainedat37°Cwith5%CO10%fetal2inRPMI1640(Invit-rogen)supplementedwithcalfserum(FCS;Hy-Clone),withoutantibiotics.MonocyticTHP-1cellsweretreatedwith50ng/mlphorbolmyristateacetate(PMA)(Sigma)for48hoursanddifferentiatedintoamacrophage-likephenotypethatcloselyresembleshumanmonocyte–derivedmacrophages.C57BL/6mice,BALB/cmice,Lewisrats,andSI-CLPϪ/Ϫmicewerekilled,andprimarymacrophagesiso-latedfromperitonealfluid(20)andbonemarrowweremain-tainedinRPMI1640.Humanperipheralbloodmononuclearcells(PBMCs)werepurifiedbyHistopaque-1077(Sigma-Aldrich)density-gradientcentrifugation(21)andmaintainedinphosphatebufferedsaline(PBS).

Antibodiesagainstp38MAPK,JNK,ERK-1/2,

phospho–p38MAPK,phospho-JNK,andphospho–ERK-1/2wereobtainedfromCellSignalingTechnology.IL-12andIL-13monoclonalantibodieswereobtainedfromAbcam.ERKinhibitor,JNKinhibitor3,JNKinhibitor2,andp38MAPKinhibitorwereobtainedfromMerck.JAKinhibitor1,JAK-2inhibitor,andJAK-3inhibitorwereobtainedfromSantaCruzBiotechnology.HumanandmouseIL-1␤,IL-4,IL-12,IL-13,andIL-6wereobtainedfromPeproTech.

PurificationofrecombinantSI-CLP.SI-CLPprotein

waspurified(22)andincubatedwithpolymyxinB–agarose(Sigma)overnighttoremoveendotoxin(23)andusedtotreatthecells,rats,andmice.ThepolyclonalantibodiesagainstSI-CLPweregeneratedasdescribedpreviously(24)andpurifiedusingCNBr-activatedSepharose4B(GEHealthcare).

PBMCsandsynovialfluidfrompatientswithRA.

PBMCsfrompatientswithRAwerecollectedatChinesePeople’sLiberationArmy169Hospital.PBMCsfrom3healthyindividualswithnosignofautoimmunedisorderswereusedascontrols.Synovialfluidsamplesfrom7patientswithRAwerecollectedatPekingUniversityPeople’sHospital,andsynovialfluidsamplesfrom2healthyindividualswithnosignofRAwereusedascontrols.Thesampleswerecentrifuged,andthesupernatantswereseparatedbysodiumdodecylsulfate–polyacrylamidegelelectrophoresisandsubjectedtoWesternblotanalyses.AllsampleswerecollectedaccordingtotheguidelinesoftheethicscommitteesatPekingUniversityPeople’sHospitalandChinesePeople’sLiberationArmy169Hospital,andallpatientsprovidedwrittenconsent.Allsyno-vialfluidsampleswerecollectedaccordingtotheguidelinesoftheEthicsCommitteeatPekingUniversityPeople’sHospital.

Magnetic-activatedcellsorting(MACS).Synovialfluid

monocyteswereisolatedfromthebuffycoatpreparationsofpatientswithRAbyFicolldensity-gradientcentrifugationtoremoveerythrocytesandgranulocytes.Monocytesintheinter-phasewerefurtherpurifiedusinganti-humanCD14magneticparticles(BDBiosciences),andapositiveMACSprocedure(inwhichcellsexpressingCD14antigenontheirsurfacewereselectedfromsynovialfluidmonocytes)wasperformedaccord-ingtothemanufacturer’sprotocols.

SI-CLPAGGRAVATESRA-RELATEDINFLAMMATIONHistologicassessment.Thepawsoftheanimalswere

removedpostmortem,storedin10%neutralformalin,decal-cifiedin20%EDTAfor6weeks,andthendehydratedandembeddedinparaffin.Sectionswerecutalongthelongitudinalaxis,mounted,andstainedwithhematoxylinandeosintoevaluatetheseverityofinflammatorycellinfiltrationinthejoint.Immunohistochemistrywasperformedtodetectimmu-noreactivityforIL-12,IL-13,andSI-CLPinthesections,usingrabbitanti-mouseIL-12antibodies(1:800),IL-13antibodies(1:250),andSI-CLPantibodies(1:200).Positivesignalsweredetectedwith3,3Ј-diaminobenzidine(R&DSystems).TheslideswerethencounterstainedwithMayer’shematoxylin(Sigma).

AnimalsandCIA.Allanimalstudieswereapprovedby

theethicscommitteesofPekingUniversityPeople’sHospitalandPekingUniversity.C57BL/6mice,BALB/cmice,andLewisratswereobtained/ϪfromVitalRiverLaboratoryAnimalTechnology.SI-CLPϪmicewereobtainedfromtheRe-searchCenterforModelOrganisms(Shanghai,China).ThesecondexonoftheSI-CLPgenewasdeletedinSI-CLPϪ/Ϫmice.ToconfirmthegenomicmanipulationinSI-CLPϪ/Ϫmice,mononuclearcellswere/Ϫisolatedfromthebonemarrowofwild-typeandSI-CLPϪmice,andtheRNAandproteinlevelsofSI-CLPwereexaminedbyreal-timepolymerasechainreaction(PCR)andWesternblotanalysis,respectively.

maceutical)LewiswasratemulsifiedmodelofCIA.inanTypeequalIIcollagenvolumeof(QiluFreund’sPhar-incompleteadjuvant(IFA;Sigma).TenfemaleLewisrats(6–8weeksold)wereimmunizedbyintradermalinjectionof0.1mltypeIIcollageninIFAondays0and7.Fromday14throughday49aftertheinitialimmunization,halfoftheratswereinjectedintheabdomenwithpurifiedSI-CLPprotein(500␮g)everyweek,andhalfwereinjectedintheabdomenwithPBSascontrol.Theratswerescoredweekly,byanindividualblindedtotreatmentassignment,forsignsofarthritisinthepaws,basedonthefollowingpreviouslydescribedscale(25):0ϭnorednessorswelling,1ϭmildswellingofthetoesandankle,2ϭmoderateswellingofthetoesandankle,3ϭsevereswellingofthetoesandankle,and4ϭentirepawswollen,withankylosis.Thescoresforeachofthe4pawsweretotaled,withafinalscoreof16indicatingmaximumseverity.FurtherevaluationofCIAwasconductedusingaradiographictech-nique.Thesourceofradiographswasaconventionalradio-graphicunit,andtheacquisitionparameterswere:voltage63kV,current8mA,andexposuretime32msec.

TwentyC57BL/6C57BL/6mousemiceand(8–9SI-CLPϪ/Ϫweeksold;mouse10modelsmalesofandCIA.

10females)and20SI-CLPϪ/ϪmiceonaC57BL/6background(8–9weeksold)wereimmunizedatthebaseofthetailwith0.1mltypeIIcollagen(Chondrex)inFreund’scompleteadjuvant(CFA),thenboostedwith0.05mltypeIIcollageninCFAonday7afterthefirstimmunization.Atthesametime,20C57BL/6mice(8–9weeksold;10malesand10females)wereinjectedwithPBSascontrol.Duringweeks5through9aftertheinitialimmunization,halfofthemicefromeachgroupwereintraperitoneallyinjectedwith150␮gpurifiedSI-CLPproteinaccordingtoapreviouslydescribedprotocol(26),andhalfwereintraperitoneallyinjectedwithPBSascontrol.Themicewerescoredweeklyinthesamemanneras

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thatusedfortherats.FurtherevaluationofCIAwascon-ductedusingaradiographictechnique.Thesourceofradio-graphswasaMikasaHF100Hunit,andtheacquisitionpara-meterswere:voltage42kV,current2mA,andexposuretime32msec.

Isolationandcultureofmurinebonemarrow–derived

macrophages.BonemarrowsamplesobtainedfromthehindlegsofthemicewereisolatedusingFicolldensity-gradientcentrifugation(17)andculturedwithRPMI1640mediumsupplementedwith10%FCSand20%ofthecellculturesupernatantofL929cells,accordingtopreviouslydescribedmethods(27,28).After7days,adherentcellsweretreatedwithSI-CLPorcytokines.

AnalysisoftheassociationofSI-CLPwiththecell

surfaceofdifferentcelllinesandpurifiedprimarycells.THP-1cellswereculturedinthepresenceorabsenceofPMA(50ng/ml)for48hoursandthencollected.Primarymacro-phagesobtainedfromC57BL/6mice,BALB/cmice,andLewisratsandPBMCsobtainedfromhumanbloodwereallfreshlyisolatedandmaintainedinRPMI1640.TheassociationofSI-CLPonthecellsurfaceofdifferentcelltypeswasexaminedbyfluorescence-activatedcellsorting(FACS).InordertodecreasethebackgroundofendogenousSI-CLPprotein,differentcontrolexperimentswereperformedwithanotherrecombinanthumansourceprotein(hCINAP;GeneID6880)expressedfromEscherichiacoliandpurifiedusingthesameproceduredescribedpreviously(22).AllanimalexperimentswereapprovedbythePekingUniversityPeople’sHospitalEthicsCommittee(FWA00001384).

RNAisolationandreal-timequantitativePCR(qPCR)

analysis.TotalRNAwasisolatedusingTRIzolreagent(Invit-rogen),andcomplementaryDNAwasgeneratedwithaRe-verseTranscriptionSystem(Promega).MessengerRNA(mRNA)expressionwasexaminedbyreal-timeqPCRwithaSYBRGreenqPCRkitinaCFX96TouchReal-timePCRDetectionSystem(Bio-Rad).Beta-actinwasusedtocorrectintersamplevariations.Theprimersusedareavailablefromthecorrespondingauthor.

Westernblotanalysisandenzyme-linkedimmunosor-bentassay(ELISA).Westernblottingwasperformedusingtheindicatedantibodies(24).TheeffectsofSI-CLPontheproteinlevelsofIL-1␤,IL-6,IL-12,andIL-13inSI-CLP–treatedhumanandmurinemacrophageswereassessedbyELISA(BioLegend).

Statisticalanalysis.Allresultsareexpressedasthe

meanϮSEM.Pvalueslessthan0.05,asdeterminedbyStudent’st-test,wereconsideredsignificant.Eachexperimentwasperformedatleast3times.

RESULTS

SI-CLPispresentinPBMCsandsynovialfluidfrompatientswithRAandaggravatesinflammationinratswithCIA.SI-CLPwaspurifiedandinjectedintoarabbittogenerateantibodiesagainstSI-CLP.Westernblotanalysisshowedthatpolyclonalanti–SI-CLPcan

1144Figure1.Stabilin-1Ϫinteractingchitinase-likeprotein(SI-CLP)ag-gravatesinflammationinratswithcollagen-inducedarthritis(CIA)A,ExpressionofSI-CLPinperipheralbloodmononuclearcellsfromrheumatoidarthritis(RA)patients01–07andcontrolsubjects01–03(top),synovialfluidfromRApatients01Ј–07Јandcontrolsubjects01Јand02Ј(middle),andCD14ϩmononuclearmacrophagesormacrophage-freecellsisolatedfromthesynovialfluidofRApatients01Љ–04Љ(bottom),asdetectedbyWesternblottingusinganti–SI-CLP.B,ExpressionofSI-CLPmRNAandproteininwild-type(WT)ratsandratswithCIA,asdetectedbyreal-timequantitativepolymerasechainreaction(top)andWesternblotanalysis(bottom).ءϭPϽ0.05versusWT.C,EffectofSI-CLPinratswithCIA.Top,SI-CLPtreatmentresultedinincreasedswellingofthepawandmoreboneerosioncomparedwithtreatmentwithsalineonly.ArrowsindicatetheswellingandbonelossinducedbyadministrationofSI-CLP.Bottomleft,SI-CLPtreatmentincreasedarthritisscores,asassessedbythedegreeofpawswelling.Bottomright,Theproteinlevelofinterleukin-1␤(IL-1␤),butnottumornecrosisfactor␣(TNF␣),wasincreasedinratstreatedwithSI-CLP.ءϭPϽ0.05versuscontrol.ValuesinBandC(bottomleft)arethemeanϮSEM.DatainC(bottomright)arepresentedasboxplots,wheretheboxesrepresentthe25thto75thpercentiles,thelineswithintheboxesrepresentthemedian,andthelinesoutsidetheboxesrepresentthe10thand90thpercentiles.Barϭ500␮m.Colorfigurecanbeviewedintheonlineissue,whichisavailableathttp://onlinelibrary.wiley.com/doi/10.1002/art.38356/abstract.

specificallyidentifyendogenousSI-CLPinTHP-1cellsandrecombinantSI-CLP(datanotshown).Thefinal

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concentrationofendotoxininthepurifiedSI-CLPpro-teinwasϽ20endotoxinunits/mg.

ToexaminetheexpressionofSI-CLPinRA,WesternblottingwasperformedtodetectSI-CLPinPBMCsandsynovialfluid(Figure1A)frompatientswithRAandhealthycontrolsubjects.SI-CLPexpressionwaselevatedinthesynovialfluidofpatientswithRAbutwasdeficientinthesynovialfluidofcontrolsubjectswithnoarthritis(Figure1A).WeisolatedCD14ϩmono-nuclearmacrophagesfromthesynovialfluidofpatientswithRAandmeasuredtheSI-CLPlevelsinCD14ϩmononuclearmacrophagesandtheothercells,includingTcellsandBcells.SI-CLPexpressionwasincreasedinCD14ϩmononuclearmacrophagescomparedwithCD14Ϫcells(Figure1A).TheseobservationsindicatedthatSI-CLPmightbecorrelatedwithRA.BecausetheanimalmodelofCIAcloselyresemblestheclinicalcharacteristicsofhumanRAandisoneofthemostcommonlyusedmodelsofhumanRA,weusedarodentmodelofCIAtofurtherstudytheroleofSI-CLPinRA.Bothreal-timeqPCRandWesternblotanalysisshowedthatSI-CLPwassignificantlyup-regulatedinmicewithCIAcomparedwithwild-typecontrols(Figure1B).

TostudytheeffectofSI-CLPinRA,ratswithCIAwereinjectedintheabdomenwithpurifiedSI-CLPprotein(PBSwasusedascontrol),andinflammationwasassessed.ComparedwithPBScontrol,SI-CLPaggra-vatedinflammationinthearthriticratsandledtogreaterboneerosionandhigherarthritisscores(Figure1C).Toelucidatewhethermoresevereswellingofthejointswasattributabletoanincreasednumberofproinflammatorycytokines,theproteinlevelsofIL-1␤andtumornecrosisfactor␣(TNF␣)inseracollectedfromtheratswithCIAwereexamined.Consistentwiththeseverityofinflam-mation,theproteinlevelofIL-1␤,butnotTNF␣,waselevatedintheSI-CLP–treatedgroup(Figure1C).

SI-CLPisspecificallyattachedtosurfaceproteinonactivatedmacrophages.TheresultsdescribedaboveindicatedthatSI-CLPmightreacttosomefunctionalcellsandregulateinflammation.Therefore,FACSana-lyseswereperformedtodetectattachmentofSI-CLPtovariousimmunecells,includingJurkat,Ramos,RAW264.7,THP-1(monocyte),andPMA-treatedTHP-1(macrophage)cells.SignificantattachmentofSI-CLPwasobservedonlyinactivatedmacrophages(Figure2A).TheattachmentofSI-CLPtoprimarymacrophagesfromC57BL/6andBALB/cmice,Lewisrats,andPBMCswasalsoexamined.SI-CLPadheredtoprimarymacrophagesfrombothmiceandrats(Figure2A),suggestingthatSI-CLPbindsselectivelytomacro-

SI-CLPAGGRAVATESRA-RELATEDINFLAMMATION1145

Figure2.Stabilin-1Ϫinteractingchitinase-likeprotein(SI-CLP)attachestothecellsurfaceofphorbolmyristateacetate(PMA)–differentiatedTHP-1cellsandprimarymacrophages.A,BindingofSI-CLPtovariouscells,includingJurkat,Ramos,THP-1,PMA-treatedTHP-1,andRAW264.7cells,humanperipheralbloodmononuclearcells(PBMCs),andprimarymacrophages(M␾)fromC57BL/6mice,BALB/cmice,andLewisrats,asdeterminedbyfluorescence-activatedcellsorting(FACS)analysis,usingpolyclonalanti–SI-CLPandfluoresceinisothiocyanate–conjugatedgoatanti-rabbitIgG.B,Attachmentofself-secreted(control)andrecombinantSI-CLPtomonocyticTHP-1cellsandPMA-differentiatedTHP-1cells,asdeterminedbyFACSanalysis.Thenumbersintheupperrightcornersarethepercentageofpositivecells.C,Time-dependentbindingofSI-CLPtothecellsurfaceofPMA-differentiatedTHP-1cells.Thenumbersintheupperrightcornersarethepercentageofpositivecells,ascalculatedonthebasisofmockcontrol.D,Up-regulatedexpressionofSI-CLPmRNAandproteininPMA-differentiatedTHP-1cells,asdeterminedbyreal-timequantitativepolymerasechainreaction(left)andWesternblotting(right).IntheWesternblots(right),lane1representsmonocyticTHP-1cellsandlane2representsPMA-differentiatedTHP-1cells.Actinwasusedtocorrectforintersamplevariations.BarsshowthemeanϮSEM.

phages.FACSanalysesfurtherconfirmedthatbothself-secretedSI-CLP(control)andrecombinantSI-CLPcouldspecificallybindtodifferentiatedTHP-1cellsbutnotunstimulatedTHP-1cells(Figure2B).Moreover,thebindingintensityincreasedinatime-dependentmannerfollowingtreatmentwithPMA(Figure2C).ThebindingactivityofrecombinantSI-CLPtomacrophageswasmuchhigherthanthatofcontrol,indicatingthatthebindingofrecombinantSI-CLPtomacrophagesresultsmainlyfromrecombinantSI-CLPbutnotendogenousSI-CLP.

THP-1cellscanbeinducedbyPMAtodifferen-tiatetomacrophage-likecellsandhavebeenwidelyusedasamodelformonocyte–macrophagedifferentiation(29–31).ThereforePMA-differentiatedTHP-1cellswereselectedforfurtherinvestigations.BoththemRNAandproteinlevelsofSI-CLPwereup-regulatedinPMA-differentiatedTHP-1cells(Figure2D).

SI-CLPinducesincreasedexpressionofIL-1␤,IL-6,IL-12,andIL-13throughactivationofERKphos-phorylationandisup-regulatedbyIL-12andIL-13throughtheJNKandJAKsignalingpathwaysinPMA-differentiatedTHP-1cells.BecauseSI-CLPcanspecif-icallyattachtothemacrophagesurface,wespeculatedthataspecificassociationmaychangecellactivities.PMA-differentiatedTHP-1cellswereincubatedwithvariousconcentrationsofSI-CLPproteins,andtheproductionofinflammation-associatedcytokineswasestimatedbyreal-timeqPCRanalyses.SI-CLPup-regulatedthemRNAexpressionofIL-1␤,IL-6,IL-12,andIL-13inmacrophages(Figure3A).

ERK,p38MAPK,andJNKareknownsignaling

1146pathwaysmediatingtheproductionofinflammation-associatedcytokinesandarthritis-relatedinflammation(32,33).ToevaluatewhetherSI-CLPpromotesproduc-tionofcytokinesinmacrophagesviatheJNK,ERK,orp38MAPKpathway,weassessedchangesinthelevelsofphosphorylatedERK,p38MAPK,andJNKinPMA-differentiatedTHP-1cellstreatedwithSI-CLP.AsshowninFigure3B,thephosphorylationlevelofERKwasincreasedafter15minutesoftreatmentwithSI-CLP,butnoobviouschangesinthephosphorylationlevelsofJNKandp38MAPKweredetected.ThisresultsuggestedthatSI-CLPregulatesinflammation-associatedcytokineproductioninmacrophagesthroughactivationofERKphosphorylationbutnotthep38MAPKorJNKpathway.

TofurtherexaminewhetherSI-CLPcouldberegulatedbyinflammation-associatedcytokines,PMA-differentiatedTHP-1cellsweretreatedwithIL-1␤,IL-4,IL-12,orIL-13,andthelevelofSI-CLPwasexamined.AsshowninFigure3C,themRNAexpressionoftheSI-CLPgenewassignificantlyincreasedinresponsetoIL-12andIL-13andwasmoderatelyincreasedbyIL-4treatment.Incontrast,themRNAlevelofSI-CLPwasslightlydecreasedbyIL-1␤.TheproteinlevelsofSI-CLPwereinaccordancewiththemRNAlevels.

Toelucidatethepathwaysinvolvedinup-regulatingSI-CLPinresponsetoIL-12andIL-13,PMA-differentiatedTHP-1cellsweretreatedwithIL-12orIL-13,andthendifferentinhibitorswereaddedindivid-uallytotheculturemedia.InIL-13–treatedcells,theJAKinhibitors,especiallytheJAK-2inhibitor,significantlydecreasedtheelevatedSI-CLPexpressioninducedbyIL-13,andtheinhibitorsofERK,p38MAPK,andJNKdidnothaveameasurableinfluence.InthecellstreatedwithIL-12,SI-CLPexpressionwasreducedbyJNKin-hibitorsbutnotbyJAKinhibitors(Figure3D).TheseresultssuggestedthatIL-13increasesSI-CLPexpressionthroughactivationoftheJAKsignalingpathway,whileIL-12functionsthroughtheJNKsignalingpathway.

AG490isaninhibitoroftheIL-13pathwaythatdecreasesthephosphorylationofSTAT-6(14).Toin-vestigatewhetherIL-13–inducedexpressionofSI-CLPoccursviatheSTAT-6signalingpathway,cellsweretreatedwithIL-13aloneortogetherwithAG490,andtheexpressionofSI-CLPwasexamined.Asexpected,theup-regulationofSI-CLPstimulatedbyIL-13wasinhibitedinthepresenceofAG490(Figure3D),sug-gestingthatIL-13stimulatesSI-CLPexpressionthroughtheJAK/STAT-6signalingpathway.

XIAOETAL

Figure3.TheeffectsofSI-CLPoncytokineexpressionandtheinfluenceofvariouscytokinesontheexpressionofSI-CLPinhumanmacrophages.A,Expressionofinterleukin-1␤(IL-1␤),IL-6,IL-12,andIL-13mRNAinPMA-differentiatedTHP-1cellstreatedwithrecom-binantSI-CLP,asdeterminedbyreal-timequantitativepolymerasechainreaction(qPCR).B,LevelsofphosphorylatedERK,JNK,andp38MAPKinPMA-differentiatedTHP-1cellstreatedwithSI-CLP,asdeterminedbyWesternblotting.C,ExpressionofSI-CLPmRNA(left)andprotein(right)inPMA-differentiatedTHP-1cellstreatedwithIL-1␤,IL-4,IL-12,orIL-13,asdeterminedbyreal-timeqPCRandWesternblotting,respectively.D,Left,ExpressionofSI-CLPinPMA-differentiatedTHP-1cellstreatedwithIL-12orIL-13andinhibitorsofERK,p38MAPK,JNK,andJAK,asdeterminedbyWesternblotting.ThenumbersshownbeneaththegelpanelsaretheratiosofSI-CLPtoactin.Right,ExpressionofSI-CLPinmono-cyticTHP-1cellsandPMA-activatedTHP-1cellstreatedwithIL-13aloneorIL-13plusAG490,asdeterminedbyreal-timeqPCR.BarsshowthemeanϮSEM.ءϭPϽ0.05;ءءϭPϽ0.01versuscontrol.SeeFigure2forotherdefinitions.

SI-CLPincreasestheexpressionofIL-1␤,IL-6,IL-12,andIL-13andisup-regulatedbyIL-12andIL-13inprimarymacrophages.Ϫ/ϪPrimarymacrophagesfromwild-typeandSI-CLPmicewereusedtofurtherconfirmtheobservationsinPMA-differentiatedTHP-1cells.ExonϪ2oftheSI-CLPgenewasdeletedtogenerateSI-CLPϪ/mice,andtheknockoutofSI-CLPwasconfirmedinbonemarrow–derivedmacrophagesfromSI-CLPϪ/Ϫmice,byreal-timeqPCRandWesternblot-ting(Figure4A).Next,bonemarrow–derivedmacro-phagesfrombothwild-typeandSI-CLPϪ/Ϫmicewere

SI-CLPAGGRAVATESRA-RELATEDINFLAMMATION1147

Figure4.Theinfluenceofstabilin-1Ϫinteractingchitinase-likeprotein(SI-CLP)oncytokineexpressionandtheeffectsofvariouscytokinesontheexpressionofSI-CLPinmousemacrophages.A,SI-CLPdeletioninSI-CLPϪ/Ϫandwild-type(WT)mice,asconfirmedbyreal-timepolymerasechainreaction(PCR)(top)andWesternblotting(bottom).B,Leftandmiddle,Expressionofinterleukin-1␤(IL-1␤),IL-6,IL-12,andIL-13mRNAinbonemarrow–derivedmacrophagesfromwild-typemice(left)andSI-CLPϪ/Ϫmice(middle)aftertreatmentwithSI-CLP,asdeterminedbyreal-timequantitativePCR(qPCR).Right,ExpressionofSI-CLPmRNAinbonemarrow–derivedmacrophagesfromwild-typemicetreatedwithIL-1␤,IL-6,IL-12,orIL-13,asdeterminedbyreal-timeqPCR.ءϭPϽ0.05;ءءϭPϽ0.01versuscontrol.C,LevelsofIL-1␤,IL-6,IL-12,andIL-13intheserumofwild-typeandSI-CLPϪ/Ϫmice,asdeterminedbyquantitativeenzyme-linkedimmunosorbentassay(ELISA).ءϭPϽ0.05;ءءϭPϽ0.01versusSI-CLPϪ/Ϫ.D,LevelsofIL-1␤,IL-6,IL-12,andIL-13inthesupernatantsofbonemarrow–derivedmacrophagesfromwild-typeandSI-CLPϪ/Ϫmicethatwereleftuntreated(control)orweretreatedwithSL-CLP,asdeterminedbyquantitativeELISA.BarsshowthemeanϮSEM.ءϭPϽ0.05;ءءϭPϽ0.01versuscontrol.#ϭPϽ0.05versusSI-CLPϪ/Ϫ.

incubatedwithvariousconcentrationsofSI-CLPpro-teins,andthelevelsofdifferentcytokinesweremea-sured.ThemRNAlevelsofIL-1␤,IL-6,IL-12,andIL-13weresignificantlyup-regulatedinbothgroups(Figures4B),andthemRNAlevelofSI-CLPinbonemarrow–derivedmacrophagesfromwild-typemicewassignificantlyincreasedinresponsetoIL-12andIL-13(Figure4B),whichisconsistentwiththeobservationsinPMA-differentiatedTHP-1cells.

SI-CLPaffectstheproductionofinflammation-associatedcytokines.TheregulationofSI-CLPininflammation-associatedcytokineproductionpromptedustoinvestigatetheinflammation-associatedcytokineswhenSI-CLPwasknockedout.Theresultsofquantita-tiveELISArevealedthatcomparedwiththelevelsinwild-typemice,thelevelsofIL-1␤,IL-6,andIL-12weresignificantlylowerinSI-CLPϪ/Ϫmice(Figure4C).

ToanalyzetheinfluenceofSI-CLPoninflamma-tion,thelevelsofinflammation-associatedcytokinesinthesupernatantsofbonemarrow–derivedmacrophagesfromwild-typeandSI-CLPϪ/ϪmiceweredetectedbyquantitativeELISA.SI-CLPtreatmentsignificantlystimulatedtheproductionofthesecytokines(Figure4D).TheseresultsindicatedthatSI-CLPisinvolvedin

1148XIAOETAL

Figure5.EffectofSI-CLPtreatmentoninterleukin-12(IL-12)andIL-13expressioninthelesionsandPBMCsofmicewithcollagen-inducedarthritis(CIA).A,IL-12andIL-13mRNAlevelsinlesions(left)andPBMCs(right),asdetectedbyreal-timequantitativepolymerasechainreaction.ValuesarethemeanϮSEM.ءءϭPϽ0.01versuscontrol.BandC,ExpressionofIL-12andIL-13(B)andSI-CLP(C)inthelesionsofmicewithCIAthatwereleftuntreatedorweretreatedwithSI-CLP.Theimagesinthefarleftandfarrightcolumnsarehigher-magnificationviewsoftheboxedareasintheimagesinthemiddlecolumns.Barsϭ100␮m.SeeFigure2forotherdefinitions.

inflammationinvivo,andthatitplaysanimportantroleinregulatingtheproductionofinflammation-associatedcytokinesinmacrophages.

SI-CLPtreatmentstimulatestheexpressionofIL-12andIL-13inthelesionsofmicewithCIA.WefurtherdetectedtheexpressionofIL-12andIL-13inboththelesionsandPBMCsofmicewithCIA,withorwithoutSI-CLPtreatment.Real-timeqPCRanalysisshowedthatIL-12andIL-13weresignificantlyup-regulatedinthelesionsandPBMCsofarthriticmicetreatedwithSI-CLP(Figure5A).Consistently,immu-nohistochemicalanalysisrevealedincreasedlevelsofIL-12andIL-13inthelesionsofSI-CLP–injectedmicewithCIA(Figure5B).Furthermore,theexpressionofSI-CLPinarthriticjointsalsoincreasedmarkedlyinSI-CLP–injectedmicecomparedwiththecontrolgroup(Figure5C).TheseresultsindicatedthatSI-CLPde-creasesinflammationbyup-regulatingtheexpressionofIL-12andIL-13andbreaksthebalancebetweeninflam-matorycytokinesandantiinflammatorycytokines.

SI-CLPAGGRAVATESRA-RELATEDINFLAMMATION1149

Figure6.Highersusceptibilitytocollagen-inducedarthritis(CIA)instabilin-1Ϫinteractingchitinase-likeprotein(SI-CLP)–knockout(KO)micecomparedwithwild-type(WT)mice.A,CumulativeincidenceofCIAinwild-typemice,SI-CLPϪ/Ϫmice,andcontrolmice(nϭ20pergroup).B,Arthritisscoresinwild-typemice,SI-CLPϪ/Ϫmice,andcontrolmicethatwereleftuntreatedorweretreatedwithSI-CLP(nϭ10pergroup).ءϭPϽ0.05;ءءϭPϽ0.01versuscontrol.#ϭPϽ0.05versusuntreatedSI-CLPϪ/Ϫmice.CandD,Pawswelling(C)andbonedestruction(D)inwild-typemiceandSI-CLPϪ/ϪmicethatwereleftuntreatedorweretreatedwithSI-CLP.ArrowsindicatethebonelossandjointdestructioninducedbySI-CLP.

SI-CLP؊/؊micehavelowersusceptibilitytoCIAcomparedwithwild-typemice.Theobservationoflowerlevelsofinflammation-associatedcytokinesinSI-CLPϪ/ϪmicepromptedustoexaminetheroleofSI-CLP–associatedinflammationinvivo.BecauseC57BL/6micebearingclassIImajorhistocompatibilitycomplexH-2bcandevelopCIAwithhighincidence(34),bothwild-typeandSI-CLPϪ/ϪmiceonaC57BL/6backgroundwereusedtogenerateamurinemodelofCIA.TheinductionofarthritisinSI-CLPϪ/Ϫmicewascomparedwiththatinwild-typemicebyassessingthecumulativeincidence,pawswelling,andbonedestruc-tion.ThedatashowedthatthecumulativeincidenceofCIAwasobviouslylowerinSI-CLPϪ/Ϫmicecomparedwithwild-typemice(Figure6A).Moreover,asignificantdecreaseinthearthritisseverityscorewasobservedinSI-CLPϪ/Ϫmice(Figure6B).ToelucidatetheeffectofSI-CLP,halfofthemicewithCIAweretreatedwithSI-CLPbyintraperitonealinjection,andhalfreceivedPBSascontrol.ComparedwithPBS,administrationofSI-CLPaggravatedinflammationinbothSI-CLPϪ/Ϫandwild-typearthriticmice,andmoresevereinflammationwasobservedinwild-typemice(Figure6B).Moreimportantly,thedegreeofpawswellingandbonede-structionwaslowerinSI-CLPϪ/Ϫmicecomparedwithwild-typemice(Figures6CandD).

1150DISCUSSION

Inthisstudy,weobservedincreasedexpressionofSI-CLPinPBMCsandthepresenceofSI-CLPinthesynovialfluidofpatientswithRA.Inaddition,adminis-trationofSI-CLPaggravatedarthritis-relatedinflamma-tioninarodentmodelofCIA.TheelevatedlevelofIL-1␤inserafromratswithCIAandtheincreasedlevelsofIL-1␤,IL-6,IL-12,andIL-13inPMA-treatedTHP-1cellsandprimarymacrophagesfrommicewithCIAindicatethatSI-CLPcanregulatetheinflammation-relatedcytokinesproducedbymacrophages.Moreim-portantly,knockoutofSI-CLPreducedthelevelsofIL-1␤,IL-6,andIL-12significantly,whichmaycorrelatewiththeϪlowsusceptibilitytoCIAobservedinSI-CLPϪ/mice.

MembersoftheGlyco_18familyhavebeenstud-iedduetotheirfunctionsintheimmunesystem.AcidicmammalianchitinasewasidentifiedasamediatorofIL-13–inducedresponsesinTh2-dominateddisorderssuchasasthma(35).TheYm1proteinwasreportedtoberegulatedbyTh2cytokinesandabundantlyexpressedbymacrophagesin“Th2-biased”chronicinflammation(36).Toourknowledge,thisisthefirstexampleidenti-fyingachitinase-likeproteinasamacrophage-associatedproteinthatcanaggravatearthritis-relatedinflamma-tion.

Stimulationofmacrophageswithvariouscyto-kinesindicatedthattheexpressionofSI-CLPwasup-regulatedsignificantlybyIL-12andIL-13.Kzhysh-kowskaetalreportedthatexpressionofSI-CLPwasstronglystimulatedbyIL-4anddexamethasoneinpri-marymacrophages(18).Takentogether,theseresultsindicatethatSI-CLPisinvolvedinTh2-associatedacti-vation,similartotheotherchitinase-likeproteins(35–40).ThecurrentstudyisthefirsttoshowthatSI-CLPwasup-regulatedbyIL-12,aTh1cytokine–associatedcytokine.Theup-regulationofSI-CLPbybothTh1andTh2cytokinessuggeststhepossibilitythatSI-CLPmaybeinvolvedinthecross-talkbetweenmacrophagesandadaptiveimmunity.ThisresultsuggestsacombinedeffectoftheTh1andTh2pathwaysinRA.Tofurtherelucidatethemolecularmechanismsinvolvedinup-regulationofSI-CLPbyIL-12andIL-13stimulation,theeffectsoftheJNKandJAK/STATpathwayinhibitorsonSI-CLPexpressionwereevaluatedinIL-12–andIL-13–treatedPMA-differentiatedTHP-1cells.Ourdataindi-catethatdifferentpathwaysareinvolvedintheup-regulationofSI-CLPbyIL-12andIL-13inmacrophages:IL-13mediatestheexpressionofSI-CLPXIAOETAL

throughtheJAK/STATpathway,andIL-12functionsviatheJNKsignalingpathway.

BecauseourresultsindicatedthatSI-CLPcouldnotonlyregulateinflammation-associatedcytokinesbutcouldalsoberegulatedbyinflammation-associatedcy-tokines,wesurmisedϪthatSI-CLPmayactasanimmuneregulator.SI-CLPϪ/micewereusedtofurtherunder-standtheroleofSI-CLPininflammation.ThereducedlevelsofIL-1␤,IL-6,andIL12inserumandthesupernatantofmacrophagesfromSI-CLPϪ/Ϫmiceindi-catethatSI-CLPhasanimportantfunctioninregulatingtheproductionofproinflammatorycytokinesinprimarymacrophages.Moreimportantly,ourresultsdemon-stratethatSI-CLPϪ/ϪmicepossesslowersusceptibilitytoCIA,andthatadministrationofSI-CLPcouldexac-erbateinflammationinCIA.Duetotheclosecorrela-tionofproinflammatorycytokinesϪ/ϪwithRA,thereducedcytokineexpressioninSI-CLPmicemayimpedethedevelopmentofchronicinflammatorydisorders,leadingtolowersusceptibilitytoRA.

AfterSI-CLPisinjectedintraperitoneallyintorats/mice,itisabsorbedthroughthecirculationandinfiltratesintothearthriticjoint,increasinginflamma-tion.SI-CLPandcytokinesregulateeachotherbyapositivefeedbackmechanism:theinflammation-associatedcytokinesup-regulatetheexpressionofSI-CLPonmacrophagesinthejointcavityandtypeAsynovialcells,andSI-CLPpromotestheinflammation-associatedcytokines,whichdisruptsthebalancebetweeninflammatorycytokinesandantiinflammatorycytokinesandexacerbatesarthritis-relatedinflammation.Thein-creasedexpressionofSI-CLPinPBMCs,theexistenceofSI-CLPinthesynovialfluidofpatientswithRA,andtheincreasedlevelofSI-CLPinthelesionsofSI-CLP–treatedmicefurtherconfirmedtheeffectofSI-CLPinRA.

AnimportantissuethatshouldbeaddressedinthefutureishowSI-CLPbindstothesurfaceofcells.TheobservationthatSI-CLPcouldbindonlytothesurfaceofmacrophagesandnotthesurfaceofTcellsandBcellssuggeststhatSI-CLPshouldbindtoamacrophage-specificreceptor.Ourstructuralandbio-chemicalanalysesofSI-CLPshowthatithasasaccharide-bindingcleftandindeedpossessesasaccharide-bindingproperty(22).Thisfeaturemaycor-relatewiththebindingbetweenSI-CLPandreceptorprotein(s).BecausenoreporthasbeenmadeaboutthereceptorsofSI-CLPandotherchitinase-likefamilyproteins,andtherecognitionofreceptorscanprovidenewpotentialtherapeuticinterventionsforRA,itis

SI-CLPAGGRAVATESRA-RELATEDINFLAMMATIONworththeefforttoidentifyandcharacterizethespecificreceptorofSI-CLPonthesurfaceofmacrophages.

OurstudyprovidesevidenceforaroleofSI-CLPinmacrophageregulation.Inautoimmunedisorderssuchasarthritis,SI-CLPisup-regulatedbyIL-12orIL-4/13andfurtherinteractswithmacrophagestoacti-vateERKphosphorylation,elevatesIL-1␤,IL-6,IL-12,andIL-13expressionandexacerbatesinflammation,whileSI-CLPknockoutresultsinlessarthritis-associatedinflammation.Theassociationbetweenchitinase-likeproteinsandimmunediseasessuchasasthmaandRA(35,41),togetherwiththefindingsofthecurrentstudy,suggestthatchitinase-likeproteinshaveanimportantfunctionindifferentautoimmunediseases.

ACKNOWLEDGMENTS

WethankDrs.ZhifangChangandChenmingSunfor

theirhelpininducingCIAinrats,andHongyuZhouandBingbingHeforprovidingPBMCsfrompatientswithchronicautoimmunedisorders.

AUTHORCONTRIBUTIONS

Allauthorswereinvolvedindraftingthearticleorrevisingit

criticallyforimportantintellectualcontent,andallauthorsapprovedthefinalversiontobepublished.Dr.Zhenghadfullaccesstoallofthedatainthestudyandtakesresponsibilityfortheintegrityofthedataandtheaccuracyofthedataanalysis.

Studyconceptionanddesign.Xiao,Meng,YanmeiZhao,T.Li,Peng,YongZhao,Luo,W.Zhao,Z.Li,Zheng.

Acquisitionofdata.Xiao,Meng,YanmeiZhao,Yuan,T.Li,Peng,YongZhao,Luo,W.Zhao,Z.Li,Zheng.

Analysisandinterpretationofdata.Xiao,Meng,YanmeiZhao,Yuan,T.Li,Peng,YongZhao,Luo,W.Zhao,Zheng.

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