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β activa tion,correlatedwithdiseaseseverityandsmallairwaywallthickeninginCOPD.Ourfindingshaveidentified TGF- aspotentialtherapeutictargetforCOPD. Chronicobstructivepulmonarydisease(COPD)isthefourth-lead ingcauseofdeathworldwideandaffectsupto50%oflong-termsmokers(1,2).ThemajorhistopathologiccorrelatesofairwayobstructioninCOPDarelossofalveolarwallsandnarrowingofthesmallairways(3).Therelativecontributionofeachofthesepathologicfeaturestoairwayobstructionisnotcompletelyunder stood(4).However,recently,airwaywallthickeninghasbeeniden tifiedasmajorindependentpredictoroftheseverityofphysio logic activationinhumanmodeloftheepithelial-mesenchymaltrophicunit(10).Inthismodel,fibroblasticintegrin v -mediatedTGF- activa tionwasfoundtoregulatethefibrogenicphenotypeaswellassynthesisandsecretionofHGF,whichinfluencestheprolifera tionofadjacentairwayepithelialcells(10). TGF- ismajorfibrogeniccytokineregulatingbothextrcellularmatrix togethermayaccountforthemajorityofTGF- activationdur development v 6,interactionwith Nonstandardabbreviationsused:BEGM,bronchialepithelialgrowthmedium;ColI, typecollagen;COPD,chronicobstructivepulmonarydisease;EDC,epidermaldiffer entiationcomplex;GOLD,GlobalInitiativeforObstructiveLungDisease;IL-1RA,IL-1receptorantagonist;LAP,latency-associatedpeptide;MT1-MMP,membranetypeI– MMP;P0,initialpassage;P2,secondpassage;SM,squamousmetaplasia.Conflictofinterest:Theauthorshavedeclaredthatnoconflictofinterestexists.Citationforthisarticle: J. Clin. Invest. 117:3551–3562(2007).doi:10.1172/JCI32526. research article 3552TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 latentTGF- resultsinconformationalalterationofLAP,allow ingcell-associatedactiveTGF- tointeractwithTGF- receptorsonimmediatelyadjacentcells(21).Inthecaseof v bindingoflatentTGF- resultsinthemetalloproteolyticcleavageofLAPandactiveTGF- surface,asanautocrineorparacrinefactor(20).Both v and v areexpressedhumanairwayepithelialcells; v (butnot v isbyairwayfibroblasts.Ofthemanycytokinesthathavebeenimplicatedinthepatho genesisofCOPD,IL-1 majorproinflammatorycytokine,isofparticularinterest(23).IL-1 hasbeenfoundinhigherlevelsinalveolarmacrophagesfromsmokersandincigarettesmoke–treat edairwayepithelialcellsfromCOPDpatients(24).TransgenicoverexpressionofIL-1 inairwayepithelialcellsleadstoairwayinflammation,emphysema,airway-wallthickeningHerewereportlinkbetweenSMofairwayepitheliumandairway-wallthickeningthroughmechanisminvolvingincreasedsecretionofIL-1 andincreasedintegrin-mediatedactivationofTGF- . SM in the small airways of COPD patientsInvolucrincomponentofthecornifiedenvelopeofstratifiedsquamousepitheliumandismarkerairwayairways,involucrinstainingwasfoundinsuperficialmetaplasticairwayepithelialcellsinareasofcuboidalairwayepitheliumdevoidofciliaaswellasinareasofmorphologicallyobviousSM,confirm inginvolucrinasrobustmarkerforSM(Figure1A).Involucrinstainingairwaysignificantlystagescompared In vitro model of human airway epithelial squamous metaplasiaHumanbronchialepithelialcellsundergoSMduringcellcultureretinoicWefoundinthelowretinoicacidconcentrationsofourcommercialairwayepithelialmediumpreparation(0.3nM;ref.27),humanbronchialphenotypicallyflattenedsquamous-likecells(Figure1C).Attheinitialpassage(P0),wedeterminedthatthehumanairway,becausemorethan95%ofthecellsexpressedmarkerdifferentiationthirddisplayedinvolucrinstainingmetaplasticsquamousepithelialcells(29).Westernblottingofconfirmedacquisitionofsquamouscellphenotypeduringserialpassage,sincehighp63lowinvolucrinwerefoundepithelialcelllysatesandlowp63andhighinvolucrinexpressionwerefoundinP3humanbronchialepithelialcelllysates(Figure1D).Thesedataareinagreementwithpreviousstudiesusingsimi larmedia,demonstratingthattheprimarycellin2Dtracheobron SM-likeSMinCOPDpatientsisnotonlycharacterizedtheonsetofepidermaldifferentiationbutisalsohyperproliferativestate(30).Thus,wecharacterizedcellproliferationandsenescenceofseri allypassagedhumanbronchialepithelialcells.ThepercentageofactivelyproliferatingcellsintheandG2/Mphasesofthecell Figure 1 SM correlates with increased GOLD stage of COPD in human lung samples, and SM can be induced during in vitro culture. ( ) Involu crin (IVL), a marker of epidermal differentiation (26), stains small air way SM. Shown is a photomicrograph of metaplastic squamous cells (arrows) of a small airway from a COPD patient stained with anti-IVL. Scale bar: 50 m. ( ) IVL-staining intensity of lung samples from nor mal versus COPD patients stratified according to GOLD (www.gold copd.com), and assessed by grading digital images (0–3 scale) with 0 cytoplasmic staining. * < 0.05. ( ) Photomicrographs of P0 compared with P3 human bronchial epithelial cells. Upper panels are stained with the basal cell marker anti-p63 and lower panels with anti-IVL. Scale bar: 50 m. ( ) Western blotting of 40 g of P0 or P3 human bronchial epithelial cells total cell lysates for p63 and IVL. ( ) Propidium iodide (PI) staining and flow cytometry of serially passaged human bronchial epithelial cells of a representative experiment ( = 3) showing the rela tive proportion of cells in the G0/G1, S, and G2/M phases of the cell cycle. ( ) Relative proportion (± SEM) of human bronchial epithelial cells ( = 3) in G0/G1 compared with S and G2/M phases of the cell cycle during serial passage. * < 0.01. ( ) Senescence-associated -gal (SA -gal) staining of serially passaged human bronchial epithe lial cells ( = 3). Shown is the percentage (± SEM) of SA -gal–positive cells. * < 0.01; ** < 0.001. research article TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 3553 cycle notsignificantly byproliferationmark edlydecreasedduetoanaccumulationofcellsintheG0/G1phasecycle1,notsignificantlybetween nificantlyinsubsequentpassages(Figure1G).WeconcludethatapproximatethehyperproliferativestateofSMinvivoandavoidtheinvitroreplicative Global analysis of gene expression during SM reveals induction of the pro - inflammatory mediators IL-1 and IL-1 WethecultureperiodfromP0toP3usinghigh-densityoligonucle otidemicroarrays.weregenesinducedand225genesrepressed(503total)thatreachedstatisticalsignificance http://www.ncbi.nlm.nih.gov/geo/Geoaccessionnumber:GSE7557).Geneontologyanalysisofthesta significantrevealed beroffunctionalcategories,includingcellmotility,cytoskeletalassembly,ectodermaldevelopment,IL-1receptorbinding,cellproliferation,andcelladhesion(SupplementalTable1;supple mentalmaterialavailableonlinewiththisarticle;doi:10.1172/JCI32526DS1).Statisticallysignificantrepressedgenesincluded(SupplementalTableOfthe503statisticallysignificantdifferentiallyexpressedgenes,93and62wereatleast2-foldinducedorrepressed,respectively.Mostnotably,IL-1 (13.4-fold),IL-1 (9.7-fold),andtheTGF- – responsivegene,plasminogen-activatorinhibitor SERPINE16.9-fold),werethefirst,third,andsixthmosthighlyinducedgenes,respectively(Table1).OthergenesknowntobeinducedbyTGF- inepithelialcellsthatwerealsodifferentiallyexpressedatleast2-foldinP3airwayepithelialcultures(seeTableandhttp:// www.ncbi.nlm.nih.gov/geo/Geoaccessionnumber:GSE7557)includedtheintegrin 6 ( ITGB6(31),thecelldifferentiationprotein MAL(32),theTGF- induciblegene BIGH3(33),andlamininA3 LAMA3(32),suggestingincreasedautocrineTGF- signalingduringserialpassage.Inaddition,numberofgenesinvolvedinepidermaldifferentiation KRT6B S100A8 GJ1A IVL DSC2, KRT14, DESC1, A2MLwerehighlyinducedduringserialpassage;ofthese KRT6 , IVLand KRT14havebeenreportedtolabelactivelyprolif eratingsuprabasalcellsinthesquamousmucosaorin(TableImmunocytochemistryconfirmedthesuprabasaldif ferentiationstateofP3humanbronchialepithelialcellssince80%,61%,and88%ofcellsexpressedkeratin6,ker atin14,andinvolucrin,respectively(SupplementalFig ure1).highpercentage(42%)oftheinvolucrin-stainedP3cellswereactivelyproliferating,sincetheywereintheorG2/Mphaseofthecellcycle(SupplementalFig ure2).Finally,76%,67%,and90%ofIL-1 –positiveP3keratin keratin14,andinvolucrin,respectively(Supplemen talFigure1).ThesedatademonstratethatP3humanbronchialepithelialcellsrepresentactivelyproliferating IL-1 squamous IL-1 IL-1 tothesameIL-1receptorandendogenoussolublerecep torantagonist(IL-1RA)butdifferinthatIL-1 issecretedwhereas IL-1 existsinbothmembrane-boundandcleaved(secreted)form.IncreasedlevelsofIL-1 andIL-1 inP3humanbronchialepithelialcellswereconfirmedbyRT-PCR(Figure2A),cytokineantibodyarray(Figure2B),andELISA(Figure2C).Theincreasein IL-1 supernatantairwaysupernatantsapproximatelylevel4-foldexcessfoundinsynovialfluidfrompatientswithrheumatoidarthritisFinally,confirmphysiological weair-liquidinterfaceweregrownfiltersbasalsurfaceofthecellswasallowedtoremainincontactwiththemedium,whichsimulatestheanatomicarrangementintheairwayinvivo(39).Humanbronchialepithelialcells (P0)grownair-liquidinterfacefordaysSM-promotingmediumconditionsadoptedsquamousmetaplasticphenotypeinvolucrin2D)andexpressedIL-1 (Figure2E).Air-liquidinterfaceculturesgrowninairway-differentiatingmediumadoptedpseudostrati fiedciliatedcolumnarphenotypeandexpressedneitherinvolucrinIL-1 (Figure2DandFigure2E). In vitro SM closely mimics metaplastic squamous epithelium in COPD in vivoWecharacterizedandconfirmedtheincreasedexpressionof IL-1 differentiationfoundoligoarraysbyimmunohistochemicalstainingoftissuesamplesofhumanairwaySMfromCOPDpatientsandcomparedthestainingwoundedsquamousmucosa,andSMfromvarietyofmucosalsites.Antibodiesto Table 1 Twenty-five most highly induced genes during human bronchial epithelial cells’ serial passage Gene Description Fold B A1 IL1B IL-1 13.42.82 KRT6BKeratin 6B IL1A IL-1 9.72.04 S100 calcium-binding protein A8 LYSMD1cDNA DKFZp564C0671 Plasminogen activator inhibitor type 1 Folylpolyglutamate synthase Rh type C glycoprotein TGF, -induced6.40.110 Parathyroid hormone-like hormone NDUFA4L2NADH:ubiquinone oxidoreductase Mal, T cell differentiation protein Connexin 43 Hypothetical protein FLJ21511 Glutathione peroxidase 3 Cytidine deaminase AREG Amphiregulin4.41.618 Desmocollin 2 N-myc downstream regulated gene 1 IVL Involucrin4.12.621 KRT14Keratin 14 Transmembrane protease, serpine 11E APOE apoE3.90.424 Endocytic receptor A2ML1 2-macroglobulin–like 1 represents the log-odds ratio where a value of greater than 0 is considered significant. research article 3554TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 IL-1 stainedbasal,suprabasal,andsuperficialcellsofmetaplasticsquamousairwayepithelium(Figure3A)butdidnotstainnormalairwaystainingwasspecific,asnostainingwasobservedafterpreabsorptionoftheinvolu crinandkeratinstainedthesuperficialandsuprabasalcellsofsquamousmetaplasticnotnormalepithelium(Figure3,andH).Keratin14immunolocal izedtothebasalandsuprabasalcellsofSM(Figure3I)andstainedairwaysuprabasalcellsofSMstainedintenselywithantibodiestointegrin lightlystained(Figure3L);theoppositewastrueofintegrin stainedintenselywithantibodiestotheproliferationmarkerKi-67(FigurefewerairwaystainedmarkerstainedcellsandmoderatelylabeledsuprabasalcellsofSM(Figure3Q)whilestainingonlybasalcellsofthenormalairway(Figure3R).patternofimmunoreactivitysimilartothatseeninSMoftheairwayssquamouscervix,andintheepidermisattheedgeofhealingwounds(Supplemen talDataSupplementalTableWeapproximateairwaywellother IL-1 released by squamous metaplastic epithelium increases v 8 inte - grin expression and v 8 -mediated TGF- activation in human airway fibroblastsWehypothesizedthatIL-1 releasedbymetaplasticsquamousepithelialcellscontributedtoprofi broticfibroblastphenotypebyincreasingfibro blastTGF- activation,largelyintegrin v 8 mediatedprocess(10).Indeed,wefoundthat v integrinexpression(Figure4,andB)and v integrin–mediatedTGF- activation(Figure4C)inhumanairwayfibroblastswasincreasedbytreatmenteitherwithrecombinantIL-1 (datanotshown)orbycoculturewithsquamousmeta plastic(P3)epithelialcells.Increasedfibroblast expressioncausedbycoculturewithsquamousmetaplasticepithelialcellswasIL-1dependent,sinceIL-1RA,physiologicalinhibitorofboth IL-1 IL-1 completely airwayepithelialcellswerethesolesourceofIL-1inthecoculturesystembecauseneitherIL-1 norIL-1 detectedairwayfibro byRT-PCR(datanotshown). IL-1 released by squamous metaplastic epithelium causes an v 8 - and TGF- –dependent switch to a fibrogenic fibroblast phenotypeIncreasedECMpro ductionandincreasedfibroblastcontractilityarehallmarksoffibroticresponsesseeninairwaywallthickening,andincreasedtypecollagen(ColI)andincreasedSMA SMA)arekeybio chemicalmarkersofthatresponse.Toassessthecontributionofautocrine v –mediatedTGF- activationtotheprofibroticfibroblastpheno type,weusedsiRNAtoknockdown expres sioninairwayfibroblasts.Treatmentofnormalairwayfibroblastswith siRNAsignificantlyreduced expressionasassessedbyRT-PCR(Figure5A)andflowcytometry(Figure5B)andcompletelyinhibited v 8 -medi atedTGF- activation(Figure5C).Theseeffectswerespecificto siRNAsincetheywerenotseenwithcontrolsiRNA(Figure5,A–C).Neutralizinganti- and siRNAblockedonlyapproxi mately50%ofthetotalTGF- activationasdeterminedusing TGF- neutralizingantibodythatinhibitsthefunctionofallTGF- isoforms(Figure5C).Thus,theincompleteinhibitionofTGF- activationcouldbeduetoactivationofTGF- byan v -independentmechanism,since v isonlyinvolvedintheactivationofTGF- and (10,20).Alternatively,theincom pleteinhibitionmaybeduetotherelativelypoorefficacyofthe 8-neutralizingantibodyandtheincompletesilencingachieved Autocrine v 8-mediatedactivationof TGF- influencedthemyofibroblastphenotype,sincetreatmentofairwayfibroblastswith siRNAreduced SMAexpression(Figure5D)andColsecretion(Figure5E).CocultureofairwayfibroblastswithP3humanbronchialepithelialcellsledtoanincreaseinColtranscriptionandproteinproductionbyairwayfibroblasts IL-1 andfibroblast 8–dependentbecausetheincreaseinCol expressioninducedbycoculturewithP3humanbronchialepi completelybycocultureswithIL-1RA(Figure5E)orbytransfectionofairwayfibroblasts 8siRNA(Figure5E). IL-1 –dependent increase in v 8 -mediated activation of TGF- is MT1- MMP dependentIL-1 whileincreasing expression,alsohasdiversefunctionaleffects,includingincreasedtranscriptionofmem - Figure 2 and IL-1 are induced during SM. ( ) RT-PCR of total RNA from P0 to P3 human bronchial epithelial cells grown in 2D culture, using primers to IL-1 and IL-1 and -actin, as a control. Shown is a representative experiment from paired samples from 3 different patients with the same results. ( ) Antibody cytokine array showing increased IL-1 and - in conditioned medium taken from P0 or P3 human bronchial epithelial cells. Upper and lower panels are duplicates. Shown is a representative experiment from 3 separate patients showing similar results. ( ) IL-1 ELISA assay showing increased IL-1 in conditioned medium from P0 compared with P3 human bronchial epithelial cells. < 0.001. ( ) Involucrin immunostaining of human bronchial epithelial cells grown in air-liquid interface culture for 21 days . Human bronchial epithelial cells grown in 2% Ultraser G (Invitrogen) produced differentiated pseudostratified ciliated columnar epithe lium (NL, upper panel ). When grown in BEGM (Clonetics), the cells adopted a squamous metaplastic phenotype and expressed involucrin (SM, lower panel ). Arrows indicate involucrin staining of basal and suprabasal squamous metaplastic epithelial cells. Scale bar: 20 m. ( ) RT-PCR of total RNA harvested from human bronchial epithelial cells grown in air-liquid interface for 3 days or 21 days in differentiating medium (USG) or SM medium (BEGM) using primers to IL-1 or -actin as controls. research article TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 3555 branetypeI–MMP(MT1-MMP)(40)andTGF- (41),eitherofwhichcouldindependentlyincreasetheefficiencyof integrin–mediated TGF- activation(20).Indeed,treatmentofairwayfibroblastswith IL-1 alsoincreasedMT1-MMPactivity(notshown).TodeterminewhethertheIL-1 –dependentincreaseinfibroblast v –mediated TGF- activationwasduetoincreased expressionortoincreasedMT1-MMPactivity,weoverexpressedeither orMT1-MMPinair wayfibroblasts.Transient transfectionsignificantlyincreased integrinexpressionand v -mediatedTGF- activation(Figure6,andB)anddidnotaffectMT1-MMPactivityorexpression(notshown).Conversely,overexpressionofMT1-MMP,whichincreased100-fold(datanotshown)effecton expression(datanotshown)or v -mediatedTGF- activation(Figure6C).However,transfectionofairwayfibroblastswithMT1-MMPsiRNA,whichinhibitedapproximately70%ofMT1-MMPsurfaceexpression(Figure6D)andactivity(notshown),markedlyinhibited v -mediatedactivationofTGF- (Figure6E).Incontrast,treatmentofairwayfibroblastswithIL-1 didnotincreaseTGF- transcription(ratioofTGF- to -actintranscript:nontreated,0.970.11;IL-1 treated,0.870.16, 3)orsecretion (TGF- activityasmeasuredbyTGF- bioassay:control,1269131; IL-1 treated,1152129, 4).TheseresultsdemonstratethattheendogenousleveloffunctionallyactiveMT1-MMPpresentinair wayfibroblastsissufficienttosupport v -mediatedactivationof TGF- andthattheIL-1 –inducedincreasesinTGF- activationareduetoincreased expressionandnotincreasedactivityofMT1-MMPor expressionofTGF- . Increased TGF- activation during serial passage of airway epithelial cellsWenextaddressedtheroleofintegrin-mediatedactivationofTGF- indrivingSM.TheTGF- –activatingintegrin v 6 showeddramaticincreaseinmRNAandsurfaceexpressionduringserialpassageofairwayepithelialcells.AtP0,airwayepi thelialcellsexpressedlittle v ByP1,highlevelsof v weredetected,whichpersistedthroughoutlaterpassages(Figure7A).TheotherTGF- –activatingintegrinexpressedbyairwayepithelialcells, v showedhighexpressioninP0cellsanddemonstratedApproximately50%ofTGF- activationbyairwayepithelialcellsfromP0toP3forby v 6 integrin,withthe v 8 integrincon tributinglittletothetotalTGF- activation(Figure7B).Thisisconsistentwithourpreviousobservationsthatairwayepithelialcellsunderthesecultureconditionsrequirestimulationtosup portfull v -mediatedactivationofTGF- (10).Anti- v and anti- v integrinantibodiesshowednoeffectsonTGF- activa (datanotshown).Airwayepithelial expressionhaspreviouslybeenreportedtobeincreasedexogenousactiveTGF- (31).Here,increased expressionwaspartiallymediatedbyautocrine v –mediatedacti vationofTGF- sincetreatmentofairwayepithelialcells(P2)withneutralizinganti- orTGF- antibodiescauseddecreasein mRNA(Figure7C).Thiseffectwasspecificto v -mediatedactiva tionofTGF- sinceanti- hadnoeffect(Figure7C).Theseresultssuggestthattheintegrin v providesself-amplifyingautocrineloopofTGF- activationduringthemetaplasticprocess. Role of autocrine and paracrine TGF- signaling in regulation of squamous differentiation of airway epithelial cellsWeinvestigatedtheroleofairway v TGF- promotingtheexpressionofpanelofgenesthatweredifferentiallyexpressedduringserialpassageandthatincludemembersofgeneclustersonchromosome1q21knownastheepidermaldifferentiationcomplex(EDC)(Tableand http://www.ncbi.nlm.nih.gov/geo/seriesacces sionGSE7557; IVL, DSC2, SPRR1A, SPRR1B, SPRR3, S100A7(42,43).Neutralizinganti- decreasedthetranscriptexpressionofalltestedmembersoftheEDC(Figure7D).Theseresultssuggestthatautocrine v –mediatedactivationofTGF- playsanactiveroleinpromotingsquamousdifferentiation.Weinvestigated v 8-dependentparacrinesecre activeTGF- byairwayfibroblastsbronchialepithelialcell expression.InP0humanbronchialepithelialcells, transcriptexpressionwasfoundtobeTGF- dependent(Figure8A).Todeterminetheparacrineroleof v 8 mediatedactivationofTGF- byairwayfibroblastsinregulation Figure 3 IL-1 is coexpressed with integrins and in hyperprolifera tive suprabasal squamous metaplastic airway epithelium of COPD patients. Immunostaining of adjacent paraffin sections from an airway with a focus of SM (Sq. Met.: , and ) compared with relatively normal airway mucosa ( , , , , , , , , and from a COPD patient using antibodies to ( and ) IL-1 ; ( and IL-1 preabsorbed with the immunogenic peptide (control); ( and ) the suprabasal markers anti-involucrin; ( and ) anti–keratin 6; and ) anti–keratin 14; ( and ) the TGF- activating integrins 6; and ) and 8; ( and ) the cell proliferation marker Ki-67; ( and ) the basal cell marker p63. Arrows point to the basement membrane. Shown is a representative experiment of 3 showing similar results. Scale bar: 100 research article 3556TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 ofhumanbronchialepithelialcell 6expression,P0humanbron chialepithelialcellswerecoculturedwithairwayfibroblasts,whichhadbeentransfectedwithcontrolor siRNA.TheP0humanbronchialepithelialcellexpressionofthe transcriptwassig nificantlyincreasedcoculturewithcontroltransfectedairwayfibroblastscomparedwithP0humanbronchialepithelialcellscul turedalone(Figure8A).Themajorityofthisincreasewasdueto TGF- blockedby anti–TGF- (Figure8A).Furthermore,theincreaseinexpression transcriptpartially v TGF- byairwayfibro blasts,sincethemajorityoftheincreaseinhumanbronchialepi -thelialcells’ byairwayfibro blockedbytransfectionfibroblasts Figure 4 Paracrine secretion of IL-1 by P3 human bronchial epithelial cells increases 8 expression and -mediated TGF- activation of adjacent airway fibroblasts in a coculture model of the epithelial-mesenchymal trophic unit. Normal adult airway fibroblasts were cultured alone (Mono) or cocultured (Co) with P3 human bronchial epithelial cells grown on filter inserts. ( ) Total fibroblast RNA was harvested and RT-PCR per formed using primers specific for 8 or -actin, as a control. Shown is a representative experiment of 3 with similar results. No cDNA indicates a no template control. ( ) Fibroblasts ( = 3) in mono- or coculture were stained with anti- 8 and analyzed by flow cytometry. Shown is mean fluorescence in arbitrary units. ( ) Monocultured fibroblasts or fibroblasts after 48-hour coculture with P0 or P3 human bronchial epithelial cells = 6) were cocultured with TGF- reporter cells in the presence of neutralizing anti- 8, or no antibody (none). Shown is fold increase in TGF- activation relative to untreated fibroblasts in monoculture. ( ) Airway fibroblasts ( = 3) were cocultured with P0 or P3 conditioned medium (CM) in the presence of no antagonist or different concentrations (5, 50, or 500 ng/ml) of IL-1RA, a naturally occurring soluble antagonist of IL-1 and . The fibroblasts were stained with anti- 8 and analyzed using flow cytometry. Shown is the fold increase in 8 expression compared with matched airway fibroblasts grown without conditioned medium. ** < 0.001. Figure 5 Autocrine -mediated TGF- activation regulates the airway fibroblast contractile phenotype and collagen production. siRNA to 8 or a control siRNA were transfected into normal adult airway fibroblasts and 72 hours following transfection were analyzed. ( ) RT-PCR using primers to 8 or -actin as a control. Shown is a representative experiment of 4 showing similar results. ( ) Flow cytometry using anti- 8. Shown is mean fluorescence ( = 4) in arbitrary units ± SEM. * < 0.05. ( ) The TGF- reporter cell line, TMLC, in the presence of anti- 8 or isotype-matched control antibodies ( = 3). TGF- β activation is shown as relative to the total TGF- activation seen in control antibody, control siRNA–treated cells. * < 0.05, ** < 0.001. ( ) Western blot (WB) using anti- SMA or RT-PCR using primers to SMA or -actin as a control. Shown is a representative experiment of 3 showing similar results. ( ) RT-PCR of fibroblasts cultured alone (monoculture) or in coculture treated with either a control antibody (control Ab) or IL-1RA using primers to Col I or -actin as a control, or Western blot using anti–Col I. Shown is a representa tive experiment of 3 showing similar results. research article 3560TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 peptide(SantaBiotechnologybiotinylatedaffin purifiedanti–IL-1 anti–pan–TGF- anti-HLAclass(1D11andW6/32,ATCC).RecombinantactiveTGF- 1, IL-1 IL-1RA(AffinityBioReagents),pan-metallopro teaseinhibitorGM6001(RyssLab),andVitrogen(Cohesion)werepur Rat-tail Plasmids, siRNA, and transfection MT1-MMP,vectorswerepreviouslydescribed(20,63).TheMT1-MMP, 8,andnegativecon trolsiRNAswerepurchased(AmbionInc.),andtransfectionofprimarywereperformedAmaxaNucleofector(Amaxa systems),usingmatchedoptimizedtransfectionkitsforairwayepithelialfibroblasts. Immunocytochemistry, immunohistochemistry, and flow cytometryImmunocytochemistry,immunohistochemistry,cellcycleanalysisandflowcytom etrywereperformedaspreviouslydescribed(44).Senescence-associated -gal(SA markerperformedhumanbronchialepithelialcells(2 10 grownon12-wellcultureplateaccordingtothemanufacturer’sinstructions -galactosidasestainingkit;Research Airway morphometryLeftoverhumanlungsamplesfrompulmonarylobectomiesorpneumonectomiesperformedattheSanFranciscoGeneralHospitalMoffit-LongHospital,UniversityCaliforniaFrancisco,forlungcancer,weregatheredduringthestudyperiod(from2003through2006).Exclusioncriteriaincludedinfection,priorradiation,orchemother apy.wereobtainednotfortransplantation,forMicrotomesectionsfrom34(22COPDand12normal)lungsampleswereimmunostainedforinvolucrinor 8.Theslideswerecodedandblind Airwaydiameterdeterminedstagemicrometer,andairwayslessthanmmindiameterwereevalu airways/case).airwaydigitally magnification(QCapture,v.Digitalwerefor immunostainingstaining;grade1,0–10%;grade2,11–30%;grade3,�30%ofairwaycellsorsubepithe fibroblastspositivestaining.Involu crinimmunostainingwasalsogradedon0–3scale,withbeingabsentstaining;grade1,0–10%;grade2,11–20%;grade3,�20%cytoplasmicstain ingofsuperficial“squamoid”cells.MeasurementsofwallthicknesswereperformedusingthemethodofHogg(5),whichexpresseswallthicknessairwaysoftwarev.1.36b).Afterrecordspatientwereretrievedandthedegreeofpulmonarydisability(stagesto4,withbeingtheworst)wasdeterminedusingGOLDcriteria.Preoperativepulmonaryfunctiontests(PFTs)wereavailablefor18of20patientsunder goingsurgery.ThenumberofpatientsineachstagewithPFTsavailablewereasfollows:stage1, 3;stage2, 11;stage3, 4;andstage4, 0. RNA isolation, polymerase chain reaction, and microarray analysis tionandRT-PCRwereperformedaspreviouslydescribed(20).Real-timePCRwasperformedusingtheSYBRgreenmethod,asdescribed(65).Prim ersequencesareavailableinsupplementaldata(SupplementalTable4).Microarraymethodologyavailablesupplementaldata. Immunoprecipitation analysis, Western blotting, zymography, and TGF- bioas - sayImmunoprecipitationsandSDS-PAGEwereperformedaspreviouslydescribed(10,63).Westernblottingofconditionedmediumofcoculturesystemwasperformedasdescribedwithminormodification(10).The TGF- bioassaysandzymographytodetermineMT1-MMPactivitywereperformed Cytokine array and IL-1 ELISAAirwayepithelialcells(3 10 wereculturedin10-cmcollagen-coatedtissuecultureplatesfor12–16hours,washedtimeswithPBS,thenincubatedinserum-freeDMEMfor72hours.CytokinesinconditionedmediumweredetectedwithCartesianArrayHumanCytokineSetfromBioSourceInternational(Invitrogen)followingthemanufacturer’sinstructions.IL-1 wasmeasuredincondi IL-1 QuantikineELISAkit(R&DSystems). Coculture model of the epithelial-mesenchymal trophic unitAirwayepithelialcells(2 10 /well)wereseededonvitrogen-coated(10 g/mlcoatingcon centration)cultureinserts(1mmporesize;BDBiosciences)in24-wellplateinBEGM(Clonetics)andcultureduntilconfluent.Airwayfibro blaststransfectedwithcontrolor siRNA(2.5 g/transfection)wereculturedfor12–16hourson24-well(2 10 /well)tissue-coatedplate(BDBiosciences)infibroblastgrowthmedium.Forthecocultureexperi ments,thecultureinsertscontainingtheairwayepithelialcellswereplacedintowellscontainingairwayfibroblasts,andmediuminboththeupperandlowerchamberswerechangedtoDMEMwithoutFCSinthepresenceorabsenceofcontrolantibody,anti–TGF- antibody(1D11), anti– integrinantibody(10D5),anti– integrinantibody(37E1),orIL-1RA.After48or72hourscoculture,integrinexpressionlevelsinairwayepithelialcellswereanalyzedbyFACSandRT-PCR.ColgeneexpressioninfibroblastswasevaluatedbyRT-PCRorbyWesternblottingofconditionedmedium. StatisticsStepwiseregressionanalysiswithcorrectionforclusteringwascompare stainingairwayStudent’s t forcomparisondatasets,for pledatasets.Tukey’sorDunn’stestwereusedforparametricandnonpara metricdata,respectively,finddifferencelay.Significancedefinedas 0.05.StatisticalsoftwareusedwasPrismv.3cx(GraphPadSoftwareStatav.9(StataCorp Figure 10 Hypothetical model of SM in the pathogenesis of airway wall thickening in COPD. (a) The normal airway epithelium when exposed to noxious stimuli responds by increas ing TGF- production (12) and increasing TGF- activation, which increases expres sion of the 6 integrin, a TGF- responsive gene (31) contributing to (b) a phenotypic switch to SM, a TGF- driven process (11). (c) Squamous metaplastic epithelial cells secrete increased IL-1 , which acts a paracrine factor with adjacent airway fibro blasts. (d) Airway fibroblasts respond to IL-1 by increasing 8 expression and mediated TGF- activation. Increased TGF- activation by airway fibroblasts causes (e) autocrine effects on the fibrogenic fibroblast phenotype by increasing Col I and SMA and (f) paracrine effects on adjacent airway epithelium, which inhibits epithelial proliferation (10, 44, 66) and contributes to the increased expression of 6 by adja cent airway epithelial cells, forming a self-amplifying loop of TGF- activation. research article TheJournalofClinicalInvestigation http://www.jci.org Volume117 Number11 November2007 3561 WewouldliketothankCharlesMcCullough(UCSFDivisionofBiostatistics)forbiostatisticalassistanceandRebeccaBarbeau,Ali ciaGoodwin,andAlanaMorrisfortechnicalassistance.ThisworkwassupportedbyNIHgrantsHL63993andHL70622,theUCSFAcademicSenate(toS.Nishimura),NIHgrantsHL72301(toD.Erle),HL53949,HL083950,HL64353,AI024674(toD.Sheppard),CA95671(toV.C.Broaddus),andDK72517andgrantfromtheCysticFibrosisFoundation(toW.Finkbeiner).WethankUCSFResearchAtakilitforprovidingReceivedforpublicationApril27,2007,andacceptedinrevisedformAugust2007.AddressStephenFranciscoGeneralHospital,Building3,Room211,1001PotreroAvenue,SanFrancisco,California94110,USA.Phone:(415)206-5906;Fax:1.Vandevoorde,J.,etal.2007.EarlydetectionofCOPD:casefindingstudyingeneralpractice. Respir. Med. 101 2.InternationalCOPDCoalition.2006.GlobalburdenofCOPDsummit2006.http://www.international - copd.org/materials/patients/learn/facts.aspx.Hogg,J.C.,Macklem,P.T.,andThurlbeck,W.M.1968.Siteandnatureofairwayobstructioninchronicobstructivelungdisease. N. Engl. J. Med. 278 Shapiro,S.D.,andIngenito,E.P.2005.Thepatho genesisofchronicobstructivepulmonarydisease:advancesinthepast100years. Am. J. Respir. Cell Mol. Biol. 32 Hogg,J.C.,al.2004.Thenatureofsmall-airwayobstructivepulmonary -ease. N. Engl. J. Med. 350 Puchelle,E.,Zahm,J.M.,Tournier,J.M.,andCoraux,Airwayrepair,afterinjuryobstructive monary Proc. Am. Thorac. Soc. 3 7.etbetween turalchangesinsmallairwaysandpulmonary- N. Engl. J. Med. 298 :1277–1281.Evans,M.J.,VanWinkle,L.S.,Fanucchi,M.V.,andPlopper,C.G.1999.Theattenuatedfibro blastsheathoftherespiratorytractepithelial-mesenchymaltrophicunit. Am. J. Respir. Cell Mol. Biol. 21 :655–657.Shannon,J.M.,Nielsen,L.D.,Gebb,S.A.,andRan dell,S.H.1998.Mesenchymespecifiesepithelialdif ferentiationinreciprocalrecombinantsofembry -oniclungandtrachea. Dev. Dyn. 212 Araya,J.,Cambier,S.,Morris,A.,Finkbeiner,W.,andNishimura,S.L.2006.IntegrinmediatedTGF- activationregulateshomeostasisofthepulmonaryepithelial-mesenchymaltrophicunit. Am. J. Pathol. 169 11.T.,etType- transforminggrowthfactoristheprimarydifferentiation-inducingserumfactorfornormalhumanbronchialepithe -lialcells. Proc. Natl. Acad. Sci. U. S. A. 83 12.deBoer,W.I.,etal.1998.Transforminggrowthfac - tor- andrecruitmentofmacrophagesandmastcellsinairwaysinchronicobstructivepulmonarydisease. Am. J. Respir. Crit. Care Med. 158 :1951–1957.Takizawa,H.,etal.2001.Increasedexpressionoftransforminggrowthfactor- insmallairwayepi theliumfromtobaccosmokersandpatientswithchronicobstructivepulmonarydisease(COPD). Am. J. Respir. Crit. Care Med. 163 ettransforminggrowth factor- (TGFB1)geneisassociatedwithchronicobstructivepulmonary Hum. Mol. Genet. 13 Lee,C.G.,etal.2004.Earlygrowthresponsegeneapoptosisfortransforminggrowthfactorbeta1-inducedpulmonaryfibrosis. J. Exp. Med. 200 Sime,P.J.,Xing,Z.,Graham,F.L.,Csaky,K.G.,andGauldie,J.1997.Adenovector-mediatedgenetrans ferofactivetransforminggrowthfactor- inducesprolongedseverefibrosisinratlung. J. Clin. Invest. 100 17.Annes,J.P.,Munger,J.S.,andRifkin,D.B.2003.TGF- activation. J. Cell Sci. 116 Rifkin,D.B.2005.Latenttransforminggrowthfac - tor- (TGF- bindingproteins:orchestratorsof TGF- availability. J. Biol. Chem. 280 19.Crawford,S.E.,etal.1998.Thrombospondin-ismajoractivatorofTGF- invivo. Cell. 93 :1159–1170.Mu,D.,etal.2002.Theintegrin v mediatesepi thelialhomeostasisthroughMT1-MMP-dependentTGF- 1. J. Cell Biol. 157 :493–507.21.Munger,J.S.,etal.1999.Theintegrin v bindsandactivateslatentTGF- 1:mechanismforregu latingpulmonaryinflammationandfibrosis. Cell. 96 Yang,Z.,etal.2007.Absenceofintegrin-mediated TGF activationinvivorecapitulatesthepheno TGF 1-nullmice. J. Cell Biol. 176 P.J.obstructivepulmonary Pharmacol. Rev. 56 Rusznak,C.,etal.2000.EffectofcigarettesmokeonthepermeabilityandIL-1 andsICAM-1releasefromculturedhumanbronchialepithelialcellsofnever-smokers,smokers,andpatientswithchronicobstructivepulmonarydisease. Am. J. Respir. Cell Mol. Biol. 23 Lappalainen,U.,Whitsett,J.A.,Wert,S.E.,Tiche laar,J.W.,andBry,K.2005.Interleukin-1 causespulmonaryinflammation,emphysema,andairway Am. J. Respir. Cell Mol. Biol. 32 Tian,D.,etal.2006.Roleofglycogensynthasekinaseinsquamousdifferentiationinducedbycigarettesmokeinporcinetracheobronchialepi -thelialcells. Food Chem. Toxicol. 44 27.Sachs,L.A.,Finkbeiner,W.E.,andWiddicombe,J.H.2003.Effectsofmediaondifferentiationofculturedhumantrachealepithelium. In Vitro Cell Dev. Biol. Anim. 39 Wang,B.Y.,etal.2002.P63inpulmonaryepithe lium,pulmonarysquamousneoplasms,andotherpulmonary Hum. Pathol. 33 Jetten,A.M.1989.Multistepprocessofsquamousdifferentiationintracheobronchialepithelialcellsinvitro:analogywithepidermaldifferentiation. Environ. Health Perspect. 80 Lee,J.J.,etal.2001.Long-termimpactofsmokingproliferationfor smokers. J. Natl. Cancer. Inst. 93 31.Wang,A.,Yokosaki,Y.,Ferrando,R.,Balmes,J.,andSheppard,D.1996.Differentialregulationofairwaybygrowth Am. J. Respir. Cell Mol. Biol. 15 Zavadil,et2001.Genetic lialcellplasticitydirectedbytransforminggrowth factor- . Proc. Natl. Acad. Sci. U. S. A. 98 :6686–6691.Skonier,etsequenceanalysisofbetaig-h3,novelgeneinducedinhumanadenocarcinomacelllineaftertreatmentwithtransforminggrowthfactor-beta. DNA Cell Biol. 11 et2001.IdentificationdifferentiationcomplexgenesbysubtractivehybridizationofentireYACstogriddedkeratinocytecDNAlibrary. Genome Res. 11 Wiszniewski,L.,Limat,A.,Saurat,J.H.,Meda,P.,andSalomon,D.2000.Differentialexpressionofconnexinsduringstratificationofhumankerati -nocytes. J. Invest. Dermatol. 115 Sedghizadeh,P.P.,etal.2006.ExpressionoftheserineproteaseDESC1correlatesdirectlywithnormalkeratinocytedifferentiationandinverselysquamouscarcinoma -gression. Head Neck. 28 37.Galliano,M.F.,etal.2006.novelproteaseinhibitorofthealpha2-macroglobulinfamilyexpressedinthehumanepidermis. J. Biol. Chem. 281 :5780–5789.Pay,S.,etal.2006.Synovialproinflammatorymetal loproteinase-3Behcet’sinterleukin-1betaplaymajorroleinBehcet’ssynovitis? Rheumatol. Int. 26 39.Gruenert,D.C.,Finkbeiner,W.E.,andWiddi combe,J.H.1995.Cultureandtransformationofhumanairwayepithelialcells. Am. J. Physiol. 268 :L347–L360.Ries,C.,etal.2007.MMP-2,MT1-MMP,andTIMP-areessentialfortheinvasivecapacityofhumanmesenchymaldifferentialbyinflammatory Blood. 109 41.K.Y.,etNF-kappaB teinresponseelementsandtheroleofhistonemodificationsinIL-1beta-inducedTGF-beta1genetranscription. J. Immunol. 176 Chidgey,M.,etal.2001.Micelackingdesmocollinshowaccompaniedbydefectsandabnormaldifferentiation. J. Cell Biol. 155 Mischke,D.,Korge,B.P.,Marenholz,I.,Volz,A.,andZiegler,A.1996.Genesencodingstructuralpro teinsofepidermalcornificationandS100calcium-proteinsformcomplexdifferentiationcomplex”)1q21. J. Invest. Dermatol. 106 Cambier,S.,etal.2000.rolefortheintegrin v negativegrowth. Cancer Res. 60 Serra,V.,etal.1989.Precociousappearanceofmarkersofsquamousdifferentiationinmetaplas endocervix. Arch. Gynecol. Obstet. 246 Smedts,F.,etal.1993.Expressionofkeratins1,6,cervicalsqua mousmetaplasia,cervicalintraepithelialneoplasia,cervicalcarcinoma. Am. J. Pathol. 142 47.Vaidyanathan,S.,etal.2003.Detectionofearlysquamousmetaplasiacordinjurypatientsbyimmunostainingforcyto keratin Spinal Cord. 41 Groves,R.W.,Mizutani,H.,Kieffer,J.D.,andKup per,T.S.Inflammatory genicmicethatexpresshighlevelsofinterleukin 1 inbasalepidermis. Proc. Natl. Acad. Sci. U. S. A. 92 Maas-Szabowski,N.,Shimotoyodome,A.,andFusenig,N.E.1999.Keratinocytegrowthregula fibroblast J. Cell Sci. 112 50.Freedberg,I.M.,Tomic-Canic,M.,Komine,M.,and