andUninvolvedSkinfromPsoriaticSubjects:AreTheyEquallyDiseased?ASSESSME - PDF document

andUninvolvedSkinfromPsoriaticSubjects:AreTheyEquallyDiseased?ASSESSMENTBYSKINTRANSPLANTEDTOCONGENITALLYATHYMIC(NUDE)MICEGERALDG.KRUEGER,DONALDA.CHAMBERS,andJANESHELBY,DivisionofDermatology,DepartmentofMedicine,UniversityofUtahSchoolofMedicine,SaltLakeCity,Utah84132;CenterforResearchinPeriodontalDiseasesandOralMolecularBiology,DepartmentofBiologicalChemistry,UniversityofIllinoisattheMedicalCenter,Chicago,Illinois60612ABSTRACTAhighlysignificant,butunanswered,questioninthepathogenesisofpsoriasisrelatestohownormalappeannganddiseasedskincancoexist,un-dergospontaneousflaresandremissions,andyetappeartobegeneticallyacquired.Aplausibleexplanationforthesedisparateobservationsisthatthereisabasicdefectinepidermalproliferationofskinofsubjectswithpsoriasisandthatdiseaseexpressionisgovernedbyotherhostfactors.Toaddressthisquestion,wecom-paredepidermalproliferationofskininvolvedandun-involvedwithpsoriasiswithnormalskinbeforeandaftertransplantationtocongenitallyathymic(nude)mice,abiologicmilieufreeofhumoralfactorsuniquetothedonorhost.Resultsdemonstratedthat(a)beforetransplant,syn-thesisofDNAbytheepidermalcellsfromskinunin-volvedandinvolvedwithpsoriasisissignificantlyhigherthannormal,1.6and3.6times,respectively;(b)6wkaftertransplantation,synthesisofDNAbyepidermalcellsisunchangedfornormalskin,increasedforuninvolvedskin,anddecreasedforinvolvedskin.Theseincreasesanddecreasesareofsuchamagnitudethatat6wkthenumberofepidermalcellssynthe-sizingDNAper1,000basalcellsisidentical,andis2.2timesthatofnormalskin.Whenremovedfromthemilieuoftheafflictedhost,skininvolvedandunin-volvedwithpsoriasisappearequally"diseased."ThesedatasupportthenotionthatthereisaberrantepidermalThispaperwaspresentedinpartattheWesternClinicalResearchMeetings,Carmel,Calif.,February1981,andattheMeetingoftheSocietyforInvestigativeDermatology,April1981(Clin.Res.29:82a.)Receivedforpublication12January1981andinrevisedform17August1981.1548proliferationinskinofpatientswithpsoriasisandthathostfactorsappeartoplayarolebothintheexpressionandnonexpressionofthisdisease.INTRODUCTIONPsoriasisischaracterizedbyscaly,unsightly,mildlypruritic,erythematousplaqueswhichare,atleastinpart,secondarytothebenignhyperproliferativestateofthegerminativelayeroftheepidermis(1).Althoughthediseasecaninvolvetheentiresurface(exfoliativeerythroderma),themajorityofpatientshaveoftheirskininvolvedwithdisease(2).Sincepsoriasisisthoughttobeageneticdisease(3,4)andcanoccuronanysiteofthebody,itislikelythatevenphenotypicallynormalskinisabnormal.Thenatureandlocationofthefactor(s)thattransformsuninvolvedskintoactivelesions,orallowsthecoexistenceofinvolvedandun-involvedskinisunknown.Studiescomparinguninvolvedskinofpsoriaticsub-jectswithskinofnormalsubjectsarelimited(5-14).Thesestudiesindicatethatalthoughuninvolvedskinappearsnormal,abnormalitiesarepresent.Examplesofsuchabnormalitiesinclude:anincreaseinthenum-berofgerminativecellsoftheepidermisundergoingDNAsynthesis(5-9),aprolongedSphaseofthegermi-nativecellsstimulatedtoproliferationbytapestripping(12),astratumcorneumwithincreasedcohesion(13),andanabnormalproliferativeresponsetotheintra-dermalinjectionofpropranololandsaline(10).Becausethesestudieshavebeenperformedinvivo,theques-tionofwhethertheabnormalitiesareinherenttotheskin,oraregeneratedbyhumoralfactorsuniquetothehost,remainsunanswered.J.Clin.Invest.(©TheAmericanSocietyforClinicalInvestigation,Inc.0021-9738/81/12/1548/10$1.00Volume68December19811548-1557 Beforegrafting,usingtechniquespreviouslydescribed(15),graftsweretrimmedand1-2-mmpiecesoftissuewerepulsedwith[3H]thymidine,specificactivity20Ci/mM(NewEnglandNuclear,Boston,Mass.)for3hat37°C.Thesespecimenswereprocessedforautoradiographytode-terminetheLI(15).Anepidermalcellwasdefinedassynthe-sizingDNAifitcontained5ormoregrains(alabeledcell)overthenucleuswhenviewedatx1,000bylightmicroscopy.TheLIwasdefinedasthenumberoflabeledcellsintheepidermisper1,000basalcellscounted.Histologicassessmentwasbyroutinelightmicroscopyoftheautoradiographicsections.Quantitationoftheepidermalmass(acanthosis)wascarriedoutwithanocularmicrometer.Parakeratosiswasquantitatedascontinuous,i.e.,throughoutthestratumcorneumverticallyandhorizontally,orintermit-tent,i.e.,presentinlocalizedareas.Backgrounddeposition'Abbreviationusedinthispaper:LI,labelingindex.ofemulsionandimproperorientationdidnotallowthistypeofassessmentoneveryspecimen.Graftingandbiopsyprocedures.Themethodologytotransplanttheskingraftstothenudemousehasbeende-scribed(15).At3and6wkaftergrafting,biopsies(2-3x3-4mm)weretakenfromeachgraftsitewhilethemousewasunderlighthalothaneanesthesia.Apreviouslyunbiopsiedgraftwasalwaysbiopsied,andwherepossiblepreviouslybiopsiedsiteswererebiopsied.Afterremovingthebiopsy,thedermiswastrimmedsothatthebiopsywas-0.4mmthick.Biopsiesweresectionedinto1-2-mmpieces,pulsedwith[3H]thymidinefor3h,andanalyzedautoradiographicallyasabove.DataarepresentedasameanLI+SDofthetotalnumberofgraftsperpatientattheindicatedtimes.Whereavailable,theLIofbiopsiesfromonegraftpregrafting,3and6wkpostgrafting,arealsopresented.AnalysisoftheLIofgraftsbiopsiedforthefirsttime,vs.thosegraftspreviouslybiopsied,revealsthatthereisnodifference,i.e.,thetraumaofabiopsytaken3wkpreviousdoesnotaffectfindings3wklater.RESULTSComparisonofepidermalLI,groupdata,normal,involved,anduninvolvedpsoriaticskinvs.time.Todeterminewhethertheepidermisofinvolvedandun-involvedskinfrompsoriaticsubjectsacquireorretainabnormalproliferationintheabsenceofhostfactors,involvedanduninvolvedskinweretransplantedtonudemice,andepidermalDNAsynthesis(LI)deter-minedat3and6wk.Theseresultswerecomparedwiththoseofnormalskintransplantedtothenudes.TableIpresentsthemeanLI+SDandtherangeoftheLIofgraftstested;themedianandmeanwereequivalent.Initially,uninvolvedskinhadahigherLIthannormal,andinvolvedskinhadaLIwhichwasgreaterthantwotimesthatoftheuninvolvedskin.AlthoughtherangeoftheLIwasconsiderable,only2of34patientshadaLIofuninvolvedskinthatwasabove100.Similarly,only4of25hadaLIofinvolvedskinthatfellbelow100.Thereason(s)whytwopatientshadaLIoftheiruninvolvedskinthatwasintherangeofthe 145±4090-213 normalskin,afourfolddifference.Contrariwise,theLIofinvolvedskinincreasedby47fromtheinitialvalue,a32%decrease.At6wk,theincreasesandde-creaseswereofsuchmagnitudethatessentiallynodif-ferencesexistedbetweentheLIofinvolvedandun-involvedskin.Thesedataprovideinsightintotwocriticalconceptsoftheclinicalexpressionsoflesionsofpsoriasis:first,thatsomefactor(s)ispresentinallskinofpsoriaticsubjectsthatpermits/causesabnormalepidermalpro-liferation;second,thatunknownhostfactorsmaybecriticaltodiseaseexpression,towit,onthehostinvolvedanduninvolvedareascoexist.Histologicassessmentandcorrelation.Forashorttimeaftergrafting,graftsofskininvolvedwithpsoria-sisusuallycanbevisuallydistinguishedfromthosegraftsfromuninvolvedsitesandthosefromnormalsubjects.Thisdistinction,baseduponathickerstratumcorneum,becomesincreasinglydifficultwithtimesothatafter3wkitisimpossibletovisuallydeterminetheoriginofgrafts.Becauseofthis,histologicassess-mentoftheautoradiographsoftheinitialgroupwasusedtoconfirmtheobservationthattheepidermisoftheuninvolvedandinvolvedskinbecomesimilar.Themostcommonhistologicaberrationsintheepidermisinvolvedwithpsoriasisare:regularacanthosis(anin-creaseintheepidermalmass),parakeratosis(delayedmaturationofthestratumcorneum),andadecreaseinthegranularlayer(absentkeratohyalinegranules).Atthetimeofgrafting,histologicassessmentshowed7/57(41%)oftheuninvolvedspecimenshadminimalacanthosis.At3wk,graftsfrom1415(93%)differentpatientshadacanthosis(arbitrarilydefinedasanepi-dermalmass�42gm(TableII).At6wk,2%4(83%)hadthisdegreeofacanthosis(cf.Figs.1and2).Fornormals,thesevalueswere/14(7%)initially,5/8(62%)at3wk,and%(50%)at6wk(cf.Figs.3and4).Forinvolvedskinatthetimeofgrafting,¾7involvedlesionsshowedregularpsoriasiformacanthosis.By6wk,thishaddecreasedto/5(TableII).ChangesinacanthosisandtheaccompanyingchangesintheLIsupporttheconceptthatbothinvolvedanduninvolvedskingraftswereevolvingtoasimilarpointwhilebeingmaintainedonanindependentbiologicsupportsystem.Thehistologicassessmentofparakeratosisprovidesamoredramaticassessmentofthechangeswhichoccur(TableIII).Ofthe14graftsfromnormals,nonehadparakeratosisbeforetransplant(Fig.3).Similarresultswerenotedintheuninvolvedskin(Fig.1);thiscon-trastswithparakeratosisin1147donorgraftsofinvolvedskin(TableIII).At6wk,noneofthenormalgraftshadFIGURE1Photomicrograph(x80)ofanautoradiographicspecimenfromasplit-thicknessgraftofuninvolvedskinofpsoriaticsubjectM.B.,pregrafting.Arrowpointstoaheavilylabeledepidermalcell.InvolvedandUninvolvedPsoriaticSkinAreEquallyDiseased1551 4177;.s{~~4_-~~~~~~~~IP*4-_10fL*s|:~~~~~~e0.:.:i*'.I.rI,k1i.IitIS,iFIGURE2Photomicrograph(x45)ofanautoradiographicspecimenfromasplit-thicknessgraftofuninvolvedskinofpsoriaticsubjectM.B.,6wkpostgrafting.Arrowsaretotheareaofparakeratosis.Notethegeneralizedincreaseinepi-dermalmass(acanthosis).parakeratosis(Fig.4).However,11/25uninvolvedgrafts(Fig.2)and'I1involvedgraftshadequaldegreesofparakeratosis.AstatisticalanalysisofthenumberofpatientswhosegraftsdevelopeddifferingamountsofparakeratosisispresentedinthelowerpartofTableIII.Dividingparakeratosisintothreecategories,none,intermittent(throughthestratumcorneumverticallyorhorizontally),andcontinuous,showedthatthehigh-estLIoccurredinthepresenceofcontinuouspara-keratosis,regardlessofwhetheritwasininvolvedort*lbFIGURE3Photomicrograph(x60)ofanautoradiographicspecimenfromasplit-thicknessskingraftofcontrolsubject43,beforegrafting.uninvolvedspecimens.Inlikefashion,adecreaseinparakeratosishadanaccompanyingdecreaseintheLI(TableIII).After3wk,amaturegranularlayerreappearedand1552G.G.Krueger,D.A.Chambers,andJ.ShelbyF1..ti..:.JYtA..W.:..0t...;: ., 84+2788+29 ofthe VIPairedComparisonoftheChangeinLI,0vs.6wkof14Controland19PsoriaticSubjectsALI,0vs.6wk*ALI,Controlidentification0vs.6wkPatientUninvolvedInvolvedl41+345N.W.+163-3034+12A.B.+107-5033+10M.A.+99-1042+9D.R.+93-3040+5A.A.+81-2938+2L.G.+70-10544+1C.D.+51-936-1G.R.+34-1537-3D.M.+31-332-3S.C.+31-6843-10D.F.+29-2431-16C.R.+24-3539-18C.V.+23-735-26R.M.+19-41E.S.+9-81S.W.+3-124B.P.+1-95L.B.-23-72K.O.-33-66MeanALI±SD0vs.6wk0.29+15"+43±48"1-47+±36Number+or-/total7/14(50%)-2/19(20%)+0/19(0%)MeanLIofthisgroupInitial47±765+20147±406wk47+10107±54101±22*ALIarerankedforbothnormalanduninvolvedspecimens.tTheALIoftheinvolvedspecimenaccompanyingtheuninvolvedvaluesarethechangeswhichoccurredinthatpatient'sinvolvedskin.§+ConnotesanincreaseintheLI0vs.6wk,-connotesadecrease."P0.002byMannWhitneyUranksumtest.ingnormallevelsin24d.OurobservationthattheLIofgraftsofnormalskinat3wkareincreasedoverbaseline(P0.005)supportshisobservations.Theper-centincreasefromtheinitialLItotheLIat3wkfornormalskinanduninvolvedskinofpatientswithpsoria-sisisquitesimilar.Thereafter,however,uninvolvedskinofpatientswithpsoriasiscontinuestoincreasewhilethatofnormalsreturnstoalevelwhichisnotdifferentfromthebase-lineLI.OurdataareinagreementwiththoseofHarperetal.(9)whonotedthattheLIofepidermisofinvolvedanduninvolvedskinwereverysimilarwhenassessedinvitro8dafterplacingskinexplantsintissueculturemedia.Intheirstudy,normalskinhadaLIthatwasalsosignificantlylowerthanthatofinvolvedorunin-volvedskinofpsoriaticsubjects.TheseexplantstudiesdidnotassessDNAsynthesisbeforeplacingtissueinexplantandthus,wereunabletodetectchangesinbothinvolvedanduninvolvedtissuethatcouldhaveoccurredduringthetimetheywereheldinculture.InarecentpreliminarycommunicationFrakietal.(17)havepartiallyconfirmedandextendedourfindings.Theydemonstratedthatfollowingtransplantationtonudemice,theLIisincreasedinuninvolvedskin,andthatplasminogenactivatoractivityincreasestothatofinvolvedskin,whilenormalskindidnotchangerelativetotheplasminogenactivatoractivity.BriggamanandWheeler(22)havealsoreportedthatgraftsoflesionalskinwithpsoriasistransplantedtothenudemousehadincreasedLIat9wk,comparedwithgraftsfromnormalsubjects.Theydidnotdopregraftanalysis,anddidnotstudyuninvolvedskin.However,usingepidermal-dermalrecombinantgrafttechniques,theydemonstratedthatabnormalLIwereonlypresentinthosecombinationsofinvolveddermisandepidermis.Ourdataconfirmthegeneralbeliefthatanincreaseinepidermalproliferation,hereindeterminedasanincreaseintheLIofepidermis,resultsinanincreaseintheepidermalmass(23).TheLIalsoappearstocorrelatewithanotherofthehistologicparametersusedtodefinepsoriasis,parakeratosis(TableIII).Thisisinharmonywithunpublisheddatafromourlabora-1556G.G.Krueger,D.A.Chambers,andJ.Shelby J.J.R.A.