A.TanoDiMarco,C.Franceschi,andG.ProdiInstitutodiPatologiaGenerale,FacoltA€diMedicinaeChinirgia,UniversitA€diBologna,40126Bologna,ItalySUMMARYTheearlyeffectofurethanoncell-mediatedimmunitywasstudiedinFischeradultratsbymeansofskinhomografts,graft-versMS-hostassays,andinvitrolymphocytestimulationbyphytohemagglutinin.AweakbutsignificantsurvivalprolongationofLewisratskingraftedinurethan-treatedFischerrats1dayaftertheendofthetreatmentwasobserved.Contrarytoexpectation,theFischerspleencellstaken1dayaftertheendofurethantreatmentshowedagraft-versws-hostreactivityabout4timesthatofthecontrolswheninjectedintoFischerXBfFthybridnewbornrats;thisreactivityrecededtovaluesslightlybelowthenormallevelsifthedonorcellsweretaken7daysaftertheendofurethantreatment.Invitroassaysdemonstratedthatcellswithheightened invivocapacitytomountagraft-versMs-hostreactionshowedalsoanincreasedpro-liferativeresponsetophytohemagglutinin.Thehypothesisissuggestedthaturethancandistinguishbetweentwomononu-cleatedspleencellpopulations.Themorenumerouswouldbedestroyed(1dayaftertreatment,thenumberofmononu-cleatedspleencellsisreducedtolessthanone-third);theother,i.e.,thatinvolvedincell-mediatedimmuneresponses,wouldbemuchlessaffectedbythecarcinogen.Itwouldresultinarelativeincreaseofthymus-dependentcellsinthespleenoftreatedanimals.INTRODUCTIONImmunodepression seemstobeawidespreadconditionduringchemicalcarcinogenesis(1,9).Sinceitisaveryearlyphenomenonaftercarcinogenadministration,itcouldactbyallowingtumorneoantigens toescape fromthehostimmunologicalsurveillance.Oneimportantmechanismbywhichthisappearstobepossibleinvolvesthedepressionofcell-mediatedimmunityoftheprimaryhost.Previousstudiesindicatethatsuchadepressionoccursduringhydrocarboncarcinogenesis(4).Thesameeffectwasalsoobservedinmiceneonatallyexposedtourethan(5,7);however,thisconditiongivesinformationmoreontheimpairmentoftheimmunologicaldevelopmentthanonthedepressionofafullyimmunocompetentsystem.Thepresentstudywasundertakentoinvestigatetheearly1SupportedbyagrantfromConsiglioNazionaledelleRicerche,Roma,Italy.ReceivedSeptember15,1971;acceptedApril13,1972.effectofurethan,giventoyoungadultrats,oncell-mediatedimmunitybymeansofskinhomograftsacrossaweakhistocompatibility barrier,GVH2assays,andinvitrolymphocytestimulationbyPHA.Theresultsofourexperimentspresenteduswithanunexpectedphenomenonnotobservedbeforewithchemicalcarcinogens,i.e.,agreatlyheightenedcapacityofthespleencellssurvivingthestronglymphocytolyticactionofurethantomountaGVHreactionandtoreactinvitrotoPHAstimulation.WeinterpretthisfindingasarelativeincreaseofT-cellsproducedbytheselectivepressureofurethanonthewholespleenpopulation.MATERIALSANDMETHODSSkinGraftExperiments.GraftswereperformedbyuseofFischer(Arsal;Pomezia,Roma,Italy)andLewis(MicrobiologicalAssociates,Inc.,Bethesda,Md.)femaleinbredrats,4to5monthsold,ashostsanddonors,respectively.ThiscombinationsharesthesamestronghistocompatibilityalAele(AgB-1),thedonor-hostincompatibility beingduetodifferencesatmultiplenon-AgBloci.Ethylcarbamate(urethan;CarloErba,Milano,Italy)wasdissolvedinsterile,distilledwaterataconcentrationof100mg/mlandinjectedi.p.intohostsin5dosesof0.5mg/gbodyweightonDays0,1,2,3,and4(atatotaldosageof2.5mg/gbodyweight)inordertoavoidtheirreversiblecomathatweobservedwhenthetotalamountofurethanwasgiveninsingledosesof1mg/gbodyweight.Allcontrolsreceivedinjectionsofsterile,distilledwater.TheFischerratsweregraftedonthe5thdayaftertreatmenthadbegun.TheprocedureusedwasessentiallythatofBillingham(2).Castswereremoved6dayslater,and thegraftswereexamineddaily appearanceofthe1stlesionsuntilcompleterejection.ThesignificanceofthedifferencesbetweentreatedanimalsandcontrolshasbeencalculatedforbothMSTandMFLTbymeansofStudent'sttest.GVHAssays.TheassayswereperformedaccordingtoSimonsen'sspleenassay(8):4-to5-month-oldFischerratsweregiveninjectionsofurethanordistilledwaterasabove,thelastinjectionbeinggiven24hror7daysbeforetheassay.Astronghistocompatibilitybarrierwastestedbyuseof6-to7-day-oldFischerXBfF,(hereaftercalledFBF,)hybrid1Theabbreviationsusedare:GVH,graft-i>mws-host;PHA,phytohemagglutinin;T-cells,thymus-dcrivedcells;MST,meansurvivaltime;MFLT,mean1stlesiontime.JULY1972 1569
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