RAW264.7macrophagesinduceapoptosisselectivelyintransformed broblasts:intercellularsignalingbasedonreactiveoxygenandnitrogenspecies

StefanieHeigoldandGeorgBauerAbteilungVirologie,Institutfu¨rMedizinischeMikrobiologieundHygiene,Universita¨tFreiburg,Germany

Abstract:Therationaleforthisstudywastode-terminewhethermacrophagesinduceapoptosisse-lectivelyintransformedcomparedwithnontrans-formed broblastsandtoelucidatetheunderlying

intercellularsignalingchemistry.Murine bro-blaststransformedbyoncogeneexpression(ras,src)ormethylcholanthrenetreatmentweresensi-tiveforapoptosisinductionbyRAW264.7mac-rophages,whereasparentalcellsandrevertantswereinsensitive.Moreover,RAW264.7macro-phagesinducedapoptosisinnormalratkidney(NRK) broblaststransientlytransformedbyepi-dermalgrowthfactor/transforminggrowthfac-tor- .Sensitivityforintercellularapoptosisinduc-tionwasbasedontargetcell-derivedsuperoxideanionsandeffectorcell-derivedperoxidaseandnitricoxide(NO).Superoxideanionsdismutatetohydrogenperoxide,whichisconvertedtoHOClbytheperoxidase.TheinteractionofHOClwithsu-peroxideanionsthengenerateshydroxylradicals.Inparallel,NOinteractswithsuperoxideanionsandgeneratesapoptosis-inducingperoxynitrite.Signalingbyreactiveoxygenandnitrogenspeciesseemstorepresentahithertounrecognizedsignal-ingprinciplefortheselectiveeliminationofpoten-tialtumorcellsbymacrophages.J.Leukoc.Biol.72:554–563;2002.

KeyWords:peroxidase nitricoxide superoxideanion

INTRODUCTION

Macrophagesrepresentanessentialpartofthenaturalanti-tumordefensesystem.Theyuseseveraldifferentsignalingmechanismsforapoptosisinductionintumorcells.Thespec-i cityofthesemechanismswithregardtothetransformedstateofpotentialtargetcellsandtheirpotentialinterdependenciesandinteractionsisnotyetcompletelyunderstood.Releaseoftumornecrosisfactor(TNF)andtransforminggrowthfactor- (TGF- )representsoneaspectofmacrophageanti-tumorac-tion[1–6].TNFinteractswithtransformedandnontransformedcells,butselectiveapoptosisinductionintransformedcellsderivesfromaselectivetargetcellresponse,whichseemstobebasedonadecreasedconcentrationofendogenousapoptosisinhibitors[7].TGF- hasbeenshowntoinduceapoptosisin

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certaintumorcelllines[8–17]andtomodulatethesensitivityofcertaintumorcellsforapoptosisinductionbytheapo/fassystem[18]orbyDNA-damagingagents[19].ThemolecularbasisofTGF- -mediatedanti-tumoractionisnotcompletelyresolved.However,thereisstrongevidenceforaninvolvementofreactiveoxygenspecies(ROS)inthisprocess[11,20].DirectapoptosisinductionbyTGF- seemsnottoberestrictedtotumorcells,asnontransformedcellshavealsobeenreportedtobeaffectedundercertainconditions[8,10,20,21].Sofar,itremainsenigmatichowsensitivitytoapoptosisinductionbyTGF- iscontrolledonthemolecularlevel.

Macrophagesreleasenitricoxide(NO),whichisknowntobeinvolvedinanti-tumordefense[22,23].Recentevidencein-dicatesthatNOmayinteractwithtransformed,cell-derivedsuperoxideanionsandtherebygeneratetheapoptosisinducerperoxynitrite[24,25].Asextracellularsuperoxideanionpro-ductionrepresentsahallmarkofthetransformedstate[26,27](forreview,seerefs[28,29]),peroxynitritegenerationbasedoninteractionofNOwithtargetcell-derivedsuperoxideanionmayrepresentthekeyforselective,NO-basedanti-tumoractionbymacrophages.Myeloperoxidase(MPO),whichisknowntobeinvolvedinthetumoricidalactivityofgranulocytes(incooperationwiththeiroxidativeburst)[30,31](forreview,seeref[32]),isnotconsideredatypicalmacrophage-speci cenzyme.However,directmeasurementsindicatedthatRAWmacrophagessynthesizeMPO,althoughinmuchlowercon-centrationthangranulocytes[33].Therefore,macrophagesmightalsousetheperoxidase/HOCl-dependentsignalingpath-wayforselectiveapoptosisinductionasrecentlydescribed[24,28,29]anddiscussedbelow.

WehaverecentlyshownthatTGF- -pretreated,nontrans-formed broblastsusetargetcell-generatedROSforselectiveapoptosisinductionintransformedtargetcells[24](forreview,seerefs[28,29]).Signalingisbasedonsuperoxideaniongenerationbytransformedtargetcellsandusestwosignalingpathways:theHOCl/hydroxylradicalandtheNO/peroxynitritepathway.TheHOCl/hydroxylradicalpathwayrequiresdismu-tationofsuperoxideanionstohydrogenperoxide.Effectorcell-derivedperoxidasethenconvertshydrogenperoxidetohypochlorousacid,whichinteractswithtargetcell-derived

Correspondence:GeorgBauer,AbteilungVirologie,Hermann-HerderStr.11,D-79104Freiburg,Germany.E-mail:tgfb@ukl.uni-freiburg.de

ReceivedJuly12,2001;revisedFebruary26,2002;acceptedApril16,2002.

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