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Pharmacological
Research
jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s
In
vivo
sex
differences
in
leukotriene
biosynthesis
in
zymosan-induced
peritonitis
Antonietta
Rossi
a,1,
Carlo
Pergola
b,1,
Simona
Pace
a,
Olof
Rådmark
c,
Oliver
Werz
b,
Lidia
Sautebin
a,∗aDepartmentofPharmacy,UniversityofNaplesFedericoII,Naples,Italy
bDepartmentofPharmaceutical/MedicinalChemistry,Friedrich-Schiller-University,Jena,Germany cDepartmentofMedicalBiochemistryandBiophysics,KarolinskaInstitutet,Stockholm,Sweden
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received19February2014
Receivedinrevisedform14May2014 Accepted21May2014
Chemicalcompoundsstudiedinthisarticle: LeukotrieneB4(PubMedCID:5280492)
LeukotrieneC4(PubMedCID:5283121)
Zymosan(PubMedCID:11375554)
Keywords: Leukotrienes Peritonitis Sexdifferences Zymosan
a
b
s
t
r
a
c
t
Leukotrienes(LTs)are5-lipoxygenase(5-LO)metaboliteswhichareimplicatedinsex-dependent inflam-matorydiseases(asthma,autoimmunediseases,etc.).Wehaverecentlyreportedsexdifferencesin LTbiosynthesisininvitromodelssuchashumanwholeblood,neutrophilsandmonocytes,dueto down-regulationof5-LOproductformationbyandrogens.Herewepresentevidencesforsex differ-encesinLTsynthesisandrelatedinflammatoryreactionsinaninvivomodelofinflammation(mouse zymosan-inducedperitonitis).Onthecellularlevel,differential5-LOsubcellularcompartmentalization inperitonealmacrophages(PM)frommaleandfemalemicemightbethebasisforthesedifferences. Sexdifferencesinvascularpermeabilityandneutrophilrecruitment(cellnumberandmyeloperoxidase activity)intoperitonealcavitywereevidentuponintraperitonealzymosaninjection,withmore promi-nentresponsesinfemalemice.ThiswasaccompaniedbyhigherlevelsofLTC4andLTB4inperitoneal exudatesoffemalecomparedtomalemice.Interestingly,LTperitoneallevelsinorchidectomizedmice werehigherthaninshammalemice.Inaccordancewiththeinvivoresults,LTformationinstimulated PMfromfemalemicewashigherthaninmalePM,accompaniedbyalterationsin5-LOsubcellular local-ization.TheincreasedformationofLTC4inincubationsofPMfromorchidectomizedmiceconfirmsarole ofsexhormones.Inconclusion,sexdifferencesobservedinLTbiosynthesisduringperitonitisinvivomay berelated,atleastinpart,toavariant5-LOlocalizationinPMfrommaleandfemalemice.
©2014ElsevierLtd.Allrightsreserved.
Introduction
Leukotrienes(LTs)arebiologicallyactivelipidmediatorswith key roles in inflammation [1], including regulation of vascular permeabilityandofleukocyteextravasationandmigration[2,3]. LTs derive from arachidonic acid (AA) through the action of
Abbreviations: 5-LO,5-lipoxygenase;AA,arachidonicacid;cys-LTs, cysteinyl-leukotrienes;DMEM,Dulbecco’smodifiedEagle’smedium;EIA,enzyme immunoas-say; ERK, extracellular signal-regulated kinases; LTs, leukotrienes; MAPKs, mitogen-activated protein kinases; MPO, myeloperoxidase; ORCH, orchidec-tomized;PM,peritonealmacrophages;PMSF,phenylmethylsulfonyl,fluoride;PG, prostaglandin.
∗ Correspondingauthorat:DepartmentofPharmacy,UniversityofNaples Fed-ericoII,ViaD.Montesano,80131Naples,Italy.Tel.:+39081678427;
fax:+39081678403.
E-mailaddresses:[email protected](A.Rossi),[email protected]
(C.Pergola),[email protected](S.Pace),[email protected](O.Rådmark),
[email protected](O.Werz),[email protected](L.Sautebin).
1 Theseauthorscontributedequally.
5-lipoxygenase(5-LO),whichconvertsAAtoLTA4,representing thefirststepinthesynthesis ofallLTs[4].Infact,LTA4 canbe hydrolyzedtothechemotacticagentLTB4byLTA4hydrolaseor con-jugatedwithreducedglutathionebyLTC4synthasetoformLTC4. LTC4isthenmetabolizedbysequential proteolytichydrolysisto LTD4 andLTE4,whicharetogetherknownascysteinyl-LTs (cys-LTs)withvascularactions.LTbiosynthesisisregulatedbydifferent mechanismsandamongthempost-translationalmodificationsof 5-LO,includingphosphorylationatserineresidues,Ca2+binding, interactionswithcertainphospholipidsandglycerides,and inter-actionswith5-LO-activatingproteinandcoactosin-likeprotein[4]. Also,LTsynthesisisstronglyinfluencedbytheintracellular local-izationof5-LOandtheamountoftrafficableenzyme.Thus,5-LOis asolubleproteininthecytosolornucleoplasmofrestingcells,and translocatestoperinuclearmembraneswhenintracellularCa2+ lev-elsincreaseor/andafteritsphosphorylationbymitogen-activated proteinkinase(MAPK)[3,5–7].
LTsareimplicatedinarangeofinflammatorydiseases,often presenting sex-related differences in the incidence and/or the http://dx.doi.org/10.1016/j.phrs.2014.05.011
course,such as asthma, allergic rhinitis, and autoimmune dis-eases[8–10].Interestingly,thesexhasemergedasafundamental variablefortheLTpathwayandwehaverecentlyreportedsex dif-ferencesinLTbiosynthesisininvitromodelssuchashumanwhole blood,neutrophils[11]andmonocytes[12].Thus,malesex hor-monesrepressedLTbiosynthesisinhumanneutrophilsbyaffecting 5-LOlocalizationviadifferentialactivationofextracellular signal-regulatedkinase (ERK) [11]. Also, sex-related differences in LT biosynthesiswereobservedinhumanmonocyteswhere,invitro, testosteronecausedarepressionofphospholipaseD,resultingin impaired5-LOproductbiosynthesisduetolackofactivating dia-cylglyceride[12]. Moreover,data fromanimal modelsshowing sex-specificattenuationofatheromaformationindual12-/15-and 5-LOknock-outmice[13]andexclusiveprotectionoffemalemice fromplatelet-activatingfactor-inducedshockafterknockingoutof theLTB4receptor1[14]suggestedamajorcontributionofLTsin thepathophysiologyoffemales.Neverthelessnoinvivodataabout sex-dependentLTbiosynthesisareavailable.
Here,weinvestigatedwhetherthesexinfluencestheproduction ofLTsinvivo,usingzymosan-inducedperitonitisasexperimental model.Thismodeldisplaysallclassicalsignsofacuteinflammation, includingincreasedvascularpermeability,edema,leukocyteinflux andreleaseofinflammatorymediators.Weshowforthefirsttime, thatLTproductionis sex-biasedinvivoand significantlyhigher intheperitoneumoffemalemicechallengedwithzymosan.The dataindicatethatthisbiascanbetracedbacktosexdifferencesin theLT-syntheticcapacitiesofperitonealmacrophages(PM)andare accompaniedbysex-specific5-LOsubcellularlocalization. Materialsandmethods
Materials
Enzymeimmunoassay (EIA) kits were from Cayman Chem-ical Company (Aurogene, Rome, Italy). [3H-PGE
2] was from PerkinElmerLifeSciences(Milan,Italy).Allotherreagentsand com-poundswereobtainedfromSigma–Aldrich(Milan,Italy).
Animals
MaleandfemaleCD-1mice(8–9weeksold,CharlesRiver,Calco, Italy)were housedin a controlled environment (21±2◦C) and providedwithstandardrodentchowandwater.Allanimalswere allowedtoacclimateforfourdayspriortoexperimentsandwere subjectedto12hlight – 12hdarkschedule.Experiments were conductedduringthelightphase.
Toinvestigatetheimpactof sexhormones,male micewere orchidectomized(ORCHgroup)andallowedtorecoverfor5weeks. Thistimeperiodensuresthatsexhormoneshavebeen metabo-lizedandarenolongerintheblood,anditissufficienttoallow theturnoverofimmunecellsgeneratedundertheinfluenceofthis reproductivehormone[15,16].Inparticular,orchidectomieswere performedthroughscrotalincision.Shamgroupswereperformed throughthesimilarprocedures,exceptthatthegonadswerenot removed. Plasma testosterone was measured with a commer-ciallyavailableEIA(CaymanChemicalCompany).Theexperimental protocolswereapprovedbytheAnimalCareCommitteeofthe Uni-versityofNaplesFedericoII,incompliancewithItalianregulations onprotectionofanimalsusedforexperimentalandotherscientific purpose(MinisterialDecree116/92)aswellaswiththeEuropean EconomicCommunityregulations(OfficialJournalofE.C.L358/1 12/18/1986).
Zymosan-inducedperitonitis
Peritonitiswasinducedbyi.p.injection(0.5mL)of zymosan (2mgmL−1 insaline,boiledandwashed;Sigma,Milan,Italy).At
selectedtimepointsafterzymosaninjection(0,15,30, 60,120, 180and240min),micewereeuthanizedandperitonealexudates werecollectedusing 2mLofPBS.Thecells werecountedusing alightmicroscopeina Burker’schamber aftervitaltrypanblue staining.Exudateswerecentrifugedat20,000×gfor20minand supernatantsandcellpelletswerecollectedandfrozenat−80◦Cfor measurementsofeicosanoidsandmyeloperoxidase(MPO) activ-ity,respectively.TheperitonealexudatelevelsofLTC4 andLTB4 weremeasuredbyEIAkitsaccordingtomanufacturer’s instruc-tions(CaymanChemical;Aurogene,Rome,Italy)andexpressedas ngmL−1.
Vascularpermeability
Evansbluedye(40mgkg−1,0.3mL;Sigma,Milan,Italy) was injectedintothetailvainfollowedbyani.p.injectionofzymosan (0.5mL;2mgmL−1).After0,15,30,60,120,180and240min,mice were euthanized and peritoneal exudates were collectedusing PBS(0.1mLg−1ofbodyweight).Aftercentrifugation,supernatants (diluted1:3)wereanalyzedforEvansBlueboundtoplasma albu-minbyreadingat610nminaplatereader(MultiskanGo,Thermo Scientific).
MPOassay
TheuseofMPOasanindexofneutrophilinfiltrationiswell doc-umented[17].Cellpelletsweredisruptedbysonicationin2mLPBS (50mM,pH6)with0.5%hexadecyltrimethylammoniumbromide, freeze-thawedthreetimesandcentrifuged(32,000×g,20min)to collectsupernatantswhichwereusedinMPOassay,performedas describedbyBradleyetal.[17].Briefly,20lsampleswereadded toa96wellplateandthereactionwasinitiatedbytheaddition of0.2mLofassaybuffercontaining0.167mgmL−1ofo-dianisidine and0.0005%hydrogenperoxide.Therateofchangeofabsorbance wasmonitoredinkineticmodebyaplatereader(IMarkmicroplate Reader,Bio-Rad,Segrate,Milan,Italy).LevelsofMPOinsamples weredetermined fromthecalibrationcurveusing human neu-trophilMPOasthereferencestandard.Thelevelsof MPOwere expressedasUmL−1.
Peritonealmacrophages
ResidentPMwereobtainedbylavageoftheperitonealcavity of micewith7mLof cold Dulbecco’s modified Eagle’smedium (DMEM) withheparin (5UmL−1).PM werethen centrifugedat 500×g4◦Cfor5min,resuspendedat1×107cellsmL−1and0.5mL ofcellsuspensionwereaddedtoa15mLconicalpolypropylene tubeforeachexperimentalcondition.Cellswerethenincubatedat 37◦CwithionophoreA23187(4.5mL,finalconcentration2.5M, 15min),zymosan(4.5mL,finalconcentration13particlespercell, 30min)orvehicle(0.25%dimethylsulfoxideinDMEMforA23187 andDMEMforzymosan).EDTAatafinalconcentrationof4mMwas addedattheendofincubationtimetoA23187-stimulatedcells.All sampleswerethencentrifugedat500×gat4◦Cfor5min. Super-natantswerecollectedandfrozenat−80◦Cformeasurementsof LTC4andprostaglandin(PG)E2byEIA(CaymanChemicalCompany; Aurogene,Rome,Italy)andradioimmunoassay(PerkinElmerLife Sciences,Milan,Italy;Sigma,Milan,Italy)respectively.Thecell pel-letswerewashedbyresuspendinginice-coldPBS,centrifugedand collectedandfrozenat−80◦Cfor5-LOevaluation.
Celldisruptionandfractionation
Totalcelllysateswerepreparedbylysisfor15minat4◦Cwith lysis buffer (20mM HEPES, pH 7.6, 1.5mM MgCl2, 0.4MNaCl, 1mMEDTA,1mMEGTA,1mMDTT,1mMphenylmethanesulfonyl fluoride(PMSF),15gmL−1soybeantrypsininhibitor,3gmL−1 pepstatinA,2gmL−1leupeptin,1%NonidetP-40,20%glycerol,
50mMNaF).Lysateswerecentrifuged(12,000×g,15min4◦C),the supernatantswerecollectedandstoredat−80◦C.
Subcellularfractionationwascarriedoutbymild-detergentlysis (0.1%NP40)accordingtoWerzandcolleagues[6].IsolatedPMwere suspendedinice-coldNP-40-lysisbuffer(10mMTris–HCl,pH7.4, 10mMNaCl,3mM MgCl2,1mM EDTA,0.1%NP-40,1mMPMSF, 60gmL−1soybeantrypsininhibitorand10gmL−1 leupeptin), vortexed(3×5s),keptonicefor10min,andcentrifuged(1250×g, 10min,4C).Resultantsupernatants(non-nuclearfractions)were transferredtoanewtubewhereasthepellets(nuclearfractions) wereresuspendedinice-coldrelaxationbuffer(50mMTris–HCl, pH 7.4,250mM sucrose, 25mM KCl, 5mM MgCl2, 1mM EDTA, 1mMPMSF,60gmL−1soybeantrypsininhibitor,10gmL−1 leu-peptin).Bothnuclearandnon-nuclearfractionswerecentrifuged (1250×g,10min,4◦C)forfurtherpurification.Lysisofcellsand integrityofnucleiwereconfirmedbylightmicroscopywith try-panblueexclusion.Nucleiinrelaxationbufferweredisruptedby sonication(3×5s).
Analysisof5-LOfromcellularfractionsbySDS/PAGEandWestern blotting
ProteinconcentrationwasdeterminedbyBio-RadProteinAssay (Bio-Rad Laboratories, Segrate, Milan, Italy). The samples were mixedwith4×gelloadingbuffer(250mMTris,8%SDS,40% glyc-erol,20%2-mercaptoethanol,10mgmL−1 ofbromophenolblue), heatedfor6minat95◦C.Eachsamplewasloadedandanalyzedby electrophoresisona10%SDS–polyacrylamidegel.Correctloading of the gel and transfer of proteins to the nitrocellulose mem-brane was confirmed by Ponceau staining. After electroblot to nitrocellulose membrane (Whatman Protran,Dassel, Germany), membraneswereblockedwith0.05% PBS/Tweencontaining5% BSAfor2hatroomtemperature.Membraneswerewashedand thenincubatedwithprimaryantibodyovernightat4◦C.5-LO (AK-7, 1551,affinity purified) and -actin antibodies wereused as 1:1000dilution.Aftertheincubation,themembraneswerewashed sixtimeswith0.1%PBS-Tween andwere incubatedfor1.5hat roomtemperaturewithhorseradishperoxidase-conjugated anti-rabbitand anti-mousesecondary antibodiesdiluted1:10,000 in 0.1%PBS-Tweencontaining5%non-fatdrymilk.Themembranes werewashedandproteinbandsweredetectedbyanenhanced chemiluminescencesystem.
Statisticalanalysis
Theresultsareexpressedasmean±S.E.M.ofthemean ofn observations,wherenrepresentsthenumberofanimalsor num-berofexperimentsperformedindifferentdays.Theresultswere analyzedbyStudentttest.Ap-valuelessthan0.05wasconsidered significant.
Results
Zymosan-inducedperitonitisinmaleandfemalemice Sexdifferencesinvascularpermeabilityandcellularinfluxinto peritoneum
Increasedvascularpermeabilityisoneoftheprimarystepsin initiating inflammationleading toplasma leakage and swelling [18].Intraperitonealinjectionofzymosaninducedanincreaseof vascularpermeabilitythatreacheditsmaximumafter60min,with asimilartime-courseinmaleandfemalemice(Fig.1A).However, significantsexdifferences wereobservedin theamountofdye extravasation,andhighervascularpermeabilitywasobservedin femalevsmalemice,particularlyafter30and60min(p=0.077at 15min;p<0.05at30and60min;Fig.1A).
Fig.1.Sex-dependentdifferencesinvascularpermeability(A)peritonealcellinflux (B) andMPO activity (C)in zymosan-inducedperitonitisin mice.Micewere treatedwithzymosan(i.p.)andsacrificedattheindicatedtimepoints.Timezero (T0)correspondedtountreatedmice.Valuesrepresentmeans±S.E.M.;(A)n=8 mice;(B)n=10mice/T0;15min;n=13mice/30min;n=13–15mice/60min;n=8 mice/120min;180minforcellinflux;n=16mice/240min;(C)n=12mice/240min.◦ p<0.05;◦◦p<0.01;◦◦◦p<0.001femalevsfemaleT0;*p<0.05;**p<0.01;***p<0.001
malevsmaleT0;#p<0.05;##p<0.01femalevsmale.
Theextravasatedplasmacontainsfirst-linechemotacticfactors (e.g.,LTs)that mediatesubsequentneutrophilinflux,leading to theiraccumulationintheinflamedtissue.Infact,zymosaninduced aslightcellularinfluxofneutrophilsintomouseperitoneumthat wassignificant120minafterzymosaninjection(p<0.05formale andfemalemice)andwassubstantialat240mininbothgenders (p<0.001formale andfemalemice)(Fig.1B).Interestingly,the numberofperitonealcellswassignificantlyhigherinfemalethan inmalemiceat240min(p<0.05),whichwasaccompaniedbya
Fig.2. Sex-dependentdifferencesinLTbiosynthesisinzymosan-induced peri-tonitisinmice.(A,B)Miceweretreatedwithzymosan(i.p.)andsacrificedatthe selectedtimepoints.Timezero(T0)correspondedtountreatedmice.Values repre-sentmeans±S.E.M.;n=8mice/T0;n=14–15mice/15min;n=11–14mice/30min; n=13–14mice/60min;n=8mice/120min,180minand 240min.◦◦ p<0.01;◦◦◦
p<0.001femalevsfemaleT0;*p<0.05;**p<0.01;***p<0.001malevsmaleT0;
#p<0.05femalevsmaleattime60min;##p<0.01femaletime15and30vsmale
time15and30.
higherMPOactivity(aneutrophilmarker)infemalemice(Fig.1C; p<0.01).
SexdifferencesinLTproduction
ZymosaninjectionresultedinamarkedproductionofLTsin theperitonealexudatesof maleand femalemice.In particular, the amountof LTC4, themain 5-LO metabolite in this experi-mentalmodel [18,19],rapidlyincreasedwithin15–30minafter zymosaninjection,andthensharplydecreasedat60min(Fig.2A). Thoughsimilarkineticswereobservedinbothgenders,LTC4 lev-elsweresignificantlyhigherinfemalemice(15–30min,p<0.01; 60minp<0.05),resultinginanabsolutedifferenceofabout2–3 fold(Fig.2A).LTB4levelsweremuchlowerascomparedtoLTC4and showedabiphasictrend,butalsoasignificantsex-dependent dif-ference.Infact,aslightbutsignificantearlyincreasewasobserved inbothgenders(p<0.001),withapeakat15minafter peritoni-tisinductioninmaleandat30mininfemale,wherelevelswere significantlyhigherthaninmales(p<0.01).Nosexdifferencewas observedinthelateLTB4increase(120–180min)(Fig.2B). EffectsoforchidectomyonLTproduction
TodeterminetherelativeimpactofsexhormonesonLT produc-tioninzymosan-inducedperitonitis,gonadsfromadultmalemice weresurgically removed.Thisproceduresignificantlydecreased
Table1
Plasmatestosteronelevels(ngmL−1).
Male 2.00±0.7
ORCH 0.050±0.006*
Female 0.055±0.008*
Valuesrepresentmean±S.E.M.;n=9–10mice/group.
*p<0.05vsmalesham.
testosteroneplasmalevelsinORCHmicethatweresimilarto lev-elsinfemalemice(Table1).Comparedwithshammalemice,which havehighertestosteronelevelsinrespecttoORCHmice, gonadec-tomycausedasignificant(p<0.05)increaseofLTC4(Fig.3A)and LTB4(Fig.3B)productionat15and30minafterzymosan injec-tion,respectively.ItisimportanttounderlinethattheLTlevelsin ORCHmiceweresimilartoLTlevelsinfemalemice(Fig.3AandB), implyinganegativeroleoftestosteroneonLTproduction. Residentperitonealmacrophagesfrommaleandfemalemice SexdifferencesinLTproduction
IthasbeenreportedthatLTC4isthemain5-LO-derivedLT syn-thesizedbyresidentPMinzymosan-inducedperitonitis[18,19]. Thus,weanalyzedwhethertheobservedsexdifferencesinLTC4 productioninvivocouldbetracedbacktoisolatedPMinvitro.To thisaimresidentPMwerecollectedfromadultmaleandfemale miceat4◦Ctominimizecellularmodification(accordingto[11]) and stimulated invitro in suspension with zymosan (13 parti-clescell−1 correspondingtoinvivocondition; 30min,37◦C)or ionophoreA23187(2.5M;15min,37◦C).BiosynthesisofLTC4in PMfromfemalemice(isolatedandtreatedside-by-sidewithmale PM)stimulatedwithzymosan(p<0.01;Fig.4A)orwithA23187 (p<0.05;Fig.4B)wassignificantlyhigherthaninPMfrommale mice,whichisinlinewiththedataobtainedintheinvivomodel.
Fig.3. EffectsoforchidectomyonLTbiosynthesisinzymosan-induced peritoni-tisinmice.ORCHandsham-operatedmiceweresacrificedat15(LTC4,A)and
30min(LTB4, B)after zymosan(i.p.)treatment, respectively.Valuesrepresent
Fig.4.Sex-dependentdifferencesinLTC4biosynthesisinmouseperitonealmacrophages.LTC4formationinresidentPMinducedby(A)zymosan(13particlescell−1;30min,
37◦C)or(B)A23187(2.5M;15min,37◦C).Dataareexpressedasmeans±S.E.M.fromn=2(zymosan)orn=4(A23187)independentexperimentsperformedintriplicates, each.#p<0.05;##p<0.01stimulatedPMfromfemalevsstimulatedPMfrommalemice.(C)5-LOproteinexpressioninresidentPMwasanalyzedbyimmunoblottingof
totalcelllysates.Resultsarerepresentativeofthreeindependentexperiments.(D,E)Subcellularlocalizationof5-LOinresidentPMfromfemaleandmalemiceinducedby zymosan(13particlescell−1;30min,37◦C)orA23187(2.5M;15min,37◦C)analyzedbyimmunodetectionof5-LOinthenuclear(Nuc)andnon-nuclear(Non-N)fractions
ofmild-detergent(0.1%NonidetP-40)-lysedcells.Dataarerepresentativeof2–4independentexperiments.
Next, we analyzed if the difference in the LTC4 production betweenactivatedfemaleandmalePMwasaccompaniedby differ-ent5-LOproteinlevelsorsubcellularlocalizationand/orobserved alsoforotherAAmetabolitesrelevantinPM(e.g.,PGE2). Interest-ingly,while theabsolute5-LO proteinlevelswerenotdifferent betweengenders(Fig.4C),adifferencein5-LOsubcellular local-ization was observed (Fig. 4D and E).In fact, 5-LO was found mainlyinthenon-nuclearfractionofrestingcellsfromfemalemice whereasinPMfrommalemice(isolatedandtreatedside-by-side withfemalePM)5-LOwasdetectedinboththenon-nuclearand inthenuclearfractionofrestingcells,indicatingasex-dependent localizationofthe5-LOenzyme.WhenPMwerestimulatedwith A23187(2.5M;15min,37◦C,Fig.4D)orzymosan(13particles cell−1;30min,37◦C, Fig.4E),translocationof cytoplasmic5-LO tothenuclearfractionwasobservedinbothgenders(Fig.4Dand E).
Importantly, the biosynthesis of cyclooxygenase-derived AA metabolite PGE2 did not vary between female and male after zymosan(13particlescell−1;30min,37◦C)(Fig.5A)orA23187 (2.5M;15min,37◦C)(Fig.5B)stimulation,alsosuggestingthat thesex-differenceinLTsynthesiswasspecificforLTs.
EffectsofsexhormonesonLTproduction
ToassesstheroleoforchidectomyonLTproductioninPMfrom adultmalemice,wefinallyanalyzedtheLTC4 levelsinPM iso-latedfromORCHmice.ComparedwithPMisolatedfromshammale mice,ORCHcausedasignificant(p<0.05)increaseofLTC4 biosyn-thesisafterstimulationofcellswithA23187(2.5M;15min,37◦C) (Fig.6).Interestingly,theinvitrotreatmentofPMwithtestosterone (100nM;30min,37◦C),5␣-dihydrotestosterone(10nM;30min, 37◦C),or17-estradiol(100nM;30min,37◦C)didnotaffectLTC4 generationinPMfrommalemice(datanotshown)suggestingthat theLTsexdifferenceisseeminglycausedbytheinvivoactionsof malereproductivesteroidhormones.
Discussion
Inflammatorydisordersareoftencharacterizedbysex-related differences,butthereisonlyalimitedknowledgeonthe mech-anisms and pathwaysinvolved. Here, we demonstrate that the biosynthesis of the pro-inflammatory LTs is sex-biased invivo, by the useof a well-establishedmodel of acute inflammation, that is, zymosan-induced peritonitisin mice [18–20]. Thus, we
Fig.5.PGE2biosynthesisinperitonealmacrophagesfrommaleandfemalemice.PGE2formationinresidentPMinducedby(A)zymosan(13particlescell−1;30min,37◦C)
or(B)A23187(2.5M;15min,37◦C).Dataareexpressedasmeans±S.E.M.fromn=2(zymosan)n=4(A23187)independentexperimentsperformedintriplicates,each.
showedthatthelevelsofLTC4andLTB4weresignificantlyhigher in the inflamed peritoneum of female than male mice, which wasfollowedand/oraccompaniedbysexdifferencesinrelevant inflammationparameters.Infact,highervascularpermeabilityand neutrophilextravasationwere observed infemales. Finally, the analysisof isolatedPMallowedtheidentificationofsex-related differencesinLTformationatthecellularlevel,accompaniedby adifferentialsubcellularlocalizationof5-LO,whichwasin agree-mentwithpreviousfindingsinvitroinhumancells[11].Taking together,our data suggest LTs and their production as critical factorsforsex-relateddifferencesininflammation,withinvivo rel-evanceindeterminingtheamplitudeoftheacuteresponse and possiblesignificanceininflammatorydiseases.
Increasedvascular permeability is one of theprimary steps involvedininitiatingtheinflammatoryprocessandis a prereq-uisitefortheleakageofplasmaintotheinflamedtissue.Thei.p. injectionofzymosanin miceinducedaninflammatory reaction characterizedbyearlyvascularpermeabilityandsteadyincrease ofproteinleakageintotheperitoneuminthefirst60min. Interest-ingly,theseeventsweremorerobustinfemalethaninmalemice, implying a more pronounced inflammatory response in female animals.Vascularpermeabilityrepresents,however,ahighly regu-latedmultistepprocessmediatedbyinflammatorymediators,and cys-LTsareparticularlyrecognizedaspotentvascular-regulating agentsthroughtheinteractionwiththecysLT1receptor[21,22].
Fig. 6.Effects of orchidectomy on LTC4 production in stimulated-peritoneal
macrophagesfrommalemice.LTC4formationinresidentPMfromorchidectomized
(ORCH)andsham-operatedmiceinducedbyA23187(2.5M;15min,37◦C).Data areexpressedasmeans±S.E.M.fromn=3independentexperimentsperformedin triplicates,each.*p<0.05,stimulatedPMfromORCHmicevsstimulatedPMfrom shammalemice.
WeobservedthatLTC4levelsrapidlyraisedafterzymosan injec-tion(peakingat15–30min)andweresignificantlyhigherinfemale mice,suggestingthatthesexdifferencesinvascularpermeability couldbedue,atleastinpart,todifferentialLTbiosynthesis.
Previousstudiesshowedthatthedominantperitoneal leuko-cytepopulationintheearlyphaseoftheinflammatoryresponse inCD1miceareresidentmacrophages (about95%,[20]), which areknowntomainlysynthesizeLTC4as5-LOproducts[18,19].In fact,ithasbeenreportedthatmacrophagedepletioncauseda pro-founddecreaseofLTC4 productionandvascular permeabilityin zymosan-inducedperitonitis[18].Itisimportanttounderlinethat residentperitonealcellsconsistofamixtureofcelltypesincluding macrophages,granulocytesandlymphocytes,buttheproportionof thesecelltypescanvary.Infact,thenumberandtypeofresident peritonealcellsdependsonthemousestrainutilized.Forexample, inoutbredCD1miceresidentcellsarepredominatelymacrophages (95%[20]),whileininbredC57BL/6micetheperitonealcellsare macrophages,Tlymphocytesand Blymphocytes[16],reflecting aprobabledifferentinflammatorymediatorproductionand sub-sequentchemotaxis,asobservedinthedifferenttime-courseof cellinfiltrationinCD1[20,presentdata]andC57BL/6mice[16]. Thoughsmallsexdifferenceshavebeenreportedinthenumber ofresidentleukocytes(macrophages,TlymphocytesandB lym-phocytes)inC57BL/6mice[16],weneitherobservedasignificant sexbiasintheamountofperitonealcellsinuntreatedCD-1mice norwithinthefirst180minafterzymosaninjection.Inparticular, underourexperimentalconditionsandaccordingtothefindings byDohertyandcolleagues[20],zymosaninjectioninducedaslight peritoneumcellularinfluxofneutrophilsuntil180mininCD1mice, aphenomenonthatwasverypronouncedinC57BL/6mice[16].In contrasttoCD1mice,intheinbredstrainthepeakofcell infiltra-tionoccurredat180minwithhighernumberofcellsinmalemice withrespecttofemalemice.Instead,cellnumbersaswellasMPO activityintheCD1mouseperitoneum(whichisanindexof neu-trophilinfiltration)weresignificantlyhigherinfemalethaninmale miceafter240min,suggestingthatchemotaxisand/ormigrationof neutrophilswassuperiorinfemalevsmaleanimalschallengedwith zymosan.
Interestingly, the extravasation and accumulation of neu-trophilsappeared asa delayedevent ascompared totherapid proteinleakage and mightbeattributable toslowmigration of neutrophilsfromthevascularsystem[20,23,24],leadingto pre-dominanceofneutrophilsintheperitoneumonlyat240minafter zymosaninjection,asobservedbyothersbefore[20].Incontrast tothesubstantialamountofrapidlysynthesizedLTC4,the con-comitantlevelsofLTB4wereabout500-foldlower(<0.3ngmL−1) and,thoughincreasedinabiphasicmannerwithadelayedpeak
(120–180min),werestillminute(<3ngmL−1)ascomparedtoLTC4 atalatertimepoint.NotethatneutrophilsproducemuchmoreLTB4 thanLTC4[11].Conclusively,wesuggestthattheLTC4peakinthe earlyphase(15and30min)isproducedbyperitoneumresident macrophageswhilethedelayedpeak(120–180min)inLTB4 syn-thesisissupportedbyaccumulatingneutrophils.Becausethelevels ofchemotacticLTB4infemaleanimalsweretwiceashighasinmale micethemoresustainedneutrophilinfluxinfemaleanimalsmay beexplainable.
AndrogenswerepreviouslyshowntosuppressLTbiosynthesis inhumanmonocytes[12]andneutrophils[11]leadingtolower LT-syntheticcapacitiesofcellsfrommales.Androgen-dependent suppressionofLTbiosynthesisduringzymosan-induced peritoni-tisisreflectedbyincreasedlevelsofLTC4andLTB4foundinmale miceafter orchidectomy which aresimilar tofemalemice and whichwe suggesttoberelatedtothelackof5-LO-suppressive androgens. It is important to underline that LT levels as well as testosterone levels were similar in male ORCH and female mice.
Asmentionedabove,residentPMareseeminglyresponsiblefor LTproductioninzymosan-inducedperitonitis.Interestingly,asex differenceinLTC4productioninPM,activatedwithdifferentstimuli suchaszymosanandA23187,wasobservedalsoinvitro, suppor-tingthatdifferentialregulationof5-LOproductsynthesis inthe cellmayaccountfortheinvivobiasofLTlevels.Interestingly, pro-tein5-LOlevelswerenotdifferentbetweenmalesandfemales,but sex-differenceswereobservedfor5-LOsubcellularlocalizationin linewithourpreviousobservationinvitroinhumanneutrophils [11].Inparticular,differencesinbasal5-LOlocalization(i.e.,higher levelsofnuclear5-LOinunstimulatedPMfrommales)resultedin areducedtrafficable5-LOproteininstimulatedmalePM, corre-latingtolower5-LOproductsynthesisvsfemalecells.HigherLTC4 syntheticcapacityofPMfrommaleORCHmicewasobserved, sug-gestingaprevalentroleofandrogensasresponsiblefactorscausing sexdifferencesinLTbiosynthesisinPM,accordingtoourprevious findingsinneutrophils[11]andmonocytes[12].Ontheotherhand, theinvitrotreatmentofPMwithtestosteronefailedtomodulateLT biosynthesis,whichcouldberelatedtocharacteristicsacquiredin thisspecificcelltypeduringcelldifferentiation.Notethatthe lev-elsofthecyclooxygenase-derivedPGE2wereinsteadequalinPM frommaleandfemalemice,indicatingselectivebiasonthe5-LO pathwaywithineicosanoidbiosynthesis.
Conclusions
Inconclusionourdatarevealsignificantsexdifferencesinthe biosynthesisofLTsinmousezymosan-inducedperitonitis,which wereassociatedwithvariantinflammatoryresponses.Onthe cel-lularlevel,differential5-LOsubcellularcompartmentalizationin PMfrommaleandfemalemicemightbethebasisforthese differ-ences.Notably,tissue-residentmacrophagesrepresentthefirstline ofdefenseagainstinvadingpathogensandactivateimmunological andinflammatory responses[25].Therefore,thedatapresented hereaddsignificantinsightsintotheunderstandingofbasicsex differences in mounting inflammatory responses, withpossible therapeuticimplicationsforasex-tailoredtreatmentofrelated dis-orders.
Acknowledgements
A.R.,C.P.,S.P.,O.W.andL.Sdesignedresearch;A.R.,C.P.andS.P. performedresearch;A.R.,C.P.,S.P.,O.R.,O.W.andL.S.analyzeddata; andA.R.,C.P.andL.S.wrotethepaper.
AppendixA. Supplementarydata
Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2014.05.011. References
[1]Peters-GoldenM,Henderson WR,Leukotrienes. N Engl JMed 2007;357: 1841–54.
[2]FunkCD,ChenXS,KurreU,GriffisG.Leukotriene-deficientmicegeneratedby targeteddisruptionofthe5-lipoxygenasegene.AdvProstaglandin Thrombox-aneLeukotRes1995;23:145–50.
[3]FunkCD.Prostaglandinsandleukotrienes:advancesineicosanoidbiology. Sci-ence2001;294:1871–5.
[4]RådmarkO,WerzO,SteinhilberD,SamuelssonB.5-Lipoxygenase:regulation ofexpressionandenzymeactivity.TrendsBiochemSci2007;32:332–4.
[5]Peters-GoldenM,BrockTG.Intracellularcompartmentalizationofleukotriene biosynthesis.AmJRespirCritCareMed2000;161:S36–40.
[6]WerzO,Klemm J,Samuelsson B,Rådmark O.Phorbolesterup-regulates capacitiesfornucleartranslocationandphosphorylationof5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes. Blood 2001;97:2487–95.
[7]WerzO,BürkertE,FischerL,SzellasD,DishartD,SamuelssonB,etal. Extra-cellularsignal-regulatedkinasesphosphorylate5-lipoxygenaseandstimulate 5-lipoxygenaseproductformationinleukocytes.FASEBJ2002;16:1441–3.
[8]WhitacreCC.Sexdifferencesinautoimmunedisease.NatMed2001;2:777–80.
[9]OsmanM.Therapeuticimplicationsofsexdifferencesinasthmaandatopy. ArchDisChild2003;88:587–90.
[10]BoumanA,HeinemanMJ,FaasMM.Sexhormonesandtheimmuneresponse inhumans.HumReprodUpdate2005;11:411–23.
[11]PergolaC,DodtG,RossiA,NeunhoefferE,LawrenzB,NorthoffH,etal. ERK-mediatedregulationofleukotrienebiosynthesisbyandrogens:amolecular basisforgenderdifferencesininflammationandasthma.ProcNatlAcadSci USA2008;105:19881–6.
[12]PergolaC,RoggeA,DodtG,NorthoffH,WeinigelC,BarzD,etal.Testosterone suppressesphospholipaseD,causingsexdifferencesinleukotriene biosynthe-sisinhumanmonocytes.FASEBJ2011;25:337787.
[13]PoeckelD, ZemskiBerry KA, Murphy RC, FunkCD. Dual 12/15- and 5-lipoxygenasedeficiencyinmacrophagesaltersarachidonicacidmetabolism andattenuatesperitonitisandatherosclerosisinApoEknock-outmice.JBiol Chem2009;284:21077–89.
[14]HaribabuB,VergheseMW,SteeberDA,SellarsDD,BockCB,SnydermanR. TargeteddisruptionoftheleukotrieneB4receptorinmicerevealsitsrolein
inflammationandplatelet-activatingfactor-inducedanaphylaxis.JExpMed 2000;192:433–8.
[15]RettewJA,Huet-HudsonYM,MarriottI.Testosteronereducesmacrophage expressioninthemouseoftoll-likereceptor4,atriggerforinflammationand innateimmunity.BiolReprod2008;78:432–7.
[16]ScotlandRS,StablesMJ,MadalliS,WatsonP,GilroyDW.Sexdifferencesin residentimmunecellphenotypeunderliemoreefficientacuteinflammatory responsesinfemalemice.Blood2011;118:5918–27.
[17]BradleyPP,PriebatDA,ChristensenRD,RothsteinG.Measurementof cuta-neousinflammation:estimationofneutrophilcontentwithanenzymemarker. JInvestDermatol1982;78:206–9.
[18]KolaczkowskaE,ShahzidiS,SeljelidR,vanRooijenN,PlytyczB.Earlyvascular permeabilityinmurineexperimentalperitonitisisco-mediatedbyresident peritonealmacrophagesandmastcells:crucialinvolvementof macrophage-derivedcysteinyl-leukotrienes.Inflammation2002;26:61–71.
[19]Rao TS, Currie JL, Shaffer AF, Isakson PC. In vivo characterization of zymosan-inducedmouse peritoneal inflammation.J Pharmacol Exp Ther 1994;269:917–25.
[20]DohertyNS,PoubelleP,BorgeatP,BeaverTH, WestrichGL,Schrader NL. Intraperitonealinjectionofzymosaninmiceinducespain,inflammationand thesynthesisofpeptidoleukotrienes andprostaglandinE2.Prostaglandins
1985;30:769–89.
[21]GriswoldDE, WebbEF,HillegassLM.Inductionofplasmaexudationand inflammatorycellinfiltrationbyleukotrieneC4andleukotrieneB4inmouse
peritonitis.Inflammation1991;15:251–8.
[22]MaekawaA,AustenKF,KanaokaY.Targetedgenedisruptionrevealstheroleof cysteinylleukotriene1receptorintheenhancedvascularpermeabilityofmice undergoingacuteinflammatoryresponses.JBiolChem2002;277:20820–4.
[23]Vinegar R, Truax JF, Selph JL. Some quantitative temporal characteris-tics of carrageenin-induced pleurisy in the rat. Proc Soc Exp Biol Med 1973;143:711–4.
[24]YamashitaT,IshibashiY,NagaokaI,KasuyaK,MasudaK,WarabiH,etal. Studiesofglycogen-inducedinflammationofmice.Dynamicsofinflammatory responsesandinfluenceofantiinflammatorydrugsandproteaseinhibitors. Inflammation1982;6:87–101.
[25]TaylorPR,Martinez-Pomares L,StaceyM,LinHH, BrownGD, Gordon S. Macrophagereceptorsandimmunerecognition.AnnuRevImmunol2005;23: 901–44.
