英文版 重症医学会

Associationsofplasmaneutrophil-derivedmicroparticleswiththeprognosisofpatientswithsepsisorsepticshock(DepartmentofCriticalCareMedicine,AffiliatedHospitalofGuangdongMedicalCollege,524001)LijunYe1*LiangZhang1*Jinlelin2HuaguoYao1LiehuaDeng1&&CorrespondingauthorWenwuZhang2&&Correspondingauthor1DepartmentofCriticalCareMedicine,AffiliatedHospitalofGuangdongMedicalCollege,Zhanjiang,Guangdong.2DepartmentofCriticalCareMedicine,People’shospitalofinBaoDistrict,Shenzhen,Guangdong.大EqualcontributorsAbstractObjective:Neutrophil-derivedmicroparticlesaresmallplasmamembranevesiclesreleasingfromstilling,activationorapoptosisneutrophils,andplayanimportantroleinregulatinginflammatoryresponse.Inthisstudy,weinvestigatedtheassociationsofplasmaneutrophil-derivedmicroparticleswiththeprognosisofpatientswithsepsisorsepticshock.Methods:Weenrolled111ICUpatients:48withsepsis,63withsepticshock.PlasmaNDMPs,TNF-a,IL-6,andsTREM-1levelsweremeasuredonICUday1,3,5,7.Atthesametimepoints,werecordedAPACHEIIscore,MODSscore,PCT.Mechanicalventilationtime,ICUhospitalizationtimeandtotalhospitalizationtimewerecounted.19healthyindividualswereenrolledactingascontrolgroup.APvalue<0.05wasconsideredsignificant.Results:NDMPswerepresentedintheplasmaofbothhealthyindividualsandpatientswithsepsisorsepticshock,andplasmaAnxA1andCD62Linsepsisandsepticshocksignificantlyexceededthoseofhealthyindividuals(P<0.05).Basically,comparedwithsepsisgroup,plasmaNDMPslevelsweresignificantlyhigherinsepticshockgroup(P<0.05).Inthecomparisonbetweensurvivalgroupandno-survivalgroup,theanalysisresultsshowedinno-survivalgroup,plasmaAnxA1wassignificantlyhigheratanytimepoint(P<0.05),plasmaCD11bweresignificantlyhigherat7d(P<0.05),plasmaCD62Lweremarkedlyhigherat3dand7d(P<0.05).Conclusions:ThestudysuggestedthatplasmaNDMPs,especiallyAnxA1andCD62Lweresignificantlyassociatedwithprognosisbypromotingthereleasingofinflammatorymediatorsinpatientswithsepsisorsepticshock.IntroductionNeutrophil-derivedmicroparticles(NDMPs)aresmallplasmamembranevesiclesreleasingfromstilling,activationorapoptosisneutrophils,whichcontaincellsurfaceproteinsandcytoplasmicmatter,withbiologicalactivities.Therearedifferentproteinmarkersonthesurface,includingthespecificproteinmarkerscellsurfaceAnnexin1(AnxA1),L-selectin(CD62L),CD11bandCD66b,andintracellularproteinsmyeloperoxidase,elastase,andotherproteolyticenzymes.Inrecentyears,microparticles(MPs)becomeafocusofattentioninresearches.AstoNDMPs,thestudiesinvitroarestillconstantlydevelopment.ResearchersobtainedNDMPsbysettingupanimalmodels,separatinghealthandthepatient'sbodyneutrophils,andanalyzedthemtoshowthebiologicalactivitiesofthesevesicles:NDMPscontributingtoanti-/pro-inflammatoryresponses[1-4],promotingthrombosis,maintainingvascularbarrierfunctionandpreventingvascularleakage[3,5,6],damagingvascularendothelialcellsandtissues[7],antibacterialeffect[8].ArareclinicalresearchbycollectingabdominalwashingsandbronchoalveolarlavagefluidfromsitesofinflammationofsepsispatientssuggestedNDMPsmayplayanimportantroleinregulatingtheinflammatoryresponsetosepsisinpatientswithcriticalillness[9],whichmaybesuggestedthatNDMPswerenotonlyinvolvedinthedevelopmentofsepsis,butalsoassociatedwiththeprognosisofit.Thisstudyfirstlycollectedtheplasmaofpatientswithsepsisandsepticshock,anddetectedplasmaNDMPs,forthesakeofassessingwhethertheNDMPscanactasbiomarkersorevensuccessfulmarkersintheprognosisassessmentofpatientswithsepsisorsepticshock.OwingtoCD62L,CD11bandAnxA1arespecialproteinmarkersofNDMPs,weonlyanalyzethethreeproteins.MaterialsandMethodsMaterialsAnti-Annexinantibody(cloneab33061),secondaryantibody(goatanti-rabbitlgGH&L,cloneab150077)andisotypecontrol(cloneab172730)arepurchasedfromabcomtradingcompanyltd.Anti-HumanCD11b(cloneCBRM1/5),anti-HumanCD62L(cloneDREG-56)andisotypecontrol(clone.1)arepurchasedfromeBioscienceAffymetrixcompany.HumanTumornecrosisfactoralpha(TNF-a)EnzymeLinkedImmunosorbentAssay(ELISA)KitandhumanInterleukin-6(IL-6)ELISAKitarepurchasedfromNeoBioscience.HumanSoubleserumtriggeringrceptorexpressedonmyeloidcells-1(sTREM-1)ELISAKitispurchasedfromRayBiotech,Inc.Allthesereagentsarestoredinthe4°Crefrigeratorfortheexperimentusing.PatientsofstudyThestudywasconductedin111patientswithsepsisindepartmentofIntensiveCareUnit(ICU),affiliatedhospitalofGuangdongMedicalCollege,ZhanjiangGuangdongandthePeople’shospitalofBao’anDistrict,ShenzhenGuangdong,betweenJanuaryandNovember2014.Thepatientsweredividedinto2groups:48patientsdiagnosedwithsepsissettingassepsisgroup,63patientsdiagnosedwithseveresepsisandsepticshocksettingassepticshockgroup.Taking19healthysubjects,fromPhysicalexaminationcenterofaffiliatedhospitalofGuangdongMedicalCollege,ZhanjiangGuangdong,setascontrolgroup.Thediagnosisandclassificationofsepsisreferredtointernationalguidelinesforseveresepsisandsepticshock2012[17].Allentrantsweresignedinformedconsentbeforeexperiment,andthisstudywasreviewedandapprovedbytheEthicsCommitteeoftheAffiliatedHospitalofGuangdongMedicalCollege(Zhanjing,China)andPeople’shospitalofBao’anDistrict(Shenzhen,China).CasesofexclusioncriteriaPatienthistorieswithmalignanttumor,orpatientsintheimmunosuppressionstateorpoisoning,werecountout.Methods2.3.1Neutrophil-derivedmicroparticlespreparationandFlowcytometryforsurfacestainingIntotal,5mlofheparinizedbloodwereobtainedfromeachpatientandhealthydonor.Thebloodsampleswereinitiallycentrifugedat1000gfor10minutes,theplasmasupernatantswerecollectedandcentrifugedat9900g,4°Cfor10minutes[9]priortofurtherultracentrifugationat15000g,4Cfor1hour[3].Themicroparticlespelletswerethensuspendedinthelastsupernatantandstoredat-80Cuntiluse.Afterthaw,theMPswereinitiallystainedwiththefollowingmAbat10pg/mL:anti-AnxA1(cloneab33061),anti-CD62L(cloneDREG-56),anti-CD11b(cloneCBRM1/5),orisotypecontrol(cloneab172730)andcloneP.1)for30minutesat37Cwithgentleshaking,afinalstainingwithsecondaryantibody(goatanti-rabbitlgGH&L)for30minutesat37Cwasconducted.TheimmunelabeledMPswerefinallyresuspendedin500p!sheathfluid.Fluorescence-activatedcellsorting(FACS)analysiswasconductedusingFACScaliburflowcytometer(BectonDickinson,SanJose,CA)usingCellQuestsoftware(BectonDickinson)[1].ELISAAssaysThelevelsofsTREM-1,TNF-aandIL-6intheplasmafromsepsisgroupandsepticshockgroupat1,3,5,7dandcontrolgroupweredeterminedusingenzyme-linkedimmunosorbentassaysaccordingtothemanufacturer'sprotocol(RayBiotech,Inc,NeoBioscience,ChinaandNeoBioscience,China,respectively).DatacollectionUponadmissionintotheICUs,thefollowingitemswererecordedforeachpatient:sourceofpatient,age,gender,primarydisease,medicalhistory,anddiagnosis.Within(firstdayofstudy)afterICUadmissionandinthemorningofdays3,5and7,recordedAcutePhysiologyandChronicHealthEvaluation(APACHE)IIscore,MultipleOrganDysfunctionSyndrome(MODS)scoreandassayresultsofProcalcitonin(PCT).Countedupthemechanicalventilationtime,ICUhospitalizationtimeandtotalhospitalizationtimeandrecordedthefinaloutcome(survivalorno-survival)afterthepatientwasdischarge.StatisticalanalysisSPSS15.0softwarewasusedforstatisticalanalysis.Allvalueswerepresentedasmeans土standarddeviation.Significantdifferencesamongthreegroupsweredeterminedbyone-wayanalysisofvariancewithsubsequentStudent-Neman-Keulstest.StatisticalanalysiswasperformedusingGraphPadPrism5.0(GraphPadSoftware,SanDiego,California,USA).APvalueoflessthan0.05wasconsideredstatisticallysignificant.Results3.1.1NDMPsAnxAl,CD11bandCD62Lweredetectedintheplasmaofsepsis,septicshockandcontrolgroupsbyFACSOnFACSanalysisoftheplasmaNDMPs,weusedasizegating(Figure1)varyingapproximatelybetween0.1and2pmtoidentifyMPs.TheresultsshowedthatallNDMPsweredetectedintheplasmaofsepsis,septicshockandcontrolgroups(Figure2).ThedistributioncharacteristicofAnxA1,CD11bandCD62Linsepsis,septicshockandcontrolgroupComparedwithcontrolgroup,AnxA1andCD62Linsepsisorsepticshockgroupweresignificantlyhigher(P<0.05).AstoCD11b,therewasnomarkedlydifferencebetweensepsisgroupandcontrolgroup(P>0.05),buttheCD11binsepticshockgroupwassignificantlyhigherthanthatinsepsisgroupandcontrolgroup(P<0.05).(showninTable1)ComparisonofvariousexperimentalvariablesbetweensepsisandsepticshockpatientsThelevelsofCD11bandCD62Linsepticshockgroupweresignificantlyhigherthanthoseinsepsisgroupat1,3,5,7dandat5,7d,respectively(allP<0.05).Comparedwithsepsisgroup,plasmaTNF-aandsTREM-1weremarkedlyhigherat3,5,7dinsepticshockgroup(P<0.05).ThelevelsofPCTandAPACHEIIscoreinsepticshockgroupweresignificantlyhigherthanthoseinsepsisgroupatanytimepoints(P<0.05).Themechanicalventilationtimeofsepticshockgroupwassignificantlylongerthanthatofsepsisgroup(P<0.05).However,therewerenodifferenceofMODSscore,ICUhospitalizationtimeandtotalhospitalizationtimebetweenthetwogroups(P<0.05).(showninTable2)DynamicchangesofAnnxin1,CD11bandCD62LwithinsepsisorsepticshockpatientsInsepsisgroup,accordingtoanalyzingthedynamicchangeoftheplasmaconcentrationofAnxA1,CD11bandCD62Latdifferenttimepoints,theresultswereshowedasfollows:i)thediscrepantlevelsofAnxA1betweenthe1dand3d,the5dand7dwerestatisticallysignificant(Figure3.1A,P<0.05).ii)ThelevelsofCD11bat3dand7dweresignificantlyhigherthanthatof1d,comparedwiththatof3d,thelevelsofCD11bat7dwerealsosignificantlyhigher(Figure3.1B,P<0.05).iii)ThelevelsofCD62Lat3,5and7dwereallmarkedlyhigherthanthatof1d,thedifferencebetweenthe3dand7dwasalsosignificant(Figure3.1C,P<0.05).Astosepticshockgroup,seemlywhetherthelevelsofCD11borCD62Lpresentedatrendofincreasingfrom1dto7d,butnotalloftheincreasingwerestatisticallysignificant,concreteanalysiswasasfollows:i)plasmaAnxA1insepticgrouppresentedthesamesignificantchangetendencywiththatinsepsisgroup.ii)At3dand5d,thelevelsofCD11bweremarkedlyhigherthanthatof1d,andfrom3dto7dtheincreasedlevelswerealsosignificant(Figure3.2E,P<0.05).iii)ThelevelsofCD62Lat3dweremarkedlyhigherthanthatof1d,andtheincreasedtrendfrom3dto7dwerealsostatisticallysignificant(Figure3.2F,P<0.05).Comparisonofvariousexperimentalvariablesinsurvivalandno-survivalpatientsAccordingtothefinaloutcomeofpatients,wedividedpatientsintotwogroups:survivalandno-survivalgroup.Comparedwithsurvivalgroup,plasmaAnxA1inno-survivalgroupwereallsignificantlyhigherat1,3,5,7d(P=0.002,P=0.000,P=0.010andP=0.000,respectively),plasmaCD11bweresignificantlyhigherat7d(P=0.000),plasmaCD62Lweremarkedlyhigherat3dand7d(P=0.028andP=0.000,respectively).TheplasmaTNF-aIL-6inno-survivalgroupweremarkedlyhigherat1,3,5,7d,howeverplasmasTREM-1inno-survivalgroupwassignificantlyhigherat1dand3d(P=0.006andP=0.033,respectively).Inno-survivalgroup,themechanicalventilationtimeandtotalhospitalizationtimeweresignificantlylongerthanthoseinsurvivalgroup(P=0.000andP=0.000,respectively).(showninTable3)DiscussionPreviousstudieshaveshowedthattheNDMPsarereleasedbyneutrophilsinvitro[1-2,5-8,10-15,18-19]andinvivo[3,9,20],inlocalandsystemicinflammation,andtheyareinvolvedininflammationanddamage.Prakashetal.havefoundNDMPsbycollectingbronchoalveolarlavagefluidandabdominaldrainagefluidintheinflamedfociofpatientswithsepsisorinfection,andconcludedNDMPswiththeroleofmodulatingtheimmuneresponsetosepsis[9].Inpresentstudy,wehaveobservedthatthethreeNDMPsAnxA1,CD11bandCD62Lnotonlygenerateintheplasmaofsepsisandsepticshockpatients,butalsodoinhealthyindividuals,what’more,thelevelsofAnxA1andCD62Lweresignificantlyincreasedinsepsisandsepticpatients.Thus,itissuggestedthatthehigh-levelofAnxA1andCD62Lsymbolizethepresenceofinflammation,evenmaybeanidealbiomarkerforearlysepsisdiagnosis.Further,ourdatahavedemonstratedthatinpatientswithsepticshock,theplasmaNDMPsareespeciallyhigherthanthoseofsepsispatients,whichsuggestthatthemoresevereofthedisease,theplasmalevesofNDMPsarehigher.IntermsofplasmaCD11b,itslevelshowedsignificantlyhigheratalltimepoints,suggestingthatitmayplayanimportantroleinthedevelopmentofsepsisandsepticshock.ThereisevidencethatAnxA1carriesanti-inflammatoryandpro-inflammatoryproperties[1],whilethelevelsofAnxA1hadnomarkedlydifferencebetweensepsisgroupandsepticshockgroup,whichmaydemonstratethatplasmaAnxA1isnotincreasingalongwithaggravatingofinflammation.Duringtheearlyperiodofthedisease,plasmaCD62Linsepticshockpatientswasnotsignificantlyhigherthanthatinsepsispatients,butgraduallypresentedmarkedlyhigheralongwiththeprogressofthedisease.ByobservingthedynamicchangesofAnxAl,CD11bandCD62Lwithinsepsisorsepticshockpatients,wehavediscoveredthatplasmaAnxA1hasnoobviouschangetrendinthetwogroups,whilebothplasmaCD11bandCD62Lpresentedincreasingtrend,whichindicatingthatAnxA1mayactasrelativelystationaryNDMPs,however,CD11bandCD62LfunctionimportantlythroughtheprocessofsepsisorsepticshockactingasrelativelyactiveNDMPs,astothefunctionofthetwoNDMPsareanti-orpro-propertiesneedtofurtherstudy.Atthelastofthestudy,bycomparingsurvivalgroupandno-survivalgroup,wehaveobservedthattherearesignificantlydifferenceinplasmalevelsofAnxA1andCD62L,suggestingthattheyarecloselyassociatedwithprognosis.Furthermore,inno-survivalgroup,mechanicalventilationtimeandtotalhospitalizationtimearealsolongerthanthatinsurvivalgroup,whichisaccordwiththepreviousstudies.Therefore,plasmaNDMPs,especiallyAnxA1andCD62L,mayprovidenewreferenceindexforprognosisassessmentofsepsis.ConclusionInsummary,ourpresentstudydemonstratedforthefirsttimethattheassociationbetweentheplasmaofNDMPsandtheprognosisofsepsisinvivostudy,asdescribedabove,theplasmaconcentrationofNDMPsindeedinvolvedinsepsis,closelyrelatedwiththeprognosisofsepsis,aggravatingstateofillnesstheseverityofillnessandorgandamage.CompetinginterestsTheauthorshavenocompetinginterests.AcknowledgementsThisstudywassupportedinpartbytheClinicalLaboratoryCenterofGuangdongMedicalCollegeAffiliatedHospitalandtheDepartmentCriticalCareMedicinePeople’shospitalofBao'anDistrict,ShenzhenGuangdong.ReferencesDalliJ,NorlingLV,RenshawD,CooperD,LeungKY,PerrettiM.Annexin1mediatestherapidanti-inflammatoryeffectsofneutrophil-derivedMicroparticles.BLOOD.2008;112(6):2512-9.GasserO,HessC,MiotS,DeonC,SanchezJC,SchifferliJA.Characterisationandpropertiesofectosomesreleasedbyhumanpolymorphonuclearneutrophils.ExpCellRes.2003;285(2):243-57.DanielL,FakhouriF,JolyD,MouthonL,NusbaumP,GrunfeldJP,etal.Increaseofcirculatingneutrophilandplateletmicroparticlesduringacutevasculitisandhemodialysis.KidneyInt.2006;69(8):1416-23.GasserO,SchifferliJA.Activatedpolymorphonuclearneutrophilsdisseminateanti-inflammatorymicroparticlesbyectocytosis.Blood.2004;104(8):2543-8.KihongLim,RonenSumagin,Young-MinHyun.ExtravasatingNeutrophil-derivedMicroparticlesPreserveVascularBarrierFunctioninInflamedTissue.ImmuneNetw.2013;13(3):102-6.ElzbietaPluskota,NeilM.Woody,DorotaSzpak,ChristieM.Ballantyne,DmitryA.Soloviev,DanielI.SimonandEdwardF.Plow.Expression,activation,andfunctionofintegrinaMP2(Mac-1)onneutrophil-derivedmicroparticles.Blood.2008;112(6):2327-35.ThassilaNogueiraPitanga,LucianadeAragaoFranga,VivianeCostaJunqueiraRocha,ThaynaMeirelles,ValeriaMatosBorges,MarildaSouzaGongalves,etal.Neutrophil-derivedmicroparticlesinducemyeloperoxidase-mediateddamageofvascularendothelialcells.BMCCellBiol.2014;15:21.TimarCI,LorinczAM,Csepanyi-KomiR,Valyi-NagyA,NagyG,BuzasEI,etal.Antibacterialeffectofmicrovesiclesreleasedfromhumanneutrophilicgranulocytes.Blood.2013;121(3):510-8.PrakashPS,CaldwellCC,LentschAB,PrittsTA,RobinsonBR.Humanmicroparticle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Table2.Comparisonofvariousexperimentalvariablesinsepsisandsepticshockpatients(x土s)variablestimensepsisgroupnsepticshockgroup4859.50±16.956361.63±19.690.483AnxAl(%)1d4810.23±3.71639.06±5.390.0003d4810.21土4.64639.95±5.850.8005d4410.37土7.685711.43±6.020.0787d3610.46±5.915411.08±7.700.623CDllb(%)1d481.85±1.75633.22±3.110.0003d482.58±4.01634.18±4.100.0495d443.07土4.01576.23±5.600.0007d364.08土4.37548.40±11.410.000CD62L(%)1d481.22土1.07631.69±1.410.1183d481.24土1.90632.34±2.140.1475d441.73