巨噬细胞吞噬凋亡中性粒细胞的实时动态观察及其转归的研究Word文档下载推荐.docx
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巨噬细胞吞噬凋亡中性粒细胞的实时动态观察及其转归的研究Word文档下载推荐.docx
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Abstract
Clearanceofapoptoticneutrophilsbymacrophagesisimportantforthesuccessfulresolutionofacuteinflammationandhomeostasis.Inthisstudy,thedynamicprocessofphagocytosisofapoptoticneutrophilswasmeasuredinrealtimeandspecialattentionwaspaidtothefatesofmacrophagesaftertheirphagocytosisinvitro,westagedtherecognitionandtethering,internalization,digestionandexocytosisstagesofphagocytosisofapoptoticneutrophils.Furthermorewefoundthatsomemacrophagesunderwentcelldeathaftertheiringestionofapoptoticcells.Thedeadmacrophagesconsistedofautophagy,apoptosisandoncosisasrevealedbytransmissionelectronmicroscopyandconfocalmicroscopycombinedwithspecificdyes.Theseresultsdemonstratedthatafteringestionofapoptoticneutrophils,macrophagesmightundergoautophagyandapoptosis.Autophagyofmacrophageafteringestionofapoptoticcellsmaybeoneofthenewmechanismsinimmuneresponse.Keywords:
macrophage;
celldeath;
phagocytosis;
time-lapseimaging
1.Introduction
Respiratoryinfectiousdiseaseisacommonandleadingcauseofmorbidityandmortalityworldwide[1].Theacuteresponsetoaninfectiousinsultisusuallyarapidrecruitmentandinfiltrationofneutrophils.Theneutrophilscaneffectivelybindandkillmicroorganismsthatmightcausedamagetotissues,thentheytimelyundergoavigilantlycontrolledprogrammedcelldeathknownasapoptosis[2].Efficientremovaloftheseintactapoptoticneutrophilsbymacrophagesisbelievedtobecriticalforthesuccessfulresolutionofacuteinflammationandmaintenanceoftissuehomeostasis[3,4].Voluminousstudieshavebeenfocusedontherecognitionanduptakeofapoptoticneutrophilsbymacrophages,butthedynamicprocessofphagocytosisandthefatesofmacrophagesaftertheiringestionofapoptoticneutrophilshavenotbeenwelldocumented[5].
Themurinemacrophage-likeRAW264.7cellshadbeenusedtoestablishassaysofphagocytosisofapoptoticJurkat(humanT-celllymphoma)cellsandCTLL-2cells[6,7].Togainabetterunderstandingofthemacrophage/apoptoticneutrophilinteractionsandthefatesofmacrophagesaftertheiringestion,wemeasuredthedynamicprocessofphagocytosisofhumanapoptoticneutrophilsbyRAW264.7cellsinrealtimeandspecialattentionwaspaidtothesubsequentchangesofmacrophagesaftertheiringestionofapoptoticneutrophilsincellculturecondition.
2.MaterialandMethods
2.1Neutrophilisolationandapoptosisinduction
10mlheparinizedvenousbloodwasobtainedfromhealthyvolunteersafterinformedwrittenconsentsignedbyeachvolunteerandapprovedbythelocalethicalcommittee.NeutrophilswereisolatedundersterileconditionsusingthePercolldiscontinuousdensitygradientcentrifugationaspreviouslydescribedwithsomemodification[8,9].Briefly,10mlheparinizedvenousbloodwasincubatedfor5minin50mlredbloodcelllysisbuffer(170mMNH4Cl,10mMKHCO3,0.1mMEDTA,pH7.3)toremoveredbloodcells.TheremainingcellswerelayeredonthetopofPercolldiscontinuousdensitygradientandcentrifuged(1000g,30min,roomtemperature)withoutbraking.Theisolatedneutrophils1ThisstudyissupportedbytheNatureScienceFoundationofChina(30670936)andDoctoralFundoftheMinistryofEducationofChina(20050366002).
-1-
werewashedthreetimeswithPBS;
culturedatadensityof1×
106cells/mlinDulbecco’smodifiedEagle’smedium(DMEM)(Gibco)supplementedwith10%fetalcalfserum(FCS),2mmol/Lglutamine,100IU/mlpenicillinand100mg/mlstreptomycin.Viabilityandpurityofneutrophilsisolatedusingthismethodwere97%and95%respectively.Spontaneousapoptosiswasachievedbyculturingfor20hat37°
Cinahumidifiedincubatorwith5%CO2[10].Atthistime,apoptosisassessedbyAnnexinV-FITCapoptosisdetectionkit(BDBioscience,USA)usingflowcytometrywas25~60%,whereasnecrosiswaslessthan3%.
2.2LivecellimagingofRaw264.7cellsengulfingapoptoticneutrophils
2×
104Raw264.7cellsgrewovernightat37°
Cwith5%CO2infeedingmediaconsistingofDMEMand10%FCSina35mm2dish.ThedishwasthenplacedinachamberonthestageofanOlympusIX-70invertedmicroscopeequippedwithaCCDcameracontrolledbySPOTRTsoftware(DiagnosticInstruments,Inc.,SterlingHeights,MI).Livecellimagingofphagocytosisassaysweredoneaspreviouslydescribed[10,11].About2×
103apoptoticneutrophilswereaddedtothedishandallowedthemtointeractwiththemacrophages.Fortime-lapsesequences,imageswereobtainedautomaticallyevery30secondsforaperiodof5—12h.TheseriesofimageswerecompiledintodigitalmoviesbyMetaMorphTM7.0ImageProcessingsoftware(UniversalImagingCorporation).
2.3Transmissionelectronmicroscopy
Macrophages(2×
105cells)weretransferredtoa35mmdishin1000µ
LDMEMwith10%FCSandallowedtogrowovernightbeforeco-incubationwithapoptoticneutrophils(4×
105cells)at37°
Cfor60minutes.Thenthenon-ingestedneutrophilswereremovedbyvigorouswashing.AfterthatRAW264.7cellswerefixedin2.5%glutaraldehyde,scrapedandpelletedbycentrifugation.Ultrathinsectionswerecutusinganultramicrotome(LKB-4,Sweden).Sectionsstainedwithuranylacetateandleadcitratewereobservedunderatransmissionelectronmicroscope(JEM-1230,Japan).
2.4Confocalmicroscopy
Afterinteractingwithculturedneutrophilslabeledwith7-AADfor60minutes,washingawaythenon-ingestedones,themacrophageswerestainedin0.05mMMDC/100µ
g/mlAO,100µ
g/mlAO/100µ
g/mlEBrespectivelyfor15min[12,13].ThenthecellswerewashedthreetimeswithPBSandscannedusingZeissLSM510confocalmicroscopywithin1hour.Monodansylcadaverine(MDC)isaspecificdyeforautophagosome,acridineorangeisacell-permeabledyethatintercalatesintoDNAandresultsagreencolorwhileethidiumbromideenterscellswithdisruptedmembraneintegrityandintercalatesintoRNAanddouble-strandedDNAtoappearorange.Thus,differentialuptakeandbindingofthesedyesallowustoidentifymacrophageswhichhadengulfedapoptoticneutrophilsinautophagy,apoptosisandoncosis.Ineachexperiment,atleast100macrophageswhichhadengulfed7-AADlabeledapoptoticneutrophilswerecounted,andtheproportionofmacrophageswithpositivegranulesstainedinMDC,AOandEBwasexpressedasapercentagerespectively.MacrophageswithoutinteractionwithapoptoticneutrophilsstainedinAO/MDC,AOandAO/EBwereusedascorrespondingcontrols.
2.5Statistics
Datawereexpressedasthemeans±
S.EandwereanalyzedforsignificantdifferencesbyIndependent-SamplesTTestandOne-WayANOVAwithSPSS10.0.DifferenceswereconsideredstatisticallysignificantifPvalue<
0.05.
3.Results
3.1Livecellimagingofthephagocytosisofapoptoticneutrophilsbymacrophage-2-
andmacrophagecelldeathafterphagocytosis
Intime-lapsesequences,wefoundthatmostneutrophilsappearedtointeractactivelywiththeramifiedmacrophages.Themacrophagescouldrecognizeandtethertheapoptoticneutrophils,andtheninternalizationensued.Analysisof30macrophagesin20independentexperimentsshowedthatmostmacrophagesengulfedintactapoptoticneutrophilswithinafewminutesto1hour.Asshowninthemovie(seesupplementarymaterial)andFig.1,werecordedthestepsofphagocytosisandthemacrophagecelldeathafteringestionofapoptoticneutrophils.Thestagesofphagocytosisweredistinguishable:
I:
recognitionandtethering:
theapoptoticneutrophilwasrecognizedandfinallytetheredtothemacrophage.Thisstagelastedabout5minutes(Fig.1image1~3).II:
internalization:
themacrophageinitiallyprotrudedpseudopodiatopreytheneutrophilandthetwocells’membranefused.Thislastedabout3minutes(Fig.1image4~6).III:
digestion:
theengulfedneutrophilwasprocessedwithinthemacrophage.Thisstagekeptonabout2minutes(Fig.1image7,8).IV:
exocytosis:
theneutrophilbecamesmallerandwasexcretedoutofthemacrophage.Thisstagelastedabout2minutes(Fig.1image9,10).Duringtheprocessofphagocytosis,themacrophagewashighlyactiveandunderwentdramaticstructuralchangesfromramifiedcelltoroundone.
Astimelapsed(1hourlater),themacrophagewhichingestedtheapoptoticneutrophilunderwentacharacteristicsequenceofmorphologicalchanges,beginningwithcellshrinkage,followedbymembraneblebbing,andultimatelyconcludingincellmembranerupture(Fig.1image11~15).Subsequentlytheneighboringmacrophagealsounderwentcellshrinkage,themembraneblebbingandrupture,justastheprecedingone(Fig.1mage16~20).
3.2Formofmacrophagecelldeathobservedbytransmissionelectronmicroscopy
AsshowninFig.2,cellscharacterizedbydepletionoforganelleswithintactnon-pyknoticnuclei,autophagosomeswereidentifiedasautophagy.Apoptoticcellscharacterizedbylossofcytoplasm,pyknoticnucleiassumedtheappearanceofcrescentshapeorirregularlyshapednucleialongwithmarginatedchromatinwerealsofound.Inaddition,weobservedthatsomemacrophagesexhibitedcellandorganelleswellingwhicharecharacteristicsofoncosis.So,afterinteractionwithapoptoticneutrophils,theformofmacrophagecelldeathwasamixtureofautophagy,apoptosisandoncosis.
3.3Confocalmicroscopeanalysisofmacrophagesafteringestionofapoptoticneutrophils
AsshowninFig.3,th
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