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

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