Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory

Jean-Marie Zahm

Description

Niveau: Supérieur, Doctorat, Bac+8
Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory Epithelium Jean-Marie Zahm,* Herve Kaplan, Anne-Laure Herard, Fabrice Doriot, Denis Pierrot, Pascal Somelette, and Edith Puchelle INSERM U314, IFR53, CHU Maison Blanche, Reims, France The respiratory epithelium is frequently injured by inhaled toxic agents or by micro-organisms. The epithelial wound repair represents a crucial process by which surface respiratory cells maintain the epithelial barrier integrity. The repair process involves both cell migration and proliferation, but as yet, the kinetic of these two mechanisms has not been extensively studied. Using an in vitro model of human respiratory epithelium wound repair, proliferative cell immunofluorescent staining and a computer-assisted technique allowing the tracking of living cells, we studied the cell proliferation and migration during the wound repair process. Respiratory epithelial cells were dissociated from human nasal polyps and cultured on a collagen I matrix. At confluency, a chemical wound was made on the culture. We observed that the cell mitotic activity peaked at 48 h after wounding (23% of the cells) and mainly concerned the cells located 160 to 400 µm from the wound edge. The migration speed was highest (35 to 45 µm/h) for the spreading cells at the wound edge and progressively decreased for the cells more and more distant from the wound edge. The temporal analysis of the cell migration speed during the wound repair showed that it was almost constant during the first 3 days of the repair mechanism and thereafter dropped down until the wound closure was completed (after 4 days)

• respiratory epithelium


• immediately after


• epithelial cells


• methods based


• epithelial cell


• wound repair


• culture medium

Sujets

• Hérard
• Doriot
• Shimizu
• Pierrot
• Kaplan
• Institut national de la santé et de la recherche médicale
• Sun Microsystems
• Respiratory epithelium
• Epithelium

Informations

• Publié par: mijec
• Nombre de lectures: 20
• Langue: English

Extrait

CellMotilityandtheCytoskeleton37:33±43(1997)

CellVitMriogrWatoiounndanRdePpraoirlifoefrtahtieonReDsupriirnagtotrhyeIn
Epithelium

Jean-MarieZahm,*HerveÂKaplan,Anne-LaureHÂerard,FabriceDoriot,
DenisPierrot,PascalSomelette,andEdithPuchelle
INSERMU314,IFR53,CHUMaisonBlanche,Reims,France
Therespiratoryepitheliumisfrequentlyinjuredbyinhaledtoxicagentsorby
micro-organisms.Theepithelialwoundrepairrepresentsacrucialprocessby
whichsurfacerespiratorycellsmaintaintheepithelialbarrierintegrity.Therepair
processinvolvesbothcellmigrationandproliferation,butasyet,thekineticof
thesetwomechanismshasnotbeenextensivelystudied.Usinganinvitromodelof
humanrespiratoryepitheliumwoundrepair,proliferativecellimmuno¯uorescent
stainingandacomputer-assistedtechniqueallowingthetrackingoflivingcells,we
studiedthecellproliferationandmigrationduringthewoundrepairprocess.
Respiratoryepithelialcellsweredissociatedfromhumannasalpolypsandcultured
onacollagenImatrix.Atcon¯uency,achemicalwoundwasmadeontheculture.
Weobservedthatthecellmitoticactivitypeakedat48hafterwounding(23%of
thecells)andmainlyconcernedthecellslocated160to400mfromthewound
edge.Themigrationspeedwashighest(35to45m/h)forthespreadingcellsatthe
woundedgeandprogressivelydecreasedforthecellsmoreandmoredistantfrom
thewoundedge.Thetemporalanalysisofthecellmigrationspeedduringthe
woundrepairshowedthatitwasalmostconstantduringthe®rst3daysoftherepair
mechanismandthereafterdroppeddownuntilthewoundclosurewascompleted
(after4days).Wealsoobservedthatovera1-hourperiod,theintra-individualand
interindividualvariationofthecellmigrationspeedwas43%and37%,respec-
tively.Theseresultsdemonstratethatcellproliferationandcellmigrationduring
respiratoryepithelialwoundrepairaredifferentlyexpressedwithregardtothe
celllocationwithintherepairingarea.CellMotil.Cytoskeleton37:33±43,
1997.
r
1997Wiley-Liss,Inc.

Keywords:airwaywound;migratingcells;proliferativecells;spreadingcells;imaginganalysis;cell
tracking

INTRODUCTION
capacityoftherespiratoryepitheliumtorepairfollowing
injury.Mostofthesemodelshavebeendevelopedinvivo
Therespiratoryepitheliumisfrequentlyinjuredbyindifferentanimalspeciesandhaveallowedthededuc-
inhaledtoxicagentsormicro-organisms,leadingtothetionthatdenudationoftheairwayepithelialsurfaceleads
alterationoftheepitheliumbarrierintegrity.Whateverthe
sourceofinjury,theresponseoftherespiratorysurface
Contractgrantsponsors:AssociationFrancËaisedeLutteContrela
epitheliumtoanacuteinjurycanbecharacterizedbya
Mucoviscidose(AFLM),PoÃleTechnologiqueRÂegionalGBM,IN-
successionofcellulareventsvaryingfromthelossof
SERM(ContratNormaliseÂd'EtudePilote),MinistÁeredel'Enseignement
surfaceepithelialimpermeabilitytothedesquamationof
SupeÂrieuretdelaRecherche.
cellsfromtheepithelium.Theepithelialwoundrepair
representsacrucialprocessbywhichtheremaining
*Correspondenceto:Jean-MarieZahm,INSERMU314,CHUMaison
surfacerespiratorycellsrestoretheepithelialbarrier
Blanche,45,rueCognacq-Jay,51092ReimsCedex,France.
integrity.Numerousmodelshavebeenusedtoexplorethe
Received19July1996;accepted18November1996.
r
1997Wiley-Liss,Inc.

34Zahmetal.

tothesurroundingepithelialcellmigrationandprolifera-inRPMI1640culturemedium(Gibco,GrandIsland,
tion[GordonandLane,1976;Keenanetal.,1982;NY).Thenasaltissuewasthendissociatedbyenzymatic
McDowelletal.,1979;Nikulaetal.,1988;Shimizuetal.,digestion(pronase0.1%)for12h.Thedissociated
1994].However,thekineticofthesetwomajorprocessessurfaceepitheliumwasremovedfromthetissuebygentle
duringthewoundrepairhasnotbeenextensivelystudied,agitationandenzymaticdigestionwasstoppedbyadding
duetothedifficultyintroducedbyinvivomodels.In10%calfserum(Seromed,Biochrom,Berlin,Germany).
addition,inalltheseanimalmodelsofinjuryandrepair,Thecellularpelletcollectedaftercentrifugationat150
3
thein¯ammatorystimulusengagescellularfactorsandgfor10minwasconstitutedbyisolatedmucouscells,
serum-derivedmoleculesintotheprocessofrepair.ciliatedorbasalcells,smallclumpsofreaggregatedcells
Toovercomethecomplexassociationoffactorsinandsmallepithelialsheets.Thesecellswereresuspended
theinvivomodels,culturemodelsofrespiratoryepithe-inaserum-freemediumandwereplatedonatypeI
lialcellsallowmoreclearlytheidenti®cationofthecollagengelmatrixpreparedfromrattailtendonsaccord-
cellularandmolecularalterationsassociatedwiththeingtothetechniquedevelopedinourlaboratoryand
woundrepairprocesses[Zahmetal.,1991,1993].AttheearlierdescribedbyChevillardetal.[1993].Thecells
molecularlevel,alterationsincytoskeletalproteinpat-wereculturedinanhumidi®edatmospherecontaining
terns,bindingofgrowthfactorsandinteractionswith
extracellularmatrixproteinsandmetalloproteinases95%airand5%CO
2
inRPMI1640mediumsupple-
throughspeci®ccellsreceptors(integrins),cangovernmentedwith1g/mlinsulin(Sigma,L'IsleD'Abeau,
thecellmigrationintherepairingarea.The¯attenedcellsFrance),1g/mltransferrin(Sigma),10ng/mlepidermal
whichmigrateintothewoundedsitechangetheirpheno-growthfactor(Serva,Heidelberg,Germany),0.5g/ml
type,becomingpoorlydifferentiatedcellsexpressinghydrocortisone(Sigma),10ng/mlretinoicacid(Sigma),
keratin14andvimentinintermediate®laments[Shimizu100U/mlpenicillin,and100g/mlstreptomycin.Theuse
etal.,1994;Buissonetal.,1996a].ThecellspreadingandoftypeIcollagengelandgrowthfactor-supplemented
migrationwhichoccurduringwoundrepairdependonculturemediumisthemostsuitableconditiontoachieve
cell-matrixinteractionsmediatedby®bronectinandonedifferentiatedculturesofhumannasalepithelialcells
ofitscellularreceptors,the
a
5
b
1integrin[HeÂrardetal.,[Chevillardetal.,1993].
1996].Recentresultshavealsodocumentedtheincrease
andlocalizationofgelatinolyticmetalloproteinasesasso-
InVitroEpithelialWounding
ciatedwiththemigratoryprocessofepithelialcellsAfter3daysinculture,whenthecellshadreached
duringtherespiratoryepitheliumrepair[Buissonetal.,con¯uency,theculturemediumwasremovedfromthe
1996b].However,uptonow,thespeci®croleofcellculturedish.A2-ldropofNaOH0.1Mwasdepositedin
migrationandcellproliferationinthewoundrepair
processoftherespiratoryepitheliumhasnotbeenexten-thecenteroftheculturedishandimmediatelydilutedin1
sivelydocumented.mlofculturemedium.ThecellsincontactwiththeNaOH
Inthepresentstudy,weusedaninvitrowoundingdropdesquamatedfromthecollagenImatrix,creatinga
modelofdissociatedrespiratoryepithelialcellsculturedcircularwoundofabout30mm
2
inarea.Thewounded
fromhumannasalpolypstoevaluatethedynamicsoftheculturewasthenrinsedwith1mlofculturemediumand
cellmigrationandproliferationduringwoundrepair.Weincubatedinairwith5%CO
2
at37°C.
alsoreportedinthepresentwork,anovelmethod,using
imageanalysis,toprovidedataonthemigrationkinetics
WoundAreaDetermination
ofeitherindividualcellsoracellpopulationduringtheEvery12hduringthewoundrepairprocess,the
woundrepairprocess.Weobservedthatthecellmitoticwoundedculturedishwasplacedonthestageofan
activitypeakedat48hafterwounding.Thetemporalinvertedmicroscope(NikonTMS-F)equippedwitha
3
1
analysisofthecellmigrationspeedshowedthat,despiteobjectiveandavideoCCDcamera(Cohu4700).The
largeintra-individualandinterindividualvariations,thevideoimageofthewoundwasdisplayedonavideo
overallmigrationspeedofthecellpopulationatthe
woundedgeremainedfairlyconstantduringthe®rst3monitorandthewoundedgewasdrawnmanually.From
daysofrepairanddecreasedprogressivelyuptothethisdrawing,thewoundareawasdeterminedandex-
woundclosure.pressedasafunctionoftime.
CellProliferation
MATERIALSANDMETHODS
Forcellproliferationquantitation,woundedcul-
RespiratoryEpithelialCellCulture
tureswerecryo®xedevery24hthroughoutthewound
Nasalpolypswereobtainedfrompatientsundergo-repairprocess.Thecentralareaoftheculture(about200
ingnasalpolypectomyandwereimmediatelyimmersedmm
2
inarea)inwhichthewoundwasperformed,wascut

CellProliferationandMigrationinWoundRepair35

outfromtheculturedish,embeddedinOCTcompound,recordedfor2severy5min,the¯uorescentnucleusof
immersedinliquidnitrogenfor5minandthenkeptatcellsinthesamemicroscopic®eldatthewoundedge.
2
80°C.Thicksections(5m)perpendiculartotheFromthesevideorecordings,weusedanimageanalysis
woundedculturewereobtainedusingaReichertCryocut,techniquethatwedevelopedtoanalyzethemigrationof
slicedonaglassslideanddehydratedinair.Forindividualcellsformingcontinuoussheets.Inoneexperi-
immunostaining,thesectionswereimmersedinmethanolment,usingphase-contrastillumination,wecontinuously
at
2
20°Cfor5minandrinsedin0.1Mphosphate-recordedthecellsatthewoundedgefora10-minperiod
bufferedsaline(PBS).Eachsectionwasthenincubatedinordertovisualizethecellspreading.
for1hwithamousemonoclonalantibody(MIB-1,
Immunotech,France)whichrecognizestheKi67nuclear
CellTrackingandCellMigrationMeasurement
antigenandwhichwasdiluted1/50in0.1MPBSand
thereafterincubatedwithabiotin-conjugategoatanti-Theimagesweredigitizedasa766
3
574pixels
mouseIgM,diluted1:25in0.1MPBSfor1h.Thecellsand24-bitarrayfromthevideorecordings,usinga
werethenstainedwithstreptavidin-FITCfor30min.TheSparc-ClassicworkstationequippedwithaXVideocard
sectionsweremountedinglycerol-PBS-1,4diazabicyclo(ParallaxGraphics,SantaClara,CA).Beforetreatment,
2-2octaneandobservedunderaZeissAxiophotmicro-theimageswerereducedto512
3
512pixelsand8-bit
scopeequippedwithepi¯