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Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

By Vincent Porter,2014-07-01 03:15
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Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

    Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in

    Early Pregnancy

Reproduction&Contraception(2000)11(1~2):33~39

    ;ModulationoftheCultureSupernatantofDecidualCells

    ;withExogenousCytokinesonKillingActivityofNatural

    ;KillerCellsinEarlyPregnancy

    ;HUDongmei(胡冬梅)WANGLili(王丽莉)HEYuanli(何援利)

    ;ObstetricsandGynecologyDepartment,ZhujiangHospital,FirstMilitaryMedical ;College,Guangzhou,510282,China

    ;ObjectiveToinvestigatetheimportantfunctionofcytokinesinearlypregnancyand ;toprovidebasicandexperimentalevidenceforunderstandingthemechanismoftheir ;action.

    ;MethodsAddinterferon7(FN7),interleukin2(L2),interleukin6(L6)and

    ;epidermalgrowthfactor(EGF)totheconfluentculturingdecidualcellswiththree ;differentconcentrationsandharvesttheculturesupernatantafter12.24and48h ;separately.ObservetheeffectofthesupernatantonkillingactivityofNKcellswith ;radioimmunologicalassayofCrimmersion.

    ;Results7Pculturesupernatantofdecidualcellscanpromotethekillingactivityof ;NKcellsinvariousdegrees,andtheeffectisindependentofthetype,concentration ;andactingtimeofcytokines.

    ;ConclusionInnormalpregnancy,decidualcytokinenetworkisinadynamicequilibri- ;um.Exogenouscytokineswouldbeharmtonormalpregnancybyinterferingtheequi- ;libriumstate,buttheexactmechanismneedsfurtherstudy.

    ;Keyword:Cytokine,Decidualcell,Naturalkillercell

    ;Duringtherecentyears,studieshavedemonstratedanimportantroleofthema

    ;ternaldeciduainpregnancy[1].Thoughdeciduacloselyadjoinsthefetalantigen.it ;wouldnotreactagainstfetus.Ascytokinesareconsideredastheintercellularsignal

    ;inglanguagefacilitatingcommunicationamongcells,thecytokinesreleasedbydecid- ;ualcellsplaymodulatingrolesinimmunologicalmicroenviromentofdecidua,oreven ;Correspondingauthor:HUDongmeiTel:08602085143691

    ;33

    ;

    ;determinestheoutcomeofpregnancyE.

    ;Owingtotheimportantbiologicalsignificanceofcytokinesinpregnancy,some

    ;scholarsconsideredtheapplicationofcytokinesforclinicalpurposeasthemethodof ;treatinginfertility,abortion,prematurebirthorcontraception,eta1..Alotoffun

    ;damentalworkhasbeendoneinthisfield.However,whatwillbetheeffectsofthe ;exogenouscytokinesonpregnancy?Woulditfeasibleandeffectiveforapplicationof ;theexogenouscytokinesinclinic?Wewillinvestigatethisproblembymeansofthe ;radioimmunologicalassayinvitro.

    ;Materialsandmethods

    ;Materials

    ;Humandecidualtissueswereobtainedfromhealthywomenwhounderwentarti

    ;ficialabortionwithin7;9weeksofpregnancyinHaidianHospitalofBeijing.Human ;peripheralvenousbloodwasprovidedbyCentralBloodStationofBeijing.K562cells ;wereofferedbyInstituteofTumor,AcademyofMedicine,Beijing.Humanrecombi

    ;nantcytokine——IFN7,IL2,IL6andEGFaretheproductsofBoehringer

    ;MannhenCompanay,German.CrwasobtainedfromAmershamCompany ;,Britain.

    ;Lymphoprepswerefrommedicalmaterialsupplyingstation

    ;,AcademyofMilitarv

    ;Medicine.

    ;Methods

    ;Decidualcells(DC)culture

    ;DecidualtissueswerewashedinHank’Ssolutionandmincedwithscissorsinto

    ;piecesassmallaspossible.IncubatedecidualtissuesinRPMI1640containing0.

    ;19,6

    ;collagenaseinatubeat37~Cwithagitation.Afterthetubestayedverticallyfor30 ;min’theupperdispersedDCwasharvested.CulturetheDCinRPMI;1640supple

    ;mentedwith10fetalcalfserumat37~C,5CO2tillconfluence.Nonadherent

    ;lymphocyteswereremovedandtheplasticadherentdecidualcellswereresusPended

    ;withfreshRPMI1640containingcytokinesrespectivelywiththreeconcentrations ;.

    ;Continueincubatingfor12,24and48hrespectively,andtheculturesupernatantsof ;decidua1cells(CSDC)wereharvested.

    ;ThetypesandconcentrationsofcytokinesaddedtOthefreshRPMI;1640wereas

    ;follows:IFN7:100,10and1U/ml;EGF:100,10and1ng/ml;IL2:100,10and

    ;1U/ml;andIL6:10,1and0.1U/m1.Theneachkindofcytokineisaddedinthree ;concentrations,withthreeharvestingtimes,andasaresultnineexperimentalgroups ;(EG)areinal1.

    ;Separationofhumanperipheralbloodlymphocytes(hPBL)

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    ;

    ;

    ;

    ;killingratioofhumanperipheralbloodNKcellstoK562cellswas38.14,26.69, ;13.08,1.13and1.13respectively.Thegroup1:25T/Ewaschosenasthe ;blankcontrolgroup.

;ThekillingratioofNKcellsstimulatedbyCSDCwithIFNy

    ;Intheconditionedcontrolgroup,astheconcentrationofIFN7was100,10and

    ;1U/ml,theratiokiUingratioofNKceilswascountedas12.749/6,16.089,6and ;18.51%respectively.Ascomparedwiththecorrespondingblankcontrolgroup,the ;killingratiooftheconditionedgroupincreased.Thefurtherincreaseofkillingratio ;ofNKcellswasobservedwhentheCSDCwasaddedtotheexperimentalgroup.The ;maximalincreasedkillingratiowas71.51(Table1).

    ;Table1ThekillingratioofNKcellsstimulatedbyCSDCwithIFN-Y ;KR:killingratio;IKR:increasedkillingratio;CCG:conditionedcontrolgroup ;ThekillingratioofNKcellsstimulatedbyCSDCwithEGF

    ;WhentheconcentrationofEGFwas100,10and1ng/ml,thekillingratiointhe ;conditionedcontrolgroupwascountedas13.220A,12.81and14.28.Comparing ;withtheconditionedcontrolgroup,thekillingactivityofNKcellsinthecorre

    ;spondingexperimentalgroupincreasedfurther,themaximalincreasedkillingratio ;being33.88(Table2).

    ;Table2ThekillingratioofNKcellsstimulatedbyCSDCwithEGF

    ;ThekillingratioofNKcellsstimulatedbyCSDCwithIL-2

    ;IL2isakindofimmunologicalactivitypromotingcytokine.WhentheIL2with

    ;theconcentration100,10and1U/mlwasaddedtothecultureNKcells,thekilling ;36

    ;

    ;activity0fNKcellswasincreasedto20.34%,16.23and15?29%repectivelya ;comDaredtothe13.O8killingactivityoftheblankcontrolgroup?Whentheam ;concentrationsoftheIL2wereaddedtothemediumofDC,theCSDCfurtheren

    ;hancedthekillingactivity.fNKcells,asc.mparedt.thec.nditi.nedc.ntrolgroup? ;Itsmaxima1increasedkillingratiowas33.3O(Table3)?

    ;ThekillingratioofNKcellsstimulatedbyCSDCwithIL6

    ;WhentheconcentrationoftheIL6inthemediumwas10,1and0?1U/ml,the

    ;killingactivityofNKcellsintheconditionedcontrolgroupwas13?98%,14?20% ;and15.64%respectively.ThoughthekillingactivityofNKcellsintheexperimenal ;groupwasfurtherincreasedascomparedtotheconditionedcontrolgrouP,itsn

    ;creased1eve1wasthelowestamongthesefourcytokines,thevalueofthelOWestone ;beingon1y0.50%,whichhadnosignificanceatal1.Themaximalonewas17?98% ;(Table4).

    ;Discussion

    ;Cytokinenetworkindecidua

    ;Cytokinesareabundantindecidualtissues.NearlyeverYkindofcytokinecanbe ;foundindecidua.Cytokinesmodulatetheintensityandefficacyofdecidualimmunity ;throughautocrineandparacrinemodeofaction.TheYinduceeachother,modulate ;37

    ;

    ;eachother’sreceptorexpressionandinfluence

    ;lishingthespecialcytokinenetworkin

    ;eachother’sbiologicalactivity,estab—

;immunologicalmicroenvironmentof

    ;decidua[.Thoughmutualinteraction,coordinationandcommunication,thosedecid

    ;ualcytokinesmakethenetworkmaintaindynamicequilibriuminnormalpregnancy, ;anychangeofasinglecytokinemightinducesuccessivereactionslikecataract,de

    ;stroytheequilibriumstareofdecidualcytokinenetwork,andeventuallywouldbe ;harmfultopregnancy.

    ;NKcellsindecidua

    ;TheNKcellindeciduaiscalledlargegranularlymphocyte(LGL),whichmakes ;up70oftotalwhitecellsindecidua.ItsphenotypeisCD56’CD16,CD2and

    ;CD3.ThefunctionofLGLisstillunclear.Itmightberelatedtocontrollingthein

    ;vasionoftrophoblastandidentifyingthefetalantigen.Innormalpregnancy,decidua ;mightreleasesomeimmuneinhibitortoinhibitthekillingactivityofNKceilstoits ;targetceilssuchasK562cellsandtrophoblast.Onceactivated,theNKceilsindecidua ;wouldreactivatethemacrophagesandtheotherinflammatoryceilsindecidua,in

    ;ducingthemtoreleasepoisonoussubstance,whichbringsdangertopregnancy ;Inthisstudy,exogenouscytokinespromotetheactionofdeciduaonthekilling ;activityofNKcellstoK562cells,implyingtheexogenouscytokinesactivatethere- ;leasingofinflammatorycytokinesindecidua,thuspromotingthecellmediatedim

    ;munity,akindofharmfulimmunitytopregnancy.

    ;Theimmunologicalfeatureofdeciduainnormalpregnancy

    ;Theimmunologicalfeatureofdeciduainnormalpregnancyistheenhancedhu

    ;motmediatedimmunityandthereducedcellmediatedimmunity.Thedecidualim

    ;munologicalmicroenviromentshowsthesamefeature,too,manifestingthatinthe ;releasingofcytokinethereleasingofThl(Thelpercells)cytokinesthatmediatethe ;cellimmunityisreduced.andthereleaseofTh2cytokinesthatmediatethehumoral ;immunityisenhanced.ThereleaseandfunctionoftheTh2cytokinessurpassaswell ;asinhibitthereleasingandfunctionofThlcytokine[.Thisisbecausethefetalanti

    ;bodytomajorhistocompatibilitycomplex(MHC)canbeabsorbedandremovedby ;theplacenta,soitwouldnotbeharmfultopregnancy.Nevertheless,theplacenta ;lackstheabilitytodefendagainstthecytotoxicityofcell—mediatedimmunity’

    ;ThoughIFN7,IL2,IL6andEGFbeingthedifferentcytokines,withthe

    ;completelydifferentfunctions,theydohavethesimilarmodulatingfunctionsofthe ;decidualimmunity.Forinstance,IFN7andIL2areThlcytokineswhichcanpro

    ;motethecytotoxicityoflymphocyteandtheactivityofNKceilsaswellasinhibitthe ;immunologicalactivityinducedbyTh2cytokines.IL-6istheTh2cytokine,andcan ;enhancetheactivityofantibodyproductingBceils.EGFplaysanimportantrolein ;occurringanddevelopingoftumors,andthemechanismmightberelatedtoitsimmu

    ;38

    ;

    ;nityinhibitingfunction.Inthosefourcytokines,somewereregardedasthepregnan- ;cyprotectingfactorsandsomeasthedestroyingones.However,whenthosefourcy

    ;tokineswereaddedtotheculturingdecidualcells,alltheculturesupernatantsshow ;thesamefunctionbypromotingthekillingactivityofNKcells.Howdidtheywork ;wasunclear.Theymightbreaktheequilibriumstateofdeciduacytokinenetworkby

;increasingthereleaseofinflammatorycytokinesandactivatingthecellmediatedim

    ;munityindecidua.Ifthisunbalancedstatecannotbemodulatedtonormalbyitself ;ormaternalendocrinesystem,thepregnancywillbedamaged.

    ;Thisfindingsuggeststhattheexogenouscytokinesmightdestroytheequilibri

    ;umstateofdeciduacytokinenetworkandthreatenthepregnancy,andthefunction ;hasnocorrelationtothetypeandactingtimeofcytokines.Thisimpliesthatthe ;complexityofcytokinemustbeconsideredwhencytokinesareappliedtoensuringits ;efficiacyandsafetyinclinica1use.

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