Characterization of a novel M-CSF induced effector macrophage [Elektronische Ressource] / vorgelegt von Sebastian Matthias Riedhammer

Description

AUS DEM LEHRSTUHL FUR INNERE MEDIZIN ABTEILUNG FUR HAMATOLOGIE UND INTERNISTISCHEONKOLOGIEPROF. DR. MED. REINHARD ANDREESENDER MEDIZINISCHEN FAKULTATDER UNIVERSITAT REGENSBURGCHARACTERIZATION OF A NOVEL M-CSF INDUCED EFFECTORMACROPHAGEInaugural-Dissertationzur Erlangung des Doktorgradesder MedizinderMedizinischen Fakult atder Universiatt Regensburgvorgelegt vonSebastian Matthias Riedhammer2010iiDekan: Prof. Dr. Bernhard Weber1. Berichterstatter: Prof. Dr. Michael Rehli2. Berich Prof. Dr. Michael MelterTag der mundlic hen Prufung: 5. Juli 2010ivfur meine ElternviContents1 Introduction 11.1 Innate and adaptive immune system . . . . . . . . . . . . . . . . 11.1.1 Innate immune system . . . . . . . . . . . . . . . . . . . . 11.1.2 Adaptive immune system . . . . . . . . . . . . . . . . . . 21.1.3 Activation of the adaptive immune system . . . . . . . . . 21.1.4 E ector functions of the adaptive immune system . . . . 31.1.5 DC and macrophage activation . . . . . . . . . . . . . . . 41.2 The macrophage: origin and di erentiation . . . . . . . . . . . . 61.2.1 Di erentiation from stem cell to monocyte . . . . . . . . . 81.2.2 Monocyte classi cation . . . . . . . . . . . . . . . . . . . 91.2.3 Experimental models of monocyte di erentiation . . . . . 111.2.4 The impact of monocyte adhesion . . . . . . . . . . . . . 121.2.5 The role of M-CSF and its receptor . . . . . . . . . . . . 142 Prework and aims 193 Material and methods 213.

Sujets

• Gesundheit

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

AUSDEMLEHRSTUHLF¨URINNEREMEDIZIN
ABTEILUNGF¨URH¨AMATOLOGIEUNDINTERNISTISCHE
OLOGIEONKPROF.DR.MED.REINHARDANDREESEN
DERMEDIZINISCHENFAKULT¨AT
DERUNIVERSIT¨ATREGENSBURG

CHARACTERIZATIONOFANOVELM-CSFINDUCED
GEOPHACRMA

Inaugural-DissertationDoktorgradesdesErlangungzurdizinMeder

deratakult¨FhenMediziniscderUniversit¨atRegensburg

SebastianvMatthiasorgelegtvRoniedhammer

2010

EFFECTOR

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Dr.

Dr.

Dr.

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Contents

onductiIntro11.1Innateandadaptiveimmunesystem................
1.1.1Innateimmunesystem....................
1.1.2Adaptiveimmunesystem..................
1.1.3Activationoftheadaptiveimmunesystem.........
1.1.4Effectorfunctionsoftheadaptiveimmunesystem....
1.1.5DCandmacrophageactivation...............
1.2Themacrophage:originanddifferentiation............
1.2.1Differentiationfromstemcelltomonocyte.........
1.2.2Monocyteclassification...................
1.2.3Experimentalmodelsofmonocytedifferentiation.....
1.2.4Theimpactofmonocyteadhesion.............
1.2.5TheroleofM-CSFanditsreceptor............

2Preworkandaims

dsmethoandMaterial33.1Basictechniques...........................
3.1.1Mediaandsolutions.....................
3.1.2Sterileprotocols.......................
3.1.3Cellcultureconditions....................

1112234689111214

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2121212121

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tstenCon

3.1.4Viablecellcountusingtrypanblue.............22
3.2Monocyteisolationthroughcounter-currentelutriation......22
3.3Monocytecultureconditions.....................23
3.3.1Classicalmonocytederivedmacrophages(MAC).....23
3.3.2Monocytederiveddendriticcells(DC)...........24
3.3.3Non-adherentlyculturedmonocytes(MMAC).......24
3.3.4AdherentlyculturedmonocyteswithadditionalM-CSF
(MAC+M-CSF).......................24
3.4Lightmicroscopyandphotographs.................24
3.5Flowcytometry............................25
3.6Shortdescriptionofthedetectedantigens.............27
3.6.1Antigensofmacrophagedifferentiation:CD14,CD16,CD71,
CD84andMAX.11......................27
3.6.2CellsurfaceantigenofDCdifferentiation:CD1a.....29
3.6.3Functionalantigenesofantigenpresentingcells:CD40,
DC-SIGN,CD80/CD86,HLA-ABCandHLA-DR....29
3.7Apoptosisassay............................30
3.8AllogenicTcellstimulationassay..................31
3.9Phagocytosisassay..........................32
3.10CytokineproductionfollowingstimulationwithLPS.......33
3.11Statistics...............................33

35Results44.1Survivalofmonocytesundernon-adherentconditions.......35
4.2Detectionofapoptoticcells.....................36
4.3Morphologyinlightmicroscopy...................39
4.4Phenotypecharacterizationwithmonoclonalantibodies.....39

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4.4.1Antigensofmacrophagedifferentiation:CD14,CD16,CD71,
CD84andMAX.11......................40
4.4.2CellsurfaceantigenofDCdifferentiation:CD1a.....41
4.4.3Functionalantigenesofantigenpresentingcells:CD40,
DC-SIGN,CD80/CD86,HLA-ABCandHLA-DR....41
4.5Phagocytosisassay..........................47
4.6ActivationofAPCwithbacterialendotoxin............48
4.7Allo-reactivTcellresponse.....................50

Discussion55.1Survival................................
5.2Differentiation............................
5.3Phagocytosis.............................
5.4Cytokineproduction.........................
5.5Tcellstimulatorycapacity.....................

Conclusion6

Zusammenfassung7

endixApp7.1Solutionsandmedia.........................
7.2Listofallcompanies.........................

Bibliography

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List

1.1

3.1

3.2

4.1

4.2

ablesTof

Mononuclearphagocytes..................

Elutriationparametersandcorrespondingcellfractions

Antibodiesusedforflowcytometry............

Survivalratesofdifferentcultureconditions.......

Distributionofvital,apoptoticanddeadcells......

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List

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List

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

Figuresof

DetectionofapoptoticcellsinFACS-Analysis...........

Morphologyinlightmicroscopy...................

Flowcytometricanalysisofdifferentiationantigens........

Expressionpatternofmacrophageantigens............

ExpressionpatternofmacrophageantigensandCD1a......

PresenceofsurfacemoleculesassociatedwithAPC........

ExpressionprofileofCD86,HLA-ABCandHLA-DR.......

Phagocytosisassay..........................

CytokineproductionuponstimulationwithLPS.........

AllogenicactivationofTcellsbyMAC,MMACandDC.....

37

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of

Figurse

ionsreviatAbb

ADCCAPCCDcDCCDPcpmCTLA-4DCDC-SIGNDNAECMAEDTELISACSAFRFcγFITCFSCGM-CSFICAMIg

Antibodydependentcellcytotoxicity
celltingpresentigenAnClusterofdifferentiation
ClassicalspleenDC
precursorDCCommonCountsperminute
CytotoxicTlymphocyte-associatedantigen4
cellDendritictegrinnon-inICAM3-grabbingecificDC-spucleinacidonxyribDesomatrixExtracellularacidylenediaminetetraaceticEthEnzyme-linkedimmunosorbentassay
Fluorescent-activatedcellsorter
ReceptorfortheFcfragmentofIgG
anatycFlouresceinisoscannerardorwFGranulocyte-monocytecolony-stimulatingfactor
moleculeadhesiontercellularInunoglobulineImm

xv

ILINFLPBLPSCMAM-CSF+CMAM-CSFMDPMFIMHCMLRCMMAMNCMPSNDNFAMPPpDCPEPIPMTPRRPSRESSSC1/2THTCR

xvi

terleukinInterferonInproteinbindingLPSherideolysaccopLipAdherentlydifferentiatedmacrophages
AdherentlydifferentiatedmacrophageswithextraM-CSF
factorulatingy-stimcolonMacrophageprecursorDCandMacrophageytensitinfluorescenceMeanMajorhistocompatibilitycomplex
reactioncytelymphoMixedmacrophagestiateddifferentlyNon-adherencellsuclearMononMononuclearphagocytesystem
teddetecNotfactorNuclearpatternmolecularciatedathogen-assoPDCPlasmacytoiderythrinycoPhdidioPropidiumPhotomultiplyertube
receptorrecognitionatternPPhosphatidylserinesystemReticulo-endothelialscannerardSidewThelpercelltype1/2
receptorcellT

TLR

TNF

ollT

elik

umorT

receptor

necrosis

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ductiIntro1on

1.1Innateandadaptiveimmunesystem

Thepurposeofthisstudywastocharacterizetheviabilityandthedifferentiation
ofhumanperipheralmonocytesculturedinsuspension.Toidentifyournovel
celltypewehavetesteditscapacitytoperformsomeoftheclassicalfunctions

ofmonocytes,macrophagesanddendriticcells(DCs).Thediversefunctionsof
monocytesanditsmoredifferentiatedformsmacrophagesandDCsareintegral

toandinterconnecttheinnateandadaptiveimmunesystems.Belowisashort
reviewoftheinnateandadaptiveimmunesystemandhowtheeffectorfunctions
ofthesecellscontributetoresolvevariousimmunologicalchallenges.

systemmuneimInnate1.1.1

Theimmunesystemisbasedontheveryancientinnateimmunesystemthat

vertebratessharewithinvertebratesandplants.Itreliesonepithelialbarriers,
microbicidalenvironmentsandcellsurfacereceptorsforconservedpathogen-
associatedmolecularpatterns(PAMPs)(Gordon,2002).Thisgerm-lineencoded
patternrecognitionreceptors(PRRs)allowaperfectself/non-selfdiscrimina-
tion,selectedoverevolutionarytime(Tayloretal.,2005).Thecellsoftheinnate
immunesystemsurveytheorganismforpathologicantigenandhavethepower
todecide,relyingontheirPRRs,whetherornottoreacttoacertainstimulus
(JanewayandMedzhitov,2002).Theinflammatoryresponseofmacrophages,

1

1ductiontroIn

DCs,polymorphonuclearleukocytes,naturalkillercells,andmastcellsresolve
theattackofpathogenicmicrobesthroughawidevarietyofmethods.But,as
microbeshavedevelopedmechanismstoevadetheinnateimmunesystem,the
meansoftheinnateimmuneresponsearenotalwayssufficienttoentirelyclear
situation.the

systemmuneimAdaptive1.1.2

Onlyvertebrateshavedevelopedanadaptiveimmunesysteminadditiontothe
innatesystem.Itconsistsoftwolymphocytesspecies,theTandtheBcells.The
diversityoftheirreceptors,Tcellreceptor(TCR)andantibodies,respectively,
istheresultofrandomreceptorgenerearrangementandsomatichypermutation
leadingtomyriadpossibleclonalTandBcells.Astherandomnatureofthis
receptorgenerationcannotexcludeself-reactingproperties,theTcellsundergo
positiveandnegativeselectioninthethymusduringontogenesis.

1.1.3Activationoftheadaptiveimmunesystem

Theadaptiveimmunesystemcanonlybeactivatedbytheinnateimmunesys-
tem.TheTCRandthemajorhistocompatibilitycomplex(MHC)molecules
linkthetwoapparentlydistinctsystems.Tcellscannotreacttoanynative
antigen;rather,theantigenhastobepresentedbyasurfaceMHCmolecule
(Unanue,2002).TherearetwoclassesofMHCmolecules.ClassImoleculesare
expressedonallnucleatedcellsandpresentfragmentsofpeptidescirculatingin
thecellcytoplasmandinteractwithclusterofdifferentiation(CD)8+Tcells.
ClassIImolecul