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

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Learn all about the services we offer
109 Pages
English

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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.

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

CHARACTERIZATIONOFANOVELM-CSFINDUCED
GEOPHACRMA

Inaugural-DissertationDoktorgradesdesErlangungzurdizinMeder

deratakult¨FhenMediziniscderUniversit¨atRegensburg

SebastianvMatthiasorgelegtvRoniedhammer

2010

EFFECTOR

ii

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1.Berichterstatter:

2.Berichterstatter:

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henundlic¨m

ufung:¨Pr

Prof.

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rof.P

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Dr.

Dr.

Dr.

Juli

Bernhard

haelMic

haelMic

2010

Wereb

Rehli

Melter

iv

¨fur

enmei

Eltern

vi

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

19

2121212121

vii

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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tstenCon

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

535354585959

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63

656567

69

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tstenCon

x

List

1.1

3.1

3.2

4.1

4.2

ablesTof

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

Elutriationparametersandcorrespondingcellfractions

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

Survivalratesofdifferentcultureconditions.......

Distributionofvital,apoptoticanddeadcells......

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23

25

35

36

xi

List

xii

of

ablesT

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

38

42

43

44

45

46

47

49

51

xiii

List

xiv

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

factor

xvii

xviii

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.
ClassIImoleculesarerestrictedtoprofessionalantigenpresentingcells(APCs),
includingmacrophages,DCsandBcells.TheseclassIImoleculespresentpep-
tidefragmentsofphagocytizedparticlesandinteractwithCD4+Tcells.To
activatetheadaptiveimmunesystem,apeptide-boundMHCmoleculemustbe

2

1.1Innateandadaptiveimmunesystem

identifiedbyaspecificTCR.ForTcellproliferationandtoavoidTcellanergy,
theco-stimulatorymoleculesCD80and/orCD86mustbeexpressedonthevery
samecellastheMHC-peptidecomplex.InAPCsthisco-stimulatorymolecules
areup-regulateduponstimulationfollowingPRRengagement(Tayloretal.,
2005).So,thestimulationoftheinnatesystemprecedestheactivationofthe
system.uneimmeadaptiv

1.1.4Effectorfunctionsoftheadaptiveimmunesystem

Theeffectorfunctionsoftheadaptiveimmunesystemare,withexceptionof
thecytotoxicCD8+killercells,mediatedbytheinnateimmunesystem.The
linkthereforearetheCD4+cells,alsocalledThelpercells,whicharedivided
intotwomajorgroups,thetype1(TH1)andtype2(TH2)cells.ATH1cell
canmaximizethekillingefficacyofamacrophagepresentingthesameMHC-
peptidecomplexwithwhichtheTcellhasbeenactivated.Theyalsoinduce
theproliferationofCD8+killercells.ATH2cellontheotherhandcanstim-
ulateaBcellthatrecognizedtheantigeninquestionbyitsmembranebound
antibody,inducingproliferation,antibodyclassswitchandhigherantibodypro-
duction.Thissecretedspecificantibodynowenablesenhancedphagocytosisof
antibody-opsonizedantigenandantibodydependentcellcytotoxicity(ADCC),
bothperformedbymacrophages.
Thedevelopmentofthesecellsisinfluencedinanautocrine/paracrinefashion
mediatedthroughtheimpactofnumerouscytokines,chemokinesandgrowth
factors.TheTH1cellsdevelopundertheinfluenceofinterferon-(INF)-γ,tumor
necrosisfactor-(TNF)-α,andinterleukine(IL)-2.They,themselves,produce
INF-γ,whichstimulatesDCsandmacrophagestoproduceIL-12,whichinturn
up-regulatesINF-γproductionbyTcells,resultinginapositivefeedbackloop.
TheTH2cellsdevelopundertheinfluenceofIL-4,IL-5,IL-6,IL-10andIL-13.

3

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TH2cellsproduceIL-4,whichisauto-regulatory,andIL-10,whichinhibitsINF-
γandIL-2productionbyTcellsandIL-12productionbyDCsandmacrophages.
Thesepositivefeedbackloopsensureprogressinthedirection,whetherTH1or
TH2,whichwasinitiallychosen.Thefactorsleadingtothisdecisionarenot
entirelyclear.TheydependonthephenotypeoftheactivatingAPCandthe
factorspresentwithinthemicroenvironment.

macrophageandDC1.1.5activation

Recognizingandsubsequentlybindinganantigenaresufficientstimuliforthe
immatureDCtomaturate.ThematureDCexpresseshighlevelsofMHC
classIIandcostimulatorymolecules,andmigratestothenextlymphnode.
Thisanatomicarchitectureoftheadaptiveimmunesystemoffersthegreatest
possibilityfortheAPCtomeetaspecific,na¨ıveTcell.AfteramatureDC
activatesaTcell,theTcellneedsnofurtheractivationsignals.
Formacrophageactivation,ontheotherhand,threedistinctactivationmodi
havebeendescribed:the’classical’macrophageactivation,the’alternative’
macrophageactivationandthemorerecentlyproposed’type2’macrophage
activation(Edwardsetal.,2006)

ClassicalmacrophageactivationTheclassicalactivationpathwayisthelongest
knownandbyfarbestcharacterized(AdamsandHamilton,1984;Gordon,
1998).Itisatwo-stepprocessinwhichthemacrophageisprimedpriorto
beingactivated.Forexample,themacrophageisprimedbyINF-γbeforeitis
exposedtoTNF-αorastimulusinducingTNF-α.Averypotentstimulatorof
TNF-αsecretionislipopolysaccharide(LPS),acomponentofthecellwallof
Gram-negativbacteria(MorrisonandRyan,1979).Althoughtherearemany
variantsofLPS,allofthemhaveaconstantregiontermedlipidA,whichalone

4

1.1Innateandadaptiveimmunesystem

canreproduceallthebiologicaleffectsofLPS.Thisactivationprocesstakes
threesteps:firstasecretedLPSbindingprotein(LPB)bindstoLPS.This
complexisrecognizedbymembraneboundCD14(Wrightetal.,1990),which
cannotbeactivatedbylipidAalone(Tobiasetal.,1999).BecauseCD14does
nothaveacytoplasmaticdomain,thiscomplexmustbedeliveredtoasignal
proteinoftheTolllikereceptor(TLR)familyasthelaststep.Experiments
inmicehaveshownthatdeletionofthegenesencodingforLPBorCD14does
onlyreducetheresponsivenesstoLPS.Incontrast,micewithouttheTLR4
geneshowatotallossofLPSreactivity(Poltoraketal.,1998).Thisindicates
thegreatimportanceofthisthirdstep(JanewayandMedzhitov,2002).This
classicalmacrophageactivationinducestheproductionofreactiveoxygenand
nitrogenradicals,whichenablethemacrophagetokillintracellularmicroorgan-
isms.Further,classicallyactivatedmacrophagesproduceIL-12,directingthe
TcellresponsetowardstheThelpertype1(TH1)response.

AlternativemacrophageactivationAbout17yearsago,Steinetal.(Stein
etal.,1992)describedafunctionallyandbiochemicallydistinctactivatedmac-
rophagephenotyperesultingfromstimulationwiththeTH2cytokineIL-4.This
alternativelyactivatedmacrophageischaracterizedbytheproductionofIL-
10andIL-1receptorantagonist,andlowantigenpresentingcapacitytowards
Tcells(Gordon,2003).Furthermore,productionofmatrix-associatedproteins
andpromotionoffibrogenesisbythesecellswassuggestedtobepossiblecon-
tributingfactorsinwoundhealingandtissuerepair(Albinaetal.,1990;Song
etal.,2000).Inthemousemodel,itcouldbeshownthatthedifferentiation
ofmonocytesintofibrocytesisregulatedbyCD4+Tcells(Niedermeieretal.,
2009).Thisphenotypechangeinalternativemacrophageactivationisepigenet-
icallyregulated(Ishiietal.,2009).

5

troIn1duction

Type2macrophageactivationAthirdtypeofactivatedmacrophagepopu-
lationcanbegeneratedbystimulationofmacrophageswithclassicalactivation
signalsinthepresenceofimmunoglobuline(Ig)Gcontainingimmunecomplexes
(Sutterwalaetal.,1998).TLRaswellasCD40activationafterligationof
receptorsfortheFcfragmentofIgG(FcγR)resultedinsuppressionofIL-12
productionandastronginductionofIL-10(Mosser,2003).Thisturnsthese
type2activatedmacrophagesintopotentanti-inflammatorycells(Gerberand
Mosser,2001).TheyalsostimulatetheproductionofTH2cells,whichproduce
highlevelsofIL-4(AndersonandMosser,2002).

NotactivatedmacrophagesMacrophagesveryimportantintheremovalof
apoptoticcellsbutdonotactivateTcellsthatrecognizeapoptoticbody-associ-
atedantigens(HensonandHume,2006).Ithasbeenshownthatmacrophages,
incontrasttoDCs,mediateanergyinTcells.Thisresultssuggestthat,dur-
ingnon-inflammatoryconditions,macrophageswhichphagocytosemainlyself-
peptides,mediateanergyinTcellsthatrecognizetheself-peptidespresented
onaMHCclassIImolecules,thushelpinginmaintainingperipheraltolerance
(Hovesetal.,2006).Brem-Exneretal.(Brem-Exneretal.,2008)identified
INF-γ-stimulatedmonocytederivedcellsinthemousemodelwhichhavecer-
tainTcell-supressiveeffectswithpotentialtherapeuticbenefitinthetreatment
inflammations.uneautoimmof

1.2Themacrophage:originanddifferentiation

ElieMechnikoffdescribedmorethan100yearsagoalargemononuclearphago-
cyticcellandnamedit’macrophage’(Karnovsky,1981).Afterthediscoveryof
differentphagocyticcellsinvarioustissuesofthebody,theideaofareticulo-
endothelalsystem(RES)waspostulatedin1924byAschoff(Aschoff,1924).

6

1.2Themacrophage:originanddifferentiation

BecausetheREScontainedcellsnotbelongingtothemononuclearlineage,van
Furthproposedthemononuclearphagocytesystem(MPS)(vanFurth,1982).
TheMPScomprisesmyeloidprogenitorcellsthatdifferentiateintobloodmono-
cytes,whichcirculateandextravasatetobecometissuemacrophages.Thedif-
ferentmacrophagesubtypesinvarioustissuesarelistedintable1.1.Intheclas-
sicalsense,celldivisionsinthislineageoccurinthemonoblastandpromonocyte
stagesandthepoolofmacrophagesintheperipheryisrenewedbycirculating
monocytesratherthanbylocalcelldivision.Thisparadigmhasbeenchallenged
asrecentfindingsshowatleastasmallpercentageofcellrenewalbylocalcell
divisionundersteadystateconditions(TackeandRandolph,2006)andevidence
exists,thatM-CSFcaninduceproliferationinmonocytes(Clanchyetal.,2006).
Recently,severalauthorsproposed,thatthemodeloftheMPSmighthaveout-
liveditsusefulness,astheremightbeasmanydifferentmacrophagesubtypes
asmarkersappliedfortheirdescription(Hume,2006,2008).Theyclaimthat
besidedifferentiatedDCandosteoclasts,allmacrophagescanchangeasaconse-
quenceoftheirmicroenviroment,continuoslyadaptingtheirfunctionalpattern
inresponsetotheprogressiveinflammatoryresponse(StoutandSuttles,2004;
2009).2005,al.,etStout

TheoriginoftheDCsandtheconnectionstothemonocytelineagearestillnot
entirelyclear(Ardavinetal.,2001;Leonetal.,2005;Foggetal.,2006).Thereis
evidencethatDCscanbegeneratedinvitrofromboth,myeloidandlymphoid
progenitors,butit’snotsure,whethertherearephysiologiccounterpartsforthis
invitrogeneratedsubtypes.AcommonprecursorformacrophageandDCwas
proclaimedbyFoggetal.(Foggetal.,2006).Iftherearedistinctmyeloidand
lymphoidDCsubsetsorifthereisjustacontinuumbetweentwoextremesis
stillpointofdiscussion(Humeetal.,2002;Hume,2006,2008).

7

troIn1duction

TissuewmarroBone

CellTissueBonemarrowPromonoMonoblastscytes
cytesMonoMacrophagesPeripheralbloodMonocytes
LivLungerAlvKupffereolarcellsmacrophages
cytesHistiotissueeConnectivSpleenRedPulpMacrophages
ThLymphymusnodeMacropMacrophageshages
clastsOsteoBoneSynoviumTypeACells
MacrophagestissuelymphoidciatiedMucosa-assoMacrophagestracttestinalGastroinCentralnervoussystemMicroglia
SkinHistiocytes/Langerhanscells
SerouscavitiesPleura/Peritonealmacrophages
InflammatorytissuesExudativEpitheloidecellsmacrophages
GranulomaMultinucleatedgiantcells
Table1.1:Mononuclearphagocytesindifferenttissues,
adaptedfrom(RossandAuger,2002)

1.2.1Differentiationfromstemcelltomonocyte

Theoriginofthebloodmonocyteisapluripotentstemcellinthebonemarrow,
dependentonstemcellfactor(orc-Kitligand).Thenextstepindifferentiation
isthecommonmyeloidprogenitorforerythrocytes,neutrophilesandmonocytes,
followedbythegranulocyte-monocytecolony-formingunit,whichcanstilldif-
ferentiateintoneutrophilesandmonocytes.Undertheinfluenceofgranulocyte-
monocytecolony-stimulatingfactor(GM-CSF)andmacrophageCSF(M-CSF)
themonoblastdifferentiatespastthepremonocytepointtomonocytes,thefirst
cellpopulationthatenterstheblood.

8

1.2Themacrophage:originanddifferentiation

1.2.2classificationcyteMono

Bloodmonocytesarenotahomogeneouspopulation,butshowatleasttwo
distinctsubtypes,aCD14highCD16−’inflammatory’monocyteandaCD14low
CD16+’resident’monocyte(Passlicketal.,1989;GordonandTaylor,2005).
TheminorCD16+monocytesubgroupseemstocontainan’intermediate’CD14+
CD16+CD64+monocytesubgroup(Grage-Griebenowetal.,2001b),whichhas
ahighphagocyticactivityandproduceslargeamountsofcytokinescomparable
tothe’classical’monocytes,butshowhighexpressionofCD86andHLA-DRand
astimulatoryactivityinmixedleukocytereactionscomparabletoDCs(Grage-
Griebenowetal.,2001a).Bothhumanmonocytesubtypescandifferentiatein
vitrointhepresenceofGM-CSFandIL-4intoDCs(Sanchez-Torresetal.,2001).
InamousemodeloftransendothelialtraffickingtheCD14+CD16+monocyte
subsetdifferentiatedtoDCmorelikelythantheCD14highCD16−monocytesub-
set(Randolphetal.,1998).ThetranscriptionalprofileoftheCD16+monocytes
suggestsamoreadvancedstageofdifferentiationcomparedtoCD16-monocytes
(Ancutaetal.,2009).Thephysiologicalroleofthismonocytesubtypesremains
unclear,astheperformedinvitroexperimentsdonotallowadirecttransferto
situation.vivointheTheidentificationofmousecounterpartsofthehumanmonocytesubsets
havecontributedtotheunderstandingoftheirfunctioninvivo.Geissmann
etal.(Geissmannetal.,2003)havedescribedtwomonocytesubsetswithdis-
tincthomingpotentialinthemouse.ThemouseGr-1+CCR2+CX3CR1lo
monocytesubset,correspondtothehumanCD14highCD16−monocytesubset,
andaGr-1−CCR2−CX3CR1himonocytesubset,correspondtothehuman
CD14loCD16+monocytesubset.Theyshowed,thattheGr-1+CX3CR1lo
subsetisrecruitedpreferentiallytoinflammatorysites,andthattheGr-1−CX3
CR1hisubsetservesasaprecursorofresidentmyeloidcells.Sunderk¨otteret

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al.(Sunderkotteretal.,2004)haveclaimedthatthedifferentmonocytesubsets
inmousearedevelopmentallyconnected.Theyhypothesizedthatmonocytes
leavethebonemarrowasGr-1hicellsandmatureoveranintermediateGr-1med
stagetotheGr-1lomonocytes.Theyshowedthataftercompletedepletiononly
Gr-1himonocytesrepopulatetheblood,andGr-1lomonocytesareseenonday
7first.TheyconfirmedthefindingsofGeissmannetal.(Geissmannetal.,
2003),inthatGr-1himonocytesarerecruitedpreferentiallytoinflammatory
sites,andshowedfurthermorethatduringinflammationGr-1himonocytesare
releasedintotheblood,analogoustotheleftshiftseenalsowithgranulocytes.
Ginhouxetal.(Ginhouxetal.,2006)furthershowedthatGr-1himonocytes
couldgiverisetoLangerhanscells(LCs)andmacrophagesininflamedskinin
vivo,confirmingtheinvitrofindingsthatmonocytescandifferentiateintoboth,
macrophagesandDCs,atleastunderinflammatoryconditions.Inthelungthe
twodistinctmonocytepopulationsGr-1hiandGr-1logiveraisetotwodistinct
tissueDCpopulations(Jakubzicketal.,2008).Usingthismousemodel,Liu
etal.(Liuetal.,2009)depictaprocursor-progenyrelationshipofmonocytes,
classicalspleen(c)DCsandplasmacytoid(p)DCs.Startingwithamyeloidpro-
genitorthatgivesrisetomacrophageandDCprogenitors(MDPs)whichcan
differentiatetomonocytesorcommonDCprogenitors(CDPs).ThisCDPsgen-
eratepre-cDCsandpDCs.Theypointoutthatmonocytesdonotdevelopinto
cDCsandcontributelittletolymphoid-organDCnetworkinsteadystatecon-
ditions.Theydefinethepointofdivergencebetweenmultipotentialprecursors
andpre-cDCsinthebonemarrow,fromwherethelattermigratethroughthe
bloodtolymphoidtissues,wheretheydivideandfilltheDCcompartment.For
theDCnetworkinthelaminapropriaofthegutthisfindingsleadtotheidea
ofadualoriginofDCsinthesteadystate.ItconsistsofDCsderivedfrom
monocytesandpre-cDCs.TheDCsthatarisefrompre-cDCsareaddressedas

10

1.2Themacrophage:originanddifferentiation

thekeysentinelsofthegutimmunesystem(Bogunovicetal.,2009;Varoletal.,
2009).ReportsthatthehumanCD14loCD16+monocytesubsetiselevatedinsepsis
(Skinneretal.,2005),rheumatoidarthritis(Kawanakaetal.,2002b)chronic
renalfailure(Kawanakaetal.,2002a;SaionjiandOhsaka,2001),cancer(Saleh
etal.,1995),tuberculosisandHIVinfection(Grage-Griebenowetal.,2001a)
leadtotheideathatthissubtypeis’inflammatory’.Takenintoaccountthat
themousecounterparts,theGr-1lomonocytesdonotmigratetositesofinflam-
mationinvivo(Geissmannetal.,2003;Sunderkotteretal.,2004),thistermhas
tobere-evaluated.

1.2.3Experimentalmodelsofmonocytedifferentiation

SincemacrophagesandDCsaredifficultlytoobtainfromhumandonorsinthe
requiredamounts,thesecellshavetobegeneratedinvitroforfurtheranalyses.
Severalinvitrosystemstodifferentiatethemfrombloodmonocyteshavebeen
developed.Whenthefirstresearchersstartedtoworkwithhumanmonocytes,
theysoonrealizedthatadhesionisaveryimportantkeystoneformonocyte
survivalanddifferentiation,butstillnotsufficientwithouthumanserum(Becker
etal.,1987).Investigatorsthentriedtodefinethebiochemicalfactorspresent
inhumanserumthatleadtomonocytematuration.Manychemicalshavebeen
identified,eitherbyusingpurifiedorrecombinantproteins,orbyinhibition
ofknownmoleculeswithspecificmonoclonalinhibitoryantibodies.Itcould,
forexample,beshownthatM-CSFisacriticalfactorformonocytesurvivalas
thetreatmentofadherentcultureswithhumanseruminthepresenceofanti-M-
CSFantibodiesinhibitedmonocytematuration(Andreesenetal.,1990;Brugger
etal.,1991).Ontheotherhand,M-CSFaloneisnotasufficientreplacementfor
humanserum.RecentlyWayetal.(Wayetal.,2009)describedtwoserum-free

11

ductiontroIn1

M-CSFcontainingcytokinecocktailstogeneratemonocytesandmacrophages
fromhumanCD34+hemopoieticstemcells.

1.2.4Theimpactofmonocyteadhesion

Beforeabloodmonocytebecomesadifferentiatedmacrophage,itleavesthebone
marrowandcirculatesintheblood,adherestotheendotheliumofabloodvessel
andtransmigratesintothesurroundingtissue.Thisinvolvesaninitialselectin-
glycoproteininteraction,resultinginmonocyterolling.Then,themonocyte
integrinshavetobeactivatedbychemokines,allowingafirmintegrin-protein
adhesion.Followingcellpolarisation,themonocytecanmigratebydiapedesis
betweentheepithelialcellsintothesubendothelialextracellularmatrix(ECM)
(ImhofandAurrand-Lions,2004).Althoughtherearemanychemokinesknown
toinduceleucocyteadhesionunderinflammatoryconditions(CravensandLip-
sky,2002),littleisknownabouttheconstitutivelyexpressedchemokineslead-
ingtomonocyteadhesioninnon-inflammatoryconditions(ImhofandAurrand-
2004).Lions,Therecentfindingsaboutdifferentmonocytesub-populationsinmicesuggest
aninflammatoryandanon-inflammatorymonocytesubset,andconfirmtheidea
thatmacrophagesintheperipheryarereplenishedbycirculatingbloodmono-
cytes(see1.2.2).Theoriginsofthemanydiverseformsofmacrophagesseenin
varioustissuesarenotmatureformsofdifferentmonocytesub-populationsbut
rathermultiplephenotypicmanifestationsofmonocytesreflectingtissue-specific
ECMandthelocalbiochemicalmicroenvironment.Onceamonocyteentersa
tissue,itsoonbecomesindistinguishablefromtheresidentmacrophages(Hume,
2006).So,thecell-cellandcell-matrixcontactscanbesuspectedtohaveanim-
pactonmonocytedifferentiation.Integrinligationcausesan”outside-in”signal

12

1.2Themacrophage:originanddifferentiation

involvingmanytranscriptionfactorscontrollingcomplexfunctionslikecellpo-
larisationandmigration.Someofthesetranscriptionfactorsinducedbyintegrin
engagementarealsoinvolvedinmonocytesurvivalanddifferentiation(Shiand
Simon,2006).InvitroexperimentsusingvariousECMmoleculesshowedthat
fibronectinseemstoinducestrongermonocytetomacrophagedifferentiation
thancollagenIorcollagenIV(Jacobetal.,2002).

Recentresultsfromthisgrouphaveclearlydemonstratedthatmonocytead-
hesioncouldinhibitapoptosis.Thisismostlikelyduetoadhesion-induced
secretionofM-CSF,whichactssubsequentlyinaautocrinemanner(Mondalet.
al,unpublisheddata).Inhibitionofapoptosisfollowingmonocytetransmigra-
tionthroughepitheliawasconfirmedbyWilliamsetal.(Williamsetal.,2009).
Similarresultswerefoundforpolymorphonuclearleukocytes,wheretransmigra-
tioninhibitedapoptosis(Huetal.,2004).

Monocyteadhesionandsubsequentintegrinengagementresultsin”outside-
in”integrinsignaling,causingtyrosinephosphorylationofcertainintracellular
proteins,namelyERK,p38andJNK.Thisresultsinrapidlyactivatedtranscrip-
tion,stabilizationoftheproducedmRNAandorganizationofthecytoskeleton
(Mondaletal.,2000).Towhatextentthistyrosinephosphorylationcascadeis
affectingthemonocytedifferentiationisincompletelydefined.Recently,Shiet
al.(ShiandSimon,2006)pointedouttheroleoftheforkheadtranscriptionfac-
torFoxp1inmonocytedifferentiation.Ontheotherhand,Himesetal.(Himes
etal.,2006)displayedtheinteractionoftheJNKandM-CSF,thusindicatinga
synergiceffectofboth,M-CSFandadhesion,onmonocytedifferentiation.M-
CSFinthiscontextisalsoknowntopromotemacrophagemotilityinaddition
toadhesiontoextracellularmatrix(Rovidaetal.,2005).

Thefirmadhesiontoplasticcouldpreventmonocytesinserumfreecultures
fromcelldeath,throughtheproductionofautocrinesurvivalfactorslikeM-

13

ductiontroIn1

CSFandTNF-α(Haskilletal.,1988).Thenot-so-firmly-adherentmoncytes
onTeflonfoilscouldberescuedfromapoptosisinserumfreemediumonlywith
additionalM-CSF.Underbothconditionsandsimilaronothersurfacecoatings,
monocytesurvivalwasneveraccompaniedwithdifferentiationunderserumfree
1990).al.,et(Andreesenconditions

1.2.5TheroleofM-CSFanditsreceptor

SincethefirstdescriptionofM-CSFanditsreceptormanyefforthasbeenmade
toelucidatethebiochemicalstructuresandtheintra-cellularsignalcascades
thatintheendleadtomorphologicchangesinthemonocyte.
M-CSFisadisulfide-linkedhomodimerandisexpressedbymanycellsinthree
isoforms:secretedglycopeptideandpeptidoglycanthataresynthesizedbyen-
dothelialcellsandenterthecirculation,andamembraneboundcell-surface
glycopeptidewhichisinvolvedinlocalregulation.Thesecretedpeptidoglycan
canalsoactlocallybybindingtoextracellularmatrices(ChituandStanley,
2006).Macrophagesderivedfrommonocytesafter5daysincultureconstitu-
tivelyproduceM-CSF(about15ng/ml)foratleast28day(Scheibenbogenand
1991).Andreesen,TheM-CSFreceptorseemstoberestrictedtomonocytesandtheirprecursors,
macrophages,DCsandosteoclastsandthefemalereproductivetract(Sasmono
etal.,2003).TheM-CSFreceptorisareceptortyrosinkinaseandcloselyrelated
tothec-Kitreceptor.BindingofM-CSFtoitsreceptorresultsinnon-covalent
dimerizationoftheM-CSFreceptor,activationofitskinaseandafirstwave
ofM-CSFreceptortyrosinephosphorylation.Aftercovalentdimerizationand
asecondwaveoftyrosinephosphorylation,affectingabout0.02%ofthetotal
cellularprotein,theligand-receptorcomplexgetsubiquitinatedandlysosomal
degraded(Stanleyetal.,1997;PixleyandStanley,2004).

14

Inresultsvitro

1.2Themacrophage:originanddifferentiation

Inearlyexperimentstogeneratemacrophagesinvitro,M-CSF,atleastinphysi-
ologicalserumconcentrations,wasfoundtobeobligatoryformonocytesurvival
anddifferentiation,asmonocytesdieinserumfreemediaorifM-CSFwasde-
pletedfromserumbyneutralizingantibodies(Beckeretal.,1987;Andreesen
etal.,1990;Bruggeretal.,1991).MacrophagesneedcontinuosM-CSF,asthe
removalofthiscytokineresultsinapoptosis(Komuroetal.,2005).Severalfac-
torsrelatedtomonocyteormacrophagesurvival,forexampleadhesion(Mondal,
unpublisheddata)induceautocrineM-CSFproduction,butalsoM-CSFitself
inducestheproductionofsurvivalanddifferentiationfactors(Komuroetal.,
2005).M-CSFcouldalsoinduceproliferationinamonocytesubset(Finnin
1999).al.,et

resultsvivoIn

ManyinsightstotheinvivofunctionofM-CSFweregainedusingaosteopetrotic
mousemodel,whichisdeficientforM-CSF(Csf1op/op).Thesemiceshownormal
bloodmonocytecountsbutareducednumberofalmostalltissuemacrophages
populations,whichcouldberestoredbydailyintracutaneousM-CSFinjection
(Stanleyetal.,1997;ChituandStanley,2006)orbyusingM-CSFtransgenes
forthedifferentisoforms(Ryanetal.,2001;Daietal.,2004;Nandietal.,
2006).Incontrast,Csf1op/opmicehavefullydifferentiatedDCsandLCs,which
leadtothehypothesisthatthesecellsdevelopindependentlyfrommononuclear
phagocytes(Wiktor-JedrzejczakandGordon,1996).
However,usingamousemodeldeficientfortheM-CSFreceptor(Csf1r-/-),
Ginhoux(Ginhouxetal.,2006)showed,thatLCsdevelopmentintheepider-
misisdependentonM-CSFandthatunderinflammatoryconditionsLCscan
ariseformmonocytesinvivo.Furthermore,MacDonald(MacDonaldetal.,

15

In1ductiontro

2005)showedthattheM-CSFreceptorisupregulatedinallDCsubsetsdur-
ingdifferentiation,supportingtheideathatmosttissueDCsareofmyeloid
origin.RecentlycouldbeshownthattheDCfavoringcytokineGM-CSFand
IL-4incombinationup-regulatetheTNF-αconvertingenzymeexpressionand
activity,causingectodomainsheddingofthemembrane-boundM-CSFreceptor
andtherebydirectingthemonocytetowardsaDClikephenotype(Hiasaetal.,
2009).

diseasesinM-CSF

BloodlevelsofM-CSFareelevatedinseveraldiseases.Highlevelswerefound
inpatientsundergoingdialysis(SaionjiandOhsaka,2001)andduringacute
renalallograftrejectioninhuman(LeMeuretal.,2004)andmouse(LeMeur
etal.,2002).ThelatterproposedusingM-CSFasaspecificmarkerofacute
allograftrejection.M-CSFseemsalsotobeelevatedinseverelyilltraumapa-
tients,whereitpossiblyimpairstheimmunologicaldefense(Deetal.,2003).In
cancer,especiallyofthefemalereproductivetract,increasedcirculatingM-CSF
canbefound(Kacinski,1997).Inthisandotherneoplasias,socalledtumor-
associatedmacrophagesseemtohaveatwoedgedrole:promotionoftumor
invasionviamacrophagerecruitmentbythesecretedM-CSFversusthestimu-
lationofdirectkillingandantigen-processingbythecell-surfaceM-CSF(Chitu
andStanley,2006).Thisoffersalsotherapeuticoptions,asM-CSFblockadeby
antisenseoligonucleotidesandsmallinterferingRNAscansuppressthegrowth
ofhumanmammarytumorxenograftsinmice(Aharinejadetal.,2004).In
rheumatoidarthritis,wheremacrophagesplayanimportantrole(Hamiltonand
Tak,2009)elevatedlevelsofM-CSFarecorrelatedwithanincreasednumber
ofCD14loCD16+monocytes(seeabove),thusindicatingthatM-CSFmaycon-
tributetothismorematuremonocytesubsetintheblood(Kawanakaetal.,

16

1.2Themacrophage:originanddifferentiation

2002b).Monocytesofseverelyinjuredtraumapatientsdonotdifferentiateinto

DCunderappropriatestimuliasasignofpreviousdifferentiationintoamore

macrophage-likecelltypecausedbyenhancedM-CSFresponsiveness(Deetal.,

2003).M-CSFreleasebyendothelialvascularcellsisup-regulatedbyCreac-

tiveproteininducingmacrophageproliferationcausingatherosclerosis(Devaraj

etal.,2009;Irvineetal.,2009).SeveraltherapeuticstrategiestargetingM-CSF

are

now

in

preclinical

studies

or

phase

I

clinical

trials

(Hamilton,

2008).

17

1

18

troIn

duction

2Preworkandaims

Variouswayshavebeendescribedtocultivateanddifferentiatehumanperipheral
bloodmonocytes,forexampleintomacrophagesorDCs.Allofthemsharean
adherentculturecondition.Ascellsofthemonocyticlineageexistalsoinbody
fluids(seetable1.1),differentiationmayoccurphysiologicallyalsoundernon-
adherentconditions.Althoughalotofresearchhasbeenpublishedconcerning
theintracellularchangescausedbymonocyteadhesion,uptonowlittleisknown
abouttheimpactofadhesiononmonocytedifferentiation.Toapproachthis,
weusedanexperimentalsettingdevelopedbymembersofthisgroupandfirst
publishedbyArndt(Arndtetal.,2007)toculturemonocytesnon-adherently.
Itcouldbeshownthatmonocytesundergoapoptosiswithin3daysifculturing
theminacontinuouslyrotatingculturesystemthatinhibitscelladhesion.This
apoptosiscouldbeinhibitedbyaone-timeadditionofexogenousM-CSFat
thebeginningofthecultureperiod.Asadhesionisinductingthesecretionof
M-CSF,thisledtothehypothesisthatadhesioninhibitsapoptosisthroughthe
autocrineactionofM-CSF(Mondaletal.,unpublisheddata).

FirstaimofthisstudywastoquantifythesurvivalrateofthisM-CSFin-
ducednon-adherentlyculturedmonocytes(MMAC)andtocompareittothatof
adherentlygeneratedmacrophages(MAC).Secondaimwastovalidatewhether
theseMMAChaddifferentiated.Tothisend,theirphenotypewascharacter-
izedindetailusingmonoclonalantibodiesandflowcytometry.Theirfunctional

19

aimsandorkPrew2

propertiesweredeterminedintermsofphagocytosis,antigenpresentationand

cytokineproduction.TheresultswerecomparedtothoseofMACandimmature

monocytederiveddendriticcells(DC).TodeterminetheinfluenceofM-CSF,

theywerealsocomparedtomonocytesthatwerecultivatedadherentlywith

additionalM-CSF(MAC+M-CSF).Theexperimentswereperformedunder

donormatchedconditions,inthatallDC,MMAC,MAC+M-CSFandMACs

weregeneratedinparallelfromfreshlyisolatedmonocytesfromthesamedonor

donorminimizeto

acteristics.

20

tendendep

ariationv

ofyabilitsurviv

tiationdifferenor

har-c

dsmethoandMaterial3

techniquesBasic3.1

andMedia3.1.1solutions

Thecompositionsofthefrequentlyusedmediaandsolutionsareprovidedin
theappendix,table7.1.Alistofallcompaniesisalsogiventhereintable7.2.

3.1.2Sterileprotocols

Allworkwithcellculturesandfunctionalanalyseswerecarriedoutundersterile
conditions,usingalaminarairflowworkbench(Hareus)andsterilesingleuse
pipettes.Ifnototherwisedeclared,cellcentrifugationwasperformedat300g
usingatablecentrifuge(Hareus)andcentrifugetubes(Falcon)with15mlor
50ml.Strictlysterileprotocolswereusedtopreventanynonspecificactivation
cells.theof

3.1.3Cellcultureconditions

Allcellsforlongtermculturingandforshorttermfunctionalanalyseswere
incubatedinanincubator(Hareus)ataconstanttemperatureof37◦C,witha
95%humidityand5%CO2concentration.

21

3Materialandmethods

3.1.4Viablecellcountusingtrypanblue

Todeterminethenumberofviableanddeadcells,thevitaldyetrypanblue
wasused.Trypanblueisactivelyexcludedbylivingcellsandstainsdeadcells
withdamagedmembranesblue.Forthis,50µlofcellsuspensionwereaddedto
50µloftrypanbluesolution(table7.1).Cellswerecountedunderamicroscope
(Axiovert25,Zeiss)at100×magnification,usingaNeubauercountingchamber
ashemacytometer.Foraccuracyallfour1mm2quadrantswerecountedand
thecellnumberwascalculatedaccordingtothefollowingformula:

Cellcount=4N∙D∙104ml−1

N=cellnumber,D=dilutionfactor

3.2Monocyteisolationthroughcounter-current
elutriation

Leukocyteenrichedbloodwasobtainedvialeukapheresis(Grawetal.,1971)
formhealthyvolunteers.Mononuclearcells(MNC)wereisolatedfromthisleuka-
pheresisconcentratesbydensitygradientcentrifugationoverFicoll-Hypaque
(Pharmacia)(Johnsonetal.,1977).MonocyteswereseparatedfromMNC
bycounter-currentelutriation(Sandersonetal.,1977)inaJ6M-Ecentrifuge
(Beckman)witha50mlchamberandaJE-5rotor.Initially,thecentrifugewas
sterilizedwith6%H2O2inH2Ofor20minutesandwashedtwotimeswithPBS
AN).(PThevariablepumpwascalibratedwithHanks’balancedsaltsolution(PAN)
atconstant1100gand4◦C.MNCsweregivenintothesystemataflowrate

22

3.3conditionsculturecyteMono

fractionflowratemaincellsinthefraction
(ml/min)1a52thrombocytes
small571bcyteslympho2a64intermediatelymphocytes
2b74intermediatelymphocytes
cyteslymphobig922ccytesmono1113Table3.1:Elutriationparametersandcorre-
fractionscellondingsp

of52ml/min,followedbycontinuousinfusionofHanks’solutionsupplemented
with6%autologousplasma.Theflowratewasincreasedstepbystepfollowing
theschemedisplayedintable3.2andmonocytesasthelargestcellsintheMNC
fractionwerecollectedinthelastfraction3.Theseelutriatedmonocyteswere
morethan80%pureasdeterminedbymorphologyandbytheexpressionofthe
CD14antigen(table3.2),measuredbyflowcytometry(3.5).

3.3Monocytecultureconditions

Becausemonocytesshowagreatdonordependentvariability,MMAC,MAC,
DCandMAC+M-CSFwerepreparedinparallelculturesfromeachdonor.

3.3.1Classicalmonocytederivedmacrophages(MAC)

Monocyteswereculturedatacelldensityof1∙106cells/mlinhydrophobicTeflon
bagsfor7daysinmacrophagemedia(table7.1)(Andreesenetal.,1983).To
harvesttheadherentmacrophagesfromtheTeflonbags,theywerecooledfor
15minutesat4◦CandthenloosenedbygentlytappingthecellsofftheTeflon
whileleavingtheplasmamembraneintact.

23

3Materialandmethods

3.3.2Monocytederiveddendriticcells(DC)

Monocyteswereculturedatacelldensityof1∙106cells/mlinventedflasks
(Costar)for7daysinDCmedia(table7.1)with6pg/mlIL-4(Immunotools)
and50pg/mlGM-CSF(Essex).Onlythenon-adherentcellswereharvested
(SallustoandLanzavecchia,1994).

3.3.3Non-adherentlyculturedmonocytes(MMAC)

Inordertoestablishaninvitrosystemtoculturemonocytesunderabsolute
non-adherentconditions,weusedasystemdescribedbyArndtetal.(Arndt
etal.,2007)inwhichmonocyteswereculturedinacontinuouslyrotating(12
U/min)flask(250mlcentrifugetubes,Falcon)withaventedcab(Costar),
usingaUniversalTurningDevice(GreinerBio-one).Thecellswereseededata
densityof1∙106cells/mlinmacrophagemedium(table7.1)supplementedwith
100ng/mlrecombinanthumanM-CSF(Cetus)andculturedfor7days.For
furtherprocessing,thesecellshadtobehandledonicetoavoidadhesiontothe
ners.taiconplastic

3.3.4AdherentlyculturedmonocyteswithadditionalM-CSF(MAC
M-CSF)+

Monocyteswereculturedandharvestedaccordingtothesameprotocolasfor
MAC(see3.3.1)withadditional100ng/mlrecombinanthumanM-CSF(Cetus)
analogoustoMMAC(see3.3.3.

3.4Lightmicroscopyandphotographs

Forphotographs,unstainedcellswerepreparedseperately.Todemonstratecell
morphologyundernon-adherentconditions,picturesofMMAC,MACandMAC

24

ytometrycwFlo3.5

+M-CSFweretakenimmediatelyafterharvesting,bysuspendinglargedroplets
ofcellsonasmallTeflonfoiltopreventcelladhesion.PicturesofMACand
MAC+M-CSFwerealsotakenadherentonTeflonpriortoharvesting.Light-
microscopicexaminationsweredoneusinganAxiovert25microscope(Zeiss)
andaFinepixS1procamera(Fujifilm)forphotographs.Thephotographswere
editedforprintingwiththefreelyavailablesoftware”TheGIMP”.

wFlo3.5cytometry

AntigenLabelIsotypeCloneCompany
primaryantibodies(allfrommouse):
IsotypeFITCIgG1X40BDBiosciences
IsotypeFITCIgGBeckmanCoulter
IsotypePEIgG1X40BDBiosciences
IsotypePEIgG2bMPC-11BDBiosciences
IgG1noneeypIsotCD1aPEIgG1BL6BeckmanCoulter
CD14FITCIgG2aMµ4BeckmanCoulter
CD16FITCIgG2a5D2PeliCluster
CD40PEIgG15C3BDPharmingen
CD71FITCIgG1YDL.1.2.2Immunotech
CD80PEIgG1L307.4BDPharmingen
CD84PEIgG12G7BDPharmingen
CD86FITCIgG12331(FUN-1)BDPharmingen
DiacloneB-F1IgG1FITCHLA-DRHLA-ABCPEIgG1G46-2.6BDPharmingen
DC-SIGNPEIgG2bDCN46BDPharmingen
MAX.11noneIgG1ownlab(Rehlietal.,1995)
secondaryantibody(fromgoat):
anti-mouseFITCIgG+MJacksonImmunoResearch
Table3.2:Antibodiesusedforflowcytometry.Conjugatedfluo-
rochromes:flouresceinisocyanat(FITC)orphycoerythrin(PE).

Thephenotypeofthecellswasdeterminedbyflowcytometryusingafluorescent-
activatedcellsorter(FACS).Flowcytometryallowsthemeasurementofthe

25

3Materialandmethods

fluorescenceofasinglecellorparticle,incontrasttorelatedtechniqueslike
spectrometry,whereonlytheabsorptionandtransmissionofthewholesample
canbedetermined.Inshort,astreamoffluidcontainingcellsflowsthroughthe
machinesuchthatasinglecellatatimepassesbyalightsource.Herethecells
interceptwithlightofacertainwavelenght,usuallyfromalasersource.Asthe
beamhitsacell,onlyapartofthelightpassesunaffected,whichisdetectedas
forwardscatter(FSC)andcorrespondstothesizeofthecell.Thepartthatis
reflected90◦offisdetectedassidewardscatter(SSC)andcorrespondstocell’s
complexityandgranularity.Ifthecellorparticlehasfluorescentproperties,
thelightbeamcanexcitethesefluorochromemoleculestoemitphotonsofthe
wavelengthcharacteristicforthefluorochrome.Forexample,fluoresceiniso-
cyanat(FITC)emitslightbetween500-550nmwavelengthandphycoerythrin
(PE)emitsfrom550-650nmwavelenght.Byusingdetectorswithdifferent
wavelengthfilters,fluorochromeswithdifferentwavelenghtscanbedetectedsi-
multaneously.Thesensitivityofthedetectors,whichmostcommonlyarephoto
multipliertubes(PMT),canbeadjustedbychangingtheirvoltage.Thedata
ofmanyevents(usually20,000)isstoredinacomputerandcanbevisualized
logarithmicallyashistogramordotplot.Tocomparetheresultsofdifferent
experiments,themeanfluorescentintensity(MFI)iscalculatedineveryhis-
togram.

Topreparethecellsforflowcytometry,theywerecentrifugedat300gfor
5minutesat4◦Candwashed2timeswithFACSwashingbuffer(table7.1).The
humanimmunoglobulinspresentintheFACSwashingbufferservestoblockthe
cells’endogenousFc-receptorsandthustominimizethenon-specificbindingof
thesubsequentlyusedantibodies.Afterthefirstwashingstep,thecellswerere-
suspendedto5∙105cells/mlandabout2.5∙105cellseachwereevenlydistributed
to5mlpolystyrenetubes(Falcon)forFACSanalysis.Afteranothercentrifuga-

26

3.6Shortdescriptionofthedetectedantigens

tion,thesupernatantwasdecantedandthepelletloosened.Thecellswerethen
incubatedwithsaturatingamountsofspecificantibodyoranisotypecontrol
(seetable3.2)for30minat4◦Cinthedark,followedbytwowashingsteps
withFACSwashingbuffer.Incaseofanun-conjugatedantibody,thecellswere
incubatedwith50µlsecondaryantibody(FITCconjugatedgoat-anti-mouse)
foranother30minutes.Aftertwofinalwashingstepsallcellswerefixedin
200µlFACSfixative(table7.1).Thecellsweresinglestainedonly.Instru-
mentsettingsweresetsothattheisotypecontrolfellinthefirstdecade.The
resultsweredisplayedlogarithmicallyashistograms.Thecellswereanalyzed
between24-48hrsusingaFACSCaliburflowcytometer(Becton-Dickinson).All
datawereanalyzedwiththeCellQuestsoftware(Becton-Dickinson),usingthe
forwardscatter(FSC)andsidewardscatter(SSC)togatecelldebrisout.

3.6Shortdescriptionofthedetectedantigens

Freshlyisolatedmonocytes,MACandMMACafter3daysincultureandMAC,
MMAC,DCandMAC+M-CSFafter7dayswerestainedforthefollowing
markers:antigenstypicallyassociatedwitheithermacrophageorDCdifferen-
tiationandantigensthatareexpressedonbothmacrophageandDC,reflecting
theirfunctionasantigenpresentingcellsandthereforeinvolvedinantigenup-
takeandTcellactivation.

3.6.1Antigensofmacrophagedifferentiation:CD14,CD16,CD71,
MAX.11andCD84

CD14isimportantforthereactionoftheorganismtoLPS.Itactsasareceptor
forLPSboundtoLBP(Wrightetal.,1990)andisexpressedonmonocytes
andmacrophages(Toddetal.,1981).CD14ismembraneboundbutlacks
acytoplasmicdomain.ThusitneedstheTolllikereceptor4(TLR4)asa

27

3Materialandmethods

coreceptortotriggerthesignalcascadeleadingtoTNF-αrelease(Poltorak
1998).al.,et

CD16orFcγIIIisalow-affinityreceptorformonomericIgG.Itisdevelopmen-
tallyup-regulatedintheinvitrodifferentiationfrommonocytetomacro-
phage(ClarksonandOry,1988;Andreesenetal.,1990).Althoughitwas
initiallythoughttoberestrictedtodifferentiatedmacrophages,amono-
cytesubpopulationexpressingCD16hasbeendescribed(Passlicketal.,
1989).

CD71ortransferrinreceptorisadifferentiationmarkerformacrophagesand
notpresentonmonocytes(Andreesenetal.,1990).Itisalsoexpressedon
intestinalDCandonmonocytederivedDCwhereitactsasareceptorfor
IgA(Pasquieretal.,2004).

CD84isamemberoftheCD2subgroupofIgsuper-family(delaFuenteetal.,
1997).ItisidenticaltotheantigenrecognizedbytheMAX.3antibodyand
isaglycoproteinexpressedonmaturemacrophages(Krauseetal.,2000).
AlthoughMAX.3boundonlytomaturemacrophages,otheranti-CD84
monoclonalantibodiesdetectingdistinctepitopesshowedaweakexpres-
siononmonocytesandDC(Zaissetal.,2003).Itisaco-stimulatory
moleculeandbindstoitself,thusenablingcontactbetweenAPCand
Tcell.TheCD84-CD84connectioninductsINF-γsecretion(Martinetal.,
2001).

TheMAX.11antibodyrecognizesCarboxypeptidaseM(Rehlietal.,1995;Krause
etal.,1998),asurfacemoleculecharacteristicformonocytetomacrophage
differentiation(Andreesenetal.,1986).

28

3.6Shortdescriptionofthedetectedantigens

3.6.2CellsurfaceantigenofDCdifferentiation:CD1a

CD1aisanantigen-presentingmoleculedistantlyrelatedtotheMHCclassI
molecules(Martinetal.,1986;Blumbergetal.,1995;Banchereauetal.,
2000).Itbindsnon-pepticselfandforeignmoleculesandpresentsthem
toTcells(DutroncandPorcelli,2002).Thusitcontributesnotonlyto
hostimmunitybutalsotoautoimmuneandanti-tumorreactions(Hunger
etal.,2004).CD1aisagoodmarkerforinvitrogeneratedblood-monocyte
derivedDCs(SallustoandLanzavecchia,1994)andepidermalLangerhans
1981).al.,et(Fithiancells

3.6.3Functionalantigenesofantigenpresentingcells:CD40,
DC-SIGN,HLA-DRandHLA-ABCCD80/CD86,

CD40isamemberoftheTNF-receptorsuper-familyandispresentonallAPCs,
particularlyonDCs(Rogersetal.,2003).Itiscriticalforpresentation
toandinductionofcytotoxicTcells.CD40isknowntobeinducedafter
activationofPRRsliketheTLRs.ActivationofCD40byitsligandCD40L
resultsinup-regulationoftheco-stimulatoryCD80andCD86andMHC
classIIviathetranscriptionfactornuclearfactor(NF)-κB(O’Sullivan
2003).Thomas,and

CD209orDC-SIGN(DC-specificICAM3-grabbingnon-integrin)isaC-type
lectinreceptorforintercellularadhesionmolecule(ICAM)3andICAM2
(Geijtenbeeketal.,2000).FurthermoreitcanactasaPRRandprovidea
mechanismforpathogens,particularlyHIV,toevadetheimmunesurveil-
lance(vanKooykandGeijtenbeek,2003).Itisconstitutionallyexpressed
onDCsandmacrophages(Soilleuxetal.,2002).

CD80andCD86orB7-1andB7-2areco-stimulatorymoleculesassociatedwith

29

3Materialandmethods

APC-Tcellinteraction.TheircounterligandsonTcellsareCD28andcy-
totoxicTlymphocyte-associatedantigen(CTLA)-4.Ithasbeenreported
thatCD28triggersTcellresponsewhereasCTLA-4seemstosuppressor
terminateTcellresponse.CD86isconstitutivelyexpressedatalowlev-
elsandisupregulateduponstimulation,whereasCD80isonlyexpressed
followingstimulation(Greenwaldetal.,2005).

HLA-ABCandHLA-DR:ForthetwogroupsofMHCreceptors,HLA-ABC
waschosenforclassI,whichisexpressedonvirtuallyeverycellsurface,
andHLA-DRforclassII,whichisrestrictedtoAPC.

3.7Apoptosisassay

Anearlyeventinapoptosisisthetranslocationofphosphatidylserine(PS)from
theinnertotheoutersideoftheplasmamembrane.InviablecellsPSislo-
catedexclusivelyontheinnerlayerofthecellmembrane,whereasinapoptotic
cellsthePStranslocatestothecellsurface.AnnexinVisamoleculethatbinds
specificallytoPS.Usingflowcytometry,thelocationofPScanbedetectedusing
FITC-labeledAnnexinV(Vermesetal.,1995).Innecroticcellsthecytoplas-
maticmembraneisdamaged,cellsbecomepermeableandallowAnnexinVto
enterthecytoplasmaandbindtoPSontheinnersideofthemembrane.Todis-
criminatebetweenearlyapoptoticcellswithintactmembraneandlatenecrotic
cellsbyflowcytometry,aDNA-stainingdyelikePropidiumiodide(PI)wasused.
Innecroticcellsthedyeentersthroughthedamagedmembraneandbindsto
thecellularDNA.ByplottingthefluorescenceofAnnexinV-FITCversusPI,
threecellpopulationscanbediscriminated:AnnexinV-FITC−/PI−viablecells,
AnnexinV-FITC+/PI−earlyapoptoticcellsandAnnexinV-FITC+/PI+late
apoptoticornecroticcells.Sinceapoptosisisabiochemicallyactiveprocess,

30

3.8AllogenicTcellstimulationassay

requiringenergy,theexperimentswereperformedstrictlyonicetohaltcellu-
laractivity.About2.5∙105cellswerewashedtwicewithicecoldPBSin5ml
polystyrenetubesandre-suspendedin200µlstainingsolution,containing2.5µl
AnnexinVand2.5µlPIin195µlAnnexinbindingbuffer(table7.1).After
20minutesincubation,cellswereanalyzedwithinonehouronaFACSCalibur
flowcytometer.Toallowforthebestcompensationparameterssampleslabeled
witheachdyesinglywerealsoanalyzed.

3.8AllogenicTcellstimulationassay

TheTcellstimulationcapacityofAPCcanbeassessedinamodelofallo-
genictransplantation,themixedleucocytereaction(MLR),whereAPCare
co-culturedwithallogenicTcells.TheTcellsrecognizetheMHCmoleculesas
non-selfandgetactivateddependingonthestimulatoryorinhibitorymolecules
presentedbytheAPC.ProliferationisagoodindicatorforTcellactivation
uponstimulation.Tomeasureproliferation,3H-methyl-thymidine(Hartmann
Analytica)wasaddedinexcesstoreplacethecells’ownthymidinepool.Thus
3H-methyl-thymidineisincorporatedintothenewlysynthesizedDNAofpro-
liferatingcells.Aftercelllysiswithdeionizedwater,theDNAwascollected
onfiberglassfilters.Theradioactivity,measuredincountsperminute(cpm),
represents3H-methyl-thymidineincorporatedintothecellularDNAandthus
proliferation.cellThedifferentstimulatorcells,MAC,MMACorDCwereexaminedfortheir
capacitytostimulateallogenicTcellsinanallogenicMLR.TheTcellswere
separatedfromtheMNCbycounter-currentelutriation(3.2)andfrozenimme-
diatelyinfreezingmediumcontaining10%DMSO(Sigma)and90%fetalcalf
serum(FCS)(PAA).ForMLR,Tcellswererapidlythawed,washedandresus-
pendedinMLRmedium(table7.1).ForMLR,5∙104Tcellswereincubatedat

31

3Materialandmethods

stimulator:responderratiosfrom1:2till1:1250.Thecellswereculturedin96-
wellroundbottomtissuecultureplates(Falcon)inatotalvolumeof200µlMLR
medium(table7.1).Asnegativecontrols,Tcellsandstimulatorcellswerealso
incubatedalone.After6days,1µCi3H-methyl-thymidine(0.037MBqspecific
activity)wasaddedtoeachwell.Followingatotalincubationof7days,cells
wereharvestedontoglassfiberfilters(PrintedFiltermatB,WallacOy)usinga
VacusafeIH280harvester(Innotech)andsubsequentlylysed.Scintillationfluid
(BetaplateScint,Wallac)wasaddedtothefiltersandtheradioactivitywasde-
terminedbyaliquidscintillationcounter(1450MicroBeta,Wallac).Allsamples
weretestedinquadruplicatesandvaluesindicatemeans±SD.

yassacytosisPhago3.9

Endocytosisisabasicfeatureofalleucaryotecells.Themostcommonwayis
pinocytosis,wheretheplasmamembranebuildssmallvesiclescontainingliquids
orsinglemolecules.Phagocytosisismorerestrictedtophagocyticcellslike
macrophages.Herethecellcanengulfbigparticleslikebacteriaorvirus,forming
phagosomeswhichlaterfusewithlysosomesthatcontainenzymestodegrade
theengulfedcontent.Thephagocyticcapacitywasassessedbythecells’ability
toingestsmallfluorescentlylabeledlatex.Thephagocytizedfluorescentbeads
couldbemonitoredbyflowcytometry.
Therefore,theharvestedcellswerewashedandresuspendedinpolypropylene
tubes(topreventadhesiontothetubes)at106cells/mlwithfreshmedium.
Thecellswereincubatedwith0.5µlFITC-labeledFlouresbriteMicrospheres
latexparticleswith1.0microndiameter(Polyscience)at37◦Cinanoscillating
waterbathtoinsurecontinuousexposureofthecellstothebeads.Tocontrol
forrandomstickingofthebeadstothecellsurface,cellstreatedwithbeads
wereincubatedonicewithadditional6mMEDTA(Sigma)tototallyinhibit

32

3.10CytokineproductionfollowingstimulationwithLPS

phagocyticuptake.After1hincubationwasstoppedbyplacingthecellsonice
andadding6mMEDTA.Following3washesanaverageof30,000cellswas
analyzedinaFACSCalibur(Beckton-Dickinson).

3.10CytokineproductionfollowingstimulationwithLPS

OnemainfeatureofmacrophageorDCactivationisincreasedcytokinesecre-
tion.Todeterminehowthedifferentcellsreacttoanactivationstimulusin
termsofcytokineproduction,theywereincubatedwithLPS,andthesuper-
natantwasanalyzedforsecretedcytokines.Tothisend,monocytesorcells
after7daysinculturewereharvested,washedand2∙106cellswereculturedin
2mlfreshmediumforadditional24hwithorwithout10ng/mlLPS.Thesu-
pernatantswerecollectedandfurthercentrifugedfor30minutesat13,000rpm
(ultracentrifuge,Beckmann)toremovecellulardebris,andstoredat−20◦C
inmicrotesttubes(Eppendorf)forlaterenzyme-linkedimmunosorbentassay
(ELISA)analyses.ThefollowingELISAswereperformedaccordingtotheman-
ufacturer’sinstructions:IL-10(Becton-Dickinson),IL-12p70(eBioscience),and
(eBioscience).TNF-α

Statistics3.11

StatisticanalyseswereperformedwithSPSS(SPSS).Fordescriptivestatistics
meanandstandarddeviation(SD)werecalculated.Alldatawascontinuousand
metricbutnotnormallydistributed,whichwastestedwiththeKolmogorov-
Smirnovtest.Therefortestsfornon-parametricdatawereappliedonfurther
statisticanalyses.Allexperimentsfromonedonorweretreatedasrelateddata,
theresultsofdifferentdonorstreatedasindependentdata.Toevaluateadiffer-
encebetweenmultiplepaireddata,Friedman-testandpost-hocWilcoxonSigned

33

3

Materialandmethods

RanksTestwereused

statisticallyaswdata

asw

34

considered

to

eb

aluateevto

t.significan

statistically

ifadifferencebetweenasetofnon-parametric

0.05≤porF

t.significan

eth

difference

beenwet

owt

aluesv

Results4

4.1Survivalofmonocytesundernon-adherentconditions

conditionmeanofvitalcells(%)SDpn
MAC50.18.9>0.05(12)
(13)13.450.5CMMAMAC+M-CSF40.89.4>0.05(3)
DC40.210.7>0.05(8)

Table4.1:Survivalratesofdifferentcultureconditions.Theviability
ofMAC,MMAC,MAC+M-CSFandDCafter7culturedayswasestimated
bytrypanbluestaining.The%vitalcellsreferstothepercentagesoftheinitial
seededmonocytesaliveafter7daysinculture.Statisticalanalysesshowedno
significantdifferences,preferstothedifferencetoMMAC.Meanvaluesofn
independentexperiments,SD:standarddeviation.

Inshort,wewantedtostudywhethermonocytesinsuspension,suchasin

peripheralbloodcansurviveandeventuallydifferentiate.Wethususedaculture

systemdevelopedearlierbymembersofthisworkgroup(Arndtetal.,2007),

wherebymonocytesaregrowncompletelyadhesion-freeandcomparedthese

cellswithconventionallydifferentiatedMAC.Theviabilityofcellsunderall

differentcultureconditionswasdeterminedbytrypanbluestaining(3.1.4)at

theendofanycultureperiodbeforefurtherprocessing.Thenumberofviable

cellswassetinratiotothenumberofcellsatthebeginningofthecultureperiod.

Table4.1showstheresultsofallexperimentsonday7.Therewasnosignif-

icantdifferencebetweenthesurvivalrateofadherentMAC(50.1%)andnon-

35

Results4

adherentMMAC(50.5%).ThepoorervaluesofMAC+M-CSF(40.8%)were
statisticallynotsignificantlowerthanMAC’sorMMAC’s.

4.2Detectionofapoptoticcells

Conditionn%vitalcells%apoptoticcells%deadcellsp
meanSDmeanSDmeanSD
Mono38416111314
MAMMACCd3d3447765152455751225213>0.05
MACd7481855114>0.05
MMACd7475765164
DCd72683131172>0.05
Table4.2:Distributionofvital,apoptoticanddeadcells.Monocytes
andfreshlyharvestedMAC,MMACandDCatdifferenttimepointswerestained
withAnnexinVFITC/PIandanalyzedwithaflowcytometer.Resultsofquad-
ranmentts.Noanalyses,signitheficanttableshodifferenceswswmeanerevfoundaluesbandetweenSDMofnMAinCdepandendenMACtorexpDC.eri-

IntheAnnexinVFITC/PIassay(3.7)freshlyisolatedmonocytesshoweda
viabilityof84%andshowedalmostnoapoptoticcells.Afteracultureperiod
of3days,theharvestedMACshowedaviabilityof77%with5%apoptotic
cellsand12%deadcells,thecorrespondingMMAChadaviabilityof65%
with5%apoptoticcellsand25%deadcells,thedifferencewasnotsignificant
forvital,apoptoticordeadcells.Afteracultureperiodof7daysMACshowed
aviablityof81%with5%apoptoticand11%deadcells,MMACwereviable
to75%with6%apoptoticand16%deadcells.DConday7wereviableto68
%with13%apoptoticand17%deadcells,alsowithnosignificantdifferences
4.2).(figure

36

Figure

4.1:

Detection

of

andfreshlyharvestedMAC

optoticap

lslec

4.2

in

Detection

of

optoticap

CS-Analysis.AF

cells

cytesMono

andMMACatdifferenttimepointswerestained

withAnnexinVFITC/PIandanalyzedwithaflowcytometer.Thefigure

showsdotplotsofonerepresentativetwo

areanalyses

ybtedrepresen

ersbumnthe

resultsanalysis,color

theinsmall

cross.

of

the

tquadran

37

4

Results

Figure

4.2:

Morphology

MACandMAC+M-CSF

DCC,MMA

cation

38

100x,

in

tghli

adherent

microscopy.

(ateflonon

andloosenedMAC+M-CSF

evnati

cells

thoutiw

staining.

(c,

d,

e

tLigh

&

&

b)

)f

microscopic

images

of

andofloosenedMAC,

in

ension.susp

Magnifi-

4.3Morphologyinlightmicroscopy

4.3Morphologyinlightmicroscopy

Themorphologyofthecellswasalsoevaluatedinparallelwiththeviabilityby
lightmicroscopy(3.4).TheMACshowedspindleshapedbodieswhenattached
totheTeflonmembrane(figure4.2a).TheextraM-CSFintheMAC+M-CSF
culturedidnotcauseanymorphologicalchanges(figure4.2b)differentfrom
thoseobservedinMAC.IntheDCculture,thenon-adherentcellsweresmaller
insizeandhadtypicaldendrites(figure4.2e).Insuspension,MMACpresented
ahighlygranularroundbodywithsmallcellplasmaprotrusions(figure4.2d).
TheloosenedMAC+M-CSFlookedsimilartotheloosenedMAC(figure4.2
c&f).Allthreemacrophagepopulations,MAC,MMACandMAC+M-CSF
attachedrapidlytotheusualglassmicroscopeslidesdisplayingaclassic’fried
form.egg’

4.4Phenotypecharacterizationwithmonoclonal
diesoantib

Afterestablishingthesimilarsurvivalratesofnon-adherentMMACandadher-
entMAC,thequestionoccuredwhetherthesurvivingcellsactuallyunderwent
differentiation.Ascelldifferentiationisassociatedwithchangesinsurfaceanti-
genexpression,thephenotypeofthesecellswasdeterminedusingfluorescently
taggedantibodiesagainstdifferentiation-associatedantigenswhichwereidenti-
fiedwithflowcytometry.
MMAChadhighervaluesinFSCandSSC,correspondingtohighergranular-
ityandcellsizecomparedtoMAC(figure4.3a).Figure4.3depictsdatafrom
onerepresentativeexperimentforMAC,MMACandDCafter7days.Ingen-
eral,MMAChadahigherauto-fluorescence,mainlyintheFITC-channel,which
madeadjustmentoftheinstrumentsettingsnecessarytobringthebackground

39

Results4

fluorescentreading(isotypecontrol)intoaminimum.Nosignificantdifferences
couldbefoundfortheadherentlyculturedMAC+M-CSF,whichhadextra
M-CSF,comparedtoMAC,whichlackedextraM-CSF.Thus,MAC+M-CSF
arepresentonthebargraphsforillustration,butarenotfurthermentionedin
paragraphs.wingfollothe

4.4.1Antigensofmacrophagedifferentiation:CD14,CD16,CD71,
MAX.11andCD84

Allisolatedmonocytes(610±109mean±SDMFI),MAC(429±172)and
MMAC(861±323)expressedCD14atahighlevelincomparisontothedown-
regulationseeninmonocytetoDC(52±39)differentiation.Still,theCD14
expressiononMMACwashigherthanonMAC,whichwasalreadysignificant
after3days.MAC(73±37)andMMAC(169±98)expressedhigherlevels
ofCD16thandidthefreshlyisolatedmonocytes(38±27).ButtheCD16
expressiononMMACwasevenhigherthanonMAC,adifferencethatwaseven
morepronouncedonday3(17±14vs.197±116).ExpressiononDCwas
veryweak(7±8).FreshlyisolatedmonocytesdidnotexpressCD71,butit
wasdetectedonalldifferentiatedcells(MAC77±51,MMAC33±9,DC35±
31).LowCD84expressionwasfoundonmonocytes(27±6)andDC(43±25),
slightly,butnotsignificanthigherlevelswereseenonbothMAC(366±151)
andMMAC(285±128),whichexpressedequivalentlevelsofCD84already
onday3(263±22vs.257±26).MAX.11wasabsentonfreshlyisolated
monocytes,butexpressedbyalldifferentiatedcells.Ahighlevelwasseenon
MAC(518±172),comparedtosignificantlyloweramountsonMMAC(133±
94)andDC(186±108).Alreadybyday3,thelevelofMAX.11apparenton
MAC(429±136)exceedthatapparentonMMAC(76±65).
Fordetailsseefigures4.4and4.5.

40

4.4Phenotypecharacterizationwithmonoclonalantibodies

4.4.2CellsurfaceantigenofDCdifferentiation:CD1a
BesidethehighexpressionofCD1aonDC(1544±1397),onlylowlevelswere
observedontheothercells.Notably,CD1aexpressiononMMACwasnot
constant:MMACofsomedonorsexpressedlowlevelsofCD1awhilethoseof
othersdidn’t,resultingintheslightly,butnotsignificanthigherlevelsseenon
MMAC(22±28)comparedtoMAC(6±6).
4.5.figurealsoSee

4.4.3Functionalantigenesofantigenpresentingcells:CD40,
HLA-DRandHLA-ABCCD80/CD86,DC-SIGN,CD40wasnotdetectedonmonocytes.ThehighestlevelwasfoundforDC(62
±20),significantlylowerlevelsforMAC(27±8)andevenloweramountswere
presentonMMAC(15±7).DC-SIGNexpressionwaslowonmonocytes(28±
21)andMAC(11±9).DC(337±185)expressedthehighestlevelswhereas
levelsonMMAC(116±118)weremoderate,significantlydistinctfromthatof
MACandDC.ThechangesinDC-SIGNexpressionseemedtobealateevent
indifferentiationpathway,asnodifferencecouldbenoticedatday3(MAC62
±27,MMAC56±5).NoexpressionofCD80wasdetectedonmonocytes,
MACorMMAC,andonlyaverylowexpressionwasseenonDC(9±13).
Comparedtofreshlyisolatedmonocytes(24±10),CD86wasequallymore
intenselyexpressedonallcelltypes(MAC56±22,MMAC75±29,DC83±
106).FreshlyisolatedmonocytesexpressedthehighestlevelsofHLA-ABC(412
±409)andmodestlevelsofHLA-DR(81±57).Allcellsdown-regulatedHLA-
ABCupondifferentiation(MAC193±321,MMAC200±249,DC48±134).
ThelevelofHLA-DRexpressionwasstableonMAC(60±51),butup-regulated
onMMAC(174±132)andevenmoreonDC(434±294)respectively.
Seealsofigures4.6and4.7.

41

4

Results

Figure

pressed

4.3:

by

dotFSC/SSC

tligh

trol,

42

grey),

GaM:

wcytometricFlo

sisanaly

donoretativrepresenone

plotswithgatesettings;(b)

MMAC(unfilledblack)andDC

second

dyotiban

of

iationtdifferen

inysda7after

ohistogramserlaidv

(unfilled

ti-mouse.goat-an

grey).

tigensan

culture.

of

IgG1:

ex-

(a)

(filledCMA

ypIsote

con-

differ-

ngridu

tigensan

macrophage

of

pattern

Expression

4.4:

Figure

meanvaluesofmeanfluorescenceintensity;

differentiation-bars:

datenofys

Mono:

ts;erimenexp

errorbars:SD;smallnumbersabovethebars:numberof

treatedun

cytes,oonm

t0;

indicates

a

difference

pwith

<

0.05.

43

4.4

diesotiban

clonalmono

with

eypPhenot

haracterizationc

4

Results

Figure

during

4.5:

Expression

ofysdatdifferen

y;tensitinorescence

ts;erimenexp

0.05.

44

Mono:

pattern

of

macrophage

tigensan

differentiation-bars:meanvalues

SD;bars:errorsmall

treatedun

cytes,mono

and

1aDC

flu-meanof

numbersabovethebars:number

t0;

indicates

a

difference

with

p

of

<

Figure

4.6:

4.4

eypPhenot

Presence

of

haracterizationc

surface

molecules

with

clonalmono

ciatedasso

with

diesotiban

APC

during

differentstagesofdifferentiation-bars:meanvaluesofmeanfluorescence

intensity;errorbars:SD;smallnumbersabovethebars:numberofexperiments;

Mono:untreatedmonocytes,t0;indicatesadifferencewithp<0.05;ND:not

detected;

45

4

Results

Figure

4.7:

Expression

tiationdifferencelling

profile

of

CD86,

-ABCAHL

and

HLA-DR

dur-

alongthemonocyticlineage-bars:meanvalues

ofmeanfluorescenceintensity;errorbars:SD;smallnumbersabovethebars:

numberofexperiments;

with

46

p

<

0.05;

Mono:

treatedun

cytes,mono

t0;

indicates

differencea

4.5Phagocytosisassay

yassacytosisPhago4.5

Figure4.8:Phagocytosisassaywithfluorescentlatexbeads.a-his-
togramsofarepresentativeexperiment,cellsafter1hincubationwithlatex
beadsat37◦C(filledgraphs)oronice(unfilledgraphs);b-percentageof
phagocyticcells,correspondingtothemarkerina;c-MFIvaluesofthecells
underthemarker;bars-meanvaluesofnindependentexperiments;errorbars:
SD;indicatesadifferencewithp<0.05;

Thephagocyticcapacitywasexaminedusingthecells’abilitytoengulffluo-
rescentlatexbeads.Theanalyzedcellshadthefluorescencedistributionshown
infigure4.8a.Thefirstbigpeakcanbeattributedtoautofluorescenceofthe
cell.Thefollowingdistinctpeaksresultfromtheuptakeofone,twoandmore
beadsbyasinglecell.Becauseinthecontrolsamplessomecellsseemedtobind
atleastonebead,amarkerwassetformorethanonebead.DC(13%±8)did
hardlytakeuplatexbeads.Thenumberofcellsthatingestedmorethanone
beaddidnotdiffersignificantlybetweenMAC(48%±17)andMMAC(61%±
15)(seefigure4.8b).ButonaverageMMACdidincorporatesignificantlymore

47

Results4

beadspercellsthanMAC.ThisdifferenceisrepresentedbyahigherMFIfor
MMAC(2152±2022)comparedtoMAC(610±420)underthemarker(see
c).4.8figure

4.6ActivationofAPCwithbacterialendotoxin

ThestimulationandsubsequentcytokinesecretionofAPCisanimportantstep
fortheactivationoftheadaptiveimmunesystem.Severalmacrophageactiva-
tionpatternshavebeendescribed,leadingeithertopropagationorinhibition
oftheinflammatoryprocess.Here,thecellswereactivatedwiththe’classical’
macrophagestimulusLPSwithoutpriorpriming.

ThesupernatantsoftheunstimulatedMMACcontainedmoreIL-10than
MAC,morepronouncedonday3(253pg/ml±266vs.17pg/ml±20)com-
paredtoday7(133pg/ml±220vs.27pg/ml±35).ThecontentsofIL-10
inDC(46pg/ml±80)cultureswerecomparabletoMMACatday7.Upon
stimulation,allcellsproducedsignificantlymoreIL-10.MMAC(3212pg/ml±
2459)onday7exceedingthesimilarvaluesofMAC(1071pg/ml±909)and
DC(1670pg/ml±1913).TheIL-12secretionwaslowforallcultureconditions
beforeandafterstimulation,theonlysignificantrisewasseenafterthestimula-
tionofmonocytes(22pg/ml±15to52pg/ml±29).ThetestingforTNF-αin
unstimulatedculturesafter7daysshowedatendencytohigherlevelsinMMAC
(703pg/ml±642)andDC(1183pg/ml±195)comparedtomonocytes(90
pg/ml±52)orMAC(91pg/ml±46).However,thesedifferenceswerenot
statisticallysignificant.StimulationwithLPSresultedinhigherTNF-αvalues
forMMAC(4885pg/ml±2448),MAC(2702pg/ml±2828)andDC(3131
pg/ml±4368),alsowithnosignificantdifference.

48

4.6

Activation

of

APC

with

bacterial

xinendoto

Figure4.9:CytokineproductionuponstimulationwithLPS.Mono-
cytes(Mono)orfreshlyharvestedMAC,MMACandDCafter3and7culture
dayswereincubatedwithorwithout10ng/mlLPSfor24handthesupernatants
analyzedwithspecificELISAsforIL-10,IL-12p70andTNF-α.Bars-meanval-
uesofnindependentexperiments,errorbars:SD,smallnumbersonthebars:
numberofindependentexperiments,indicatesadifferencewithp<0.05.

49

Results4

4.7Allo-reactivTcellresponse

TheativationofTcellsbyAPCiscriticalforthefunctionoftheadaptive

immunesystem.ThestimulatorycapacityofthedifferentAPCswastestedina

MLR(3.8).ProliferativecapacityofallogenicTcellsincocultureswithMMAC

(ratio1:115347cpm±22343mean±SD,ratio1:28545cpm±13339)was

strongerthanincocultureswithMAC(ratio1:12389cpm±1493,ratio1:22347

cpm±1528).However,MMACdidnotinducethehighproliferationactivity

seenincocultureswithDC(ratio1:132463cpm±31481,ratio1:218792cpm

±8996).ThedifferencebetweenMAC,MMACandDCweresignificantfor

boththe1:1andthe1:2ratio.Proliferationofthestimulatororrespondercells

culturedalonewasnotseen.

50

4.7

onserespcellTAllo-reactiv

Figure4.10:AllogenicactivationofTcellsbyMAC,MMACand
DC.AllogenicTcellswereincubatedwitheithermacrophages,MMACorDC
atthedifferentstimulatorresponderratiosindicated.Onday6,3H-methyl-
thymindinewasadded.After24hoursthecocultureswereharvested.Values
indicatemeansofcell-associatedradioactivity+/-SDofquadruplicates.One
representativeoutof6experimentsisshown.

51

4

52

Results

Discussion5

Survival5.1

Theinvitrodifferentiationofmonocytestomacrophagesusuallyimplicates
anadherentculturecondition,whichiscausedparticularlybythenatureof
monocytestoadheretothecommonlyusedcultureplates.Onedisadvantageof
thisculturemethodisthatsuchdifferentiatedcellsfirmlysticktotheirtissue
plates,demandingquiteaforcetoharvestthemforfurtheranalyses.Thus,
Andreesenetal.(Andreesenetal.,1983)culturedmonocytessemi-adherenton
hydrophobicTeflonfoilsfromwheretheycouldbeharvestedwithoutdamaging
thecells’integrity.However,intheabsenceofhumanserum,theseTeflongrown
monocytesrequiredexogenousM-CSFforsurvival(Andreesenetal.,1990)in
contrasttomonocytesculturedfirmlyadherentonplastic(Haskilletal.,1988).
ItcouldlaterbeshownthatthequantityofM-CSFphysiologicallypresentin
humanserumisenoughtoassuremonocytesurvivalonTeflonfoils(Brugger
etal.,1991).Toelucidatetheimpactofadhesiononmonocytesurvivalor
apoptosis,anon-adherentculturesystemdevelopedbymembersofthisgroup
andpublishedbyArndtetal.(Arndtetal.,2007)wasusedforthisstudy
(3.3.3).IthasbeenshowedbyMondal(unpublisheddata)thatnon-adherently
culturedmonocytesundergoapoptosiseveninthepresenceofhumanserumas
aresultoftherapidlossoftheanti-apoptoticproteinMcl-1whichcouldbe
inhibitedbyadditionofexogenousM-CSF.

53

Discussion5

ThesimilarsurvivalratesofMMACandMAC(4.1)underlinetheproposed
anti-apoptoticeffectofM-CSFonmonocytesanddemonstratethatadhesion
isnotanabsoluteconditionformonocytesurvival.TheadditionalAnnexin
V/PIstainingconfirmedtheresultsofthetrypanbluestaininginthatnoearly
apoptoticcellswerecountedasvital.Focusingonapoptosis,responsiblefor
thelossofabout50%ofseededcellsunderallconditions,itwasremarkable
thatonlyabout10%deadcellswerefoundattheendofthecultureperiod.It
cannotberuledoutthattheremainingones,inboth,adherentandnon-adherent
conditions,ingestedtheapoptoticcells.Humanbloodmonocyteshavebeen
showntophagocytiseapoptoticcellsalreadyaftertheirthirddayincirculation
(Mikolajczyketal.,2009).

Differentiation5.2

Monocytesgiverisetoalldifferentkindoftissuemacrophages,DCsubsetsand
osteoclasts.Althoughspecificmonocytesubsetshavebeendescribedtodiffer-
entiatepreferablytoDC(Randolphetal.,2002),nospecificmonocytechar-
acteristicshavebeenfoundthatleadtothevariousmacrophagetypesinthe
differenttissues(Hume,2006,2008).Itseemsmorelikelythatthemonocytes
enterthetissuesrandomly,butretainacertainplasticitytoreacttothelocal
microenviromentratherthanexhibitingmultiplebutdistinctsubpopulations
(StoutandSuttles,2004).Asaninflammatoryprocessisneverstatic,mac-
rophagescouldchangetheirphenotypeaccordingtothestageoftheprocess
(Stoutetal.,2005,2009).DCsandosteoclastsmightbeterminallydifferen-
tiatedexceptions(Paluckaetal.,1998).Themicro-enviromentisdefinedby
stromalandlymphoidcells,ECM,andsolublefactors.Thisimplicatesthevery
importantroleforcell-cellandcell-substrateinteractions,influencingabroad
rangeofcellularcharacteristics,asreviewedbyShietal.(ShiandSimon,2006).

54

tiationDifferen5.2

Ontheotherhand,monocytesanddifferentiatedmacrophagesarealsofound
inbodyfluidslikeblood,peritoneal,pleuralandsynovialfluid.Afterhaving
establishingthatmonocytescansurviveinsuspensionculture,itremainedto
beproventhatthesecellshadalsodifferentiated.Infact,thesurvivingcellsin
thesuspensionculturedidnotremainstatic,rathertheyunderwenttremendous
.angeshcmorphological

Bylightmicroscopy,MMACinsuspensiondidnotshowpseudopodiaasdid
theadherentMACandMAC+M-CSF.ComparedtoMACandMAC+M-CSF
insuspension,theonlydistinguishingfeatureofMMACwerelittlecytoplasmic
protrusions.AsthischaracterizedonlyMMACandnotMAC+M-CSF,this
couldbecausedbythenon-adherentculturecondition.Furtherdemonstrated
MMACastrongcapacitytoattachtotheusualglassslides,asdidMACin
ension.susp

Byflowcytometry,MMACwereobservedaslarge,highlygranularcellswith
highvaluesintheFSCandSSC,accordingtothelightmicroscopicobservations.
ThetwodifferentpopulationsshownintheMMACFSC/SSCdotplotdidnot
revealanydistinguishingfeaturesinfurtheranalyses.Moreover,allanalyzed
cellswerefixedinparaformaldehyde.InthevitalstainingwithAnnexinV/PI,
wherecellswereprocessedimmediatelywithoutfixation,thisfeaturecouldnot
bereproducedandMMACshowedanuniquepopulationintheFSC/SSCdot
plot.Thusthiswasmostlikelyanartifactduetothefixationprocedure.MMAC
alsoweremoreauto-fluorescentcomparedtoMAC.Likewise,Njoroge(Njoroge
etal.,2001)describesanon-adherentcellpopulationinanadherentmonocyte
culture,resemblingMMACwithhighauto-fluorescenceandanabilityforre-
t.hmenattac

FlowcytometricanalysiswithmonoclonalantobodiesdemonstratedthatMMAC
didnotonlysurvive,butclearlyhaddifferentiatedintoamacrophage-likephe-

55

Discussion5

notype.Notably,theadditionalM-CSFintheMAC+M-CSFculturedid
notcauseanysignificantdifferencetoMAC.MMACupregulatedCD14,CD16,
CD71,CD84andMAX.11comparedtomonocytes,asdidMAC.Allthese
markersareknowntoberelatedtomonocytetomacrophagedifferentiation
1990).al.,et(Andreesen

However,MMACshowedaremarkableelevationofCD16expressionasan
earlyeventafter3days,atatime-pointwhenadherentMACshowedstilllow
CD16expression.AsimilarhumanCD14+CD16+monocytesub-population
wasdescribedinseveraldiseases,suchasAIDS(Cassoletal.,2006),rheumatoid
arthritis(Kawanakaetal.,2002b)andchronicrenalfailure(Kawanakaetal.,
2002a).ThisCD16+subsetisalsoexpandedaftertreatmentwithM-CSFin
bothhumans(Salehetal.,1995)andprimates(Munnetal.,1996).Thesecells
havebeendescribedasamorematuretypeofbloodmonocytes,differentiated
inthebloodundertheinfluenceofelevatedlevelsofserumM-CSFandother
inflammatorycytokines(Kawanakaetal.,2002b;SaionjiandOhsaka,2001).
SincethecultureconditionsofMMACresembledinpartthesituationinthe
blood,theseinvitroresultssuggestthatmonocytesinanon-adherentcondition
candifferentiateintoamacrophage-likeeffectorcell.

Eventhoughadhesionisnotanabsoluteconditiontomonocytedifferentia-
tion,itundoubtedlyhasaninfluenceonthecells’characteristics.Adherence,
forexampletocollagenmolecules,inducesCD14down-regulation(Jacobetal.,
2002).AsMMAClackadhesion,thesefindingsstandinagreementwiththe
resultsthatCD14expressioninMMACremainedatahighlevelincontrastto
thedown-regulationinadherentMAC.

TakenintoconsiderationthatbloodmonocytescangiverisealsotoDCs,
MMACwerecomparednotonlytoMACbutalsotoDC.Consideringthelight
microscopicphenotypeandthemarkersofmacrophagedifferentiationdiscussed

56

Differen5.2tiation

above,MMACshowedclearlyamacrophage-likephenotype.Inadditiontothat,
noexpressionoftheDC-markerCD1awasdetectedinanyculturecondition
DC.except

Toaccountfortheantigenpresentingfunctionoftheevaluatedcells,the
expressionpatternofantigenpresentingandcostimulatorymoleculesthatcor-
relatewiththeirfunctionasAPCsweredetermined.Upondifferentiationfrom
themonocyte,theexpressionofMHCclassImoleculesdiminishedequallyin
MMACandMAC.Incontrast,thelevelofMHCclassIImoleculeswasstable
inMAC,butincreasedinMMACandtoanevenhigherdegreeinDC.Nosig-
nificantdifferencewasfoundforthecostimulatorymoleculesoftheB7family.
CD80wasveryweaklyexpressedonDCandnotdetectedonanyoftheother
cells.CD86wasequallyupregulatedonallcellsinthedifferentiationprocess
frommonocytes.However,significantdifferencewasfoundintheexpression
oftheTcelladhesionmoleculesCD40andDC-SIGN.Bothmoleculeswere
presentmostlyonDC.ButwhereasCD40expressionwaslowerinMMACthan
onMAC,theoppositewasfoundforDC-SIGN,wheretheexpressionwashigher
onMMACthanonMAC.ThecharacterizationofDC-SIGNasaDCmarkerhas
beenchallengedbythediscoverythatinpatientswithleprosy,themacrophages
expressDC-SIGNandCD16,whereastheDC,characterizedbyCD1lackfDC-
SIGN(Krutziketal.,2005).Morerecently,CD14+DC-SIGN+macrophages
inthelaminapropriaweredescribedaspotentantigenpresentigcellsandare
supposedtoplayanimportantroleinmaintainingtheimmunologicalbalance
inthegut(Kamadaetal.,2009).

Conclusively,thephenotypeofMMACisclearlymacrophage-like,withsome
APC-relatedmarkersmoreaccentuated.Inpart,MMACresemblethepheno-
typeofperitonealmacrophages,whichwasreportedforotherM-CSFdifferen-
tiatedmonocytesbefore(Xuetal.,2007;Akagawaetal.,2006).Inpatientsun-

57

Discussion5

dergoingperitonealdialysis,themonocytesinvadingtheperitoneumexpressed
highCD14,CD16,CD71andHLA-DR(Andreesenetal.,1990;Brauneretal.,
1998).TheM-CSFlevelsintheperitonealfluidarehighereveninhealthyin-
dividuals(Weinbergetal.,1991),butriseinaccordancetohighM-CSFblood
levels,forexampleinpatientsundergoingdialysis(SaionjiandOhsaka,2001).

cytosisPhago5.3

Endocytosisisperformedbyalleucaryoticcellsinformofuptakeofliquidsand
singlemoleculesintosmallplasmamembraneenclosedvesicles,whichisreferred
toaspinocytosis.Theengulfmentofbigparticleslikebacteriaisrestrictedto
professionalphagocytes.Allantigen-presentingcellsarealsophagocytesbut
thevarioussubtypesdifferconsiderablyintheirphagocyticcapacity.DCsare
verygoodantigenpresentingcellsbutpoorphagocytes,macrophagesinstead
areverygoodphagocytesbutweakantigenpresentingcells.Macrophagesare
distinguishedbytheirabilitytoingestlargeparticles.Theyevencanmerge
togetherandformmultinucleargiantcellsinordertodigestalargerforeign
.dyob

Functionally,MMACappeartobeamoreefficientMAC.Itsphagocyticca-
pacityismorerobustthanthatofanadherently-generatedMAC.Againthis
couldbeduetotheextraM-CSFintheculturemedia,asM-CSFisknownto
enhancephagocytosis(Akagawa,2002;Nemunaitis,1998).Moreover,thestrong
expressionofCD14,CD16,andDC-SIGNonMMAC,whichallareinvolvedin
antigenuptake,couldalsoaccountforthiscaracteristic.Therewasnouptake
ofbeadsinDCasexpected.

58

ductionropCytokine5.4

ductionproCytokine5.4

Anotherkeystonetotheactivationoftheimmunesystemiscell-cellinterac-
tionviacytokines.UponstimulationwithLPS,allcellssecretedhighamounts
ofIL-10andTNF-α,confirmingpreviousresultsofthisgroupforMACand
DC(Ammonetal.,2000).MMAChoweverproducedmoreIL-10andTNF-α
comparedtoMACuponstimulation.Incontrasttomonocytes,whichsecreted
IL-12followingstimulation,noincreaseofthiscytokinewasfoundinMACand
C.MMAMMACconstitutivelyproducedIL-10.Accordingly,Smithetal.(Smith
etal.,1998)foundthesamecytokinesecretionpatternwithhighIL-10andlow
IL-12secretionforMAC,DCandMAC+M-CSF.Theyfoundthatconstitutive
IL-10secretionofM-CSF-inducedmacrophagesdidnotaccountforlowIL-12
levels,asitoccurredalsointhepresenceofinhibitoryanti-IL-10-antibodies.
ConstitutiveIL-10secretionwasalsofoundforM-CSFinducedmacrophages
thatalsowerehighlyactiveinphagocytosis(Xuetal.,2006),althoughinthis
report,thesemacrophagesdidnotproduceTNF-αasdidMMAC.Conclusively
theCD14+CD16+monocyteswereidentifiedasmainproducersofIL-10inthe
blood(Skrzeczy´nska-Monczniketal.,2008).

5.5Tcellstimulatorycapacity

CoculturesofallogenicTcellswithMMACcausedaTcellproliferationmuch
morepronouncedthanwithMAC,butnotaspotentaswithDC.Thisisin
accordancewithhigherexpressionofMHCclassIImoleculesandDC-SIGNon
MMAC.DC-SIGNisimportantfortheearlyTcell-APCinteractionandresults
inprofoundTcellstimulation(Geijtenbeeketal.,2002;Gijzenetal.,2007).
DC-SIGNcanbeinducedbycyclicnucleotides,whichinhibitDCgeneration

59

Discussion5

andfavoracelltypeexpressinghighMHCclassIIandCD86molecules,and

ahighproliferativeTcellresponseinMLR(Giordanoetal.,2003).Theweak

proliferationofTcellsincoculturewithMACisinlinewiththeinhibitory

proprietiesofnon-activatedMAC,whichhaverecentlybeenstated(Hovesetal.,

2006).TheM-CSFinMMACculturescouldberesponsibleforamoreactivated

celltype,asM-CSFseemstofavorthealternativeactivationpathway(Martinez

etal.,2006).AlsoIL-10couldhaveaninfluenceontheTcellactivation,asit

inhibitsTH1response,favoringthegenerationofTH2cells(Contietal.,2003).

Interestingly,MMACshowedhigherphagocytosisratesandhigherstimula-

torycapacitytowardTcells,twoitemsthatgenerallyaredivergent.Monocytes

loosetheirphagocyticcapacityandgainbetterantigenpresentingupondiffer-

entiationtowardsDC.

60

6Conclusion

Theresultsofthisstudyestablishthatmonocyteadhesion,whichundoubtedly

isresponsibleformanysubstantialchangesinthemonocyte,isnotessential

formonocytesurvivalanddifferentiationtoamacrophage-likecell.Asmono-

cytesandtheirdifferentiatedformsarefound,notonlyintheextracellular

matrix,butalsoinsuspensioninthebloodandtheperitonealfluid,thiscan

helptounderstandtheirfunctionunderthesecircumstances.Manydetailshave

beenrevealedforthedifferentmonocytesubsets.Thisstudygivesnowfurther

substanceforthehypothesisthattheCD16+monocytesareamoremature

subset,differentiatedinthebloodundertheinfluenceofM-CSF.Moreover,

MMAChaveasauniquefeaturetheupregulationofboth,phagocytosisand

Tcellstimulatorycapacity,twofunctionsthatusuallyarenotup-regulatedin

parallel.WhetherthisnewmacrophagetypehasitsownplaceintheMPS,or

itstandsforanotherpossiblephenotypeofachameleonlikecelltype,knownas

themacrophage,remainstobeelucidated.

61

6

62

Conclusion

Zusammenfassung7

MonozytenunddieausihnenhervorgehendenMakrophagenunddendritischen
Zellen(DC)habenimImmunsystemdesMenscheneinebesondereRolleals
BindegliedzwischendemangeborenenunddemadaptivenImmunsystem.In
vielenExperimentenzurDifferenzierungvonMakrophagenausMonozytener-
wiessichderWachstumsfaktorM-CSFals¨uberlebensnotwendig.Dabeiwurden
jedochdieMonozytenstetsadh¨arentkultiviert,sodassderEinflussvonM-CSF
aufdieDifferenzierungnieunabh¨angigvondenAuswirkungenderAdh¨asion
betrachtetwerdenkonnte.M-CSFkommtauchinnennenswertenMengenim
BlutgesunderIndividuenvorundkannbeibestimmtenKrankheitenindeutlich
h¨oherenKonzentrationennachgewiesenwerden.DaMonozytenaufihremWeg
vomKnochenmarkzumGewebeeinigeZeitimBlutzirkulierenundsichdort
unterdemEinflussvonM-CSFver¨andernk¨onnten,wurdeversuchtdieseUm-
gebunginvitronachzuformen.Daf¨urwurdenhumaneBlutmonozytenineiner
SuspensionzurVermeidungvonZell-ZelloderZell-Substrat-Kontaktenundun-
terZugabevonrekombinantemM-CSFineinerrotierendenFlaschekultiviert.
DiesedurchM-CSFinduziertenMakrophagen(MMAC)exprimiertensigni-
fikanth¨ohereWertederOberfl¨achenantigeneCD14,CD16,HLA-DRundDC-
SIGNimVergleichzuadh¨arentgeneriertenMakrophagen(MAC).ImVergleich
zuDCzeigtensieniedrigereWertederMarkerCD1aundDC-SIGN,jedoch
h¨ohereWertederMarkerCD84undHLA-ABC.NachStimulationmitLipopo-

63

Zusammenfassung7

lysaccharid(LPS)produziertenMMAC,analogzudenDC,Interleukin(IL-)10

undTumornekrosefaktor(TNF-)alpha.ImVergleichzuMACzeigtenMMAC

einedeutlicherh¨ohtePhagozytoserateundTZellstimulierendeEigenschaften.

ZusammenfassendzeigtensichMMACalsMakrophagen-¨ahnlicheZellenmit

agten¨ausgepr

ten.

64

haften,Effektoreigensc

die

cjedoh

thnic

an

die

der

DC

-hheranreic

endixApp

mediaandSolutions7.1

V-Annexinbuffer:binding

medium:DC

Fe:fixativCSA

FACSwashingbuffer:

10ml1MHEPES(Sigma)
8.12gNaCl(Merck)
0.28gCaCl2(Merck)
ad1000mlultrapurewater

500mlRPMI1640(Biochrom)
5mlL-Glutamine(Biochrom)
5mlSodiumpyruvate(100mM,Gibco)
5mlNon-essentialaminoacids(100x,Gibco)
2mlVitamins(100x,Gibco)
2.5mlPenicillin-Streptomycin(104U/ml,Gibco)
0,5ml2-Mercaptoethanol(50mM,Gibco)
add10%fetalcalfserum(FCS,PAA)rightbeforeuse

AN)(PPBSml50050mlParaformaldehyde(10%w/vinPBS,Sigma)

AA)(PPBSml5005mlImmunoglobulin(60mg/ml,SandozPharma)
5mlSodiumacide(10%w/vinPBS,Sigma)

65

endixApp

medium:Macrophage

medium:MLR

500mlRPMI1640(Biochrom)

5mlL-Glutamine(Biochrom)
2.5mlPenicillin-Streptomycin(104U/ml,Gibco)

add2%pooledAB-groupserum(Cambrex)rightbeforeuse

500mlRPMI1640(Biochrom)

5mlL-Glutamine(Biochrom)

5mlSodiumpyruvate(100mM,PAN)

5mlNon-essentialaminoacids(100x,PAN)

2mlVitamins(100x,PAN)
2.5mlPenicillin-Streptomycin(104U/ml,Gibco)

0,5ml2-Mercaptoethanol(50mM,Gibco)

add10%pooledmalehumanABgroupserum(PAN)rightbeforeuse

Trypanbluesolution:0.4%(w/v)

66

0.9in(w/v)%

(Sigma)bluerypanT

NaCl(Merck)inultrapurewater

allofList7.2companies

escnBioscieBDPharmingenBDCoulterkmanBeckmannBeckinsonBecton-DichromcBiobrexCamCorp.CetusCostarDiacloneeBioscienceendorfEppEssexalconFBDujifilmFcoGibBio-oneGreinerHareusAnalyticaHartmannhunotecImmolsunotoImmhInnotechunoResearcImmksonJackMercAAP

companiesallofList7.2

USACA,Jose,SanUSACA,go,DieSanUSACA,ullerton,FyGermanh,MunicUSAA,CJose,SanyGermanBerlin,USANJ,Rutherford,EastUSACA,Emeryville,UKbridge,CamranceFcon,Besan¸USACA,Diego,SanyGermang,burHamyGermanh,MunicyGermanerg,HeidelbDu¨sseldorf,Germany
Karlsruhe,Germany
Frickenhausen,Germany
yGermanHanau,yGermanh,MunicranceFMarseille,yGermanythe,riesoFSwitzerlandon,DottikUKSuffolk,yGermanDarmstadt,AustriaLinz,

67

endixApp

68

ANP

eliClusterP

Pharmacia

olyscienceP

SandozGAPharma

Sigma

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allacW

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yGermanDeisenhof,

Chicago,USA

UKKeynes,Milton

urku,TFinnland

yGermanJena,

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on

umanh

enslaufLeb

Pers¨onlicheDaten:
meramRiedhMatthiasSebastiangeborenam10.Februar1981inLandshut

ulbildung:hSc1987-1992BesuchderJosefineHaasGrundschuleinBurglengenfeld
1992-2000BesuchdesJohannMichaelFischerGymnasiumsinBurg-
lengenfeldmitErreichenderAllgemeinenHochschulreifeim
2000Juni

Zivildienst:2000-2001AbleistungdesWehrersatzdienstesalsZivildienstleistender
imgensburg,pflegerischenAbteilungBereicf¨urhHdes¨amatologieKlinikumsundderOnkUnivologieersit¨atRe-

ulstudium:hhsccHo2001-2008StudiumderHumanmedizinanderUniversit¨atRegensburg
9/2003¨ArztlicheVorpr¨ufung
2/2007-2/2008PraktischesJahr
6/20082.Abschnittder¨ArztlichenPr¨ufung
ArztalsApprobation7/2008

PraktischeT¨atigkeit:
2/2004-3/2004FamulaturKrankenhausBurglengenfeld,InnereMedizin
8/2004FamulaturKrankenhausBurglengenfeld,Chirurgie
9/2005FamulaturinHelsinki,Finnland,Allgemeinmedizin
8/2006-9/2006FamulaturKlinikSt.HedwigRegensburg,Kinder-undJu-
gendmedizin

BeruflicheT¨atigkeit:
10/2008-6/2009AssistenzarztinderchirurgischenAbteilungdesEvange-
lischenKrankenhausinRegensburg
7/2009-12/2009AssistenzarztinderkinderchirurgischenAbteilungdesKin-
derkrankenhausesSt.MarieninLandshut
seit1/2010Assistenzarztinderp¨adiatrischenAbteilungdesKinder-
krankenhausesSt.MarieninLandshut

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Danksagung

MeinDankgiltPhDKrishnaMondalf¨urdieinteressanteThemenstellungmeiner
Dissertation.Dar¨uberhinausstandsiemirsowohlw¨ahrendderZeitimLaborals
auchbeimVerfassendiesesManuskriptesjederzeitmitgutemRatzurSeite.Sie
f¨uhrtemichmotivierendandaswissenschaftlicheArbeitenheranundgestaltete
meineZeitimLabor¨außerstangenehmundlehrreich.
IchdankeProf.Dr.MichaelRehli,dassichdieseArbeitunterseinerLeitung
durchf¨uhrenkonnte,f¨urdiewertvolleUnterst¨utzungbeiderGestaltungund
AuswertungderVersucheunddiefreundlicheAufnahmeinseineArbeitsgruppe.
EbensogiltmeinDankProf.Dr.MichaelMelterf¨urdiehilfreicheUnterst¨utzung
beiderschriftlichenAusarbeitung.DankeauchandiegesamteMakrophagen-
gruppe,dirmiroft¨uberdievielenkleinenFallendesLaboralltagshinwegge-
holfenhat.InsbesondereMTAAlexandraM¨ullerbinichf¨urihreHilfebeider
IsolationderMonozytensehrdankbar.F¨urdieEinf¨uhrungimUmgangmitdem
Durchflusszytometersowief¨urdievielenkonstruktivenDiskussionendankeich
Dr.SabineHoves.AußerdemdankeichallenMitarbeiterinnenundMitarbei-
ternausdemForschungsbauH1derUniversit¨atRegensburg,diemirbeider
ErstellungdieserArbeitgeholfenhaben.NichtzuletztgiltmeinDankmeiner
FreundinMarieUlrichundmeinerFamilie,diemichaufdemlangenWegvon
denerstenVersuchenbiszumfertigenManuskriptimmerwiedermotivierten
undohnederenUnterst¨utzungdieseArbeitnichtm¨oglichgewesenw¨are.

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