Effect of low ethanol concentrations on the production and stability of interferon {γ [gamma] [Elektronische Ressource] / vorgelegt von Senja Sauter

Senja

Sujets

• Biologie

Informations

• Publié par: universitat_hohenheim
• Publié le: 01 janvier 2008
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Effectoflowethanolconcentrationsonthe
productionandstabilityofInterferon γ


DissertationzurErlangungdesDoktorgrades
derNaturwissenschaft(Dr.rer.nat)

FakultätNaturwissenschaften
UniversitätHohenheim

InstitutfürErnährungsmedizin180b
FachgebietErnährungsphysiologie


vorgelegtvon
SenjaSauter


ausGeislingen/Steige
März2008





Dekan: Prof.Dr.H.Breer
Berichterstatter,1.Prüfer: Prof.Dr.Ch.Bode
Mitberichterstatter,2.Prüfer: Prof.Dr.A.Parlesak
3.Prüfer: Prof.Dr.T.Grune


Einreichtam 02.04.2008
TagdermündlichenPrüfung: 01.07.2008











DievorliegendeArbeitwurdeam03.06.2008vonderFakultätNaturwissenschaftender
UniversitätHohenheimals“DissertationzurErlangungdesDoktorgradesder
Naturwissenschaften“angenommen.







Thisstudywasfinanciallysupportedbythe





















Tableof contents

Tableofcontents V
Listofabbreviations IX
1 Introduction 1
1.1 Alcohol–damagetotheimmunesystem 3
1.1.1 Humandiseasesrelatedtoalcoholabuse 4
1.1.2 Alterationsoftheimmunesystem 6
1.1.3 Mechanismofalcohol8inducedmodulationofimmuneresponse 8
1.1.4 EffectsofethanolonIFN8γproduction 14
1.2 Interferonγ–signals,mechanismsandfunctions 23
1.2.1 CharacteristicsofInterferonγ 23
1.2.2 Interferonγreceptor 24
1.2.3 IFN8γ8associatedsignaltransduction 25
1.2.4 MoleculareffectsofInterferonγ 30
1.2.5 Interferonγ–roleinhostdefense 33
1.2.6 Interferonγindiseaseandtherapeuticapplication 35
1.3 MechanismsinregulationofInterferonγexpression 37
1.3.1 CellularsourceofInterferonγ 37
1.3.2 RegulationofInterferonγproductionbyInterleukin12 39
1.3.3 Regulatorypathways 42
1.3.4 RegulationofInterferonγgeneexpression 43
2 Objectives 49
3 MaterialandMethods 51
3.1 Material 51
3.1.1 CelllineandPBMC 51
3.1.2 Chemicalsandreagents 51
3.1.3 Equipmentandexpendableitems 57
3.2 Software 60
3.3 Methods 61
3.3.1 Cellsandcellcultivation 61
3.3.2 Cellincubationexperiments 63
3.3.3 Cellviabilitymeasurement 65
3.3.4 ELISA–Enzyme8LinkedImmunosorbentAssay 67
3.3.5 Flowcytometry(FACS) 70
3.3.6 Samplepreparation 72
3.3.7 Proteinassay 75
3.3.8 Westernblotting 76
3.3.9 RT8PolymeraseChainReaction(RT8PCR) 80
3.3.10 Statisticalevaluation 84
VI TABLEOF CONTE N TS

4 Results 85
4.1 InterferonγproductionbyhumanPeripheralBloodMononuclearCells 85
4.1.1 EffectofPHAactivation,LPSstimulationandethanoltreatmentonIFN8γ
productionbyhumanPeripheralBloodMononuclearCells(PBMC) 85
4.2 OthercytokinesproducedbyhumanPeripheralBlood
MononuclearCells 89
4.2.1 EffectofPHAandLPSstimulationontheproductionofseven
cytokinesbyhumanPeripheralBloodMononuclearCells(PBMC) 89
4.2.2 Effectofethanolonsevencytokinesproducedby
PeripheralBloodMononuclearCells(PBMC) 91
4.3 ProliferationofhumanPeripheralBloodMononuclearCells(PBMC) 103
4.3.1 EffectsofPHAactivationandethanoltreatmentonproliferation
ofhumanPeripheralBloodMononuclearCells(PBMC) 103
4.4 FluorocytometricanalysisofhumanPeripheralBlood
MononuclearCells(PBMC) 106
4.4.1 EffectofPHA8activationonmorphologicalparametersofPeripheralBlood
MononuclearCells(PBMC) 106
4.4.2 ExpressionofClustersofDifferentiation(CD)inPBMC 106
4.4.3 TheeffectofPHAstimulationoncellcompositionofPeripheralBlood
MononuclearCells 107
4.4.4 EffectofPHAandethanolonIL812receptordensityonPBMC 110
4.5 InterferonγproductionbyNK@92cells 113
4.5.1 EffectofIL812ontheInterferonγproductionbyNK892cells 113
4.5.2 EffectofethanolonIL8128inducedIFN8γproductionbyNK892cells 116
4.6 Effectofethanolincubationoncellviability 119
4.7 IL@12@inducedactivationoftranscriptionfactorsinNK@92cells 120
4.7.1 KineticsofIL8128inducedSTAT4phosphorylation 120
4.7.2 EffectofethanolonIL8128inducedSTAT4phosphoryaltion 121
4.7.3 EffectofethanolonSTAT4translocation 122
4.7.4 IL8128inducedIFN8γproductionbyNK892cellsisntomediatedbyAP81 123
4.8 IL@12@inducedIFN@γmRNAexpressioninNK@92cells 124
4.8.1 KineticsofIL8128inducedIFN8γmRNAexpressioninN K892cells 124
4.8.2 EffectofethanolonIL8128inducedIFN8γmRNAexprsesion
inNK892cells 126
4.9 IntracellularIFN@γconcentrationofNK@92cells 128
4.9.1 EffectofIL812onintracellularIFN8γconcentrationofNK892cells 128
4.9.2 EffectofethanolonintracellularIFN8γconcentrationofIL812
stimulatedNK892cells 130
4.10 DetectabilityofIFN@γinthepresenceethanol@treatedNK@92cells
ortheircell@freesupernatant 133
4.11 Ethanol@inducedsecretionofsolubleIFN@γreceptor 138
4.11.1 EffectofIFN8γreceptorantibodyontheethanol8mediatedchangeofIFN8γ
concentrationinNK892cellsupernatant 138
4.11.2 EffectofethanolontheIFN8γreceptor1concentrationin
NK892cellsupernatant 140
4.12 Effectofaproteaseinhibitorcocktailontheethanol@mediated
changeofIFN@γconcentrationinNK@92cellsupernatant 142



TABLEOF CONTE N TS VII
5 Discussion 145
5.1 DifferentcellsasamodeltoinvestigateIFN@γexpression 145
5.1.1 IsolatedhumanPeripheralBloodMononuclearCells(PBMC) 145
5.1.2 NK892cellsinculture 146
5.2 Stimulationoflymphocytes 147
5.2.1 HumanTcellactivationwithphytohemagglutinin 147
5.2.2 InductionofcytokineproductioninhumanTlymphocytesby
lipopolysaccharide(LPS) 148
5.2.3 EnhancementofNK892cellactivitybyInterleukin12 149
5.3 Ethanol@inducedchangesincellactivationandcytokinepattern
seeninactivatedPBMCandNK@92cells 153
5.3.1 Ethanolincellculture:maintenanceofconstantethanolconcentrations 153
5.3.2 InfluenceofethanolonPeripheralBloodMononuclearCells 154
5.3.3 Ethanol8inducedsuppressionofIFN8γexpressioninN K892cells 161
5.3.4 Cellviability 163
5.3.5 Physiologicalrelevanceofappliedethanolconcentrations 163
5.3.6 EthanoleffectsontranscriptionalactivationofIFN8γinNK892cell 164
5.3.7 Posttranscriptionaleventsinfluencedbyethanol 168
5.4 Outlook 174
6 Summary 175
7 Zusammenfassung 177
References 179
Publications 207
CurriculumVitae 209
Acknowledgements 211
Declaration/EidesstattlicheErklärung 213 Listof abbreviations

% percent
°C degreecentigrade
‰ permille
ADCC antibodydependentcellularcytotoxicity
ADH alcoholdehydrogenase
ALDH aldehydedehydrogenase
ANOVA analysisofvariances
AP activatedprotein
APC antigenpresentingcell
APS ammoniumpersulfate
ATF activatingtranscriptionfactor
BCG Mycobacteriumbovis
bidest. doubledistilled
bp basepairs
BSA bovineserumalbumin
cAMP cyclicAMP
CD clusterofdifferentiation
cDNA complementaryDNA
ConA ConcanavalinA
CREB cAMPresponseelementbindingprotein
CREB8ATF CREB8activationtranscriptionfactor
C thresholdcycleT
d day
dd doubledistilled
DEPC diethylpyrocarbonate
DNA deoxyribonucleicacid
DMEM Dulbeccomodifiedeaglemedium
dNTP desoxyribonukleosidtriphosphate
DTT dithiothreitol
EDTA ethylenediaminetetraaceticacid
EGF epidermalgrowthfactor
ELISA enzyme8linked8immunosorbentassay
EMSA electrophoreticmobilityshiftassay
EPO erythropoetin
EtOH ethanol
FACS fluorescenceactivatedcellsorting
FCS fetalcalfserum
Fig. figure
FI fluorescenceintensity
g gram
GADD growtharrestandDNA8damageinducible
GAF γ8activatingfactor
GAPDH glycerinaldehyde3phosphatedehydrogenase
GAS IFN8γ8activatedsequence
GH growthhormoneX LISTOFA BBRE VI AT IONS

GRB2 growthfactorreceptor8boundprotein2
h hour
H SO sulfuricacid2 4
HCl hydrochlorideacid
HEPES (N8[28hydroxylethyl]piperazine8n’8[28ethaneslfuonicacid])
HRP horseradishperoxidase
HS horseserum
ICAM intercellularadhesionmolecule
ICSPB IFNconsensusbindingprotein
IFN interferon
IFNAR1 IFN8α8receptor
IFNGR2 IFN8γ8receptor
IFN8γ interferongamma
Ig immunoglobulin
IgG immunoglobulinG
IL interleukin
IL12Rβ1 IL8128receptorsubunitβ 1
IL12Rβ2 IL8128receptorsubunitβ 2
iNOS induciblenitricoxidesynthase
IRAK insulinreceptoractivatedkinase
IRF81 IFN8regulatorfactor1
IRS insulinreceptorsubstrate
IκB inhibitorofκB
Jak januskinase
JH Jakhomologous
JNK c8Jun8NH28terminalkinase
KCl potassiumchloride
KH PO potassiumdihydrogenphosphate2 4
l liter
LPS lipopolysaccharide
M mol
MAPK mitogen8activatedproteinkinase
MEF2C myocyteenhancerfactor2c
MEK MAPK8kinase
MEKK MAPKK8kinase
MEOS microsomalethanoloxidizingsystem
MHC majorhistocompatibilitycomplex
MKK Mitogen8activatedproteinkinasekinase
mRNA messengerRNA
MyD88 myeloiddifferentiationprimaryresponsegene88
n.c. negativecontrol
Na CO sodiumcarbonate2 3
NaCl sodiumchloride
NaHCO sodiumhydrogencarbonate3
NFAT nuclearfactorofactivatedTcells
nuclearfactorkappaBNFκB
NKcells naturalkillercells
NKSF naturalkillercellstimulatoryfactor
OD opticaldensity
p.c. positivecontrol
p38 serin8threonin8kinase