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Tolerance induction in the liver after
T and NKT cell activation
Toleranzinduktion in der Leber nach T- und NKT-
Zellaktivierung
Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
vorgelegt von
Annette Erhardt
aus Münchberg Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 11.07.2008
Vorsitzender der Prüfungskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Gisa Tiegs
Zweitberichterstatter: PD Dr. Robert Slany













































M einem D addy CONTENTS
CONTENTS
Publication List
Abbreviations
1 Introduction ........................................................................7
1.1 The liver: anatomy, physiology, and diseases........................................... 7
1.2 Animal models of immune-mediated liver injury ..................................... 11
1.3 Immunological tolerance and the outstanding role of the liver.............. 14
1.4 General overview of regulatory T cell subsets......................................... 17
1.5 Gender-specific differences in autoimmunity.......................................... 25
1.6 Aims of this study....................................................................................... 28
2 Materials and Methods.....................................................30
2.1 Mice.............................................................................................................. 30
2.2 Animal treatment ........................................................................................ 31
2.2.1 Treatment schedules and Con A administration ................................ 31
+2.2.2 Depletion of cells (KCs and CD25 T )........................................... 31 regs
2.2.3 Blockade of the IL10-receptor............................................................ 32
2.3 Sampling of material .................................................................................. 32
2.4 Isolation of cells.......................................................................................... 33
2.4.1 Isolation of primary hepatocytes ........................................................ 33
2.4.2 Isolation of intrahepatic mononuclear cells and splenocytes ............. 34
+ +2.4.3 Isolation of CD4 CD25 T and responder cells ............................. 34regs
2.5 In vitro experiments.................................................................................... 36
2.5.1 Co-culture of responder cells and T .............................................. 36 regs
2.5.2 Specific inhibition of cAMP by a PKA inhibitor ................................... 36
2.5.3 CFSE labelling................................................................................... 37 CONTENTS_______________________________________________________
2.5.4 Neutralization of IL-10........................................................................ 37
2.6 Analysis of plasma transaminases ........................................................... 38
2.7 Real time RT- PCR ...................................................................................... 38
2.8 Cytokine determination by enzyme-linked immunosorbent assay
(ELISA)....................................................................................................... 39
2.9 Flow cytometry ........................................................................................... 40
2.10 Immunofluorescent staining and confocal laser imaging..................... 41
2.11 Haematoxylin/eosin staining of liver sections ....................................... 41
2.12 Analysis of hCD2-ΔkTβRII mice by tail biopsies .................................... 42
2.13 Statistical analysis.................................................................................... 42
3 Results ..............................................................................43
3.1 Characterization of Con A-induced tolerance.......................................... 43
3.1.1 Con A pretreatment results in reduction of transaminase levels after
Con A rechallenge ........................................................................... 43
3.1.2 Con A pretreatment ameliorates Con A-induced necrosis................. 44
3.1.3 Induction of an anti-inflammatory cytokine profile.............................. 45
3.1.4 Determination of the frequency of cell subpopulations ...................... 48
3.1.5 Investigation of the time point of tolerance induction ......................... 51
3.1.6 Induction of Con A tolerance ex vivo ................................................. 54
3.2 Identification of IL-10 as central mediator of Con A tolerance ............... 55
3.2.1 Loss of Con A-mediated tolerance in male IL10 KO mice and after
anti-IL10R-treatment........................................................................ 55
3.2.2 Detection of gender-related differences in IL10 KO mice .................. 60
3.3 Importance of Kupffer cells as IL-10-producing cells ............................. 61
+ +
3.4 Involvement of CD4 CD25 regulatory T cells during Con A tolerance . 62
3.4.1 Identification of T as source of IL-10 ............................................. 62 regs
3.4.2 Special characteristics of tolerized T ............................................. 65 regs CONTENTS
3.4.3 Therapeutic potential mediated by tolerized T .............................. 72 regs
3.4.4 Dispensability of IL-10 on T activity in vitro .................................... 74 reg
3.5 Oppositional regulation of IL-10 and IL-17 during Con A tolerance...... 77
3.6 Relevance of NKT cells in Con A hepatitis and during tolerance........... 79
4 Discussion ........................................................................81
+ + +4.1 The role of IL-10-producing CD4 CD25 FoxP3 regulatory T cells......... 81
4.1.1 ...as cellular immunotherapy in vivo................................................... 81
4.1.2 ...as suppressor cells in vitro.............................................................. 86
4.2 The conversion of Kupffer cells from type I to type II MΦ....................... 91
4.3 Proposed mechanism of Con A-mediated tolerance............................... 93
4.4 Outlook ........................................................................................................ 95
5 Summary...........................................................................98
References
Deutschsprachige Zusammenfassung
Danksagung
Lebenslauf PUBLICATION LIST
PUBLICATION LIST
Abstracts:
Biburger M, Erhardt A, Tiegs G. Concanavalin A induced tolerance in a murine
+
model of immune mediated hepatitis is a multifactorial process involving CD4
+
CD25 regulatory T cells but not depending on Interleukin-10. Immunobiology
2005; 210(6-8):400 (Abstract E.7)
Biburger M, Erhardt A, Tiegs G. The central role of tumor necrosis factor in the
murine –galactosylceramide model of immune mediated hepatitis.
Immunobiology 2005; 210(6-8):493 (Abstract L.20)
Erhardt A, Biburger M, Tiegs G. Concanavalin A-induzierte Toleranz im Maus-
+ +Immunhepatitis-Modell wird unter Beteiligung von CD4 CD25 regulatorischen T-
Zellen, Kupffer-Zellen und IL-10 vermittelt. Z Gastroenterol 2006; 44:128 (Abstract
4.38).
Erhardt A, Biburger M, Tiegs G. Con A-induced tolerance involves T , Kupffer regs
cells, IL-10 and non-responsiveness in IL-2 producing cells. J Hepatol 2006; 44
(Suppl 2): S9 (Abstract 16).
Erhardt AL, Biburger M, Tiegs G. Untersuchungen zum Zeitverlauf der
Toleranzinduktion im Concanavalin A-Immunhepatitis-Modell. Z Gastroenterol
2007; 45:123 (Abstract 4.09).
Erhardt A, Biburger M, Tiegs G. IL-10 und regulatorische T-Zellen sind die
Hauptmediatoren der Concanavalin A-induzierten Toleranz im Maus-
Immunhepatitis Modell. Z Gastroenterol 2008; 46: 142 (Abstract 4.51).
DD PUBLICATION LIST
Erhardt A, Biburger M, Tiegs G. Oppositional effects of IL-10 and IL-17 during
immunological tolerance against concanavalin A. J Hepatol 2008; 48 (Suppl 2):
S69 (Abstract 152).
Journal article:
Erhardt A, Biburger M, Papadopoulos, T, Tiegs G. IL-10, regulatory T cells, and
Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice.
Hepatology 2007; 45(2):475-485.
Further presentations:
Erhardt A, Biburger M, Tiegs G. Immunological tolerance against concanavalin A
thinvolves T , Kupffer cells, IL-10, and impaired IL-2 production. 16 European regs
Congress of Immunology, Paris 2006
Erhardt A, Biburger M, Tiegs G. Long-lasting tolerance against concanavalin A
st
involves regulatory T cells, Kupffer cells and IL-10. 1 World Immune Regulation
Meeting, Davos 2007
Erhardt A, Biburger M, Tiegs G. IL-10 and regulatory T cells – the main mediators
thof immunological tolerance against concanavalin A. 37 Annual Meeting of the
German Society of Immunology, Heidelberg 2007