La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres

Partagez cette publication







The Role of Sensory Neuropeptides
during Experimental Hepatitis


Die Rolle von sensorischen Neuropeptiden
in der experimentellen Hepatitis







Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades



vorgelegt von
Irena Kröger
aus Hamburg

Als Dissertation genehmigt von den Naturwissenschaftlichen
Fakultäten
der Universität Erlangen-Nürnberg


















Tag der mündlichen Prüfung: 13.03.2009
Vorsitzender der Prüfungskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Gisa Tiegs
Zweitberichterstatter: Prof. Dr. Robert Slany




























„Freunde sind wie Sterne. Du kannst sie nicht immer sehen, aber du
weißt, sie sind immer für dich da.“
CONTENTS
Contents
1 Introduction......................................................................1
1.1 The Liver: anatomy and physiology............................................................ 1
1.2 Immune - mediated liver injury and the role of cytokines............................ 4
1.2.1 Viral hepatitis .................................................................................... 7
1.2.2 Autoimmune hepatitis ....................................................................... 8
1.2.3 Toxic liver injury ................................................................................ 9
1.3 Animal models of apoptotic and immune-mediated liver injury................... 9
1.3.1 Concanavalin A Model.................................................................... 10
1.3.2 Galactosamine/anti-CD3 Model ...................................................... 11
1.3.3 Galactosamine/Lipopolysaccharide Model...................................... 11
1.3.4 Galactosamine/ Tumor-necrosis factor α Model ............................. 11
1.4 Neurogenic inflammation.......................................................................... 12
1.5 Calcitonin gene related peptide (CGRP) and its receptor......................... 14
®1.6 CGRP receptor antagonists and CGRP-binding Spiegelmer .................. 17
1.7 Aim of the study........................................................................................ 19
2 Materials and Methods ..................................................21
2.1 Mice.......................................................................................................... 21
2.2 Animal Treatment ..................................................................................... 21
2.2.1 Treatment schedules ...................................................................... 21
®2.2.2 Administration of receptor antagonists, CGRP-binding Spiegelmers
(NOX-504P) or rat αCGRP ............................................................. 23
2.3 Sampling of Material................................................................................. 23
2.4 Real Time RT-PCR................................................................................... 23
2.5 Agarose Gel Electrophoresis of DNA ....................................................... 25
2.6 Gel Band Purification and Sequencing..................................................... 25
2.7 Analysis of liver enzymes ......................................................................... 26
2.8 Cytokine determination by enzyme-linked immunosorbet assay
(ELISA)..................................................................................................... 26
2.9 Determination Caspase-3 ELISA.............................................................. 27
I CONTENTS
2.10 CGRP Immunoassay................................................................................ 27
2.11 Isolation of cells........................................................................................ 28
2.11.1 Isolation of primary hepatocytes ..................................................... 28
2.11.2 Isolation of non-parenchymal cells.................................................. 29
2.12 Flow – Cytometric Analysis....................................................................... 29
2.13 Haematoxylin/eosin staining of liver sections ........................................... 31
2.14 In vitro experiments .................................................................................. 31
2.14.1 Primary hepatocytes cultures.......................................................... 31
2.14.2 RAW cells cultures.......................................................................... 32
2.14.3 Cell viability test .............................................................................. 32
2.15 Statistical Analysis.................................................................................... 33
3 Results............................................................................34
3.1 Characterization of CGRP and its receptor in murine liver ....................... 34
3.1.1 CRLR, RAMP1 and RCP were found in liver tissue by RT-PCR..... 34
3.1.2 CGRP-receptor expression on different cell types .......................... 35
3.1.3 Increased CRLR expression on hepatocytes and macrophages
during GalN/LPS-induced liver injury .............................................. 38
3.1.4 CGRP concentrations in liver tissue are reduced during GalN/LPS
induced liver injury .......................................................................... 40
3.2 Blockade of CGRP receptor with different antagonists and a CGRP-
®binding Spiegelmer ................................................................................. 41
3.2.1 Pretreatment with the CGRP antagonist had no effect on 8-37
GalN/LPS induced liver injury ......................................................... 41
3.2.2 Pretreatment with BIBN4096BS had no effect on GalN/LPS induced
liver injury........................................................................................ 42
®3.2.3 Pretreatment with CGRP-binding Spiegelmer had no effect on
GalN/LPS induced liver injury ......................................................... 43
3.3 Pretreatment with exogenous αCGRP reduced immune-mediated
liver injury ................................................................................................. 45
3.3.1 GalN/LPS induced liver injury was reduced by exogenously
administrated CGRP....................................................................... 45
II CONTENTS
3.3.2 ConA induced liver injury was reduced by exogenously administrated
CGRP ............................................................................................. 49
3.3.3 CGRP pretreatment reduces GalN/anti-CD3- induced liver injury... 52
3.3.4 αCGRP pretreatment failed to reduce GalN/TNFα-induced liver injury
........................................................................................................ 54
3.4 Possible mechanisms of the αCGRP effect.............................................. 55
3.4.1 Lack of in vitro effects of αCGRP.................................................... 55
3.4.1.1 αCGRP had no influence on apoptotic primary hepatocytes .. 56
3.4.1.2 CGRP had no influence on LPS stimulated RAW 264.7 cells. 57
3.4.2 αCGRP acts in an IL-10 independent manner................................ 58
3.4.3 Reduced plasma TNFα levels correlated with CGRP-induced up-
regulation of ICER expression ........................................................ 59
3.4.4 Determination of the frequency of cell populations in the liver after
αCGRP pretreatment...................................................................... 61
3.5 Immune-mediated liver injury in αCGRP knockout mice .......................... 63
-/-3.5.1 ConA induced liver injury in αCGRP mice .................................... 64
3.5.2 GalN/LPS induced liver injury in αCGRP knockout mice. ............... 66
4 Discussion......................................................................69
4.1 CGRP and its receptor were found in murine liver tissue ......................... 70
4.2 CGRP had a protective effect in immune-mediated liver injury................. 71
4.3 Possible mechanism of the protective effect of αCGRP........................... 73
-/- 4.4 CGRP mice differently reacted to immune-mediated liver injury............. 76
4.5 Outlook ..................................................................................................... 77
5 Summary.........................................................................78
6 Deutschsprachige Zusammenfassung........................81
7 References......................................................................84
8 Danksagung .................................................................102
9 Lebenslauf....................................................................104
III CONTENTS
Figure legend
Fig. 1: Immune cells in the liver. ............................................................................ 1
Fig. 2: Different models and point of attacks in apoptotic and immune-mediated
liver injury. .................................................................................................... 10
Fig. 3: Structure of CGRP. ................................................................................... 14
Fig. 4: Model of the functional CGRP receptor. ................................................... 15
Fig. 5: CGRP receptor expression in murine liver. . ............................................. 35
Fig. 6: CGRP-receptor expression on different cell types and in different mouse
strains. .......................................................................................................... 36
Fig. 7: Histograms of fluorescence intensity of CRLR. .. ...................................... 37
Fig. 8: GalN/LPS treatment increases CRLR expression on different cell-types. 38
Fig. 9: Flow cytometry analysis of GalN/LPS-stimulated cells. ............................ 39
Fig. 10: Time course of CGRP amount during GalN/LPS induced liver injury. .... 40
Fig. 11: Pretreatment with CGRP had no effect on GalN/LPS induced liver 8-37
injury. ........................................................................................................... 42
Fig. 12: BIBN4096BS pretreatment had no effect on GalN/LPS induced liver injury.
...................................................................................................................... 43
® Fig. 13: CGRP-binding Spiegelmer pretreatment had no effect on GalN/LPS
induced liver injury. ...................................................................................... 44
Fig. 14 αCGRP pretreatment in the GalN/LPS mouse model. ............................ 45
Fig. 15: Induction of an anti-inflammatory cytokine profile upon αCGRP
pretreatment. ................................................................................................ 46
Fig. 16: Histological alterations of liver tissue of αCGRP pretreated mice. ......... 47
Fig. 17: αCGRP pretreatment in the GalN/LPS mouse model. ........................... 48
Fig. 18: Induction of an anti-inflammatory cytokine profile upon αCGRP
pretreatment. ................................................................................................ 49
Fig. 19: Protection from ConA-induced liver injury by αCGRP pretreatment. ...... 50
Fig. 20: Cytokine response of ConA treated mice with or without αCGRP
pretreatment. ................................................................................................ 51
Fig. 21: Protection from GalN/anti- CD3-induced liver injury by αCGRP
pretreatment. ................................................................................................ 52
Fig. 22: Cytokine response of GalN/anti-CD3 treated mice with or without αCGRP
pretreatment. ................................................................................................ 53
IV CONTENTS
Fig. 23: Caspase-3 activities were reduced in pretreated mice. .......................... 54
Fig. 24: Lack of protection of αCGRP in the GalN/TNFα-induced liver injury. ..... 55
Fig. 25: Effect of CGRP on ActD/TNFα induced toxicity in primary hepatocytes
cultures. ........................................................................................................ 56
Fig. 26: Histogram of fluorescence intensity of CRLR. . ....................................... 57
Fig. 27: Effect of αCGRP on cytokine release and viability of LPS stimulated RAW
264.7 cells. ................................................................................................... 58
-/-Fig. 28: αCGRP pretreatment prevented GalN/LPS induced liver injury in IL-10
mice. ............................................................................................................ 59
Fig. 29: αCGRP induces ICER Expression in GalN/LPS-treated mice. ............... 60
Fig. 30: αCGRP pretreatment induced phenotypic changes of intrahepatic cell
populations during GalN/LPS induced liver injury. ....................................... 62
+ +Fig. 31: αCGRP pretreatment did not influence FoxP3 /CD4 T cells during ConA
induced liver injury. ...................................................................................... 63
-/- Fig. 32: ConA treatment of αCGRP and C57BL/6 mice. . .................................. 64
Fig. 33: Expression of cytokines in plasma and liver tissue after ConA treatment. .
...................................................................................................................... 65
-/- Fig. 34: Caspase-3 measurement and histopathology in C57BL6 and αCGRP
mice after ConA challenge. .......................................................................... 66
Fig. 35: αCGRP-/- mice are protected from GalN/LPS induced liver injury. ........ 66
Fig. 36: Cytokine profile of C57BL6 and αCGRP-/- mice treated with GalN/LPS. 67
-/-Fig. 37: Caspase-3 measurement and histopathology in C57BL6 and αCGRP
mice after GalN/LPS challenge. ................................................................... 68











V CONTENTS
Table legend
Tab. 1: Characteristics of cytokines involved in liver diseases ............................... 6
Tab. 2: Nucleotide sequences of the specific primers used in RT- PCR. ............. 24
Tab. 3: Antibodies used for flow-cytometric analysis............................................ 30






























VI CONTENTS
Abbreviation
Ab antibody
ActD Actinomycin D
AIH autoimmune hepatitis
ALT alanine aminotransferase
ANOVA analysis of variances
APP acute-phase protein
APR acute-phase response
BSA bovine serum albumine
cAMP cyclic adenosine monophosphate
cDNA copy DNA
CGRP calcitonin gene-related peptide
ConA concanavalin A
CRLR calcitonin receptor like receptor
CRP C-reactive protein
Cy3 indocarbocyanin
DCs dentric cells
DNA desoxyribonucleic acid
dNTP deoxynucleosidtriphospahte
EIA enzyme-linked immunoassay
ELISA enzyme-linked immunosorbent assay
FACS fluorescence-activated cell sorter
FCS fetal calf serum
FITC fluorescein isothiocyanate
FoxP3 forkhead box 3
GalN D-galactosamine
HE haematoxylin/eosin
HAV hepatitis A virus
HBSS Hanks balanced salt solution
HBV hepatitis B virus
HCC hepatocellular carcinoma
HCV hepatitis C virus
HDV hepatitis D virus
HEV hepatitis E virus
VII

Un pour Un
Permettre à tous d'accéder à la lecture
Pour chaque accès à la bibliothèque, YouScribe donne un accès à une personne dans le besoin