The role of sensory neuropeptides during experimental hepatitis [Elektronische Ressource] = Die Rolle von sensorischen Neuropeptiden in der experimentellen Hepatitis / vorgelegt von Irena Kröger

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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 ...............................................................
Publié le : jeudi 1 janvier 2009
Lecture(s) : 27
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2009/1301/PDF/IRENA_KROEGERDISSERTATION.PDF
Nombre de pages : 117
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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

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