Role of monocyte subsetes in chronic liver injury and liver fibrosis [Elektronische Ressource] / vorgelegt von Karlin Raja Karlmark

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Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Role of monocyte subsets in chronic
liver injury and liver fibrosis
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation
vorgelegt von
Master of Science
Karlin Raja Karlmark
aus Trichy, Indien
Berichter:
Herr Privatdozent
Dr. med. Frank Tacke
Herr Universitätsprofessor
Dr. rer.nat. Lothar Elling
Tag der mündlichen Prüfung: 07. Dezember 2009
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Table of contents

Table of contents I
1. Introduction, 1
1.1 Biology of the Liver 1
1.2 Intrahepatic immune cells 2
1.2.1 Innate immune effector cells in the liver 3
1.2.2 Antigen presenting cells and adaptive T cell
responses in the liver 5
1.3 Chemokines 5
1.3.1 Chemokines in acute liver disease 6
1.3.2 Chemokines in chronic liver disease 9
1.4 Immune cell infiltration in liver diseases and
experimental liver injury models 12
1.4.1 NKT cell infiltration 12
1.4.2 T cell infiltration 12
1.4.3 Monocyte/Macrophage infiltration 13
1.5 Aims of this study 14
1.5.1 Analysis of monocyte subset infiltration in acute and
chronic liver injury 14
1.5.2 Role of the chemokine receptors CCR2 and CCR6 for
monocyte subset recruitment in acute and chronic livery 15
1.5.3 Functional role monocyte subset infiltration during liver
fibrogenesis and molecular mechanisms of these
actions 15

2. Materials and Methods 17

2.1 Materials 17
2.1.1 Chemicals 17
2.1.2 Instruments and equipment 18
2.1.3 Analytical chemicals, Reagents and Kits 19
2.1.4 Primers for genotyping and RT-PCR 20
2.1.5 Antibodies 22
I Table of contents

2.1.5.1 Primary antibodies 22
2.1.5.2 Secondary antibodies 23
2.1.6 Enzymes 23
2.1.7 ELISA kit 23
2.1.8 Cytometric bead array 23
2.1.9 Buffers and Solutions 23

2.2 Animal experimental methods 29
2.2.1.1 Animal breeding 29
2.2.1.2 Animals 29
2.2.2 Acute carbon tetrachloride (CCl ) induced liver injury 30 4
2.2.3 Chronic liver injury and liver fibrogenesis induced by
carbon tetrachloride (CCl ) administration 30 4
2.2.4 Depletion of circulating monocytes 30
2.2.5 Nucleic acids 30
2.2.5.1 Isolation of RNA from liver samples 30
2.2.5.2 Isolation of DNA from tail tips 31
2.2.5.3 cDNA synthesis 31
2.2.5.4 Real time PCR 32
2.2.5.5 End point PCR with genomic DNA 33
2.2.6 Proteins 34
2.2.6.1 Isolation and quantification of protein from liver samples 34
2.2.6.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
(SDS-PAGE) 34
2.2.6.3 Westernblot (semi-dry Gel transfer) 35
2.2.6.4 ELISA 36
2.2.7 Leukocytes and Tissues
2.2.7.1 Isolation of blood and splenic leukocytes 37
2.2.7.2 Isolation of intrahepatic leukocytes and tissues 37
2.2.7.3 Surface antibody staining of blood and liver Leukocytes 38
2.2.7.4 Intracellular antibody staining of liver leukocytes 39
2.2.7.5 Fluorescence activated cell sorting (FACS) 40
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2.2.7.6 Isolation of monocytes, lymphocytes for adoptive transfer 40
2.2.7.7 Monocyte adoptive transfer experiment 41
2.2.7.8 Co-culture experiments 41
2.2.8 Microscopy 42
2.2.8.1 Immunohistochemistry 42
2.2.8.2 Histology
2.2.8.2.1 Haematoxylin/Eosin (H&E) staining 43
2.2.8.2.2 Ladewig staining 43
2.2.9 Further biochemical analyses 44
2.2.9.1 Aminotransferase activities in serum 44
2.2.9.2 Hydroxyproline assay 44
2.2.10 Statistics 45

3 Results 47

3.1.0 Liver injury and immunopathogenesis 47
3.1.1 Acute liver injury by CCl and leukocyte migration Into the liver 47 4
3.1.2 Monocyte infiltration into the injured liver 48
hi low3.1.3 Gr-1 but not Gr-1 monocytes are massively
recruited into the injured Liver 50
+3.1.4 CCR2 is critical for recruitment of Gr-1 intrahepatic
monocyte-derived cells 52
+3.1.5 Intrahepatic Gr-1 monocyte-derived cells are also Increased
during liver fibrogenesis, and their recruitment is CCR2-dependent 56
+3.1.6 Reduced infiltration of Gr-1 monocyte derived cells in
the Ccr2 and Ccr2/Ccr6-knockout animals is associated with
reduced liver fibrosis 59
+3.1.7 Hepatic Gr-1 monocyte-derived cells preferentially constitute
proinflammatory iNOS-producing Macrophages and interplay
in T cell differentiation 61
+3.1.8 The infiltrating Gr-1 monocyte subpopulation promotes
profibrogenic actions at different stages of liver fibrosis 63
III Table of contents

+3.1.9 Intrahepatic Gr-1 monocytes directly activate HSCs in a
TGFβ1-dependent manner 67

4 Discussion 69

4.1 Monocyte infiltration during acute liver injury 69
4.2 Role of CCR2 for monocyte subset infiltration in liver injury 70
4.3 Monocyte infiltration during chronic liver injury 72
4.4 Monocyte/macrophage differentiation during liver fibrosis 73
4.5 Monocyte subset function during liver fibrogenesis 74
4.6 Dissection of molecular mechanism underlying monocyte-
mediated hepatic stellate cell activation and fibrogenesis 74
4.7 Open questions and outlook 75

5 References 78

Summary 94
Zusammenfassung 96
Appendix-Abbreviations VII
Acknowledgement XI
Curriculum vitae XIII

















IV Table of contents

Tables

Table 1.0 Overview about the chemokines and their corresponding receptors 7
Table 2.0 bout the chemokines that can be expressed upon liver
injury by different cell-types within the liver 10
Table 2.1.1 Primer sequences 21
Table 2.1.2 Primary antibodies 22
Table 2.1.3 Secondary antibodies 23
Table 2.2 Ratio of oil and CCl mix according to body weight 30 4
Table 2.3 Reverse transcription protocol 32
Table 2.4 PCR Program 33
Table 2.5 Antibody panel for the cell surface staining 38
Table 2.6 Antibody panels for surface- and intracellular staining 40

Figures

Figure 1: Liver microanatomy 1
Figure 2: Intrahepatic immune cell populations 2
Figure 3: Monocyte subsets 13
Figure 4: Liver pathology and liver infiltrating leukocytes after CCl - induced injury 47 4
Figure 5: Real Time gene expression analysis of monocyte attracting chemokines 48
Figure 6: Immunofluorescence analysis of liver infiltrating monocytes 48
Figure 7: Kinetics of monocyte chemoattractant MCP-1 in serum 49
Figure 8: FACS analysis of monocyte subsets in the liver 50
Figure 9: Kinetics of infiltrating monocyte subsets during CCl -mediated acute liver 50 4
injury
Figure 10: Monocyte infiltration is dispensable for initiation of acute CCl liver injury 52 4
Figure 11: Role of chemokine receptors CCR2, CCR6 in acute liver injury 53
+Figure 12: Hepatic Gr-1 monocyte subset recruitment is dependent on CCR2 54
+ + +Figure 13: Statistics of liver infiltrating CD11b F4/80 Gr-1 cell during acute liver injury 54
Figure 14: Role of CCR2 and CCR2/6 receptors on monocyte populations in the blood 55
Figure 15: Experimental design of CCl mediated chronic liver injury 56 4
Figure 16: Long term administration of CCl resulted in liver fibrosis 56 4
+ + +Figure 17: Statistics of liver infiltrating CD11b F4/80 Gr-1 cell during chronic liver injury 57
+Figure 18: Reduced Gr-1 monocyte subset recruitment during chronic
liver injury is associated with reduced liver fibrogenesis 57
+ + +Figure 19: Reduced CD11b F4/80 Gr-1 monocyte subset recruitment is reflected
by decreased profibrogenic factor TGF-β1 levels in Ccr2-and Ccr2/Ccr6-
knockout mice 59

V Table of contents

+ + +Figure 20: Reduced CD11b F4/80 Gr-1 monocyte subset recruitment is
reflected by decreased intrahepatic expression of matrix
metalloproteinases (MMPs) in Ccr2- and Ccr2/Ccr6-knockout mice 59
+ + +Figure 21: Hepatic CD11b F4/80 Gr-1 derived cells differentiate into classically
activated state or alternative state and modulate liver fibrogenesis 60
Figure 22: Th1/Th2 alterations in liver fibrosis 61
Figure 23: Experimental design for adoptive monocyte transfers during fibrogenesis 62
Figure 24: Isolation of monocytes for adoptive transfer 62
+Figure 25: Adoptive transfer of Gr-1 monocytes promotes liver fibrogenesis
during early and late stages of chronic liver injury 63
Figure 26: Total liver hydroxyproline quantification after monocyte transfer in