Proinflammatory activation of human macrophages by the serine protease plasmin [Elektronische Ressource] : characterization of signaling pathways / submitted by Qun Li

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• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Ulm University
Institute of Pharmacology of Natural Products and Clinical Pharmacology
Head: Prof. Dr. Th. Simmet







Proinflammatory activation of human macrophages
by the serine protease plasmin
Characterization of signaling pathways









Thesis
Presented to the Faculty of Medicine, Ulm University,
to obtain the degree of a Doctor of Human Biology
Submitted by
Qun Li
From China
2008






























Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin
1. Berichterstatter : Prof. Dr. Thomas Simmet
2. Berichterstatter : Prof. Dr. Johannes M. Weiss
Tag der Promotion : 12.12.2008 1
TABLE OF CONTENTS__________________________________________________1
ABBREVIATIONS ______________________________________________________3
1. INTRODUCTION _____________________________________________________7
1.1. IMMUNE CELLS______________________________________________________7
1.1.1. Macrophages and monocytes_______________________________________8
1.1.2. Macrophages and monocytes in the immune response ___________________8
1.1.3. The role of macrophages in atherosclerosis ___________________________9
1.2. IL-6 FAMILY CYTOKINES ______________________________________________9
1.2.1. IL-6 family cytokines and their receptors _____________________________9
1.2.2. JAK⁄STAT signaling pathways _____________________________________13
1.3. PLASMIN _________________________________________________________19
1.3.1. Structure of plasmin _____________________________________________19
1.3.2. Functions of plasmin ____________________________________________20
1.3.3. Plasmin receptors ______________________________________________21
1.3.4. Plasmin-induced cell activation____________________________________24
1.4. PLASMIN IN HUMAN DISEASES _________________________________________27
1.4.1 A role for plasmin in atherosclerosis ________________________________27
1.4.2. Plasmin and cancer _____________________________________________28
1.5. AIM OF THE STUDY__________________________________________________29
2. MATERIALS AND METHODS ________________________________________31
2.1. ANTIBODIES _______________________________________________________31
2.2. CHEMICALS AND KITS _______________________________________________32
2.3. CELL PREPARATION AND CULTURES_____________________________________34
2.4. ANALYSIS OF MRNA EXPRESSION ______________________________________34
2.4.1. Reverse transcription and polymerase chain reaction analysis (RT-PCR) ___34
2.4.2. Quantitative real time PCR (qPCR)_________________________________36
2.5. ANALYSIS OF PROTEIN EXPRESSION _____________________________________37
2.5.1. Western blotting ________________________________________________37
2.5.2. Analysis of STAT and NF-κB transcription factor activation _____________40
2.5.3. Flow cytometry_________________________________________________41
2.5.4. Enzyme-linked immunoSorbent assay (ELISA) ________________________41
2.6. INHIBITION OF GENE EXPRESSION BY ANTISENSE ODN ______________________42
2.7. IMMUNOFLUORESCENCE MICROSCOPY ___________________________________43
2.8. EQUIPMENT _______________________________________________________43
2.9. STATISTICAL ANALYSIS ______________________________________________44
3. RESULTS ___________________________________________________________45
3.1. THE SERINE PROTEASE PLASMIN TRIGGERS PROINFLAMMATORY GENE EXPRESSION IN
HUMAN MONOCYTE DERIVED MACROPHAGES- _________________________________45
3.1.1. Phenotypic characterization of macrophages _________________________45
3.1.2. Human macrophages express the annexin A2 heterotetramer ____________47
3.1.3. Plasmin triggers signaling in macrophages __________________________49
3.1.4. Plasmin induces nuclear translocation of STAT and NF-κB transcription
factors in macrophages _______________________________________________54
3.1.5. Plasmin elicits expression of proinflammatory cytokines in macrophages ___55
3.1.6. Plasmin-induced activation of JAK1, p38, ERK1/2, and NF-κB is indispensable
for the cytokine expression in macrophages _______________________________59
3.1.7. The annexin A2 heterotetramer is a plasmin receptor in macrophages _____61
2
3.1.8. Summary of plasmin-mediated activation of human macrophages _________63
3.2. A ROLE FOR THE ANNEXIN A2 N-TERMINAL PEPTIDE (A2NP) IN THE PLASMIN-
INDUCED ACTIVATION OF HUMAN PERIPHERAL MONOCYTES ______________________65
3.2.1. A2NP-induced signaling is similar to that of plasmin ___________________65
3.2.2. A2NP-induced expression of MCP-1 is similar to that of plasmin _________67
3.2.3. Effect of inhibition of protease-activated receptor 1 (PAR1) on plasmin-
induced cytokine release in human monocytes _____________________________68
3.2.4. Expression of members of gp130 family receptors in human monocytes ____69
3.2.5. Down-regulation of IL31Ra but not gp130 impairs the plasmin- __________71
dependent STAT3 activation ___________________________________________72
3.2.6. Plasmin-mediated activation of human monocytes. Summary II___________74
4. DISCUSSION ________________________________________________________75
4.1. PLASMIN A PROINFLAMMATORY ACTIVATOR OF HUMAN MONOCYTES-DERIVED
MACROPHAGES ________________________________________________________75
4.2. MECHANISM OF PLASMIN-INDUCED MONOCYTE ACTIVATION _________________78
5. SUMMARY _________________________________________________________83
6. REFERENCES_______________________________________________________85
ACKNOWLEDGEMENTS_______________________________________________97
CURRICULUM VITAE _________________________________________________98
PUBLICATIONS _______________________________________________________99

3

Abbreviations

2ME 2-mercaptoethanol
A2Ct Annexin A2 C-terminus
A2NP Annexin A2 N-terminus peptide
A2Nt Annexin A2 N-terminus
AA2t Annexin A2 heterotetramer
AB Acrylamide bisacrylamide
AG490 Alpha-cyano- (3,4-dihydroxy) -N-benzylcinnamide
AKβBA Acetyl-11-keto-β-boswellic acid
AP-1 Activating protein-1 (transcription factor)
APS Ammonium persulfate
bp Base pair
BSA Bovine serum albumin
cDNA Complementary DNA
CIS-1 Cytokine-inducible SH containing protein 2
CLC Cardiotrophin-like cytokine
CNTF Ciliary neurotrophic factor
CT-1 Cardiotrophin-1
CTA Committee on thrombolytic agents
DC Dendritic cells
DMEM Dulbecco's modified eagle medium
DMSO Dimethyl sulfoxide
DNase Deoxyribonuclease
DNTP Deoxynucleoside triphosphates
DTT Dithiothreitol
EB Elution buffer
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
ERK Extracellular signal-regulated kinases
FACS Fluorescence activated cell scan/sorter
Foetal calf serum FCS
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GB Gel buffer
4
GPL Gp130-like receptor
HEPES 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid
HRP Horseradish peroxidase
ICAM-1 Intercellular adhesion molecule-1
IEC Intestinal epithelial cells
IFNγγγγ Interferon-γ
IL Interleukin
iNOS Inducible nitric oxide synthase
IPTG Isopropyl- β-D-thiogalactoside
JAK Janus kinases
LB-medium Luria-bretani medium
LDL Low-density lipoproteins
LIF Leukaemia inhibitor Factor
LPS Lipopolysaccharide
MAb Monoclonal antibody
MAPK Mitogen-activated protein kinase
MCP-1 Monocytes chemoattractant protein-1
M-CSF Macrophage-colony stimulating factor
MHC Major histocompatibility complex
MIP-3α Macrophage inflammatory protein-3α
MMLV-RT Moloney murine leukaemia virus reverse transcriptase
MMP Matrix metalloproteinases
MPS Mononuclear phagocyte system
NES Nuclear export signal
NF-κB Nuclear factor κ-B
ODN Oligodeoxynucleotides
Oligo (dT) Oligo-deoxythymidine
OSM Oncostatin M
PAb Polyclonal antibody
PAI Plasminogen activator inhibitors
PAMPS Pathogen associated molecular patterns
PAR1 Protease activated receptor 1
PBMC Peripheral blood mononuclear cells
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PDK1 Phosphoinositide-dependent protein kinase 1
5
PE Phycoerythrin
PEG Polyethylene glycol
Pi3K Phospho