The MAPK phosphatase DUSP1 [Elektronische Ressource] : an IL-10-induced negative regulator of macrophage activation / Michael Hammer

Micha Girdwoyn

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
der Technischen Universität München


The MAPK phosphatase DUSP1: An IL-10-induced negative
regulator of macrophage activation


Michael Hammer


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften (Dr. rer. nat.)


genehmigten Dissertation.




Vorsitzende(r): Univ.-Prof. Dr. Dirk Haller

Prüfer der Dissertation: 1. Univ.-Prof. Dr. Hannelore Daniel
2. Priv.-Doz. Dr. Roland H. Lang
3. Univ.-Prof. Dr. Stefan Bauer, Philipps Universität Marburg



Die Dissertation wurde am 16.05.2007 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 14.11.2007 angenommen. Table of Contents 2
Table of Contents
TABLE OF CONTENTS..............................................................................................1
INDEX OF FIGURES...................................................................................................5
INDEX OF TABLES.....................................................................................................6
ABBREVIATIONS........................................................................................................7
1 INTRODUCTION...................................................................................................12
1.1 Innate and adaptive immunity .................................................................................. 12
1.2 Toll-like receptors.......................................................................................................13
1.2.1 TLR signalling pathways........................................................................................15
1.2.1.1 MyD88-dependent pathways.........................................................................16
1.2.1.2 MyD88-independent ......................................................................16
1.2.2 Negative regulation of TLR signalling...................................................................17
1.3 Function and activation of macrophages.................................................................. 18
1.3.1 Deactivation of macrophages.................................................................................21
1.4 Interleukin-10 – The ‘macrophage deactivating factor’ ......................................... 24
1.4.1 Structure and expression of IL-10 ..........................................................................24
1.4.2 IL-10 receptor and signalling26
1.4.3 Target genes of IL-10 .............................................................................................27
1.5 Dual-specificity MAPK phosphatases.......................................................................28
1.5.1 Dual-specificity phosphatase 1...............................................................................30
2 AIMS OF THE STUDY..........................................................................................32
3 MATERIAL AND METHODS..............................................................................33
3.1 Material....................................................................................................................... 33
3.1.1 Equipment...............................................................................................................33
3.1.2 Consumable items, kits and enzymes.....................................................................34
3.1.3 Reagents..................................................................................................................35
3.1.4 Primers and probes .................................................................................................37
3.1.5 Antibodies used for Western blot ...........................................................................39
3.1.6 Antibodies used for flow cytometry .......................................................................40
3.2 Methods....................................................................................................................... 40
3.2.1 Cell biology............................................................................................................40
3.2.1.1 Media used for eukaryotic cell culture ..........................................................40
3.2.1.2 Culture of RAW 264.7 macrophages ............................................................40
3.2.1.3 Cryopreservation of RAW 264.7 macrophages.............................................41
3.2.1.4 Stable transfection of RAW 264.7 macrophages...........................................41
3.2.1.5 Generation of murine bone marrow-derived macrophages ...........................41
3.2.2 Mice........................................................................................................................42 Table of Contents 3
3.2.3 Animal experiments................................................................................................42
3.2.3.1 Removal of organs.........................................................................................42
3.2.3.2 LPS-induced shock........................................................................................42
3.2.3.3 Colon ascendens stent peritonitis (CASP).....................................................42
3.2.3.3.1 Bacterial counts.......................................................................................43
3.2.3.4 Generation of bone marrow-chimeric mice...................................................43
3.2.4 Protein biochemistry...............................................................................................44
3.2.4.1 Buffers and solutions.....................................................................................44
3.2.4.2 Cell lysis........................................................................................................45
3.2.4.3 SDS-polyacrylamid gel electrophoresis (PAGE) of proteins........................46
3.2.4.4 Western blot analysis46
3.2.4.5 Luciferase reporter gene assay ......................................................................47
3.2.5 Molecular biology...................................................................................................47
3.2.5.1 Buffers and solutions47
3.2.5.2 Basic tools.....................................................................................................48
3.2.5.3 Agarose gel electrophoresis...........................................................................49
3.2.5.4 PCR................................................................................................................49
3.2.5.5 Generation of DUSP1 reporter constructs.....................................................50
3.2.5.6 Total RNA extraction ....................................................................................50
3.2.5.7 First strand cDNA synthesis..........................................................................51
3.2.5.8 Relative quantitation of gene expression by real-time PCR analysis............51
3.2.5.9 Microarray analysis of gene expression ........................................................52
3.2.5.10 Northern blot.................................................................................................53
3.2.6 Immunology............................................................................................................54
3.2.6.1 Enzyme linked immunosorbent assay (ELISA) ............................................54
3.2.6.1.1 ELISA buffers and solutions ...................................................................54
3.2.6.1.2 Determination of cytokine and chemokine levels by ELISA..................54
3.2.6.2 NO-Assay......................................................................................................55
3.2.6.3 Flow cytometry..............................................................................................56
3.2.6.3.1 Flow cytometry buffers ...........................................................................56
3.2.6.3.2 Analysis of cell surface antigens by flow cytometry...............................56
3.2.6.4 Magnetic activated cell sorting (MACS).......................................................57
4 RESULTS.................................................................................................................58
4.1 Expression and regulation of DUSP1........................................................................ 58
4.1.1 Mining of microarray datasets for regulation of phosphatases in macrophages ....58
4.1.2 Validation of the microarray results for DUSP1 ....................................................60
4.1.3 Sustained activation of p38 MAPK in IL-10 knockout macrophages....................62
4.1.4 Stability of DUSP1 mRNA.....................................................................................63
4.1.5 Activation of DUSP1 reporter by LPS but not by IL-10........................................64
4.1.6 LPS-induced DUSP1 expression