Analysis of macrophages and neutrophils from Vav-mutant mice [Elektronische Ressource] : roles for Vav3 in phagocytes ; Vav-mutant phagocytes in in vivo models of inflammation / vorgelegt von Francesca Mannella

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2010
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

FAKULTÄT FÜR NATURWISSENSCHAFTEN

UNIVERSITÄT ULM



Analysis of macrophages and neutrophils from Vav-mutant mice


Roles for Vav3 in phagocytes

Vav-mutant phagocytes in in vivo models of inflammation




DISSERTATION

Zur Erlangung des Doktorgrades (Dr. rer. nat.) an der Fakultät für
Naturwissenschaften der Universität

vorgelegt von
Francesca Mannella

geboren in Rom, Italien


Ulm, 2010



Amtierender Dekan:

Prof. Dr. Axel Groß



Erstgutachter:


Prof. Dr. Thomas Wirth, Institut für Physiologische Chemie, Universität Ulm



Zweitgutachter:





Tag der Promotion:




Die Arbeiten im Rahmen der vorliegenden Dissertation wurden am Institut für Physiologische
Chemie der Universität Ulm durchgeführt und von Herrn Prof. Dr. Klaus-Dieter Fischer
betreut.



Erklärung



Ich versichere hiermit, dass die vorliegende Arbeit von mir selbständig angefertigt wurde und
ich keine anderen als die angegebenen Quellen und Hilfsmittel benutzt sowie wörtlich oder
inhaltlich übernommene Textpassagen als solche gekennzeichnet habe.




_________________________
(Francesca Mannella)




Ulm, den _________________





TABLE OF CONTENTS 1
TABLE OF CONTENTS
TABLE OF CONTENTS ..............................................................................................1
FIGURES .................................................................4
SUMMARY...............................................................5
ZUSAMMENFASSUNG ...........................................6
ABBREVIATIONS ....................................................7
INTRODUCTION.........................................................................................................9
1 Phagocytes: monocytes, macrophages and neutrophils ..................................9
1.1 Monocytes ............................................10
1.2 Macrophages...........................................................................................11
1.3 Neutrophils ..............................................................................................12
2 Phagocytes in vivo..........................................................................................13
2.1 Peritonitis model of inflammation.............................................................13
2.2 Wound Healing model of inflammation and tissue repair.........................14
3 Phagocyte recruitment.................................16
3.1 Integrins...................................................................................................16
3.2 Rho GTPases..........................................................................................17
4 Vav family of RhoGEFs ...............................18
4.1 Structure...............................................18
4.2 Vav Catalytic activity...............................18
4.3 Expression...............................................................................................19
5 Vav in signaling ...........................................19
5.1 Multiple receptors activate Vav................................................................19
5.2 Vav proteins in integrin signaling.............................................................20
5.3 Vav-activated signaling pathways............................................................21
5.4 Vav regulation of actin.............................................................................21
6 Vav roles in T cells and B cells .......................22
7 Vav roles in macrophages and neutrophils..............23
7.1 Vav in macrophages................................................................................23
7.2 Vav in neutrophils....................................................................................23




TABLE OF CONTENTS 2
RESULTS..................................................................................................................26
8 Vav1 and Vav2 not required for basic macrophage and neutrophil ................26
functions .............................................................26
8.1 Vav2 expression is low in myeloid cells...................................................26
8.2 Vav1 and Vav2 not required for Fc!R phagocytosis ................................26
8.3 Vav1 is not required for basic cytoskeletal functions in neutrophils.........29
9 Roles for Vav3 in macrophages and neutrophils ............................................31
9.1 Expression of Vav3 protein in various cells and tissues ..........................31
9.2 Vav3 regulates migration, actin polymerization and ROS in neutrophils .32
10 Vav3 regulates macrophages and neutrophils in thioglycolate-induced......35
peritonitis ...............................................................................................................35
11 Vav3 regulates macrophages and neutrophils in wound healing ................41
11.1 Vav3 required for normal wound healing and macrophage recruitment ..42
11.2 Vav3 regulates adhesion, phagocytosis and TGF- ! release.................44 1
11.3 Vav3 is required for TGF-" in vivo and ROS production ........................45 1
11.4 Vav3 is a downstream target of " -integrin-dependent macrophage 2
adhesion.............................................................................................................47
DISCUSSION.........................................................50
11.5 Specific roles for Vav1 in phagocytes......................................................50
11.6 Specific roles for Vav3 in phagocytes..................52
11.7 Vav3 in peritonitis ....................................................................................54
12 Vav3 is required in wound healing ..............................................................55
12.1 Vav3 required for recruitment of macrophages to wound sites................56
12.2 Vav3 required for macrophage phagocytosis of neutrophils....................57
12.3 Vav3 regulates TGF-" and ROS production...........................................57 1
12.4 Vav3 signals from " -integrins.................................................................58 2
12.5 Future Perspectives.................................................................................59
MATERIALS..............................................................................................................60
13 Chemicals and reagents .............................................................................60
13.1 Cell lines...............................................62
13.2 Antibodies for Western Blotting ...............................................................62
13.3 Antibodies for Flow cytometry ........................62


TABLE OF CONTENTS 3
METHODS ................................................................................................................63
14 Mice .........................................................63
15 In vivo mouse models .................................................................................63
15.1 Thioglycolate (TG)-induced acute peritonitis model.................................63
15.2 Wound healing model..............................................................................63
16 Primary cell preparations ............................................................................64
16.1 Bone marrow (BM)-derived macrophages isolation.................................64
16.2 Fresh and apoptotic neutrophil preparation.............................................65
16.3 Flow cytometry ........................................................................................66
17 Functional assays ....................................67
17.1 In vitro macrophage adhesion to and phagocytosis of apoptotic
neutrophils.......................................................67
17.2 In vitro bone marrow-derived neutrophil adhesion assay ........................67
17.3 In vitro cell migration assay .....................................................................68
17.4 In vitro macrophage phagocytosis of latex beads....................................69
17.5 In vitro macrophage phagocytosis of apoptotic neutrophils.....................69
17.6 In vitro macrophage adhesion assay.......................................................69
18 Biochemistry ...............................................................................................70
18.1 Enzyme-based immunoassay (ELISA)-Based Cytokine Detection..........70
18.2 ROS production by bone marrow-derived neutrophils.............................72
18.3 Actin polymerization detection.................................................................73
18.4 Western blo