Plasma Cell Homeostasis in the TPO-retrogenic Mouse Model [Elektronische Ressource] / Martin Szyska. Betreuer: Rudolf Manz

Martin Szyska

Sujets

• Biologie

Informations

• Publié par: technische_universitat_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

Deutsches Rheuma-Forschungszentrum Berlin
Plasma Cell Homeostasis in the
Tpo-retrogenic Mouse Model
von der Fakultät III - Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
– Dr. Ing. –
genehmigte Dissertation
vorgelegt von
Dipl.-Ing. Martin Szyska
Promotionsausschuss:
Vorsitzender: Prof. Dr.-Ing. Vera Meyer
Berichter: Prof. Dr. rer. nat. Rudolf Manz
Berichter: Prof. Dr. rer. nat. Roland Lauster
Tag der wissenschaftlichen Aussprache: 03.06.2011
Berlin 2011
D83Contents
Contents i
1 Introduction 1
1.1 Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Adaptive Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.1 B cell development . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2 Immunological memory . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.3 Break of tolerance . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3 The Bone Marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.1 Bone marrow structure . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.2 Cells of the bone marrow . . . . . . . . . . . . . . . . . . . . . . 9
1.3.3 The plasma cell niche . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4 The Megakaryocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.4.1 Megakaryocyte and platelet function . . . . . . . . . . . . . . . 15
1.4.2aryocyte development . . . . . . . . . . . . . . . . . . . . 16
1.5 Aim of this Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5.1 Retrogenic mouse model . . . . . . . . . . . . . . . . . . . . . . 18
1.5.2 Microdissection . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2 Materials and Methods 20
2.1 Tpo-retrogenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.1 Generation of retroviral expression vector . . . . . . . . . . . . . 20
2.1.2 of retrovirus producer cell lines . . . . . . . . . . . . 22
2.1.3 HSC transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2 Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.2.1 Reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . 24
2.2.2 PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2.3 Cloning procedures . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.1 Cells for virus production . . . . . . . . . . . . . . . . . . . . . 32
2.3.2 32D cells – TPO assay . . . . . . . . . . . . . . . . . . . . . . . 36
2.3.3 HSC-culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.4 Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39CONTENTS ii
2.4.1 Preparation of single cell suspensions . . . . . . . . . . . . . . . 39
2.4.2 Staining of cell suspensions . . . . . . . . . . . . . . . . . . . . . 39
2.4.3 Analyzing platelet numbers . . . . . . . . . . . . . . . . . . . . 40
2.4.4 Analyzing megakaryocyte ploidy . . . . . . . . . . . . . . . . . . 40
2.4.5 Channels and compensation . . . . . . . . . . . . . . . . . . . . 42
2.5 ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.6 ELISPOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.7 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.7.1 Freezing of organs . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.7.2 Sectioning of organs . . . . . . . . . . . . . . . . . . . . . . . . 47
2.7.3 Staining of tissue sections . . . . . . . . . . . . . . . . . . . . . 48
2.8 Laser Capture Microdissection . . . . . . . . . . . . . . . . . . . . . . . 49
2.8.1 Preparation of sections for LCM . . . . . . . . . . . . . . . . . . 50
2.8.2 Fixing and staining of sections for LCM . . . . . . . . . . . . . 50
2.8.3 LCM – procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.8.4 Isolation of RNA from megakaryocytes . . . . . . . . . . . . . . 51
2.9 Analysis software and graphic presentation . . . . . . . . . . . . . . . . 53
2.10 General Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3 Results 55
3.1 Generation of TPO-retrogenic mice . . . . . . . . . . . . . . . . . . . . 55
3.1.1 Cloning of retroviral TPO expression vector . . . . . . . . . . . 55
3.1.2 Generation of Thpo retrovirus packaging cell line . . . . . . . . 57
3.1.3 of TPO-dependent 32D cells . . . . . . . . . . . . . 59
3.1.4 Retrogenic HSC-transfer . . . . . . . . . . . . . . . . . . . . . . 62
3.1.5 General features of TPO-retrogenic mice . . . . . . . . . . . . . 64
3.2 Plasma Cells in TPO-retrogenic Mice . . . . . . . . . . . . . . . . . . . 66
3.2.1 Immunization of mice . . . . . . . . . . . . . . 68
3.2.2 Transfer of antigen-specific cells into TPO-retrogenic mice . . . 74
3.2.3 Retrogenic cell transfer into Ova-immunized mice . . . . . . . . 78
3.3 Gene Expression Analysis of Megakaryocytes . . . . . . . . . . . . . . . 83
3.3.1 LCM of bone marrow megakaryocytes . . . . . . . . . . . . . . 84
3.3.2 RT-PCR analysis of LCM-isolated megakaryocytes . . . . . . . 86
4 Discussion 91
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.2 Methods Used in this Work . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2.1 Working with mouse models . . . . . . . . . . . . . . . . . . . . 92
4.2.2 Induction of Megakaryopoiesis by retrovirally transgenic TPO . 92
4.2.3 The cell line 32D-Mpl displays high sensitivity for TPO . . . . . 93
4.2.4 Laser capture microdissection . . . . . . . . . . . . . . . . . . . 94
4.3 Discussion of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.3.1 Effects of increased TPO-levels by retrogenic cell transfer . . . . 95CONTENTS iii
4.3.2 Antibody titers in TPO retrogenic mice . . . . . . . . . . . . . . 99
4.3.3 Plasma cells in TPO-retrogenic mice . . . . . . . . . . . . . . . 102
4.3.4 The germinal center reaction in TPO-retrogenic mice . . . . . . 104
4.3.5 Gene expression analysis of murine megakaryocytes . . . . . . . 105
4.3.6 Proposed interplay between immunity and megakaryopoiesis . . 106
4.4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5 Summary 109
References 112
Appendix I
Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I
Danksagung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III
Erklärung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX1. Introduction
The immune system is a highly complex assembly of multiple lymphoid organs. It
comprises a great host of different cell types and a dedicated system of vessels called
the lymphatic system. Its main function is the protection of the body from the entirety
of potentially pathogenic microorganisms, multicellular parasites and toxic substances
as well as the clearance of defect and transformed cells that could result in tumors.
The immune system of higher vertebrates is one of the most evolved biological systems,
matched in complexity of cell types and their interactions only by the central nervous
system of higher mammals. Its flawless function is rooted in a fine-tuned balance. On
the one hand, it effectively removes potentially harmful substances and cells from the
body. On the other hand, the virtually unlimited number of tissues and cell types that
make up a healthy individual are recognized as self and left unharmed.
Continuous evolutionary re-adjustment is required to maintain this balance on all lev-
els of organization, as any deviation will tip the balance of immunity toward either
immunodeficiency or autoimmunity. In the latter, the immune system fails to recog-
nize certain host components as self and therefore attacks them, resulting in disease
as displayed e.g. by systemic lupus erythematosus (SLE) (62).
The immune system can be broadly subdivided into innate (or unspecific) immunity
and adaptive (or specific) immunity, the two of which contribute to the immune system
in a predetermined order of events.
1.1 Innate Immunity
1
At first encounter with a pathogen , the mechanisms of innate immunity are initiated.
Steps of innate immunity
2
First, soluble factors in the serum called complement factors initiate host protection
by facilitating phagocytosis of microbes via opsonization and by direct killing via the
membrane-attack complex.
Next, tissue-residentmacrophagesphagocytoseencounteredpathogensandsecretevar-
ious cytokines and chemokines to attract monocytes, neutrophils, NK cells and later
eosinophils and lymphocytes to the site of infection (40), all of which participate in
variouswaysinhostdefense. Attractedmonocytesdifferentiateintomoremacrophages
1Starting here, the word pathogen is used for any microorganism, transformed cell or substance,
that display antigenic epitopes, recognizable as non-self
2The complement system is also calle