Die Rolle von Chemokinen bei der Rekrutierung von Leukozyten durch die Blut-Hirn-Schranke [Elektronische Ressource] / vorgelegt von Carsten Alt

Carsten Alt <[e-mail]>

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 51 Mo

Extrait

Aus dem
Max-Planck Institut fur klinische und physiologische Forschung /
Kerckho Institut, Bad Nauheim
Direktor: Prof. Dr. Werner Risau†
und dem
Max-Planck Institut fur vaskul are Biologie, Munster
Direktor: Prof. Dr. Dietmar Vestweber
Die Rolle von Chemokinen
bei der Rekrutierung von Leukozyten
durch die Blut-Hirn-Schranke
Inaugural-Dissertation zur Erlangung des Doktorgrades der Humanbiologie
dem Fachbereich Humanmedizin der Philipps-Universit at Marburg vorgelegt von
Carsten Alt
aus Friedberg / Hessen
Marburg 2003iiFrom the
Max-Planck Institute for Clinical and Physiological Research /
Kerckho Institute, Bad Nauheim
Director: Prof. Dr. Werner Risau†
and
Max-Planck Institute for Vascular Biology, Muns ter
Director: Prof. Dr. Dietmar Vestweber
The Role of Chemokines
in Leukocyte Recruitment
across the Blood-Brain Barrier
Inaugural-Dissertation to obtain the degree of Doctor of Human Biology
submitted to the Faculty of Medicine at the Philipps-University Marburg by
Carsten Alt
from Friedberg / Hessen
Marburg 2003iv
Angenommen vom Fachbereich Humanmedizin der Philipps-Universitat Marburg
am 09.06.2004, gedruckt mit Genehmigung des Fachbereichs.
Accepted by the Faculty of Medicine at the Philipps-University Marburg
06/09/2004, printed with permission of the Faculty.
Dekan: Prof. Dr. med. Bernhard Maisch
Referent: Prof. Dr. Britta Engelhardt
1. Correferent: Prof. Dr. Klaus Michael Heeg
2. Correferent: Prof. Dr. Jurg en WestermannContents
1 Introduction 1
1.1 The blood-brain barrier. . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Multiple sclerosis and experimental autoimmune encephalomyelitis . . 4
1.3 Multi-step model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.4 Chemokines and chemokine receptors . . . . . . . . . . . . . . . . . . 8
1.5 Chemokine involvement in EAE . . . . . . . . . . . . . . . . . . . . . 13
1.6 The aim of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2 Materials and Methods 16
2.0.1 Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.0.2 Expendable materials . . . . . . . . . . . . . . . . . . . . . . . 18
2.0.3 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.0.4 Bu ers and Solutions . . . . . . . . . . . . . . . . . . . . . . . 18
2.1 Tissue culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1.1 Tissue culture media and solutions . . . . . . . . . . . . . . . 18
2.1.2 Primary cell preparation . . . . . . . . . . . . . . . . . . . . . 20
2.1.3 T cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.4 Endothelial cell culture . . . . . . . . . . . . . . . . . . . . . . 25
2.1.5 GP+E86 cell culture . . . . . . . . . . . . . . . . . . . . . . . 27
2.1.6 Chemotaxis assay . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1.7 Transmigration assay . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.8 Frozen section assay . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.9 Cytospin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.1.10 Flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.1 Experimental autoimmune encephalomyelitis . . . . . . . . . . 30
vvi CONTENTS
2.2.2 Intravital microscopy . . . . . . . . . . . . . . . . . . . . . . . 32
2.3 Morphological methods . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.2 Tissue preparation and sectioning . . . . . . . . . . . . . . . . 38
2.3.3 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . 38
2.3.4 Immuno uorescence . . . . . . . . . . . . . . . . . . . . . . . 39
2.3.5 In situ hybridization . . . . . . . . . . . . . . . . . . . . . . . 39
2.4 Molecular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.4.1 Bu ers and Solutions . . . . . . . . . . . . . . . . . . . . . . . 44
2.4.2 Molecular DNA cloning techniques . . . . . . . . . . . . . . . 47
2.4.3 Subtractive suppression hybridization . . . . . . . . . . . . . . 52
2.4.4 High-throughput DNA mini preparation and sequencing . . . 53
2.4.5 Genotyping by polymerase chain reaction . . . . . . . . . . . . 54
2.4.6 Molecular RNA preparation and analysis . . . . . . . . . . . . 55
2.4.7 Gene array analysis . . . . . . . . . . . . . . . . . . . . . . . . 58
2.5 Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.5.1 Preparation of protein samples from animal tissue . . . . . . . 59
2.5.2 Bicinchoninic acid (BCA) protein assay . . . . . . . . . . . . . 59
2.5.3 Polyacrylamide gel electrophoresis (PAGE) . . . . . . . . . . . 60
2.5.4 Coomassie staining of SDS-gels . . . . . . . . . . . . . . . . . 61
2.5.5 Semi-dry electroblot . . . . . . . . . . . . . . . . . . . . . . . 61
2.5.6 Immunoblotting (western blotting) . . . . . . . . . . . . . . . 62
2.5.7 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3 Results 66
3.1 Expression and functional involvement of chemokines in lymphocyte
recruitment across the endothelial BBB . . . . . . . . . . . . . . . . . 66
3.1.1 Preparation of chemokine cDNA clones . . . . . . . . . . . . . 66
3.1.2 FunctionalexpressionofthelymphoidchemokinesCCL19and
CCL21 at the blood-brain barrier . . . . . . . . . . . . . . . . 67
3.1.3 CCL19 and CCL21 are present in in ammatory cu s in the
CNS of mice a icted with EAE . . . . . . . . . . . . . . . . . 73CONTENTS vii
3.1.4 Quanti cation of CCL19 and CCL21 in whole brain lysate of
mice a icted with EAE . . . . . . . . . . . . . . . . . . . . . 74
3.1.5 CCR7andCXCR3arepresentonencephalitogenicTlympho-
cytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.1.6 Encephalitogenic T lymphocytes express L-selectin in vivo . . 76
3.1.7 Encephalitogenic T lymphocytes speci cally chemotax to-
wards CCL19 and CCL21 . . . . . . . . . . . . . . . . . . . . 79
3.1.8 Transmigration of encephalitogenic T cells towards chemokines 79
3.1.9 Reduced binding of encephalitogenic T lymphocytes to in-
amed brain venules in vitro . . . . . . . . . . . . . . . . . . . 82
3.1.10 Functional involvement of CCL19 and CCL21 in lymphocyte
recruitment across the endothelial BBB in vivo . . . . . . . . 82
3.2 Gene and protein expression pro ling of cerebral microvessels in EAE 85
3.2.1 Gene chip analysis of cerebral microvessels . . . . . . . . . . . 85
3.2.2 Subtractive suppression hybridization analysis of cerebral mi-
crovessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3.2.3 Proteomic analysis of cerebral microvessels . . . . . . . . . . . 112
3.3 Gene expression pro ling of encephalitogenic T cells . . . . . . . . . . 114
3.3.1 Gene array analysis of encephalitogenic T cells . . . . . . . . . 115
3.3.2 Subtractive-suppressionhybridizationanalysisof encephalito-
genic T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.4 InvolvementofDARCinleukocyterecruitmentacrosstheendothelial
blood-brain barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3.4.1 Expression of DARC during EAE . . . . . . . . . . . . . . . . 120
3.4.2 Experimental autoimmune encephalomyelitis in DARC-
de cient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
3.4.3 Endothelioma cell lines lacking DARC . . . . . . . . . . . . . 123
4 Discussion 125
4.1 Functional expression of CCL19 and CCL21 at the endothelial BBB . 125
4.2 Gene and protein pro ling of cerebral microvessels . . . . . . . . . . . 130
4.3 Gene expression pro ling of encephalitogenic T cells . . . . . . . . . . 140viii Table of Contents
4.4 InvolvementofDARCinTcellrecruitmentintotheCNSduringEAE
pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.5 Modelforchemokineinvolvementinlymphocyterecruitmentintothe
CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
A Bibliography 145
B Summary 175
C Zusammenfassung 177
D List of abbreviations 179
E List of gures 183
F List of tables 185
G Lebenslauf 186
H Akademische Lehrer 188
I Danksagung 189
J Ehrenw ortliche Erklarung 191Chapter 1
Introduction
The highly sophisticated network of cooperating neurons within the central nervous
system (CNS) requires stringent homeostasis of its environment for proper function.
Thishomeostasisismaintainedbytheendothelialblood-brainbarrier(BBB),which
protects the CNS from the changing milieu of the bloodstream by strictly limiting
passage of molecules and cells. However, another highly sophisticated network of
cells, which composes the immune system, requires tra cking of its cellular compo-
nents through the whole organism to guarantee immunosurveillance. Tra cking of
lymphocytesfromthebloodstreamintotissuesisnotrandom,butstrictlyregulated.
Taken together, the necessity of stringent regulation of the CNS micromilieu on the
one hand and immunosurveillance on the other hand suggests unique mechanisms
for lymphocyte recruitment across the endothelial BBB into the CNS.
1.1 The blood-brain barrier
Normal function of neurons within the brain and spinal cord requires a homeostatic
microenvironment, which is maintained by the blood-brain bar