Dendritic cells and dietary antigens [Elektronische Ressource] / vorgelegt von Maryna Nikulina

De
Dendritic cells and dietary antigens Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Maryna Nikulina aus Temirtau November 2006 Aus dem Deutsches Diabetes-Zentrum an der Heinrich-Heine Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. H. Kolb Koreferent: Prof. Dr. D. Riesner e.m. Koreferent: Prof. Dr. D. Willbold Tag der mündlichen Prüfung: 21.12.2006 Contents i Contents Contents ……………………………………………………………………………. i Abbreviations……………………………………………………………………… iv 1 Introduction……………………………………………………………………… 1 1.1 Gut immune system……………………………………………………...1 1.2 Dendritic cells……………………………………………………………4 1.3 Oral tolerance………………………………………………………….. 12 1.4 Food antigens………………………………………………………….. 16 1.4.1 Wheat gluten……………………………………………………..16 1.4.2 Hsp 60……………………………………………………………19 1.5 Diabetes, food antigens and gut immune system……………………… 23 1.5.1 Type 1 diabetes mellitus (T1DM) ……………………………… 23 1.5.
Publié le : dimanche 1 janvier 2006
Lecture(s) : 28
Tags :
Source : DOCSERV.UNI-DUESSELDORF.DE/SERVLETS/DERIVATESERVLET/DERIVATE-3605/1605.PDF
Nombre de pages : 165
Voir plus Voir moins




Dendritic cells and dietary antigens



Inaugural-Dissertation
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf



vorgelegt von
Maryna Nikulina
aus Temirtau


November 2006
Aus dem Deutsches Diabetes-Zentrum
an der Heinrich-Heine Universität Düsseldorf











Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. H. Kolb
Koreferent: Prof. Dr. D. Riesner e.m.
Koreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 21.12.2006




Contents i
Contents

Contents ……………………………………………………………………………. i

Abbreviations……………………………………………………………………… iv

1 Introduction……………………………………………………………………… 1
1.1 Gut immune system……………………………………………………...1
1.2 Dendritic cells……………………………………………………………4
1.3 Oral tolerance………………………………………………………….. 12
1.4 Food antigens………………………………………………………….. 16
1.4.1 Wheat gluten……………………………………………………..16
1.4.2 Hsp 60……………………………………………………………19
1.5 Diabetes, food antigens and gut immune system……………………… 23
1.5.1 Type 1 diabetes mellitus (T1DM) ……………………………… 23
1.5.2 BB rats…………………………………………………………...24
1.5.3 Association between T1DM and celiac disease (CD) …………..25
1.5.4 Diet is a risk factor for T1DM………………………………….. 26
1.6 Aims of the current study……………………………………………… 28
2 Materials and methods………………………………………………………….29
2.1 Materials………………………………………………………………..29
2.1.1 Chemicals………………………………………………………. 29
2.1.1.1 Chemicals……………………………………………… 29
2.1.1.2 Inhibitors, agonists and antagonists…………………… 30
2.1.2 Materials for SDS-PAGE and Western blot……………………. 30
2.1.2.1 Antibodies……………………………………………… 30
2.1.2.2 Markers………………………………………………….31
2.1.2.3 Others…………………………………………………... 31
2.1.3 Antibodies for Fluorescence Activated Cell Sorting (FACS)
analysis…………………………………………………………. 31
2.1.3.1 Primary antibodies…………………………………….. 31
2.1.3.2 Secondary antibody……………………………………. 32
2.1.3.3. Blocking antibody……………………………………… 32
2.1.4 Materials for Enzyme-Linked Immunosorbent Assay (ELISA)... 32
2.1.4.1 ELISA Sets……………………………………………. 32
2.1.4.2 Others…………………………………………………. 34
2.1.5 Materials for Limulus Amebocyte Lysate (LAL) Assay………. 35
2.1.6 Materials for protein determination……………………………. 35
2.1.7 Medium, buffers and solutions………………………………… 35
2.1.7.1 Media and serum for cell culture……………………… 35
2.1.7.1.1 Media for dendritic cell culture……………. 35
2.1.7.1.2 Media for macrophage culture………………35 Contents ii
2.1.7.1.3 Investigated stimuli for dendritic
cell culture and macrophage culture……….. 36
2.1.7.2 Buffer for cell lysate preparation……………………… 37
2.1.7.3 Solutions, buffers and gels for SDS-gel
electrophoresis and electrotransfer…………………… 38
2.1.7.4 Solutions for immunological determination
of proteins by Western blot…………………………… 39
2.1.7.5 Solutions for FACS analysis………………………….. 39
2.1.7.6 Buffers and solutions for ELISA……………………… 39
2.1.8 Labware………………………………………………………… 41
2.1.9 Equipment……………………………………………………… 42
2.1.10 Animals………………………………………………………. 43
2.1.11 Diet…………………………………………………………… 43
2.2 Methods……………………………………………………………….. 44
2.2.1 Cell culture……………………………………………………… 44
2.2.1.1 Isolation and culture of dendritic cells (DCs)………….. 44
2.2.1.2 Culture of macrophages…………………………………45
2.2.2 Biochemical methods…………………………………………… 45
2.2.2.1 Solubilization of wheat gluten…………………………. 45
2.2.2.2 Isolation of gut tissue samples and preparation
of tissue homogenates………………………………….. 45
2.2.2.3 Preparation of cell lysates……………………………… 46
2.2.2.4 Protein determination………………………………….. 46
2.2.2.5 SDS-Polyacrylamide Gel Electophoresis
(SDS-PAGE) of proteins………………………………. 46
2.2.2.6 Electrophoretic transfer………………………………... 48
2.2.3 Immunological methods…………………………………………49
2.2.2.1 Immunological determination of proteins
by Western blot………………………………………… 49
2.2.3.2 FACS analysis…………………………………………. 49
2.2.3.3 ELISA…………………………………………………. 51
2.2.4 LAL assay………………………………………………………. 53
2.2.5 Statistics and graph computer programmes…………………….. 54
3 Results…………………………………………………………………………... 55
3.1 In vitro study of the effects of food components on innate immunity… 55
3.1.1 Wheat gluten……………………………………………………. 55
3.1.1.1 Wheat gluten stimulates maturation of BMDC………... 55
3.1.1.2 Wheat gluten-induced maturation of BMDC
is not due to bacterial contamination……………………56
3.1.1.3 Wheat gluten induces cytokine and
chemokine production in BMDC………………………..60
3.1.1.4 Wheat gluten-stimulated chemokine production
in BMDC is not secondary to IL-1β secretion………… 63 Contents iii
3.1.1.5 Wheat gluten signals in BMDC through p38,
extracellular signal-regulated kinases (ERK 1/2) and
NFκB, but not c-Jun NH -terminal kinase (JNK)……… 65 2
3.1.1.6 The active compound in wheat gluten preparation has
a peptidic nature………………………………………... 66
3.1.1.7 None of the investigated synthetic gliadin peptides
reproduce stimulatory activity of wheat gluten
α-chymotryptic digest on BMDC……………………… 67
3.1.1.8 PEP does not affect the stimulatory capacity of active wheat gluten peptide(s) on BMDC……………70
3.1.2 Hsp60…………………………………………………………… 71
3.1.2.1 Hsp60 induces maturation of BMDC………………….. 71
3.1.2.2 Hsp60 induces cytokine and chemokine
production in BMDC……………………………………73
3.1.2.3 HSP60-induced maturation of BMDC is partly due
to LPS………………………………………………….. 75
3.1.2.4 The involvement of TLR4 in Hsp60 signaling
in BMDC………………………………………………. 79
3.1.2.5 MAPK signalling in BMDC after stimulation
with Hsp60………………………………………………82
3.2 In vivo study of the effect of dietary wheat gluten on gut immunity…. 83
3.2.1 The level of IFN-γ in duodenum of BB rats fed the WG,
NTP-2000 or HC diets………………………………………….. 83
3.2.2 The level of IL-10 and of the ratio IFNγ/IL-10 in gut
duodenum of BB rats fed the WG, NTP-2000 or HC diets…….. 86
3.2.3 MCP-1 content in gut duodenum of BB rats fed the WG, 92
3.2.4 MIP-2 level in duodenum of BB rats fed the WG, 95
3.2.5 IFNγ, IL-10 and MCP-1 levels in duodenum of small
intestine of BB rats before and right after weaning……………. 98

4 Discussion………………………………………………………………………. 101
5 Conclusions…………………………………………………………………….. 124
6 Summary (in English and in German)……………………………………….. 125
7 Literature………………………………………………………………………. 128
Acknowledgements……………………………………………………………….. 155 Abbreviations iv
Abbreviations

ANOVA Analysis of variances
AP Alkaline phosphatase
AP-1 Activating protein-1
APC Antigen-presenting cell Allophycocyanin
APS Ammoniumpersulfate
ATP Adenosine triphosphate
BB Bio-Breeding
BMDC Bone-marrow-derived dendritic cell
CD Celiac disease
CP Cryptopatch
α-CT α-Chymotrypsin
CTL Cytotoxic T lymphocytes
CTLA Cytotoxic-T-lymphocyte-associated antigen
DC Dendritic cell(s)
DC-SIGN DC-specific ICAM-3 grabbing nonintegrin
ELISA Enzyme-linked immunosorbent assay
ERK 1/2 Extracellular signal-regulated kinases
EU Endotoxin units
Fig. Figure
FACS Fluorescence-activated cells sorter
FAE Follicle-associated epithelium
FCS Fetal calf serum
FITC Fluorescein isothiocyanate
Flt3L Fms-like tyrosine kinase 3 ligand
Fox Forkhead box
GAD Glutamic acid decarboxylase
GALT Gut-associated lymphoid tissue
Gln ine Abbreviations v
GM-CSF Granulocyte macrophage colony stimulating factor
HC Hydrolyzed casein
HEPES N-(2-Hydroxyethyl)piperazine-N`-2-erhane sulfonic acid
HLA Human leukocyte antigen
HRP Horseradish peroxidase
HSP Heat shock protein
HMW High molecular weight
IBD Inflammatory bowel disease
ICAM Intercellular adhesion molecule
IDO Indoleamine 2,3-dioxygenase
IEL Intraepithelial lymphocytes
IENK Intraepithelial natural killer cells
IFN Interferon
Ig Immunoglobulin
IKDC Interferon-producing killer dendritic cell
IL Interleukin
IL-1R in-1 receptor
ILFs Isolated lymphoid follicles
JNK c-Jun NH -terminal kinase 2
KC Keratinocyte-derived cytokine
LAL Limulus amebocyte lysate
LAP Latency-associated peptide
LMW Low molecular weight
LN Lymph node
LP Lamina propria
LPS Lipopolysaccharide
MALP Macrophage-activating lipopeptide
MALT Mucosal-associated lymphoid tissue
MAPK Mitogen-activated protein kinase
MFI Mean fluorescence intensity
MHC Major histocompatibility complex Abbreviations vi
MIP Macrophage inflammatory protein
Mkk3 Mitogen-activated protein kinase kinase 3
MLN Mesenteric lymph node
MAdCAM Mucosal addressin cell adhesion molecule
MCP Monocyte chemoattractant protein
MyD88 Myeloid differentiation factor 88
NFκB Nuclear factor kappa B
NOD Non-obese diabetic
NOS Nitric oxide synthetase
pNA p-Nitroaniline
OVA Ovalbumin
PAGE Polyacrylamide gel electrophoresis
PAMP Pathogen-associated molecular pattern
PBS Phosphate buffered saline
PE Phycoerythrin
PEP Prolyl endopeptidase
PmB Polymyxin B
PP Peyer’s patch
Pro Proline
PRR Pattern recognition receptor
RANTES Regulated on activation, normal T cell expressed and secreted
SDS Sodium dodecylsulfate
SED Subepithelial dome
TBS Tris buffered saline
TBST TBS/Tween20
TCA Trichloracetate
T1DM Type 1 diabetes mellitus
TEMED N,N,N’,N’ -Tetramethylethylendiamine
TECK Thymus-expressed chemokine
tTG Tissue transglutaminase
TGF Transforming growth factor Abbreviations vii
TLR Toll-like receptor
TMB 3,3’,5,5’-Tetramethylbenzidine
TNF Tumor necrosis factor
TSLP Thymic stromal lymphopoeitin
VCAM Vascular cell adhesion molecule
WF Wistar-Furth
WG Wheat gluten 1. Introduction 1
1 INTRODUCTION
1.1 Gut immune system
Striking feature of the mammalian gut immune system is the ability to distinguish
between harmful and non-pathogenic flora and to be tolerant to a great variety of food
antigens. Mammals and other vertebrates successfully coexist with a diverse and
abundant non-pathogenic intestinal microflora: the lower intestine of mammals contains
12an enormous load of commensal bacteria with a density of 10 organisms per ml of
luminal content that are representative of about 1000 species, mostly anaerobes (Mackie,
R. et al., 1999; Macpherson, A.J. et al., 2005). The intestine is the biggest lymphoid
12tissue in the body. There are 10 lymphoid cells per meter of human small intestine, and
the number of immunoglobulin (Ig)-secreting cells in murine or human gut exceeds by
several fold their amount found in all other lymphoid organs together (Mestecky, J., et.
al., 1987; van der Heidjen, P.J., et al., 1987).
Gastrointestinal associated lymphoid tissue referred to as GALT for Gut-Associated
Lymphoid Tissue, or MALT for Mucosa-Associated Lymphoid Tissue, can be divided
into loosely organized effector sites, i.e. the layer of connective tissue in a mucosa
immediately beneath the epithelium called the lamina propria (LP) and intraepithelial
lymphocytes (IEL), and more organized structures - mesenteric lymph nodes (MLNs),
Peyer’s patches (PPs), isolated lymphoid follicles (ILFs) and cryptopatches (CPs)
(Newberry, R.D., 2005).
The surface area of intestinal mucosa exceeds in several folds the skin area and is
2estimated to be 300 m in the human small intestine (Moog, F., 1981). Such a large
mucosal surface area in the small intestine is generated by means of evagination into
plicae and villi and short tubular invaginations, the crypts. Plicae and villi help to
increase gut contact with luminal content, and the crypts provide a protected site for stem
cells that are located along the crypt length. The intestinal mucosa is a major
immunological surveillance site, and all cell types required for antigen-presenting cell
(APC)/T cell interactions are found here. APC are defined as highly specialized cells that
can process antigens and display their peptide fragments on the cell surface together with
molecules required for T cell activation (Janeway, C.A.Jr., et al., 2001). The immune
cells that can express both major histocompatibility complex (MHC) class I and class II

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.