Cellular recognition of microbial patterns through toll-like receptor (TLR) 2 [Elektronische Ressource] : analysis of molecular requirements and monoclonal antibody mediated blockage / Guangxun Meng

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 17
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

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


Cellular recognition of microbial patterns through
toll-like receptor (TLR) 2: Analysis of molecular
requirements and monoclonal antibody mediated
blockage


Guangxun Meng


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.


Vorsitzender: Univ.-Prof. Dr. Rudi F. Vogel

Prüfer der Dissertation: 1. Univ.-Prof. Dr. Siegfried Scherer
2. Priv.-Doz. Dr. Stefan Bauer


Die Dissertation wurde am 30. 07. 2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 11. 10. 2004 angenommen. Table of contents

Abbreviation list 4

1 Introduction 10
1.1 Septic shock and Innate immunity 10
1.2 Drosophila Toll receptor and Mammalian TLRs 13
1.2.1 Drosophila Toll 14
1.2.2 Mammalian Toll-like receptors 17
1.3 TLR2 Structure 21
1.4 TLRFunction 25
1.4.1 Recognition of microbial agonists 25
1.4.2 Recognitiendougenous ligands 28
1.4.3 Cellular activation and Phagocytosis 29
1.5 TLR2 Polymorphism and Expression 30
1.5.1 Genomic organization and Genetic polymorphism 30
1.5.2 Expression and Regulation
1.6 Perspectives 32
1.7 Objectives 34
1.7.1 Analysis of structural requirements for the TLR2 extracellular domain
in specific pattern recognition 34
1.7.2 Identification and characterization of TLR2 specific monoclonal antibody 34

2 Materials and Methods 35
2.1 Materials 35
2.1.1 Buffers and solutions
2.1.2 KIT-systems 38
2.1.3 Media 39
2.1.4 Antibodies and antibody conjugates 41
2.1.5 Plasmids 42
2.1.6 Oligonucleotides 42
2.1.7 Reagents 47
2.1.8 Cell lines 48
2.1.9 Mice 48
2.2 Methods 49
2.2.1 Site directed mutagenesis 49
2.2.2 Restrict digestion and ligation of DNA 51
2.2.3 Agarose-gel electrophoresis 51
12.2.4 Transformation of E. coli 51
2.2.5 DNA plasmid preparation from E. coli 52
2.2.6 Silver and coomassie brilliant blue staing of protein resolved on gel 52
2.2.7 Cytochemical staining 53
2.2.8 Cell culture 54
2.2.9 Transient and stable transfection of HEK 293 cells 55
2.2.10 Electroporation 56
2.2.11 Protein isolation 57
2.2.12 Electro mobility shift assay (EMSA) 57
2.2.13 Immunoprecipitation 57
2.2.14 SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) 58
2.2.15 Immunodetection of proteins 59
2.2.16 Biosensor based binding analysis
2.2.17 Generation, identification and purification of TLR2 specific antibodies 60
2.2.18 FACS analysis 63
2.2.19 Luciferase reporter assay 64
2.2.20 Enzyme linked immuno sorbent assay (ELISA) 67
2.2.21 Inhibition of TLR2 dependent cell activation in vitro and in vivo 68
2.2.22 Systemic shock induction 68

3 Results and Discussion (I) 69
3.1 Mutagenesis 69
3.2 Functional analysis of TLR2 ecd mutant constructs 70
3.3 Cellular localization of wild-type and mutant TLR2 72
3.4 DNA binding of NF-κB and phosphorylation of cellular kinase Akt, as well as of
MAP kinases p38, ERK 1/2, and JNK mediated by TLR2 and mutant receptors 72
3.5 Interaction of mutant TLR2 constructs with wild-type TLR2 or MyD88 73
3.6 Epitope difference between TLR2 ecd mutants 74

4 Results and Discussion (II) 76
4.1 Identification and characterization of murine TLR2 specific mAbs 76
4.2 Application of murine mAb T2.5 for expression analysis in vitro 76
4.3 Inhibitory effects of T2.5 on TLR2 specific cell activation 79
4.4 Abrogation of TLR2ECD ligand-binding by T2.5 in a SPR biosensor system and
analysis of T2.5 epitope localization 83
4.5 Surface and intracellular TLR2 expression ex vivo as analyzed immediately upon
primary cellpreparation 85
4.6 Antibody mediated interference with TLR2 specific immune responses towards
systemic challenge 87

25 Summary 92

6 References 93

Acknowledgments 101

Curriculum vitae 102

Appendix 106
App. I Meng G, Grabiec A, Vallon M, Ebe B, Hampel S, Bessler W, Wagner H, Kirschning CJ.
Cellular recognition of tri-/di-palmitoylated peptides is independent from a domain
encompassing the N-terminal seven leucine-rich repeat (LRR)/LRR like motifs of TLR2.
J Biol Chem. 2003 Oct 10; 278 (41): 39822-9.
App. II Meng G, Rutz M, Schiemann M, Metzger J, Grabiec A, Schwandner R, Luppa PB, Ebel
F,Busch DH, Bauer S, Wagner H, Kirschning CJ.
Antagonistic antibody prevents Toll-like receptor 2 driven lethal shock-like syndromes.
J Clin Invest. 2004 May 15; 113:1473-81.

3
Abbrevation list

A
AA Amino acid
Ab Antibody
Ag Antigen
AP-1 Activating protein-1
APC Antigen presenting cell
APC Allophycocyanin
APS Ammonium peroxidedi sulfate
ARDS Adult respiratory distress syndrome

B
β-gal β-galactosidase
BH Brain heart
β−ΜΕ β−Mercaptoethanol
BSA Bovine serum albumin

C
C5αR C5α Receptor
CD Cluster of differentiation
CHO hinese hamster ovary
CLP Cecal Ligation and Puncture
CMV Cytomegalovirus
CREB cAMP response element binding (protein)

D
DC Dendritic cell
D-gal D-galactosamine
DID Death inducing domain
DMEM Dulbecco’s modified eagle medium
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
dNTP Desoxynucleotidetriphosphate
DTT Dithiothreitol

E
EB Ethdium bromide
4ECD Extracellular domain
ECL Enhanced chemoluminescence
E. coli Escherichia coli
EDTA Ethylendiamintetraacetate
EGTA Ethylenglycoltetraacetate
ELAM-1 Endothel cell-leucocyte-adhesion molecule-1
ELISA Enzyme linked immunosorbent assay
EMA Photoactivated ethidium monozide
EMSA Electro mobility shift assay
ER Endoplasmic reticulum
ERK1/2 Extracellular-signal-regulated kinase 1/2
EtOH Ethanol

F
FACS Fluorescence activated cell sorting
FADD Fas associated death domain
FC Flow cell
FCS Fetal calf serum
FITC Fluorescein Isothiocyanate

G
GBS Grop B Strptococci
GIPL Glycoinositol phospholipid
GPI ycosyl-phosphatidyl-inositol (anchor)

H
H Hour
HAT Hypoxanthine, aminopterin, thymidine (hybridoma selection supplement)
HBS Hepes buffered saline
HEK 293 Human embryonic kidney 293
HFCS Hybridoma fusion and cloning supplement
HGPRT Hypoxanthine-guanine phospho-ribosy I transferase
h.i.B.s. heat inactivated Bacillus subtilis
h.i.E.c. Escherichia coli
HMGB1 High Mobility Group Box 1 (protein)
HRP Horse radish peroxidase
HSP60 Heat shock protein 60

I
ICU Intensive Care Unit
5ICD Intracellular domain
IFN-β Interferon-β
I-κB Inhibitor of NF-κB
IKKβ I-κB kinase β
IL Interleukin
IL-1RI IL-1 receptor I
Imd Immune deficiency
IP munoprecipitation
i.p. intra-peritoneal (injection)
IRAK IL-1-receptor-associated kinase
IRF3 Interferon regulatory factor 3

J
JNK c-Jun NH -terminal kinase 2

K
kDa Kilodalton

L
LAM Lipoarabinomannan
LB Luria Bertani
LBP LPS binding protein
LPS Lipopolysaccharide
LRR Leucine rich repeat
LRRCT LRR C-terminal
LTA Lipoteichoic acid
LSM Laser scanning microscope

M
M mol/l
MAb Monoclonal antibody
Mal (TIRAP) MyD88 adaptor-like
MALP-2 Macrophage-activating lipopeptide, 2 kDa
MAPK Mitogen-activated protein kinase
MCP-1 Monocyte chemoattractant protein-1
MEF ouse embryonic fibroblast
MI Macrophage migration Inhibitory Factor
Min Minute
MKK AP kinase kinase
MOF Multiple organ failure
6Mut Mutant
MyD88 Myeloid differentiation marker 88
M/SAPK Mitogen/stress activated protein kinase

N
Na Ac Sodium acetate
NF-κB Nuclear factor κB
NGS Normal goat serum
NO Nitric Oxide
NP40 Nonidet P-40

O
OD Optical density
Omp (Osp) Outer membrane protein
o.n. Over night
Osp (Omp) Outer surface protein

P
p Plasmid
PAGE Polyacrylamide gel electrophoresis
PAI-1 asminogen-activator inhibitor type-1
PAMP Pathogen associated molecular pattern
PBL Peripheral blood lymphocyte
PBMC pheralblmonocyt
PBS Phosphate buffered saline
PBT