More than a danger Signal - the versatility of the heat shock protein Gp96 in innate and adaptive immunity [Elektronische Ressource] = Mehr als ein Alarmsignal - die vielfältigen Rollen des Hitzeschockproteins Gp96 in der angeborenen und erworbenen Immunität / vorgelegt von Norbert Hilf

Noh

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2003
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

More than a Danger Signal –
The Versatility of the Heat Shock Protein Gp96 in
Innate and Adaptive Immunity

Mehr als ein Alarmsignal -
Die vielfältigen Rollen des Hitzeschockproteins Gp96 in der
angeborenen und erworbenen Immunität



D I S S E R T A T I O N

der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften

2003

vorgelegt von
Norbert Hilf GP96 – MORE THAN A DANGER SIGNAL















Meinen Eltern gewidmet.




























Tag der mündlichen Prüfung: 30. April 2003

Dekan: Prof. Dr. H. Probst
1. Berichterstatter: Prof. Dr. H.-G. Rammensee
2. Berichterstatter: PD Dr. H. Schild
GP96 – MORE THAN A DANGER SIGNAL
Contents
Abbreviations...................................................................................................................5
Summary .........................................................................................................................7
Zusammenfassung ..........................................................................................................8
Chapter 1 General Introduction...............................................................................9
Chapter 2 The Heat-Shock Protein Gp96 Links Innate and Specific
Immunity (Introduction Part II)
Int. Journal of Hyperthermia 18[6], 521-533 [Review] (2002)...............................31
Outline of this Thesis .....................................................................................................48
Chapter 3 Cross-presentation of Gp96-Associated Antigens on MHC
Class I Molecules Requires Receptor-Mediated Endocytosis
Journal of Experimental Medicine 191, 1965-1974 (2000)..................................49
Chapter 4 The Heat Shock Protein Gp96 Induces Maturation of
Dendritic Cells and Down-Regulation of its Receptor
European Journal of Immunology 30, 211-2215 (2000) .....................................71
Chapter 5 Human Platelets Express Heat Shock Protein Receptors and
Regulate Dendritic Cell Maturation
Blood 99[10], 3676-3682 (2002) ...................................................................83
Chapter 6 The ER-Resident Heat Shock Protein Gp96 Activates
Dendritic Cells Via the TLR2/4 Pathway
Journal of Biological Chemistry 277[23], 20847-20853 (2002)...........................101
Chapter 7 Relevance of Toll-like Receptor Signaling for Priming of
Cytotoxic T-Lymphocytes in vivo
(submitted) ............................................................................................119
Chapter 8 Discussion and Outlook......................................................................133
References ..................................................................................................................139
List of publications .......................................................................................................167
Danksagungen - Acknowledgements ..........................................................................168
Curriculum vitae...........................................................................................................170
Akademische Lehrer....................................................................................................171
Lebenslauf...................................................................................................................172
4
Abbreviations
For peptide sequences the one- or three-letter amino acid code was used. SI units
and standard abbrevations are not explained in the table.

ABTS 2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)
ACAMP Apoptotic cell-associated molecular patterns
APC Antigen presenting cell
BMDC Bone marrow-derived DC
BSA Bovine Serum Albumin
CD Cluster of differentiation
CLIP Class II-associated invariant chain peptide
CTL Cytotoxic T-lymphocyte
DC Dendritic cell
DMSO Dimethylsulfoxid
dsRNA Double-stranded ribonucleic acid
ELISA Enzyme-linked immunosorbent assay
ER Endoplasmic reticulum
FACS fluorescence-activated cell sorter
FCS Fetal calf serum
FITC Fluorescein isothiocyanate
GM-CSF Granulocyte macrophage colony stimulating factor
Gp96 96 kDa glucose-regulated protein
HAU haemagglutinating units
HLA Human leukocyte antigen
HSP Heat shock protein
Ig Immunglobulin
IL Interleukin
Imd gene Immune deficiency gene
IMDM Iscove’s Modified Dulbecco’s Medium
IRAK IL-1receptor-associated protein kinase
ITAM Immunoreceptor tyrosine-based activation motif
Jnk c-Jun N-terminal kinase
LBP LPS-binding protein
LPS Lipopolysaccharide
mAb Monoclonal antibody
MAPK mitogen-activated protein kinase
MBL Mannan-binding lectin
MDC Monodansylcadaverine
MSR Macrophage scavenger receptor
MyD88 Myeloid differentiation factor 88
OAS 2’-5’-oligoadenylate synthase (OAS)
OVA Ovalbumin
Pam Cys (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine 3
PAMP Pathogen-associated molecular pattern
PBL Peripheral blood lymphocytes
PBMC eral blood mononuclear cell
PBS Phosphate-buffered saline
5GP96 – MORE THAN A DANGER SIGNAL
PDI Protein disulfide-isomerase
PE Phycoerythrin
Poly(I:C) Polyinosine-polycytidylic acid
PRP Platelet rich plasma
PRR Pattern recognition receptor
SAPK Stress-activated protein kinases
TAP Transporter associated with antigen processing
TCR T cell receptor
TIR domain Toll/IL-1 receptor domain
TIR domain containing adaptor protein TIRAP
TNF-α Tumor necrosis factor-α
TRAF TNF receptor activated factor
TRIF TIR domain-containing adapter inducing IFN-β

Figures are numbered for each chapter separately. If not otherwise stated, the
mentioned figure numbers refer to the figures in the same chapter.

6
Summary
Immunization with tumor lysates induces tumor protection against the original tumor
in mouse models. Surprisingly, all tumor antigens purified from the lysates belong to
the heat shock protein (HSP) family and are not mutated compared to the proteins in
normal tissue. Instead, their immunogenicity is based on tumor-specific peptides
bound to the HSPs. The investigation of the mechanisms that allow the induction of
efficient immune responses with low doses of HSP:peptide complexes was the focus
of this work, which was performed using mainly the ER-resident HSP Gp96.
We were able to show (chapter 3) that HSP-mediated immunity depends on its
receptor-mediated uptake by professional antigen presenting cells (APCs). This is a
prerequisite for the efficient shuttling of the associated peptides to the MHC
molecules of the APC. Moreover, specific immune responses require the unspecific
activation of APCs by danger signals. These lead to the upregulation of costimulatory
molecules and the secretion of pro-inflammatory cytokines. Danger signals can be
pathogen-derived as well as endogenous substances. Here we provide evidence that
Gp96 itself is a danger signal (chapter 4). Gp96 triggers APC activation via the Toll-
like receptors (TLRs) 4 and 2 pathways which are also used by pathogen-derived
danger signals (chapter 6). TLR-mediated activation of the immune system is of
importance for the specific immune response (chapter 7). Priming of cytotoxic T-
+lymphocytes (CTLs) after a viral challenge depends on either CD4 T cells or TLR-
mediated signals. If both are missing, CTL priming is impaired. For minor H specific
CTL responses, TLR signaling is strictly required. A similar importance of TLR-
mediated signals can be expected for Gp96-induced immune responses.
The strong inflammatory signals triggered by Gp96 have to be controlled in vivo. In
chapter 5, it is shown that human platelets are able to control the immunostimulatory
capacities of Gp96. Platelets bind high amounts of Gp96, especially after their
activation by thrombin. Thereby, free Gp96 is withdrawn resulting in a lower APC
activation. This mechanism might have a physiological role during wound healing to
prevent the adverse effects of chronic inflammation.
HSPs are more than endogenous danger signals released from dying cells. They
also deliver detailed information about the events that caused the death. They
combine everything in a single protein that is required for the induction of an efficient
and specific immune response.
7GP96 – MORE THAN A DANGER SIGNAL
Zusammenfassung
Die Injektion von Tumorlysaten bewirkt im Mausmodell eine protektive Immunität
gegen den verwendeten Tumor. Alle in den Arbeiten von P. Srivastava identifizierten
Tumorantigene gehören jedoch zur Familie der Hitzeschockproteine (HSPs) und sind
nicht gegenüber dem Protein im Ursprungsgewebe verändert. Die Immunogenität
rührt vielmehr von an das HSP gebundenen, tumorspezifischen Peptiden. Die
Aufkl&#