The role of galectins in clearance and immunogenicity of apoptotic and secondary necrotic cells [Elektronische Ressource] = Die Rolle der Galektine in der Clearance und Immunogenität apoptotischer und sekundär nekrotischer Zellen / vorgelegt von Kerstin Sarter

Schulzce

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2010
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

The Role of Galectins in Clearance and Immunogenicity of Apoptotic and
Secondary Necrotic Cells
(Die Rolle der Galektine in der Clearance und Immunogenität apoptotischer
und sekundär nekrotischer Zellen)


Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg






zur Erlangung des Doktorgrades



vorgelegt von
Kerstin Sarter
aus Schwetzingen


                                                                                                                                                    























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Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-
Alexander-Universität Erlangen-Nürnberg










Tag der mündlichen Prüfung: 16.04.2010


Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Lars Nitschke
Zweitberichterstatter: PD Dr. Reinhard Voll




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4 Table of Contents
Table of Contents

Abstract 7
Zusammenfassung (german summary) 9

1. Introduction 11
1.1. Galectins - Introduction 11
1.1 1. Gene organization 12
1.1.2. Structure 12
1.1.3. Binding specificity 14
1.1.4 Non-classical secretion pathway 14
1.1.5 Expression sites and regulated secretion 16
1.1.6 Ligands/counterreceptors 16
1.2. Biological functions of galectins 18
1.2.1. Cell adhesion 19
1.2.2. Cell homeostasis 20
1.2.3. Inflammation 23
1.3. Apoptosis, clearance and development of autoimmunity 24
1.3.1. Apoptosis – programmed cell death 24
1.3.2. Clearance of apoptotic cells and clearance defects 26
1.3.3 Apoptosis failure and autoimmunity 30
1.3.4 Role of galectins in development and pathogenicity of autoimmunity 31

2. Material and Methods 35
2.1. Materials 35
2.2. Methods 41
2.2.1 Galectins 41
2.2.2 Cells and culture conditions 42
2.2.3 ELISA 45
2.2.4 Assays for LPS detection 48
2.2.5 SLE and RA study group 49
2.2.6 Phagocytosis Assay 50
2.2.7 Immunization experiments 51

5 Table of Contents
3. Results 55
3.1. Detection and chromatographic removal of lipopolysaccharide in preparations of
galectins 55
3.2. Human Galectins as sensors for apoptosis/necrosis-associated surface changes
of granulocytes and lymphocytes 62
3.3. Influence of Galectins on the immunogenicity of apoptotic and secondary
necrotic cells 70
3.4. Autoantibodies against galectins 78
3.5 Interaction with phospholipids of galectins 85

4. Discussion 91
4.1. Detection and chromatographic removal of lipopolysaccharide in preparations of
recombinant multifunctional galectins 91
4.2. Human Galectins as sensors for apoptosis/necrosis-associated surface changes
of granulocytes and lymphocytes 93
4.3. Influence of Galectins on the immunogenicity of apoptotic and secondary
necrotic cells 95
4.4 Role of human Galectins in the development and pathogenicity of autoimmune
diseases 100
4.5 New binding mechanism of galectins 102

5. Concluding Remarks 105

6. Acknowledgements 107

7. References 109

Curriculum vitae 125









6Abstract
Abstract
The objective of this Ph.D. thesis was to investigate the role of galectins in clearance
and immunogenicity of apoptotic and secondary necrotic cells
We first monitored the potential LPS contamination of galectin preparations. We
observed that almost all recombinant produced galectins were contaminated with
LPS. We then tested a convenient and effective method for decontaminating the
galectins. This was important to continue with further functional assays. To
investigate the role of galectins during clearance, we first had to determine the
binding characteristics of galectins to viable, apoptotic, secondary apoptotic ad
necrotic cells. We observed that galectins display an inhomogeneous binding pattern
with an increase of binding towards late stages of apoptosis. Since galectins
differentially recognize apoptotic cells they may play a potentially non-redundant role
in the recognition and opsonization of apoptotic cells for phagocytic clearance. This
assumption was supported by the findings that opsonization of apoptotic cells with
galectins increased the rate of phagocytic uptake. Furthermore, opsonization with
galectins of apoptotic and secondary necrotic cells was able to shift and decrease the
respective immune response, indicating that galectins may act as opsonins for dying
and dead cells and play a pivotal role in directing the outcome of phagocytosis.
Furthermore, pre-incubation of irradiated tumor cells with galectins decreased or
even abrogated the protection from tumor development induced by tumor
vaccination. This further underlined our assumption that galectins act as pivoting
opsonins for dying and dead cells capable of interfering with the clearance process.
Anti-galectin autoantibodies were present in sera of patients with the autoimmune
diseases SLE and RA. Since the development of SLE is connected to deficiencies in
clearance, the presence of anti-galectin autoantibodies may play a role in the
7Abstract
pathogenicity of this autoimmune disease. Further examination of the functional role
of galectins may reveal new aspects within the surveillance system to protect against
auto-inflammation. In summary, the presented results demonstrate that galectins can
actively influence the recognition and clearance of dying and dead cells and thus play
a role in the inflammatory outcome of apoptotic cells uptake.