The guanylate binding protein-1 [Elektronische Ressource] : a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells / vorgelegt von Clara Lubeseder-Martellato

Lubeseder-Martellato, Clara

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

The guanylate binding protein-1:
a molecular marker of the inflammatory cytokine-
activated phenotype of endothelial cells












Dissertation
der Fakultät für Biologie der Ludwig-Maximilians-Universität in München
zur Erlangung des Grades Doktor der Naturwissenschaften
- Dr. rer. nat.-















vorgelegt von
Clara Lubeseder-Martellato
aus Venedig, Italien

2003













































Eingereicht am: 26. Juni 2003
Erstgutachter: Prof. Dr. E. H. Weiß
Zweitgutachter: PD Dr. H. Weiher
Sondervotum: Prof. Dr. M. Stürzl
Tag der mündlichen Prüfung: 28. Juli 2003 Table of contents
TABLE OF CONTENTS

ABBREVIATIONS……………………………………………………………….……... 1
SUMMARY……………………………………………………………………………... 3

INTRODUCTION
1 Function of the quiescent endothelium ………………………………………………. 5
2 Pathophysiological activation of the endothelium during inflammation …………….. 6
2.1 Vessel sprountig (angiogenesis)…….............……………………………….......... 7
2.2 The role of angiogenic growth factors in endothelial cell activation.….…………. 8
2.2.1 Basic fibroblast growth factor (bFGF) ………………………………………... 8
2.2.2 Vascular endothelial growth factor (VEGF) ………………………………….. 8
2.3 Recruitment of leukocytes………………………………………………….……... 9
2.4 The role of inflammatory cytokine in endothelial cell activation…………….…… 10
2.4.1 Interferon-gamma (IFN-)………………………………………………….…. 10
2.4.2 Interleukin-1 (IL-1) …………………………………………………………... 10
2.4.3 Tumor necrosis factor-alpha (TNF-)………..……………………………….. 11
2.5 The role of inflammatory cytokines in inflammatory skin diseases……….…...…. 12
2.5.1 Adverse drug reactions of the skin and drug eruption……….….……….……. 13
2.5.2 Psoriasis……….….……….….……….….……….….……….….……….…... 13
2.5.3 Urticaria……….….……….….……….….……….….……….….……….…... 14
2.5.4 Atopic dermatitis……….….……….….……….….……….….……….….…... 15
2.5.5 Erythema exudativum……….….……….….……….….……….….……….… 15
2.5.6 AIDS-associated Kaposi´s Sarcoma ………………….…………………….… 16
3 Molecular markers of endothelial cell activation……………………………….…….. 18
3.1 Complexity and redundancy of endothelial cell activation……………………….. 19
3.2 The Guanylate Binding Protein-1: a molecular marker of inflammatory
cytokine activated endothelial cells ………………………………………………. 20
4 Goals of the project……………………………………………………………………. 24

MATERIALS AND METHODS
1 Materials……………………………………………………………………………….. 25
1.1 Chemical reagents………………………………………………………………… 25
1.2 Other solutions…………………………………………………………………….. 25
1.3 Oligonucleotides……………………………………………………………. …….. 26
1.4 Enzymes and reagents for molecular biology…………………………………… 26
1.5 Kits………………………………………………………………………………... 26
1.6 Media and supplements…………………………………………………………… 26
1.7 Cytokine and growth factors…………………27
1.8 Vectors……………………………………………………………………… . …… 27
1.9 Bacterial strains…………………………………………………………………… 27
1.10 Eukaryotic cells……………………………………………………………..…… 28
1.11 Paraffin-embedded tissues………………………………………………….….… 28
1.12 Blood samples……28
1.13 Antibodies and lectine…………………………………………………………… 28
1.14 Columns……………………………………………………………………..…… 29
1.15 Equipment…………………………………………………………………...…… 29
1.16 Other material……………………………………………………………….…… 29
1.17 Centrifuges and rotors……………………………………………………….…… 29
1.18 Computer programms………………………………………………………….… 30
1.18 Buffers…………………………………………………………………………… 30
ITable of contents
2 Methods………………………………………………………………………….….… 30
2.1 Cell biological methods…………………………………………………………… 30
2.1.1 Mammalian cell culture…………………………………………………..…… 30
2.1.2 Cell stimulation with different factors……………………………………….… 31
2.1.3 Inhibition of secretory pathways……………………………………………… 31
2.1.4 Freezing of cells………………………………………………………….…… 31
2.1.5 Proliferation assay………………………………………………………..…..… 31
2.1.6 Chemotaxis assay……………………………………………………………… 32
2.1.7 Metabolic labeling of cells……………………………………………………… 32
2.1.8 Determination of cell viability……………32
2.2 Molecular biological methods…………………………………………………...… 33
2.2.1 Preparation of plasmid DNA………………………………………………...… 31
2.2.2 Restriction digest…………………………………………………………….… 33
2.2.3 Agarose gel electrophoresis ………………………………………………...… 33
2.2.4 Isolation of DNA fragments from agarose gels………………………….…..… 33
2.2.5 Oligonucleotide primers……………………………………………………..… 34
2.2.6 Polymerase chain reaction (PCR) …………………………………………..… 34
2.2.7 Purification of PCR products………………………………………………..… 34
2.2.8 DNA ligation…………………………………………………………………….…...... 34
2.2.9 Site-directed mutagenesis ………………………………………………………….. 35
2.2.10 Cloning of GST-GBP1-His………………………………… 35
2.2.11 His-GBP-1, His-GBP-2, His-mGBP-1……………………………………..….... 35
2.2.12 Preparation of electroporation competent cells……………………………….. 35
2.2.13 Transformation of electrocompetent cells. ………………………………..…. 35
2.2.14 Heat-shock transformation of E. coli.………………………………………... 36
2.2.15 Screening for positive E. coli transformants…………………………………. 36
2.2.16 E. coli permanent cultures……………………………………………….…... 36
2.2.17 Determination of induction kinetics………………………………………….. 36
2.2.18 Determination of protein solubility…………………………………………… 36
2.3 Biochemical methods………………………………………………………………. 37
2.3.1 Inhibition of protease activity …………………………………………………. 37
2.3.2 Preparation of cellular extracts using RIPA buffer…………………………….. 37
2.3.3 Thaw-lysis of cells…………………………………………………………….. 37
2.3.4 Triton extraction of cellular proteins………………………………………….. 37
2.3.5 Precipitation of proteins by TCA…………38
2.3.6 Determination of protein concentration………………………………………… 38
2.3.7 Western blotting…………………………………………………………….… 38
2.3.8 Coomassie Blue staining of proteins………39
2.3.9 Sypro Orange staining of proteins…………………………………………….. 39
2.3.10 Silver staining of proteins……………………………………………………. 39
2.3.11 Protein purification through NiTA affinity chromatography………….…….. 39
2.3.12 Protein purification of GST-GBP-1-His through NiTA affinity
chromatography followed by glutathione affinity chromatograph………….. 40
2.3.13 Dialysis……………………………………………………………………… 40
2.3.14 Desalting…………………………………………………………………….. 40
2.3.15 Immunoprecipitation………………………………………………………… 40
2.4 Production of anti GBP-1 antibodies.……………………………… …………… 41
2.4.1 Monoclonal antibodies……………………………………………………….. 41
2.4.2 Polyclonal antibodies………………………………………………………….. 41
2.5 Immuncyto- and immunohisto-chemistry………………………………………… 41
2.5.1 Immunohistochemistry on paraffin embedded sections………………………. 41
II Table of contents
2.5.2 Indirect immunofluorescence on paraffin embedded sections………………… 42
2.5.3 Immunocytochemistry…………………………………………………………. 42
2.5.4 Indirce on fixed cells…………………………………… 43
2.6 Enzyme-linked immunoassay (ELISA) ………43
2.7 Statistical analysis…………………………………………………………………. 43

RESULTS
1 Generation of mono- and poly-clonal antibodies against GBP-1…………………… 45
1.1 Expression cloning and purification of recombinant GBP-1 proteins……………. 45
1.2 Production of anti-GBP-1 antibodies…………………………………………..… 48
1.3 Characterization of anti-GBP-1 antibodies……………………………………… 49
2 Characterization of GBP-1 expression in inflammatory cytokine-activated
endothelial cells in vitro………….………………………………………………….. 52
2.1.1 Effects of inflammatory cytokines on GBP-1 expression …………………… 52
2.1.2 Effects of angiogenioc growth factors on inflammatory cytokine-
induced GBP-1 expression …………………………………………………… 55
2.1.3 Effects of different factors on GBP-1 expression………………………………. 57
2.2. Studies of GBP-1 subcellular localization in HUVEC……………….……………. 60
2.3 Colocalization studies of GBP-1 with markers for different organelles…………… 61
2.4 Studies of GBP-1 association with detergent-resistant membranes……………….. 63
2.5 Studies of IFN--induced GBP-1 expression in different
cell types ………………………………………………………..………….……… 64

Summary of chapters 1 to 2 : IC-induced GBP-1 expression in vitro………………..… 65

3 Characterization of inflammatory cytokine activation of endothelial cells in vivo…… 66
3.1 Expression of GBP-1 in normal human tissue………………………………….… 66
3.2 GBP-1 expression in endothelial cells in skin diseases with
a high-inflammatory component………………………………………………….. 67
3.3 GBP-1 expression in Kaposi´s sarcoma …………………………………………. 71
3.4 GBpression in non-proliferating vessel endothelial cells …………………… 72

Summary of chapter 3: GBP-1 is a marker of inflammatory cytokine-activated
endothelial cells in vivo……………………………………………………………………. 75

4 Studies of GBP-1 secretion ………………………………….….………. 75
4.1 Studies of GBP-1 secreted by HUVEC in the absence of cell death ………..……. 78
4.2 Development of an anti-GBP-1 enzyme-linked
immunoadsorbent assay (ELISA)…………………………………………..……… 80
4.3 Modulation of GBP-1 secretion………………………………………………….… 82
4.4 Studies of cell specifi