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Digitale Dissertation

Jens Albrecht Ernst Geginat :
Abhängigkeit der zellulären Proliferation von der Adhäsion
Die Rolle des alphaL/beta2 Integrins LFA-1 in der T-Zell-Antigenrezeptor-abhängigen Proliferation primärer humaner T Lymphozyten
Interdependence between adhesion and proliferation

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Abstract

Summary In this study, the role of the alphaL/beta2-integrin leukocyte function antigen-1(LFA-1) in the proliferation of T cell antigen receptor(TCR)-stimulated primary human T lymphocytes was analyzed. Co-engagement of LFA-1 was found to be a prerequisite for proliferation in TCR-stimulated cells in the absence of other receptor-ligand interactions. The effect of LFA-1 on cell cycle progression can not simply be explained with enhanced adhesion-dependent TCR triggering, but is mediated by TCR-independent signal transduction. As reported for non-lymphoid, anchorage-dependent cell types, the integrin-mediated signal transduction and proliferation critically depend on an intact actin-based cytoskeleton and a spread cell shape rather than on receptor aggregation. The pro-mitotic effects of LFA-1 act at two distinct points on cell cycle progression: In the Gap 0 phase of the cell cycle, LFA-1-mediated spreading synergizes with the TCR on tyrosine phosphorylation, leading to enhanced mitogen-activated protein(Map) kinase activation, immediate early gene expression, cell cycle entry and the induction of responsiveness to the T cell growth factor Interleukin 2 (IL-2). However, LFA-1 was found to be as well a required late component of TCR-dependent proliferation: Prolonged LFA-dependent spreading, in the context of intercellular contact, is a prerequisite for the production of the mitogenic cytokine Interleukin-2. LFA-1-dependent IL-2 production was found to be cyclosporin A-resistant and was paralleled by the enhanced binding of transcription factors to the NF-AT and the CD28RE/AP-1 site of the IL-2 promoter in vitro. The alternative costimulatory receptor CD28 is able to bypass this step in an adhesion-independent way. IL-2 in turn triggers the expression of the a-chain of the IL-2 receptor (CD25), and leads to the activation of cyclin-dependent kinases (CDKs), probably due to enhanced cyclin D3 expression and to the downregulation of the CDK inhibitor p27kip1. The activation of the CDKs leads to the inactivating phosphorylation of the retinoblastoma protein (pRb) and to the progression into the synthesis(S) phase of the cell cycle. Anchorage-dependent T cell growth is therefore characterized by a sequential action of signals conveyed by integrins which together with the activating antigenic stimulus effects both cell cycle entry and G1 to S transition.

Table of Contents

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Cover and Contents

1. Introduction

1.1 Interdependence between adhesion and proliferation: The role of integrins in anchorage-dependent proliferation of non-lymphoid cells 6
1.2 T lymphocyte activation 12
1.3 The role of integrins and other costimulatory receptors in T cell activation 16
1.4 Aims of the study 19

 2. Material and methods

2.1 Purification of primary human T cells 20
2.2 Stimulation of primary T cells 21
2.3 Propidium iodide staining 22
2.4 Immunofluorescence 23
2.5 F-actin staining 23
2.6 Cloning of the glutathione-S-transferase - intercellular adhesion molecule 1 fusion protein (GST-ICAM) 23
2.7 Competence induction and transformation of E. coli 25
2.8 Expression, purification and protease cleavage of GST fusion proteins 26
2.9 Purification of monoclonal antibodies 27
2.10 Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), band shift assay, Coomassie- staining and immunoblotting 27
2.11 Protein determination 28
2.12 Preparation of sub-cellular fractions 29
2.13 Immunprecipitation 29
2.14 In-vitro kinase assays 30
2.15 RNA preparation and Northern blotting 31
2.16 Enzyme Linked Immuno-Sorbent Assay (ELISA) 32
2.17 Electroporation of primary T cells 33
2.18 Electrophoretic mobility shift assay 34

 3. Results

3.1 Proliferation in response to TCR cross-linking requires LFA-1-dependent spreading in quiescent human T cells. 36
3.2 The antibody-induced internalization of the TCR does not correlate with LFA-1-dependent proliferation 39
3.3 Tyrosine phosphorylation is synergistically induced by TCR triggering and LFA-1-mediated spreading 41
3.4 Regulation of MAP kinases by LFA-1 and the TCR in primary human T cells 43
3.5 Effects of LFA-1 on the expression of the immediate early genes c-fos and c-jun. 47
3.6 LFA-1-mediated spreading is a required late component for S phase entry in TCR-stimulated cells 48
3.7 Sustained LFA-1-dependent spreading promotes pRb-inactivation 50
3.8 LFA-1-dependent Interleukin-2 production mediates G1 to S transition. 52
3.9 CD28 aggregation can bypass the late LFA-1- dependent step of anchorage-dependent T cell growth 55
3.10 Co-engagement of either CD28 or LFA-1 induces cyclosporin A-resistant proliferation 56
3.11 The in vitro binding of transcription factors to the IL-2 promoter is not differentially affected by cyclosporin A in TCR- and costimulated cells 58

 4. Discussion

4.1 Summary 61
4.2 The experimental system 62
4.3 The role of tyrosine phosphorylation in LFA-1-dependent signalling and proliferation 65
4.4 The role of spreading-dependent Map kinase activation in T cell proliferation 66
4.5 Costimulation mediates cyclosporin A-resistant G1 to S transition: The role of IL-2 69
4.6 The presumed role of LFA-1 in T cell proliferation in a physiological intercellular contact 71

 Abbreviations 75

Acknowledgements 78

References 79

Zusammenfassung 93

Curriculum vitae 95

More Information:

Online available: http://www.diss.fu-berlin.de/1999/35/indexe.html
Language of PhDThesis: english
Keywords: T cell activation, integrins, cell cycle, Map kinases, Interleukin 2
DNB-Sachgruppe: 30 Chemie
Date of disputation: 04-May-1999
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Eberhard Riedel
Second Referee: Prof. Dr. Werner Reutter
Third Referee: Prof. Dr. Ferdinand Hucho, Prof. Dr. Menzel
Contact (Author): geginat@mail.bii.ch
Contact (Advisor): pardi.ruggero@mail.hsr.it
Date created:16-Jun-1999
Date available:24-Aug-2000

 

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