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The Investigation of Chemokine-Mediated Signalling, Regulation and Receptor Recycling in Lymphocytes

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The Investigation of Chemokine-Mediated Signalling, Regulation and Receptor Recycling in Lymphocytes University of Bath PHD The investigation of chemokine-mediated signalling, regulation and receptor recycling in lymphocytes Wain, Clare M. Award date: 2004 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 05. Oct. 2021 THE INVESTIGATION OF CHEMOKINE-MEDIATED SIGNALLING, REGULATION AND RECEPTOR RECYCLING IN LYMPHOCYTES submitted by Clare M. Wain for the degree of PhD of the University of Bath 2004 COPYRIGHT Attention is drawn to the fact that copyright of this thesis res author. This copy of the thesis has been supplied on condition that anyone who suits it is understood to recognise that its copyright rests with its author and that no quotatiorf from the thesis and no information derived from it may be published without the prior written consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purposes of consultation. UMI Number: U177B40 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U177B40 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 U N IV ER SE r BATH % i r * L. i D <4-0 2 9 JUL 2004 _ . l i a ...... In memory ofQemma Sharp Acknowledgements I, Bob, would like to show my appreciation to my supervisor Dr Stephen Ward for his support and guidance throughout. Also, a big thank you goes to my industrial supervisor Professor John Westwick of Novartis Pharmaceuticals for his support and generosity. My fellow Wardettes I love you all. Boys you have educated me with music, pool, lab rounders and bomb construction I thank you, I think! Girls what would I have done without you. Karj (otherwise known as Bird), words cannot express my gratitude for your friendship; you are one in a million. I would particularly like to thank you for your hospitality and for getting me through the difficult periods of my thesis write up. Others that should not go un-mentioned include: Dr Zarin Brown, Jenny Graham and Chris Rothwell of Novartis Pharmaceuticals; Professor Ann Richmond and Assistant Professor Guo-Huang Fan of Vanderbilt University, Nashville USA, who were all very helpful and kind during my placements. One special person who is such an inspiration in my life is my dearest friend Gemma Sharp, who sadly lost her life at the age of 19.1 dedicate this thesis in memory of you. Finally and most importantly, I would like to thank my family. Yes Sarah, that includes you! My parents have been a rock, bringing me up to believe that hard work will always pay off in the end. They have shown me so much love and support despite all that I have thrown their way, and I will always be in their debt for this. I may be off to Boston but you will always be close in my heart. Abstract The research within this thesis primarily concentrates on the two chemokine receptors CCR8 and CXCR4. The chemokine family is divided into 4 groups, where CCR8 and CXCR4 fall into each of the two main subfamilies. The first section focuses on the signalling pathways mediated by the CCL1/CCR8 chemokine/chemokine receptor pair. CCL1 is described as a potent Th2 lymphocyte chemoattractant and is therefore under investigation as a potential treatment for asthma. Thus, examination of the ambiguous biochemical and functional pathways elicited by CCL1 would provide a novel insight for future drug developments. This study demonstrated for the first time that ligation of CCR8 leads to the activation of both the ERK1/2 MAPK and PI3K/PKB pathways. In addition, the CCL1-stimulated migration of a leukaemic T cell line exhibited classic bell-shaped, concentration-dependent characteristics, a phenomenon widely associated with chemokine receptors in general. Taken together, these data show that CCR8- mediated responses appear to be analogous with that of other well-defined chemokine receptors. The following 2 sections examined the chemokine receptor CXCR4 and its ligand CXCL12. CXCR4 is fundamental in organ development and has been identified as a co­ receptor for HIV. The first section aimed to determine the role of the inositol-lipid phosphatase SHIP in the inhibitoy regulation of chemokine signalling. Investigation into the negative regulation of chemokine-mediated signalling pathways by SHIP is ill- defined. Failure to initiate inhibitory feedback mechanisms results in profound immune defects, as seen in autoimmunity. This study demonstrates in B cells that co-ligation of the BCR and FcyRIIb receptor can not only attenuate BCR-mediated responses but also those induced by chemokine receptors. In addition, a T cell model that expresses a tetracycline-regulated, constitutively active SHIP mutant showed that SHIP can impinge on CXCL12-induced PI(3,4,5)P3 levels, PKB phosphorylation and chemotaxis. Thus, these data implicate a regulatory role for SHIP in chemokine-mediated responses. The concluding section investigates the role of the class II PI3K-C2p in chemokine receptor recycling, which has been shown to be essential for chemokine-mediated cell migration. In summary, these data suggest that class II PI3Ks may co-localise with CXCR4 and AP2 and hence contribute to the local phosphoinositide levels that play a central role in receptor sequestration. Abstract i CONTENTS CHAPTER 1: INTRODUCTION 1 1.1 Introduction 1 1.2 Chemokines 2 Classification of Chemokines 4 ELR Chemokines 4 Chemokines that act predominantly on T cells 5 Constitutive/Homeostatic Chemokines 7 CXCL12/SDF-1 and CXCR4 7 Inducible Chemokines 8 CCR8 and its ligand CCL1 9 1.3 Chemokine Receptors 11 1.4 The Process of Leukocyte Extravasation 15 Chemotaxis 17 1.5 Tissue-specific Lymphocyte homing Signals 17 1.6 Genetically Modified Mice, Chemokines and Disease 19 1.7 Differential expression of chemokine receptors within T cell subsets 19 Memory T Cells 19 T Helper Cells 20 1.8 Chemokine Signalling 23 1.9 Phosphoinositide Lipids 24 1.10 Phospholipase C (PLC) 25 Contents ii 1.11 Phosphoinositide 3-kinase 27 Classification of Phosphoinositide 3-kinase 27 Class I PI3Ks 28 Class II PI3Ks 30 Class III PI3Ks 30 1.12 Protein Kinase B (PKB) 31 Role of PKB in Cell Growth 33 Components of apoptotic machinery - targets of PKB 33 Possible link between PKB and Raf protein kinase 35 1.13 PI3K dependent signalling 35 Chemokine-mediated Lipid Accumulation 36 The role of PI3K isoforms in chemokine-mediated PI(3,4,5)P3 Accumulation 37 1.14 Regulation of PI3K-dependent pathways 38 SHIP 1/2 39 PTEN 39 Regulation of PI3K within a B Cell Model 39 Regulation of the MAP Kinase Pathway 41 1.15 Calcium 43 Calcium Release from Internal Stores 44 1.16 MAP-kinase Pathway 45 The Link between GPCRs and MAPK Pathways 47 1.17 CXCL12 mediated Signalling through CXCR4 49 1.18 CCL1 mediated Signalling through CCR8 50 Contents iii 1.19 Receptor Desensitization 51 Regulation of Endosomes by Rab GTPases 53 Internalisation of CXCR4 53 The role of Class I PI3K in GPCR internalisation 55 Class II PI3K in the regulation of membrane trafficking 56 1.20 Aims of the study 57 CHAPTER 2: MATERIALS 61 2.1 Antibodies and Plasmids 61 2.2 Consumables 62 2.3 Reagents 64 2.4 Cells, Bacteria and Media 68 2.5 Kit Assays and Inhibitors 69 CHAPTER 3: TECHNIQUES 70 3.1 Freezing Down of Cells 70 3.2 Thawing of Cells 70 3.3 Cell Culture 71 Suspension Cells 71 Semi Adherent Cells 72 Adherent Cells 72 3.4 Transient Transfection of HEK293 cells 73 3.5 Detection of CCR8 using the Fluorokine System 74 Contents iv 3.6 Cell Stimulations 74 Whole Cell Lysates 75 Immunoprecipitation from Cell Lysates 75 3.7 Western Blotting 76 SDS-PAGE 76 Immuno staining 77 3.8 Stripping of Membranes 78 3.9 Calcium Mobilization 78 Suspension Cells - Flow cytometric analysis 81 3.10 Chemotaxis Assays 82 ChemoTx Chamber 82 Boyden Chamber 84 3.11 Chemotactic Index 86 3.12 Statistics 86 3.13 Measurement of D-3 Phosphoinositide Lipids in A20 cells 87 3.14 GFP-CXCR4 Construct 88 Polymerase Chain Reaction (PCR) 88 PCR Primers 89 Purification of PCR product 90 Digestion of PCR product and EGFP-N1 Vector 91 Gel Purification 92 Ligation Step 92 Transformation into Bacteria 93 Purification of Plasmid DNA 93 Mini Prep using a Qiaquick kit 93 Contents v 3.15 Digestion of plasmid DNA 94 Maxi Prep of positive clones 94 Purification of Plasmid from Maxi Prep 95 Digestion of Plasmid DNA 95 3.16 Preparation of GST Fusion Proteins 96 GST-AP2 Purification 96 Incubation of lysates with GST alone or GST-AP2 97 3.17 Co-localisation Assay by Confocal Microscopy 97 3.18 Co-localisation during Chemotaxis 98 CHAPTER 4 99 4.1 Introduction 99 4.2 Analysis of CCR8 Signal Transduction in RBL Cells 100 Surface Expression of CCR8 on RBL Cells.
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