Chemokine Receptor Expression by Human Brain Endothelial Cells
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Open Research Online The Open University’s repository of research publications and other research outputs Chemokine and chemikine receptor expression by human brain endothelium :an in vitro and in situ study Thesis How to cite: Subileau, Eva-Anne (2006). Chemokine and chemikine receptor expression by human brain endothelium :an in vitro and in situ study. PhD thesis The Open University. For guidance on citations see FAQs. c 2006 Eva-Anne Subileau https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.0000fe62 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk i ^ L 'J ~ ’ Chemokine and chemokine receptor expression by human brain endothelium: An in vitro and in situ study A thesis submitted for the degree of doctor of philosophy Eva-Anne Subileau Supervised by Doctor Ignacio A. Romero and Professor David K. Male Immunology and Cell Biology Research Group Department of Biological Sciences The Open University Walton Hall Milton Keynes, MK7 6AA, England and by Professor John Greenwood Endothelial and Epithelial Cell Biology Research Unit Institute of Ophthalmology 11 - 43 Bath Street, London, EC IV 9EL, England Submitted in August 2005 jy X ' $ 2q<£ ■ ■ jCAu/\/,P- ProQuest Number: 13917279 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 com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13917279 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Acknowledgements I would first like to thank my supervisors without whom this thesis could not have been possible. Thanks to Nacho for giving me the opportunity to make a PhD, for his availability and advice during the four years. Thanks to David for his enthusiasm and much valued ideas and knowledge. On a more personal level, I am very grateful for the hospitality I have received when I came to London (thanks as well to Margot). Finally, thank you to John for his useful overview of my work and his input. This work would not have been possible without a certain number of people. Amongst them, special thanks to the members of the immunology group, Jane and Payam for their help and enthusiasm, Karen, Monica, Sandeep, Hadassah, Suzanne, Sarah and Jackie for being such a brilliant and easy people to work with as well as good friends. I would like to thank Filipe and Seema for the good time in the N0076 office, and all the passionate discussions about chemokines. Thanks to Marc for his help with the statistics. Thanks as well to the group of the Institute Cochin in Paris in particular Babette Weksler who carried out the viral work for the immortalisaion of brain endothelial cells and Pierre- Olivier Couraud who made it possible to develop the immortalised human brain endothelial cells. Heather and Frances, who did the ultrathin sections, of the Electron Microscopy group for sharing their expertise and for giving me invaluable help with all the electron microscopy experiments as did Julio, Chris and Howard. Finally, the frustration that arise with the uncertainty of sciences would have been much harder if it were not for the presence and care of my favourite housemate Yoseph, for all my sport friends who kept me healthy among who Dnyanesh, Dinar, Vanessa, Vee, and co., and finally my friends and family back in France and around Europe. 2 Abstract The infiltration of leukocytes across the BBB is thought to be mediated by chemokines released in the CNS. This study investigated the production of chemokines and expression of chemokine receptors by human brain endothelial cells (HBEC) as well as the consequences of CXCL10 stimulation on brain endothelial function in terms of leukocyte migration and endothelial permeability. Purified primary HBEC as well as a novel human brain endothelial cell line, called hCEMC/D3 and generated in collaboration with the Institute Cochin, were used in the present study. Characterisation of hCMEC/D3 cells was effected by determining the expression and localisation of HBEC markers such as tight junction and adherens junction proteins. Both primary and immortalized HBEC were tested for the production of four chemokines (CCL2, CCL5, CXCL8 and CXCL10) considered to play a role in multiple sclerosis. CXCL8 and CCL2 were constitutively released, whereas CXCL10 and CCL5 could not be detected at basal levels. By contrast, all these chemokines were up-regulated in response to either TNF-a or IFN-y or a combination of both. TNF-a had the most striking effect, up-regulating the production of CCL2, CCL5 and CXCL8, while IFN-y up- regulated CXCL10 exclusively. The chemokine receptors CXCR1 and CXCR3, whose ligands include CXCL8 and CXCL10, were expressed constitutively by HBEC both in vitro and in vivo, and only CXCR3 was up-regulated in response to cytokine stimulation in vitro. Furthermore, CXCR3 on endothelial cells is functional as CXCL10 induces phosphorylation of p42/44 MAP kinase, p38 MAP kinase and JNK/SAPK. However, activation of HBEC by CXCL10 did not induce any permeability change nor did it modulate leukocyte adhesion. These results demonstrate that endothelial cells could play an important role in chemokine production and hence leukocyte infiltration during inflammatory pathologies such as MS. The role of chemokine receptors requires further investigation. Table of Contents Acknowledgements .................................................................................................................... 2 Abstract....................................................................................................................................... 3 Table of Contents ........................................................................................................................4 List of Figures ........................................................................................................................... 11 List of Tables ............................................................................................................................ 16 Abbreviations ............................................................................................................................ 18 1. Chapter 1: General Introduction ..................................................................................... 21 1.1 Multiple sclerosis ..................................................................................................... 22 1.1.1. Aetiology ...........................................................................................................22 1.1.2. The symptoms of multiple sclerosis ................................. .............................. 25 1.1.3. The pathology of multiple sclerosis ................................................................ 26 1.1.3.1. Demyelination ...........................................................................................29 1.1.3.2. Gliosis ....................................................................................................... 30 1.1.3.3. Inflammation .............................................................................................30 1.1.3.4. Blood-brain barrier dysfunction ...............................................................32 1.2 The blood-brain barrier ............................................................................................ 32 1.2.1. Brain endothelial properties .............................................................................33 1.2.2. Role of astrocytes in barrier phenotype induction ............... 37 1.2.3. Diapedesis across the Blood-brain barrier ...................................................... 38 1.2.3.1. Capture and rolling ....................................................................................39 1.2.3.2. Firm adhesion ............................................................................................40 1.2.3.3. Transendothelial migration ......................................................................41 1.2.4. The blood-brain barrier in multiple sclerosis ..................................................43 1.2.4.1. Alterations in adhesion molecule expression ..........................................43 1.2.4.2. Changes in blood-brain barrier permeability ..........................................44 4 1.3 Chemokines...............................................................................................................45 1.3.1. Structure and function .......................................................................................45 1.3.2. Chemokine receptors .........................................................................................47 1.3.3. Physiological functions of chemokines ........................................................... 49 1.3.4. Coordination of the immune system ................................................................50