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Mitogen-Activated Protein Kinases in Dendritic Cell Maturation and Death

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Mitogen-Activated Protein Kinases in Dendritic Cell Maturation and Death Mitogen-Activated Protein Kinases in Dendritic Cell Maturation And Death A thesis submitted by Matthew Handley for the degree of Doctor of Philosophy in the University of London 2005 Department of Immunology fit Molecular Pathology University College London Gower Street London WC1T 4JF UMI Number: U593587 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 U593587 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 Abstract Dendritic cells (DC) sense infection in their microenvironment and undergo a dynamic process of changes in phagocytic capacity, morphology and migratory activity in order to induce optimum T cell immunity. The acquisition of these properties is termed “ DC maturation” . In this study, key selected aspects of this DC maturation process have been explored. One aspect of DC maturation is the signalling pathways that DC use to respond to micro­ environmental signals. DC respond to conserved microbial structures in their micro­ environment via pattern recognition receptors including Toll-like receptors (TLRs). Generally it is thought that this leads to activation of the mitogen-activated protein kinase (MAPK) pathways, p38, ERK, and JNK. JNK and p38 MAPK are also activated by reactive oxygen species, including hydrogen peroxide (H202), known to be present in the inflammatory DC milieu. Therefore the ability of H202 to stimulate DC, or modulate their response to TLR ligands such as lipopolysaccharide (LPS) was examined. Activation of JNK, associated with inhibition of tyrosine phosphatases, is linked to induction of DC apoptosis. JNK inhibition partially protected the DC from the pro-apoptotic effects of H202. Furthermore, H202 and LPS synergise in inducing JNK activation, and DC apoptosis. These studies suggest that excessive DC maturation, which may induce pathogenic T cell activation may be limited by DC apoptosis. The second section of the thesis explores the mechanisms leading to MAPK activation upon TLR ligation. Biochemical studies demonstrated that mixed lineage kinase 3 (MLK3), a kinase not previously implicated in DC maturation, was phosphorylated upon TLR3 and 4, but not TLR2 ligation. Studies using a selective MLK inhibitor supported a role for the mixed lineage kinase (MLK) family of MAPK kinase kinase (MAP3K) proteins in regulation of DC signalling, phenotype and cytokine secretion, in response to TLR3 and 4, but not TLR2 ligation. Selective activation of MAP3K may therefore contribute to the heterogeneity and flexibility of DC/pathogen interaction. A further feature of the DC sensory role is antigen uptake, but the relationship between morphology and phagocytic capacity has not been defined. Therefore a simple and flexible assay to examine this relationship was developed. DC with long dendritic processes were less efficient at antigen internalisation than round DCs. 1 To Mom, Dad, Sally and James for always supporting me 2 Acknowledgements Undertaking the work presented in this thesis has been extremely enjoyable and for this I owe a debt of thanks to a large number of individuals. Firstly, I would like to say a special thank- you to my supervisors Prof David Katz and Prof Benny Chain for their kind assistance, advice, and support at all times. I would also like to thank many people who started as colleagues in the lab and became great friends, including Gabriele Pollara, Paul Kaye, Emma Willoughby, Paul Free, Cheryl Chiang, Rino Tulone, Pippa Newton, Ali Al-Shangiti, Jane Rasaiyaah, Ariel Rad, Claire Swetman, Antonia Kwan, and Ian Gerrard. Special thanks also goes to both of the BSc students that I had the pleasure of supervising, Manish Thakker, and James Barnett, both of whom contributed to some of the experiments presented in this thesis. I am also gratefully indebted to the many individuals who have kindly donated blood, without whom none of this work would have been possible. Final thanks goes to my family; Mom, Dad, James, and Sally, for always being there. 3 Publications Data presented in thesis has been published in the following reports (attached at the end of the thesis): Handley, M. E., Thakker, M., Pollara, G., Chain, B. M., and Katz, D. R. (2005). JNK activation limits dendritic cell maturation in response to reactive oxygen species by the induction of apoptosis. Free Radic. Biol. Med. 38, 1637-1652. Handley, M. E., Pollara, G., Chain, B. M., and Katz, D. R. (2005). The use of targeted microbeads for quantitative analysis of the phagocytic properties of human monocyte-derived dendritic cells. J. Immunol. Methods 297, 27-38. Submitted for publication: Handley, M. E., Barnett, J., Thakker, M., Pollara, G., Katz, D.R., and Chain, B.M. The Role of Mixed Lineage Kinases in Dendritic Cell Responses to Different Stimuli 4 Table of Contents Abstract .............................................................................................................................. 1 Acknowledgements ............................................................................................................ 3 Publications ....................................................................................................................... 4 Table of Contents ...............................................................................................................5 Table of Figures ................................................................................................................10 Table of Abbreviations ......................................................................................................12 Chapter 1 Introduction............................................................................................................................... 15 1.1 Introduction To Dendritic Cells ...............................................................................16 1.1.1 The role of DC in adaptive immunity ..................................................................16 1.1.2 DC subtypes ......................................................................................................19 1.1.3 DC maturation .................................................................................................. 19 1.1.3.1 Antigen sampling ........................................................................................19 1.1.3.1.1 Receptor-Mediated Endocytosis..............................................................20 1.1.3.1.2 Phagocytosis ...........................................................................................20 1.1.3.1.3 Macropi nocytosis ................................................................................... 21 1.1.3.2 Induction of DC maturation ........................................................................21 1.1.3.3 Migration of DC .......................................................................................... 22 1.1.3.3 DC - T Cell interaction ............................................................................... 23 1.2 Signaling Pathways Regulating DC Maturation ...........................................................24 1.2.1 Introduction ...................................................................................................... 24 1.2.2 Toll-like receptors and induction of intracellular signaling ................................ 24 1.2.2.1 Introduction ............................................................................................... 24 1.2.2.2 TLR functions .............................................................................................25 1.2.2.3 Interaction of TLRs with other receptors ....................................................26 1.2.2.4 TLR signaling ..............................................................................................28 1.2.2.5 IRAKs......................................................................................................... 29 1.2.2.6 TRAFs........................................................................................................33 1.2.3 Limitations to studying intracellular signaling pathways in DC ...........................34 1.2.4 Mitogen-activated protein kinases ......................................................................36 1.2.4.1 Introduction ............................................................................................... 36 1.2.4.2 Control of DC maturation ........................................................................... 39 1.2.4.3 Effector T cell polarization .........................................................................42 5 1.2.4.4 Mechanisms of AAAPK action in DC .............................................................. 45 1.2.2.5 Survival.....................................................................................................47 1.2.5 PI3K................................................................................................................. 48 1.2.6 NF-kB...............................................................................................................49
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