REGULATION OF ACUTE INFLAMMATION BY ONCOSTATIN M RECEPTOR-P Emily Hams BSc (Hons) Thesis presented for the degree of Philosophiae Doctor October 2008 Medical Biochemistry and Immunology Tenovus Building School of Medicine Cardiff University Heath Park Cardiff CF14 4XN UMI Number: U584337 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 U584337 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 DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree o f T.KD. .....................(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed . ..rrr..............................................................(candidate) Date ..\Q(o7rlQ.°\................ STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed . (candidate) Date AGl.&hl.QZ.................. STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed . .............................................................(candidate) Date .A Q .lp ^io................. ^ STATEMENT 4 - BAR ON ACCESS APPROVED I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access approved by the Graduate Development Committee. Signed (candidate) Date .. Dedication This thesis is dedicated to my family. Acknowledgements There are many people to whom I owe a great deal of thanks for their help and support throughout the past three years. Firstly I would like to take this opportunity to thank my supervisors Nick and Simon for their support, guidance, and above all patience during my PhD, and their invaluable assistance with the preparation of this thesis. I would also like to thank all my colleagues from both the Institute of Nephrology and Tenovus for their help, advice and continued friendship. Special thanks must go to Ann, Ceri, Chantal, Vikki, John and Rob for their technical assistance. I would also like to thank Daz and John, without you I would never have completed my PhD. Finally I would like to thank my family for their continued love and support. Thank you! Summary Although the interleukin (IL)-6 related cytokine Oncostatin M (OSM) affects a variety of inflammatory events associated with disease progression, the function of OSM in the face of an inflammatory challenge remains unclear. In this thesis a peritoneal model of inflammation, in association with in vitro studies using human primary cell lines, has been used to define the influence of OSM on chemokine-mediated leukocyte recruitment. When compared to wild type mice (WT) the induction of peritoneal inflammation in Oncostatin M receptor-p deficient mice (OSMR-KO) resulted in enhanced monocytic cell trafficking, with no differences in neutrophil or lymphocyte recruitment observed, suggesting that OSM control of leukocyte recruitment is functionally distinct from that of IL-6. Subsequent in vitro studies and an in vivo appraisal of inflammatory chemokine expression following peritoneal inflammation inferred that OSM regulation of CCL5 might account for the observed difference in monocytic cell trafficking. The OSM-mediated control of CCL5 is clearly distinct from the actions of IL-6, which acts as a more prominent in vivo regulator of CCL2 expression than OSM. Mechanistically, these studies inferred a hitherto unidentified interplay between OSM-mediated STAT signalling and NF-kB activation. In this respect, EMSA analysis of nuclear extracts from peritoneal membranes isolated during course of the inflammatory response showed that OSMR-KO mice display an enhanced profile of NF-kB activation as compared to WT mice. Initial in vivo appraisal of the role of OSMRP-mediated signalling in repeated episodes of inflammation and associated tissue damage suggest that OSM continues to regulate monocytic cell trafficking throughout recurrent inflammatory episodes and does not play a significant role in inflammation-associated peritoneal tissue damage, again a finding clearly distinct from the observed effects of IL-6 in tissue injury. These findings suggest that activation of gp 130 by IL-6 and OSM trigger distinct inflammatory responses to affect individual aspects of leukocyte trafficking. v Publications and Presentations Publications Hams E, Colmont CS, Dioszechy V, Hammond VJ, Fielding CA, Williams AS, Tanaka M, Miyajima A, Taylor PR, Topley N, Jones SA. Oncostatin M receptor-beta signaling limits monocytic cell recruitment in acute inflammation. J Immunol 2008; 181(3): 2174-80. Hams E. Topley N, Jones SA. Interplay between JFN-y and OSM- and IL-31- mediated control of chemokine expression. Currently in progress. Fielding CA, McLoughlin RM, Williams AS, Colmont CS, Jones G, Coles B, Hammond VJ, Hams E. Kift-Morgan AP, McLeod L, Nowell MA, Williams G, Jenkins B, Jones SA and Topley N. IL-6 driven fibrosis requires gamma-IFN mediated STAT1 signalling. Submission in progress. Presentations Hams E. Topley N and Jones SA (2006). The role of Oncostatin M in chemokine regulation (poster). 6th International Cytokine Conference, Vienna, August 27-31st 2006. Hams E. Topley N and Jones SA (2006). The role of Oncostatin M in chemokine regulation (poster). 21st Annual Postgraduate Research Day, Cardiff University, November 24th 2006. Hams E. Colmont CS, Fielding CA, Kelly CE, Dioszechy V, Hammond VJ, Topley N and Jones SA. Defining the inflammatory role of Oncostatin M receptor p signalling (oral). Infection, Immunity and Inflammation IRG meeting, Gregynog, Wales, July 13-14th 2007. Hams E. Colmont CS, Fielding CA, Kelly CE, Dioszechy V, Hammond VJ, Topley N and Jones SA. Defining the inflammatory role of Oncostatin M receptor P signalling (oral). 22nd Annual Postgraduate Research Day, Cardiff University, November 25th 2007. vi Hams E. Dioszechy V, Colmont C, Hammond VJ, Fielding CA, Williams AS, Tanaka M, Miyajima A, Taylor PR, Topley N and Jones SA (2007). Oncostatin M receptor signalling regulates monocytic cell trafficking during acute inflammation (poster). 15th Annual meeting of The International Cytokine Society, San Francisco, October 26-30th 2007. vii Abbreviations ADAM A Disintegrin and Metalloproteinase APC Antigen Presenting Cell APC Allophycocyanin Bel (2/x l ) B-Cell Lymphoma (2/x l ) BCR B Cell Receptor BDCA-2/4 Blood Dendritic Cell Antigen-2/4 BSA Bovine Serum Albumin Fraction V BSF-3 B-Cell Stimulating Factor-3 CD Cluster of Differentiation cDNA Complementary DNA CCR CC Chemokine Receptor CD40L CD40 Ligand CLC Ciliary Neurotrophic Factor-Like Cytokine CNTF Ciliary Neurotrophic Factor CNTFR Ciliary Neurotrophic Factor Receptor CRP C Reactive Protein CT-1 Cardiotrophin-1 Ctl Unstimulated Control CTLD C-Type Lectin-Like Domain CXCR CXC Chemokine Receptor DARC Duffy Antigen Receptor for Chemokines DC Dendritic Cell DC-SIGN Dendritic Cell-Specific Intracellular Adhesion Molecule 3-Grabbing Nonintegrin DD Death Domain DMEM Dulbecco’s Modified Eagle’s Medium dNTP Deoxynucleotide Triphosphate DS-sIL-6R Differentially Spliced Soluble Interleukin-6 Receptor DTT Dithiothreitol EAE Experimental Autoimmune Encephalomyelitis ECM Extracellular Matrix EDTA Ethylenediaminetetraacetic Acid ELISA Enzyme Linked Immunosorbent Assay EMSA Electrophoretic Mobility Shift Assay EMT Epithelial-to-Mesenchymal Transition ERF Established Renal Failure ERK1/2 Extracellular signal Related Kinase 1/2 FACS Fluorescence Activated Cell Sorting FCS Fetal Calf Serum FITC Fluorescein Isothiocyanate FoxP3 Forkhead box P3 GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase GAT A3 GATA binding protein 3 G-CSF Granulocye Colony Stimulating Factor GDP Guanosine 5’-Diphosphate GLR gp 13 0-Like Receptor GM-CSF Granulocyte-Macrophage Colony Stimulating Factor gpl30 glycoprotein 130 GPI Glycosylphosphatidylinisotol GROa gro protein a Grb2 Growth Factor Receptor Bound Protein 2 GTP Guanosine 5 ’ -Triphosphate HHV Human Herpesvirus HIV Human Immunodeficiency Virus HPMC Human Peritoneal Mesothelial Cell HRP Horseradish Peroxidase Hrs Hours ICAM-1 Intracellular Adhesion Molecule-1 IFN-y Interferon-y IFNAR Interferon ot/p Receptor IFNGR Interferon y Receptor Ig Immunoglobulin IKK Inhibitor of Nuclear Factor-xB Kinase Ik B Inhibitor of Nuclear Factor-icB IL-(1 -31) Interleukin-1-31 IL-6KO Interleukin-6-deficient mice IL-11KO Interleukin-11-deficient mice IL-(1-31)R Interleukin-(1-31) Receptor IL-1RAP IL-1 Receptor Accessory Protein iNOS Inducible Nitric-Oxide Synthase Ip Intra-peritoneal IP-10 lOkDa Interferon-y induced Protein IRAK IL-1-Receptor Associated Kinase IRF3 Interferon-Regulatory Factor-3 I-TAC Interferon-inducible T cell A Chemoattractant iTreg Inducible Regulatory T Cell Jak Janus Kinase JNK Jun N-terminal