THE ROLE OF SPHINGOSINE KINASE IN INFLAMMATORY DISEASES LAI WENQI (B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 I Acknowledgements First and foremost, I would like to express my immense gratitude to my supervisor, Dr Bernard Leung, without whom the completion of this project would not have been possible. Throughout these four years of study, Dr Bernard has found time to supervise my work in spite of his busy schedule; he is always around to offer advices, guidance and encouragements. Without his remarkable vision and generous support, this work would not have been accomplished. It was a huge honour and blessing to have worked in his lab. Thank you very much, boss. Next, I would like to extend my gratitude to my co-supervisor, A/P Alirio Melendez, who has been a source of encouragement and positivity. I am also extremely grateful to the members of the Cytokine Biology Lab and the Molecular and Cellular Immunology Lab for their assistance and friendships throughout these four years. They have made the research work so much more lively and fun. Very special thanks belong to the following for their contributions to this work: Mr Goh Hong Heng and Ms Anastasia Windy Irwan, who have since left the lab; Prof Fred Wong and Dr Bao Zhang from the Department of Pharmacology, NUS. I would like to acknowledge my deepest gratitude to my family: mummy, daddy, pongpong and winson, and my best friends, for their unwavering love, patience, and support throughout my life. Thank you being there for me, each step of the way. Last but not the least, Yaozhong, thank you for being the sunshine of my life. II Table of Contents Acknowledgements I Table of Contents II Summary IX List of Tables XI List of Figures XI Abbreviations XV Chapter 1.1 1.2 1.3 General Introduction Sphingolipids 1.1.1 Sphingolipid Metabolism 1.1.2 Sphingosine-1-Phosphate 1.1.3 Sphingosine Kinase and 1.1.4 Mechanism of SphKs activation in mammalian cells S1P signalling in the immune responses 1.2.1 S1P and immune cell trafficking 1.2.2 S1P and vascular barrier integrity 1.2.3 SphK/S1P and cell adhesion molecules expression Role of SphKs in specialised immune and other inflammatory cells 1.3.1 Role of SphK in lymphocytes 1.3.2 Th17 and SphK/S1P 11 1.3.3 Role of SphK in neutrophils 12 1.3.4 Role of SphK in mast cells 13 III 1.3.5 Role of SphK in monocytes and macrophages 14 1.4 Role of SphK1 versus SphK2 in disease 15 1.5 Rheumatoid Arthritis 18 1.5.1 Etiology of RA 18 1.5.2 Pathogenesis of Rheumatoid Arthritis: interactions 21 between immune cells and synoviocytes 1.5.3 Role of Cytokines in RA pathogenesis 1.6 1.7 24 1.5.3.1 TNF-α 25 1.5.3.2 IL-1β 25 1.5.3.3 IL-6 26 1.5.3.4 IL-17 27 1.5.4 Cell contact in RA 28 Asthma 32 1.6.1 Asthma Pathogenesis 32 1.6.1.1 IL-4 36 1.6.1.2 IL-13 36 1.6.1.3 IL-5 37 1.6.1.4 Eotaxin 37 1.6.1.5 IL-17 38 1.6.2 Asthma therapy 39 1.6.2.1 IL-4 and IL-13 therapy 39 1.6.2.2 IL-5 therapy 40 Rationale: The expanding view of S1P/SphKs and their functions in inflammation 41 IV Chapter 2.1 Materials and Methods Preparation of DMS, SKI2, antisense oligonucleotides, and 44 45 siRNA 2.2 Patient and clinical samples 45 2.3 Cell culture 46 2.3.1 Cell line maintenance 46 2.3.2 Peripheral Blood Mononuclear Cells (PBMC) preparation 47 2.3.3 Synovial fluid preparation 48 2.3.4 Murine lymph node cell preparation 48 2.3.5 Antigen-specific in vitro culture 48 2.3.6 Cell contact protocol 49 2.3.7 Treatment protocol 50 2.3.8 Degranulation Assay 51 2.3.9 Proliferation Assay 51 2.4 Animal model of inflammation 52 2.4.1 Induction of collagen induced arthritis (CIA) in mice 52 2.4.2 CIA model: Treatment protocols 53 2.4.3 Monitor of progression of CIA 54 2.4.4 Quantification of paw histology 54 2.4.5 Induction of Asthma in mice- Sensitisation and challenge with 55 OVA 2.4.6 Asthma model: Treatment protocols 56 2.4.7 BAL process 56 2.4.8 Lung histology 57 2.4.9 Measurements of AHR 58 V 2.5 2.4.10 Serum collection 59 ELISA assays 59 2.5.1 Measurement of S1P by ELISA 59 2.5.2 Measurement of antigen-specific serum IgG isotypes by 60 ELlSA 2.5.3 2.6 2.7 General ELISA protocol 61 Western blot 62 2.6.1 Lysis with RIPA buffer 62 2.6.2 General protocol for Western Blot 62 Statistical analysis Chapter The Role of Sphingosine Kinase in Murine Model of 63 66 Allergic Asthma Introduction 67 Results 3.1 DMS reduces BAL inflammatory infiltrates in OVA-induced 70 asthma mice 3.2 DMS suppresses inflammatory infiltration and mucus production 72 in the lung tissue 3.3 DMS treatment reduces Th2 cytokine levels in BAL 74 3.4 DMS suppresses OVA-specific responses in vitro 76 3.5 DMS treatment reduces lung resistance in vivo 78 3.6 Treatment with SphK1-siRNA suppresses eosinophilic airway 80 inflammation 3.7 SphK1 siRNA treatment reduces Th2 cytokine levels in BAL 83 VI 3.8 SphK1 siRNA treatment reduces serum IgE levels 85 Discussion Chapter 84 Anti-Inflammatory Effects of Sphingosine Kinase 89 Modulation in Inflammatory Arthritis Introduction 90 Results 4.1 Detection of S1P in RA synovial fluid 92 4.2 SphK inhibitors reduce Jurkat T cell contact-induced cytokine 94 production via cognate interactions in human monocytic cell line U937 and promyelocytic cell line HL-60 4.3 Cell contact with inserts 97 4.4 DMS inhibits cell contact-induced cytokine production via 99 cognate interactions in PBMCs derived from RA patients 4.5 DMS did not affect cell viability in cell-contact assays 101 4.6 DMS inhibits cell contact-induced MMP-9 production 102 4.7 Treatment with DMS did not affect cell contact-triggered 104 degranulation 4.8 Treatment with DMS inhibits the development of murine CIA 106 4.9 Treatment with DMS reduced inflammatory infiltrate into the 108 synovium and articular destruction 4.10 Effect of DMS on serum cytokines, S1P and anti-CII Ab 110 production in vivo 4.11 DMS reduced in vitro CII-specific pro-inflammatory immune responses 114 VII 116 Discussion Chapter Distinct Roles of Sphingosine Kinase and in Murine 121 Collagen-Induced Arthritis 122 Introduction Results 5.1 Downregulation of SphK1 via specific antisense oligonucleotides 124 reduce Jurkat T cell contact-induced cytokine production via cognate interactions in human promyelocytic cell line HL-60 5.2 SphK1, but not SphK2 downregulation suppressed development 127 of CIA 5.3 Treatment with SphK1, but not SphK2 siRNA, reduced 131 inflammatory infiltrate into the synovium and articular destruction 5.4 Effect of SphK siRNA treatment on serum cytokines and S1P 133 levels 5.5 SphK1-siRNA suppresses collagen-specific pro-inflammatory 137 immune responses in vitro 5.6 Anti-collagen antibody (Ab) production in vivo 142 Discussion Chapter 140 General Discussion 147 Conclusion 157 Future Studies 159 Bibliography 160 VIII Appendix I Preparation of Buffers 204 Appendix II Publications 207 IX Summary Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). S1P is an important bioactive signalling molecule that has both intracellular and extracellular functions, mediating cellular events such as calcium mobilisation, proliferation, migration, and cytokine production. Increasingly, roles of SphK/S1P have been demonstrated in several inflammatory responses and pathophysiologic conditions, rendering these molecules an excellent target of investigation in various disease models. Of interest to our research are the chronic inflammatory diseases Asthma and Rheumatoid Arthritis (RA). For asthma, the role of SphK in a murine model of allergic asthma was examined. In mice previously sensitized to OVA, intraperitoneal administration of N,Ndimethylsphingosine (DMS), a potent SphK inhibitor, significantly reduced the total inflammatory cell infiltrate and eosinophilia and the IL-4, IL-5, and eotaxin levels in bronchoalveolar lavage fluid in response to inhaled OVA challenge. In addition, DMS significantly suppressed OVA-induced inflammatory infiltrates and mucus production in the lungs, and airway hyperresponsiveness to methacholine in a dose-dependent manner. OVA-induced lymphocyte proliferation and IL-4 and IL-5 secretion were reduced in thoracic lymph node cultures from DMS-treated mice. Moreover, similar reduction in inflammatory infiltrates, BAL IL-4, IL-5, eotaxin, and serum OVAspecific IgE levels was observed in mice with SphK1 knock-down via small interfering RNA approach. Together, these data demonstrate the therapeutic potential of SphK modulation in allergic airways disease. 192 236. Greenfeder, S., S. P. Umland, F. M. Cuss, R. W. Chapman, and R. W. Egan. 2001. Th2 cytokines and asthma. The role of interleukin-5 in allergic eosinophilic disease. Respir Res 2:71. 237. Rothenberg, M. E., and S. P. Hogan. 2006. The eosinophil. Annu Rev Immunol 24:147. 238. Foster, P. S., S. P. Hogan, A. J. Ramsay, K. I. Matthaei, and I. G. Young. 1996. Interleukin deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 183:195. 239. Hamelmann, E., G. Cieslewicz, J. Schwarze, T. Ishizuka, A. Joetham, C. Heusser, and E. W. Gelfand. 1999. Anti-interleukin but not anti-IgE prevents airway inflammation and airway hyperresponsiveness. Am J Respir Crit Care Med 160:934. 240. Tournoy, K. G., J. C. Kips, C. Schou, and R. A. Pauwels. 2000. Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness. Clin Exp Allergy 30:79. 241. Birrell, M. A., C. H. Battram, P. Woodman, K. McCluskie, and M. G. Belvisi. 2003. Dissociation by steroids of eosinophilic inflammation from airway hyperresponsiveness in murine airways. Respir Res 4:3. 242. Li, L., Y. Xia, A. Nguyen, Y. H. Lai, L. Feng, T. R. Mosmann, and D. Lo. 1999. Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J Immunol 162:2477. 243. Sanz, M. J., P. D. Ponath, C. R. Mackay, W. Newman, M. Miyasaka, T. Tamatani, B. F. Flanagan, R. R. Lobb, T. J. Williams, S. Nourshargh, and P. J. Jose. 1998. Human eotaxin induces alpha and beta integrin-dependent 193 eosinophil accumulation in rat skin in vivo: delayed generation of eotaxin in response to IL-4. J Immunol 160:3569. 244. Shinkai, A., H. Yoshisue, M. Koike, E. Shoji, S. Nakagawa, A. Saito, T. Takeda, S. Imabeppu, Y. Kato, N. Hanai, H. Anazawa, T. Kuga, and T. Nishi. 1999. A novel human CC chemokine, eotaxin-3, which is expressed in IL-4stimulated vascular endothelial cells, exhibits potent activity toward eosinophils. J Immunol 163:1602. 245. Bettelli, E., T. Korn, and V. K. Kuchroo. 2007. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 19:652. 246. Ivanov, II, B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121. 247. Bullens, D. M., E. Truyen, L. Coteur, E. Dilissen, P. W. Hellings, L. J. Dupont, and J. L. Ceuppens. 2006. IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 7:135. 248. Henness, S., E. van Thoor, Q. Ge, C. L. Armour, J. M. Hughes, and A. J. Ammit. 2006. IL-17A acts via p38 MAPK to increase stability of TNF-alphainduced IL-8 mRNA in human ASM. Am J Physiol Lung Cell Mol Physiol 290:L1283. 249. Rahman, M. S., A. Yamasaki, J. Yang, L. Shan, A. J. Halayko, and A. S. Gounni. 2006. IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways. J Immunol 177:4064. 194 250. Schnyder-Candrian, S., D. Togbe, I. Couillin, I. Mercier, F. Brombacher, V. Quesniaux, F. Fossiez, B. Ryffel, and B. Schnyder. 2006. Interleukin-17 is a negative regulator of established allergic asthma. J Exp Med 203:2715. 251. Hellings, P. W., A. Kasran, Z. Liu, P. Vandekerckhove, A. Wuyts, L. Overbergh, C. Mathieu, and J. L. Ceuppens. 2003. Interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell Mol Biol 28:42. 252. Karin, M., Y. Yamamoto, and Q. M. Wang. 2004. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 3:17. 253. Kumar, S., J. Boehm, and J. C. Lee. 2003. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2:717. 254. Borish, L. C., H. S. Nelson, M. J. Lanz, L. Claussen, J. B. Whitmore, J. M. Agosti, and L. Garrison. 1999. Interleukin-4 receptor in moderate atopic asthma. A phase I/II randomized, placebo-controlled trial. Am J Respir Crit Care Med 160:1816. 255. Gavett, S. H., D. J. O'Hearn, C. L. Karp, E. A. Patel, B. H. Schofield, F. D. Finkelman, and M. Wills-Karp. 1997. Interleukin-4 receptor blockade prevents airway responses induced by antigen challenge in mice. Am J Physiol 272:L253. 256. Shanafelt, A. B., C. P. Forte, J. J. Kasper, L. Sanchez-Pescador, M. Wetzel, R. Gundel, and J. M. Greve. 1998. An immune cell-selective interleukin agonist. Proc Natl Acad Sci U S A 95:9454. 257. Leckie, M. J., A. ten Brinke, J. Khan, Z. Diamant, B. J. O'Connor, C. M. Walls, A. K. Mathur, H. C. Cowley, K. F. Chung, R. Djukanovic, T. T. Hansel, 195 S. T. Holgate, P. J. Sterk, and P. J. Barnes. 2000. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 356:2144. 258. Kips, J. C., B. J. O'Connor, S. J. Langley, A. Woodcock, H. A. Kerstjens, D. S. Postma, M. Danzig, F. Cuss, and R. A. Pauwels. 2003. Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent asthma: a pilot study. Am J Respir Crit Care Med 167:1655. 259. Flood-Page, P. T., A. N. Menzies-Gow, A. B. Kay, and D. S. Robinson. 2003. Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 167:199. 260. Jacobson, J. R., and J. G. Garcia. 2007. Novel therapies for microvascular permeability in sepsis. Curr Drug Targets 8:509. 261. Nofer, J. R., M. van der Giet, M. Tolle, I. Wolinska, K. von Wnuck Lipinski, H. A. Baba, U. J. Tietge, A. Godecke, I. Ishii, B. Kleuser, M. Schafers, M. Fobker, W. Zidek, G. Assmann, J. Chun, and B. Levkau. 2004. HDL induces NO-dependent vasorelaxation via the lysophospholipid receptor S1P3. J Clin Invest 113:569. 262. Deutschman, D. H., J. S. Carstens, R. L. Klepper, W. S. Smith, M. T. Page, T. R. Young, L. A. Gleason, N. Nakajima, and R. A. Sabbadini. 2003. Predicting obstructive coronary artery disease with serum sphingosine-1-phosphate. Am Heart J 146:62. 263. Ammit, A. J., A. T. Hastie, L. C. Edsall, R. K. Hoffman, Y. Amrani, V. P. Krymskaya, S. A. Kane, S. P. Peters, R. B. Penn, S. Spiegel, and R. A. Panettieri, Jr. 2001. Sphingosine 1-phosphate modulates human airway 196 smooth muscle cell functions that promote inflammation and airway remodelling in asthma. Faseb J 15:1212. 264. Kitano, M., T. Hla, M. Sekiguchi, Y. Kawahito, R. Yoshimura, K. Miyazawa, T. Iwasaki, H. Sano, J. D. Saba, and Y. Y. Tam. 2006. Sphingosine 1phosphate/sphingosine 1-phosphate receptor signalling in rheumatoid synovium: regulation of synovial proliferation and inflammatory gene expression. Arthritis Rheum 54:742. 265. Yanagawa, Y., K. Sugahara, H. Kataoka, T. Kawaguchi, Y. Masubuchi, and K. Chiba. 1998. FTY720, a novel immunosuppressant, induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats. II. FTY720 prolongs skin allograft survival by decreasing T cell infiltration into grafts but not cytokine production in vivo. J Immunol 160:5493. 266. Maki, T., R. Gottschalk, N. Ogawa, and A. P. Monaco. 2005. Prevention and cure of autoimmune diabetes in nonobese diabetic mice by continuous administration of FTY720. Transplantation 79:1051. 267. Srinivasan, S., D. T. Bolick, D. Lukashev, C. Lappas, M. Sitkovsky, K. R. Lynch, and C. C. Hedrick. 2008. Sphingosine-1-phosphate reduces CD4+ Tcell activation in type diabetes through regulation of hypoxia-inducible factor short isoform I.1 and CD69. Diabetes 57:484. 268. Kitabayashi, H., M. Isobe, N. Watanabe, J. Suzuki, Y. Yazaki, and M. Sekiguchi. 2000. FTY720 prevents development of experimental autoimmune myocarditis through reduction of circulating lymphocytes. J Cardiovasc Pharmacol 35:410. 269. Kohno, T., T. Tsuji, K. Hirayama, R. Iwatsuki, M. Hirose, K. Watabe, H. Yoshikawa, A. Matsumoto, T. Fujita, and M. Hayashi. 2005. A novel 197 immunomodulator, FTY720, prevents development of experimental autoimmune myasthenia gravis in C57BL/6 mice. Biol Pharm Bull 28:736. 270. Okazaki, H., D. Hirata, T. Kamimura, H. Sato, M. Iwamoto, T. Yoshio, J. Masuyama, A. Fujimura, E. Kobayashi, S. Kano, and S. Minota. 2002. Effects of FTY720 in MRL-lpr/lpr mice: therapeutic potential in systemic lupus erythematosus. J Rheumatol 29:707. 271. Matsuura, M., T. Imayoshi, and T. Okumoto. 2000. Effect of FTY720, a novel immunosuppressant, on adjuvant- and collagen-induced arthritis in rats. Int J Immunopharmacol 22:323. 272. Kieseier, B. C., H. Wiendl, B. Hemmer, and H. P. Hartung. 2007. Treatment and treatment trials in multiple sclerosis. Curr Opin Neurol 20:286. 273. Zemann, B., B. Kinzel, M. Muller, R. Reuschel, D. Mechtcheriakova, N. Urtz, F. Bornancin, T. Baumruker, and A. Billich. 2006. Sphingosine kinase type is essential for lymphopenia induced by the immunomodulatory drug FTY720. Blood 107:1454. 274. Mansoor, M., and A. J. Melendez. 2008. Recent trials for FTY720 (fingolimod): a new generation of immunomodulators structurally similar to sphingosine. Rev Recent Clin Trials 3:62. 275. Kahan, B. D., J. L. Karlix, R. M. Ferguson, A. B. Leichtman, S. Mulgaonkar, T. A. Gonwa, A. Skerjanec, R. L. Schmouder, and L. Chodoff. 2003. Pharmacodynamics, pharmacokinetics, and safety of multiple doses of FTY720 in stable renal transplant patients: a multicenter, randomized, placebo-controlled, phase I study. Transplantation 76:1079. 276. Tedesco-Silva, H., G. Mourad, B. D. Kahan, J. G. Boira, W. Weimar, S. Mulgaonkar, B. Nashan, S. Madsen, B. Charpentier, P. Pellet, and Y. 198 Vanrenterghem. 2005. FTY720, a novel immunomodulator: efficacy and safety results from the first phase 2A study in de novo renal transplantation. Transplantation 79:1553. 277. Kahan, B. D. 2008. Frontiers in immunosuppression. Transplant Proc 40:11. 278. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, and et al. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315. 279. Trentham, D. E., A. S. Townes, and A. H. Kang. 1977. Autoimmunity to type II collagen an experimental model of arthritis. J Exp Med 146:857. 280. Joosten, L. A., E. Lubberts, P. Durez, M. M. Helsen, M. J. Jacobs, M. Goldman, and W. B. van den Berg. 1997. Role of interleukin-4 and interleukin-10 in murine collagen-induced arthritis. Protective effect of interleukin-4 and interleukin-10 treatment on cartilage destruction. Arthritis Rheum 40:249. 281. Duan, W., J. H. Chan, C. H. Wong, B. P. Leung, and W. S. Wong. 2004. Antiinflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol 172:7053. 282. Peebles, R. S., Jr., R. Dworski, R. D. Collins, K. Jarzecka, D. B. Mitchell, B. S. Graham, and J. R. Sheller. 2000. Cyclooxygenase inhibition increases interleukin and interleukin 13 production and airway hyperresponsiveness in allergic mice. Am J Respir Crit Care Med 162:676. 283. Spiegel, S., and S. Milstien. 1995. Sphingolipid metabolites: members of a new class of lipid second messengers. J Membr Biol 146:225. 199 284. Spiegel, S., and S. Milstien. 2003. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4:397. 285. Melendez, A., R. A. Floto, A. J. Cameron, D. J. Gillooly, M. M. Harnett, and J. M. Allen. 1998. A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium. Curr Biol 8:210. 286. Stam, J. C., F. Michiels, R. A. van der Kammen, W. H. Moolenaar, and J. G. Collard. 1998. Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signalling. Embo J 17:4066. 287. Rosenfeldt, H. M., Y. Amrani, K. R. Watterson, K. S. Murthy, R. A. Panettieri, Jr., and S. Spiegel. 2003. Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells. Faseb J 17:1789. 288. Roviezzo, F., A. Di Lorenzo, M. Bucci, V. Brancaleone, V. Vellecco, M. De Nardo, D. Orlotti, R. De Palma, F. Rossi, B. D'Agostino, and G. Cirino. 2007. Sphingosine-1-phosphate/sphingosine kinase pathway is involved in mouse airway hyperresponsiveness. Am J Respir Cell Mol Biol 36:757. 289. Kurashima, Y., J. Kunisawa, M. Higuchi, M. Gohda, I. Ishikawa, N. Takayama, M. Shimizu, and H. Kiyono. 2007. Sphingosine 1-phosphatemediated trafficking of pathogenic Th2 and mast cells for the control of food allergy. J Immunol 179:1577. 290. Vlasenko, L. P., and A. J. Melendez. 2005. A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo. J Immunol 174:6456. 291. Jia, G. Q., J. A. Gonzalo, A. Hidalgo, D. Wagner, M. Cybulsky, and J. C. Gutierrez-Ramos. 1999. Selective eosinophil transendothelial migration 200 triggered by eotaxin via modulation of Mac-1/ICAM-1 and VLA-4/VCAM-1 interactions. Int Immunol 11:1. 292. Gleich, G. J. 2000. Mechanisms of eosinophil-associated inflammation. J Allergy Clin Immunol 105:651. 293. Kunisawa, J., Y. Kurashima, M. Gohda, M. Higuchi, I. Ishikawa, F. Miura, I. Ogahara, and H. Kiyono. 2007. Sphingosine 1-phosphate regulates peritoneal B-cell trafficking for subsequent intestinal IgA production. Blood 109:3749. 294. Nishiuma, T., Y. Nishimura, T. Okada, E. Kuramoto, Y. Kotani, S. Jahangeer, and S. Nakamura. 2008. Inhalation of sphingosine kinase inhibitor attenuates airway inflammation in asthmatic mouse model. Am J Physiol Lung Cell Mol Physiol 294:L1085. 295. Roviezzo, F., B. D'Agostino, V. Brancaleone, L. De Gruttola, M. Bucci, G. De Dominicis, D. Orlotti, E. D'Aiuto, R. De Palma, F. Rossi, R. Sorrentino, and G. Cirino. 2009. Systemic Administration of Sphingosine-1-phosphate Increases Bronchial Hyper-responsiveness in the Mouse. Am J Respir Cell Mol Biol. 296. Melendez, A. J. 2008. Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets. Biochim Biophys Acta 1784:66. 297. McInnes, I. B., and F. Y. Liew. 2005. Cytokine networks--towards new therapies for rheumatoid arthritis. Nat Clin Pract Rheumatol 1:31. 298. Katz, Y., O. Nadiv, and Y. Beer. 2001. Interleukin-17 enhances tumour necrosis factor alpha-induced synthesis of interleukins 1,6, and in skin and synovial fibroblasts: a possible role as a "fine-tuning cytokine" in inflammation processes. Arthritis Rheum 44:2176. 299. Koenders, M. I., L. A. Joosten, and W. B. van den Berg. 2006. Potential new targets in arthritis therapy: interleukin (IL)-17 and its relation to tumour 201 necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis 65 Suppl 3:iii29. 300. Ribbens, C., J. M. Dayer, and C. Chizzolini. 2000. CD40-CD40 ligand (CD154) engagement is required but may not be sufficient for human T helper cell induction of interleukin-2- or interleukin-15-driven, contact-dependent, interleukin-1beta production by monocytes. Immunology 99:279. 301. Pi, X., S. Y. Tan, M. Hayes, L. Xiao, J. A. Shayman, S. Ling, and J. Holoshitz. 2006. Sphingosine kinase 1-mediated inhibition of Fas death signalling in rheumatoid arthritis B lymphoblastoid cells. Arthritis Rheum 54:754. 302. Jovanovic, D. V., J. Martel-Pelletier, J. A. Di Battista, F. Mineau, F. C. Jolicoeur, M. Benderdour, and J. P. Pelletier. 2000. Stimulation of 92-kd gelatinase (matrix metalloproteinase 9) production by interleukin-17 in human monocyte/macrophages: a possible role in rheumatoid arthritis. Arthritis Rheum 43:1134. 303. Seki, N., Y. Sudo, T. Yoshioka, S. Sugihara, T. Fujitsu, S. Sakuma, T. Ogawa, T. Hamaoka, H. Senoh, and H. Fujiwara. 1988. Type II collagen-induced murine arthritis. I. Induction and perpetuation of arthritis require synergy between humoral and cell-mediated immunity. J Immunol 140:1477. 304. Kamada, K., N. Arita, T. Tsubaki, N. Takubo, T. Fujino, Y. Soga, T. Miyazaki, H. Yamamoto, and M. Nose. 2009. Expression of sphingosine kinase in synovial fibroblasts of rheumatoid arthritis contributing to apoptosis by a sphingosine analogue, FTY720. Pathol Int 59:382. 305. Sekiguchi, M., T. Iwasaki, M. Kitano, H. Kuno, N. Hashimoto, Y. Kawahito, M. Azuma, T. Hla, and H. Sano. 2008. Role of sphingosine 1-phosphate in the pathogenesis of Sjogren's syndrome. J Immunol 180:1921. 202 306. Kappos, L., J. Antel, G. Comi, X. Montalban, P. O'Connor, C. H. Polman, T. Haas, A. A. Korn, G. Karlsson, and E. W. Radue. 2006. Oral fingolimod (FTY720) for relapsing multiple sclerosis. N Engl J Med 355:1124. 307. Lai, W. Q., H. H. Goh, Z. Bao, W. S. Wong, A. J. Melendez, and B. P. Leung. 2008. The role of sphingosine kinase in a murine model of allergic asthma. J Immunol 180:4323. 308. Tan, S. Y., L. Xiao, X. Pi, and J. Holoshitz. 2007. Aberrant Gi protein coupled receptor-mediated cell survival signalling in rheumatoid arthritis B cell lines. Front Biosci 12:1651. 309. Zhao, C., M. J. Fernandes, M. Turgeon, S. Tancrede, J. Di Battista, P. E. Poubelle, and S. G. Bourgoin. 2008. Specific and overlapping sphingosine-1phosphate receptor functions in human synoviocytes: impact of TNF-alpha. J Lipid Res 49:2323. 310. Ishii, M., J. G. Egen, F. Klauschen, M. Meier-Schellersheim, Y. Saeki, J. Vacher, R. L. Proia, and R. N. Germain. 2009. Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature 458:524. 311. Brennan, F. M., and I. B. McInnes. 2008. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 118:3537. 312. Hammad, S. M., H. G. Crellin, B. X. Wu, J. Melton, V. Anelli, and L. M. Obeid. 2008. Dual and distinct roles for sphingosine kinase and sphingosine phosphate in the response to inflammatory stimuli in RAW macrophages. Prostaglandins Other Lipid Mediat 85:107. 313. Lai, W. Q., A. W. Irwan, H. H. Goh, H. S. Howe, D. T. Yu, R. Valle-Onate, I. B. McInnes, A. J. Melendez, and B. P. Leung. 2008. Anti-inflammatory 203 effects of sphingosine kinase modulation in inflammatory arthritis. J Immunol 181:8010. 314. Rivera, J., R. L. Proia, and A. Olivera. 2008. The alliance of sphingosine-1phosphate and its receptors in immunity. Nat Rev Immunol 8:753. 315. Kase, H., Y. Hattori, T. Jojima, T. Okayasu, A. Tomizawa, K. Suzuki, N. Banba, T. Monden, H. Satoh, K. Akimoto, and K. Kasai. 2007. Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells. Life Sci 81:939. 316. Dorner, T., and G. R. Burmester. 2008. New approaches of B-cell-directed therapy: beyond rituximab. Curr Opin Rheumatol 20:263. 317. Michaud, J., M. Kohno, R. L. Proia, and T. Hla. 2006. Normal acute and chronic inflammatory responses in sphingosine kinase knockout mice. FEBS Lett 580:4607. 318. Mizugishi, K., T. Yamashita, A. Olivera, G. F. Miller, S. Spiegel, and R. L. Proia. 2005. Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol 25:11113. 319. Roman-Blas, J. A., and S. A. Jimenez. 2006. NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis. Osteoarthritis Cartilage 14:839. 320. Hait, N. C., A. Bellamy, S. Milstien, T. Kordula, and S. Spiegel. 2007. Sphingosine kinase type activation by ERK-mediated phosphorylation. J Biol Chem 282:12058. 321. Pitson, S. M., P. A. Moretti, J. R. Zebol, H. E. Lynn, P. Xia, M. A. Vadas, and B. W. Wattenberg. 2003. Activation of sphingosine kinase by ERK1/2mediated phosphorylation. Embo J 22:5491. 204 Appendix I Buffers PBS (10x stock) 80g NaCl 11.6g NaH2PO4 2g KCl 2g KH2PO4 PBS/0.05% Tween-20 0.5 ml Tween 20 1000 ml PBS (1x) Coating buffer for ELISA 0.1 M NaHCO3, pH 8.2 TBS/1% Tween-20 for Western Blot ml Tween 20 1000 ml PBS (1x) 1% Paraformaldehyde 1g PFA 100 ml PBS 50 µl 1M NaOH Heat to 60°C then gently mix until solution clears. Adjust pH to 7.4 with HCl. 205 RIPA buffer for total cell lysate preparation 50 mM Tris-HCl, pH 7.4 150 mM NaCl mM EDTA 0.25% sodium deoxycholate 1% NP-40 12% resolving gel for SDS-PAGE Distilled water 30% bis-acrylamide 1.5 M Tris, pH 8.8 10% SDS 10% APS TEMED 5% Stacking gel for SDS-PAGE Distilled water 30% bis-acrylamide 1.0 M Tris, pH 6.8 10% SDS 10% APS TEMED Running buffer for SDS-PAGE 25 mM Tris base 206 250 mM glycine, pH 8.3 0.1% SDS Transfer buffer for SDS-PAGE 48 mM Tris base 39 mM glycine 0.037% SDS 20% methanol 1x SDS gel-loading buffer for SDS-PAGE 50 mM Tris-HCl, pH 6.8 100 mM β-Mercaptoethanol 2% SDS 0.1% bromophenol blue 10% glycerol Substrate solution for degranulation assay mM p-nitrophenyl-N-acetyl-D-glucosominide 0.1 M sodium citrate buffer, pH 4.5 207 Appendix II Publications Lai, W. Q., H. H. Goh, Z. Bao, W. S. Wong, A. J. Melendez, and B. P. Leung. 2008.The role of sphingosine kinase in a murine model of allergic asthma. J Immunol 180:4323. Lai, W. Q., A. W. Irwan, H. H. Goh, H. S. Howe, D. T. Yu, R. Valle-Onate, I. B. McInnes, A. J. Melendez, and B. P. Leung. 2008. Anti-inflammatory effects of sphingosine kinase modulation in inflammatory arthritis. J Immunol 181:8010. Lai, W. Q., A. W. Irwan, H. H. Goh, A. J. Melendez, I. B. McInnes, and B. P. Leung. 2009. Distinct roles of sphingosine kinase and in murine collagen-induced arthritis. J Immunol 183:2097. . analysis Chapter 3 The Role of Sphingosine Kinase in Murine Model of Allergic Asthma Introduction Results 3.1 DMS reduces BAL inflammatory infiltrates in OVA-induced asthma mice 3.2 DMS suppresses inflammatory. THE ROLE OF SPHINGOSINE KINASE IN INFLAMMATORY DISEASES LAI WENQI (B.Sc. (Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF. Preparation of Buffers Appendix II Publications 204 207 IX Summary Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine- 1-phosphate