Anti-Inflammatory Effects of Sphingosine Kinase Modulation in Inflammatory Arthritis 1 Wen-Qi Lai, 2 * Anastasia Windy Irwan, 2 * Hong Heng Goh,* Hwee Siew Howe, † David T. Yu, ‡ Rafael Valle-On ˜ ate, § Iain B. McInnes, ¶ Alirio J. Melendez,* and Bernard P. Leung 3 * Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphin- gosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various patho- logic conditions. Recently, S1P 1 receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P sig- naling via S1P 1 enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E 2 production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-, IL-6, IL-1, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-, IFN-, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in re- sponse to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflam- matory cytokines. The Journal of Immunology, 2008, 181: 8010 – 8017. S phingolipids are sources of important signaling molecules in addition to their role as structural components of the eukaryotic cell membranes. In particular, sphingolipid metabolites such as ceramide and sphingosine-1-phosphate (S1P) 4 have emerged as a new class of potent bioactive mes- sengers involved in an array of cellular processes, including angiogenesis, proliferation, and apoptosis (1, 2). Recently, in- terest in S1P has focused on two distinct cellular roles, namely its function as an intracellular second messenger, or extracel- lularly as a specific and high-affinity ligand for a family of G protein-coupled receptors previously known as the endothelial differentiation gene (EDG) family. To date, five S1P receptors in the endothelial differentiation gene family have been identi- fied, including EDG-1, EDG-3, EDG-5, EDG-6, and EDG-8, now collectively known as S1P 1–5 (2– 4). Sphingosine kinase (SphK) is a key enzyme in the sphingo- lipid metabolic pathway, responsible for phosphorylating sphin- gosine into S1P. To date, two mammalian SphK isoenzymes, SphK1 and SphK2, have been identified and characterized (5– 7). As an intracellular second messenger, S1P was found to play a role in calcium signaling and mobilization (8, 9), cell prolif- eration, and survival (10). Activation of various plasma mem- brane receptors, such as the fMLP receptor (11), the C5a re- ceptor (12, 13), and TNF- receptor (14), leads to rapid increase in intracellular S1P level via SphK stimulation. We have previously shown that inhibition of SphK activity by N,N- dimethylsphingosine (DMS), a potent SphK inhibitor, leads to reduced Ca 2 mobilization, enzyme release, and chemotaxis, cytokine, and chemokine production in human neutrophils, monocytes, and macrophages (12, 13, 15). Moreover, by em- ploying a specific antisense knock-down approach, SphK1 was found to be a critical regulator in TNF--mediated IL-1 and IL-6 proinflammatory responses in human monocytes (14). Elevated levels of proinflammatory cytokine production char- acterize rheumatoid arthritis (RA) synovial inflammation (16, 17). In particular, within inflamed RA synovial membrane, the levels of proinflammatory cytokines (namely TNF-, IL-1, and IL-6) exceed those of anti-inflammatory agents (IL-1RA and IL-10), and this likely contributes directly to cartilage and bone erosion through promoting matrix metalloproteinase (MMP) production and dysregulated chondrocyte/osteoclast function (16 –18). Moreover, successful therapeutic targeting of cytokines in RA, particularly TNF-, had demonstrated their critical pathogenic importance (17–19). There are multiple pathways that drive cytokine production in RA synovium with *Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore; † Department of Rheumatology, Al- lergy and Immunology, Tan Tock Seng Hospital, Singapore, Republic of Singapore; ‡ Rheumatology Division, University of California, Los Angeles, CA 90095-167022; § Division of Rheumatology, Hospital Militar Central/Universidad de la Sabana, Bogota ´, Colombia; and ¶ Division of Immunology, Infection and Inflammation, Glas- gow Biomedical Research Centre, University of Glasgow, Glasgow, U.K. Received for publication July 2, 2008. Accepted for publication October 2, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Office of Life Sciences, National University of Singapore. W.-Q.L. is supported by a scholarship from the National University of Singapore, Republic of Singapore. 2 W.-Q.L. and A.W.I. contributed equally to this study. 3 Address correspondence and reprint requests to Dr. Bernard P. Leung, Department of Physiology, 2 Medical Drive, MD9, National University of Singapore, Singapore 117597, Republic of Singapore. E-mail address: phslplb@nus.edu.sg 4 Abbreviations used in this paper: S1P, sphingosine-1-phosphate; CIA, collagen- induced arthritis; CII, type II collagen; DMS, N,N-dimethylsphingosine; MMP, ma- trix metalloproteinase; OA, osteoarthritis; PB, peripheral blood; RA, rheumatoid ar- thritis; siRNA, small interfering RNA; SphK, sphingosine kinase. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org