Abstract
Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P(1) receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling 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-alpha, IL-6, IL-1beta, 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-alpha, IFN-gamma, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response 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-inflammatory cytokines.
Highlights
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We show that synovial fluid of rheumatoid arthritis (RA) patients exhibited higher levels of S1P than did those of osteoarthritis (OA) patients and in RA-derived human cells in which DMS suppressed cytokine and matrix metalloproteinase (MMP)-9 release by PBMCs and by monocytes following cell contact-dependent interaction with activated T lymphocytes
S1P levels were measured by a competitive ELISA, and up to 17.51 Ϯ 4.23 M of S1P was detected in RA synovial fluids, significantly higher than those observed in OA fluids (3.45 Ϯ 0.85 M, p Ͻ 0.05, Mann-Whitney U test)
Summary
S1P, sphingosine-1-phosphate; CIA, collageninduced arthritis; CII, type II collagen; DMS, N,N-dimethylsphingosine; MMP, matrix metalloproteinase; OA, osteoarthritis; PB, peripheral blood; RA, rheumatoid arthritis; siRNA, small interfering RNA; SphK, sphingosine kinase. 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). We show that synovial fluid of RA patients exhibited higher levels of S1P than did those of osteoarthritis (OA) patients and in RA-derived human cells in which DMS suppressed cytokine and MMP-9 release by PBMCs and by monocytes following cell contact-dependent interaction with activated T lymphocytes. The potential clinical relevance of these observations is illustrated by the ability of DMS and SphK1 small interfering RNA (siRNA) to effectively suppress murine collagen-induced arthritis (CIA) both in vivo and in vitro via specific suppression of the pathologic proinflammatory and Th1 responses
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