Sphingosine 1-Phosphate Elicits Proinflammatory Responses in ARPE-19 Cells

PURPOSE. To investigate the effects of sphingosine 1-phosphate (S1P) on the production of inflammatory mediators and the signaling pathways involved in S1P-mediated production of cytokines by ARPE-19 cells. METHODS. Expression of S1P receptors was examined using RT-PCR and real-time PCR. ARPE-19 cells were stimulated with S1P or TNF-alpha, and by coculturing with S1P in the presence or absence of pertussis toxin (PTX) or a series of kinase inhibitors. The induction of inflammatory cytokine production was determined by ELISA. Western blot analysis was used to detect the activation of signaling mediators and S1P(3) receptor. RESULTS. ARPE-19 cells express all the known receptors for S1P. Moreover, exogenously applied S1P induces ARPE-19 cell secretion of interleukin-8 (IL-8) in a dose-and time-dependent manner, but not IL-6 and monocyte chemotactic protein-1 (MCP-1). S1P-mediated IL-8 secretion is regulated by PTX, extracellular regulated protein kinases 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Interestingly, treatment of ARPE-19 cells with TNF-alpha increases S1P(3) expression and correlates with the enhancement of S1P-induced IL-8 and IL-6 production. CONCLUSIONS. This study demonstrates that S1P significantly promoted ARPE-19 cells to secrete inflammatory mediators. extracellular regulated protein kinases 1/2 (ERK1/2), p38 MAPK, and G(i)-dependent pathways are important signaling components in S1P-mediated IL-8 secretion by ARPE-19 cells. Moreover, these results provide evidence that S1P stimulation of RPE cells may play a role in regulating leukocyte function during ocular inflammation. (Invest Ophthalmol Vis Sci. 2012; 53: 8200-8207) DOI:10.1167/iovs.12-1096