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JOURNAL ARTICLE
RESEARCH SUPPORT, NON-U.S. GOV'T
Sphingosine 1-phosphate elicits proinflammatory responses in ARPE-19 cells.
Investigative Ophthalmology & Visual Science 2012 December
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-α, 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-α 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.
METHODS: Expression of S1P receptors was examined using RT-PCR and real-time PCR. ARPE-19 cells were stimulated with S1P or TNF-α, 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-α 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.
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