FTY720 inhibits release of prostanoids and arachidonic acid independent of CDLA₂ translocation and ERK activation. (A) Effect of FTY720 on LPS-stimulated PGE₂ production in RAW264.7 cells. RAW264.7 cells were treated for 2 hours with the indicated concentrations of FTY720, stimulated without (□) or with LPS (1 μg/mL, ▪) for 1 hour, and PGE₂ secretion was determined by ELISA. Data are means ± SD of triplicate determinations. (Inset) Duplicate cultures were treated without (□) or with 1 μM FTY720 (▪), and cell viability was measured. (B) FTY720 inhibits Ag-induced arachidonic acid release from mast cells. RBL-2H3 mast cells (5 × 10⁴) were sensitized with anti–DNP IgE (1 μg/mL) in the presence of 1 μCi (0.037 MBq)/mL [³H]AA for 18 hours, washed, and incubated further in the absence or presence of FTY720 (1 μM) in 0.5% BSA-EMEM. Cells were then stimulated without or with Ag (10 ng/mL) or ionomycin (5 μM) for 15 minutes, and [³H]AA release was measured by scintillation counting. Data are means ± SD of quadruplicate determinations expressed as percent of total AA released. (A) *P < .05; **P < .01. (C) Lack of effect of FTY720 on Ag-induced translocation of cPLA₂α. Sensitized RBL-2H3 cells were treated without (vehicle) or with 1 μM FTY720 for 18 hours and then stimulated with Ag for the indicated times. Membranes were isolated from cell lysates by centrifugation at 100 000g, and equal amounts of proteins were analyzed by immunoblotting with anti-cPLA₂. Blots were stripped and reprobed with anti–α_V-integrin to demonstrate equal loading. (D-E) FTY720 does not inhibit Ag-induced ERK1/2 activation. RBL-2H3 (D) or LAD2 (E) mast cells were sensitized with IgE, treated with FTY720 (1 μM) for 2 hours (D) or 18 hours (E), and then stimulated with Ag (10 ng/mL) for the indicated times. Cells were lysed and phospho-ERK1/2 activation was determined by immunoblotting. Blots were stripped and reprobed with anti–β-tubulin to demonstrate equal loading. Similar results were obtained in 3 independent experiments.