FTY720 attenuates Ag-induced secretion of PGD₂. (A) RBL-2H3 cells (5 × 10⁴) were treated with vehicle, S1P (1 μM), FTY720 (1 μM), C1P (2.5 μM), or FTY720-P (1 μM) for 2 hours or sensitized overnight without or with anti–DNP IgE (1 μg/mL), washed, and then treated with Ag (10 ng/mL) for 15 minutes. PGD₂ release was measured by enzyme-linked immunosorbent assay (ELISA) and data are means ± SD of quadruplicate determinations. (B) RBL-2H3 cells were sensitized overnight with anti–DNP IgE (1 μg/mL), treated for 2 hours with FTY720 (0.1 or 1 μM, ▪), and then stimulated with Ag (10 ng/mL) for 15 minutes, and PGD₂ release was determined by ELISA. Data are expressed as means ± SEM (n = 4). (Inset) Duplicate cultures were treated without (□) or with 1 μM FTY720 (▪) and cell viability was measured with the WST-1 assay. Data are the means of A₄₅₀ ± SD of triplicate determinations. (C) Sensitized RBL-2H3 cells were treated with the indicated concentrations of FTY720 for 2 hours, and then stimulated with Ag for 15 minutes, and PGD₂ release was determined. Data are expressed as percent inhibition. (D) LAD2 human mast cells were sensitized overnight with anti–DNP IgE, pretreated with the indicated concentrations of FTY720 for 2 hours, and then stimulated without or with Ag (10 ng/mL) for 15 minutes, and secreted PGD₂ was determined. (Inset) Duplicate cultures were treated without (□) or with 1 μM FTY720 (▪), and cell viability was measured. (B-D) *P < .05; **P < .01. (E) Sensitized RBL-2H3 cells were treated with vehicle, S1P (1 μM), or Ag (10 ng/mL) for the indicated times, and equal amounts of lysate proteins were immunoblotted with anti–COX-2 antibody. Blots were stripped and reprobed with antitubulin as a loading control. (F) Sensitized LAD2 cells were treated with vehicle, 1 μM each of S1P, FTY720, or FTY720-P, or Ag (10 ng/mL) for the indicated times, and proteins were immunoblotted with anti–COX-2 antibody or antitubulin. Similar results were obtained in 3 independent experiments.