Figure 3.

S1P₂ antagonism or down-regulation of S1P₂ decreases S1P- and Ag-induced degranulation and chemokine secretion from CB-MCs. (a and b) Purified CB-MCs were stimulated with vehicle, 100 nM S1P, or 1 µM ionomycin, or sensitized overnight with IgE, and then stimulated with 30 ng/ml Ag for 2 h. CB-MCs were pretreated for 30 min with vehicle or 1 µM JTE013 before stimulation, as indicated. Degranulation (a) and secretion of CCL2 (b) were measured. *, P ≤ 0.01; ^#, P ≤ 0.05, compared with vehicle treatment. (c–e) CB-MCs transfected with control siRNA or S1P₂ siRNA were stimulated with vehicle, 100 nM S1P, or 1 µM ionomycin, or sensitized overnight with IgE, and then stimulated with 30 ng/ml Ag for 2 h. (c) RNA was isolated and mRNA levels of S1P₁, S1P₂, SphK1, SphK2, and GAPDH were determined by quantitative real-time PCR. Degranulation (d) and secretion of CCL2 (e) were measured. *, P ≤ 0.01; ^#, P ≤ 0.05, compared with scrambled siRNA. Data are the means ± SD of triplicate determinations. Similar results were obtained in three independent experiments with CB-MC from three different donors.