Figure 6.
Chemotaxis of mast cells toward Ag is dependent on SphK1, Gi, and S1P1. (A) RBL-2H3 cells were sensitized with 1 μg/ml anti-DNP IgE in the absence (white bars) or presence of 200 ng/ml pertussis toxin (PTX, black bars) and allowed to migrate toward the indicated concentrations of Ag or S1P (1 nM) for 3 h. (B) RBL-2H3 cells were transfected with S1P1 antisense (diagonally striped bars) or scrambled oligonucleotides (white bars) for 48 h, sensitized with anti-DNP IgE, and allowed to migrate toward 10 nM S1P, 10 ng/ml Ag, or 20 μg/ml fibronectin. (C) RBL-2H3 cells stably expressing GFP-S1P1 (black bars) or GFP-vector (white bars) were sensitized with anti-DNP IgE and allowed to migrate for 1.5 h toward 10 nM S1P, 10 ng/ml Ag, or 20 μg/ml fibronectin. Random motilities of vector and S1P1 transfectants were 82 ± 11 and 219 ± 6, respectively. RBL-2H3 cells stably expressing GFP-S1P2 (gray bars) or GFP-vector (white bars) were allowed to migrate toward the same agents for 3 h. Random motilities of vector and S1P2 transfectants were 157 ± 9 and 174 ± 29, respectively. (inset) Western blot of cell lysates showing expression of GFP-S1P1 and GFP-S1P2 visualized by ECL with anti-GFP. Lanes 1, 2, and 3 are lysates of cells overexpressing GFP vector, GFP-S1P1, and GFP-S1P2, respectively. (D) IgE-sensitized RBL-2H3 cells were treated without (white bars) or with 1 μM DMS (black bars) for 20 min before the addition of the indicated concentrations of Ag or S1P. (E) RBL-2H3 cells were transfected with control siRNA (white bars), siRNA targeted to SphK1 (black bars), or siRNA targeted to SphK2 (gray bars) as described in Materials and Methods, sensitized with 1 μg/ml anti-DNP IgE for 6 h, and allowed to migrate toward vehicle (None), 10 ng/ml Ag, 10 nM S1P, or 20 μg/ml fibronectin for 3 h. Results are expressed as means ± SD. *, P < 0.05 by Student's t test. #, P < 0.05 by ANOVA.