Figure 3. ATX in the presence of LPC induces polarization of T cells.

(A) TK1 cells were allowed to settle on ICAM-1 substratum in the presence of the indicated concentration of ATX with (1 μM, 10 μM) or without LPC in solution. □denotes response to 5 μg/ml of T210A with 10 μM LPC. After 7 min, percent of cells that became polarized was determined by CD44 accumulation in uropods. For all ATX concentrations except 5 μg/ml: 10 μM LPC > 1 μM > 0 LPC with p < 0.01 by ANOVA. For 5 μg/ml ATX: 1 and 10 μM LPC > 0 LPC with p < 0.001 by ANOVA. (B) Time course of TK1 polarization response to 1 μg/ml ATX and 1 μM LPC in solution. ■indicates response to 1 μM LPC alone. (C) TK1 polarization response to ATX (5 μg/ml) plus LPC (1 μM) or to LPA (1 μM) in the presence of either BrP-LPA (10 μM) or HA130 (0.3 μM). Uropod response was measured after 7 min. CRTL, no additions. *** denotes p < 0.001 for comparisons of ATX/LPC vs. ATX/LPC + HA130 and ATX/LPC + BrP-LPA. (D) TK1 polarization response to ATX (5 μg/ml) plus LPC (1 μM) when cells were treated with pertussis toxin (200 ng/ml) or Y27632 (10 μM). *** denotes p < 0.001 for comparison of ATX/LPC vs. ATX/LPC + Y27632. (E) Naïve T cell polarization response (CD43 uropod assay) to the indicated concentration of ATX in solution with 1 μM LPC present. The substratum consisted of ICAM-1 with or without co-immobilized CCL21 (200 ng/ml). ** denotes p < 0.01 for comparison of uropod formation with CCL21 vs. without CCL21. (F) Human blood T cells (left) and neutrophils (right) were allowed to settle on ICAM-1 coated wells in the presence of LPA or ATX (1 μg/ml) with 1 μM LPC. Uropod formation was determined by staining for the accumulation of CD44. In all panels, means and SDs are shown and are based on 3 replicate wells. Data are representative of 3 independent experiments with the exception of panel F which was performed twice.