Engagement of S1PR2 with exogenous S1P leads to RhoA activation and inhibition of phagocytosis. (A) S1P levels in bronchoalveolar lavage fluid (BALF) from wild-type (WT, S1pr2^+/+) and S1pr2^−/− mice, as well as in the supernatants of cultured bone marrow–derived macrophages (BMDMs), at different time points (as indicated) after challenge with live Escherichia coli. Data are presented as the mean ± SD and were analyzed by Student t test. n = 6 per group. (B) Phagocytosis of fluorescent E. coli by BMDMs challenged with various doses of S1P. Data are presented as mean ± SD from three independent experiments and analyzed by two-way ANOVA with Bonferroni corrections. FI = fluorescent intensity. (C) Activation of S1PR2 with S1P prevents phagocytosis of fluorescent E. coli (red) in BMDMs. WT and S1pr2^−/− BMDMs were starved and pretreated with or without S1P (100 nM) for 30 min and then incubated with fluorescent E. coli for 30 min. F-actin (green) and nuclei (blue) were fluorescently stained. (D) S1P activates small RhoA GTPase in BMDMs through S1PR2. RhoA-GTP level was detected in E. coli-stimulated WT and S1pr2^−/− BMDMs using glutathione-S-transferase pull-down assay. Total RhoA protein was used a loading control. Data are presented as mean ± SD from three independent experiments and were analyzed by two-way ANOVA with Bonferroni corrections. S1PR2 = sphingosine 1-phosphate receptor 2. *P < 0.05, **P < 0.01, and ***P < 0.001.