Figure 7. S1PR2 mediates migration inhibition in macrophages.

(A and B) Effects of S1P and MCP-1 on transwell migration in serum-starved peritoneal macrophages isolated from S1pr2^+/+Apoe^–/– and S1pr2^–/–Apoe^–/– mice. Macrophages were placed in the upper chamber. Various concentrations of S1P were added to either the lower chamber (A) or the upper chamber (B) in the presence or absence of MCP-1 (100 ng/ml) in the lower chamber. *P < 0.05 compared with the value in the absence of S1P (n = 4 each). (C) Effects of S1P and MCP-1 on Rac activity. Peritoneal macrophages were not treated or treated with either S1P (0.1 μM) alone for 10 minutes, MCP-1 alone (100 ng/ml) for 3 minutes, or the combination of S1P and MCP-1. In the combination treatment, S1P was added 7 minutes before the addition of MCP-1. Amounts of GTP-bound Rac were determined by the pulldown assay (n = 3 each). *P < 0.05, compared with nontreated macrophages; ^#P < 0.05, compared with MCP-1–treated macrophages. (D) Western blot analysis of VLA-4 (integrin α₄β₁), LFA-1 (integrin α_Lβ₂), and CCR2 expression on peritoneal macrophages, with α-tubulin as internal control. (E) Macrophage homing assay. Peritoneal macrophages isolated from S1pr2^+/+GFP-Tg and S1pr2^–/–GFP-Tg mice were injected via the tail vein into S1pr2^+/+Apoe^–/– mice fed HCD (n = 3 each). Sections of the aortic sinus were made and GFP-expressing cells infiltrating into the intima were observed under a fluorescent microscope (asterisks show the lumen), and GFP-positive cells were counted. Sections were counterstained with DAPI. Representative images (left) and quantified data are shown (right). **P < 0.01. Scale bars: 50 μm. Data are expressed as mean ± SEM.