Fig. 3

LPA₁ is essential for LPA-induced neutrophil migration. a Ly-6G-positive neutrophils from murine spleen were incubated with LA-01 (0, 1, 10 nM) at 37 °C for 30 min in RPMI 1640. Cells were added to the upper wells of the transwell, LPA (10 μM) was loaded into the lower wells, and cells were incubated at 37 °C for 2 h. b Ly-6G-positive neutrophils from the spleen of WT or LPA₁-deficient mice were loaded into the upper wells, LPA (10 μM) was added to the lower wells of the transwell, and cells were incubated at 37 °C for 2 h. The number of neutrophils that migrated into the lower well was counted by flow cytometry. Triplicates of three independent experiments were performed. Data are mean ± SEM; *P < 0.05 vs vehicle or WT mice. c, d Fluorescence-labeled Ly-6G-positive neutrophils from the spleen of WT mice were transferred into CAWS-induced vasculitis WT mice on day 21. The recipient mice were treated with LA-01 (100 mg/kg) or saline as a control 12 h and 30 min before and 12 h after the transfer. Migrated labeled cells were evaluated histologically 24 h after the transfer (c), and the number of labeled cells that migrated into the vascular walls was counted (d). e, f Fluorescence-labeled Ly-6G-positive neutrophils from the spleen of LPA₁-deficient or WT mice were transferred into WT vasculitis mice on day 21. Migrated labeled cells were evaluated histologically 24 h after the transfer (e), and the number of labeled cells that migrated into the vascular walls was counted under a fluorescent microscope (f). Arrows indicate migrated labeled cells. Original magnification × 200 (c, e). Data are the mean ± SEM; *P < 0.05 vs vehicle or WT mice (d, f)