Effect of LTB4 and an LTB4 receptor antagonist on the ERK activity and the neutrophil recruitment. (A) FRET and CFP images of the lamina propria of the intestinal mucosa in Eisuke mice. The top left panel shows a schematic view of this image. At time 0 during time-lapse imaging, LTB4 was injected intravenously at 7.5 µg/kg. Gamma, 1.14. Image is a representative view field of a mouse in three independent experiments. (B) Time courses of the ERK activity of intravascular and interstitial neutrophils plotted against time. 10 neutrophils in each of three mice were randomly selected in the CFP images and examined for ERK activity in the corresponding FRET/CFP ratio image. Error bars indicate the one SD. (C) Migration velocity of interstitial neutrophils was measured before and after LTB4 treatment. 20 neutrophils each from three mice were randomly selected in the CFP images and examined for migration velocity during 5-min time-lapse imaging. Dots and bars indicate the migration velocity of each neutrophil and the mean values, respectively. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (D) FRET and CFP images in Eisuke mice. The bottom left panel shows a schematic view of this image. Cr, crypt; Ly, lymphatic vessel; Ve, venule. At time 0 during time-lapse imaging, LY293111, an LTB4 receptor antagonist, was injected intravenously at 4 mg/kg. Gamma, 1.7. Image is a representative view field of a mouse in three independent experiments. Bars: (A) 50 µm; (D) 30 µm. (E) Time courses of the ERK activity of intravascular and interstitial neutrophils plotted against time. 10 neutrophils in each of three mice were randomly selected in the CFP images and examined for ERK activity in the corresponding FRET/CFP ratio image. Error bars indicate the one SD. (F) Neutrophils on the endothelial cells were classified into the four steps of the recruitment cascade before and after LY293111 treatment. Before and after LY293111 injection, 428 and 386 neutrophils, respectively, were scored in three mice. Error bars indicate SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (G) Effect of LY293111 on the migration velocity of interstitial neutrophils. 20 neutrophils in each of three mice were randomly chosen in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Red bars indicate mean values. ***, P < 0.001 (Mann–Whitney U test). (H) Schematic view of the regulation of neutrophil migration. ERK plays a central role in controlling cell migration, whereas PKA regulates cell migration via ERK regulation.
