Figure 6.
Figure 6. Inhibition of the recruitment to endothelial cells and the migration of neutrophils by an EP4 agonist. (A) Time courses of the PKA activity of intravascular and interstitial neutrophils are plotted against time. An EP4 agonist, ONO-AE1-329, was injected intravenously (0.25 mg/kg) at time 0. 10 neutrophils in each group were randomly selected in the CFP images and examined for PKA activity in the corresponding FRET/CFP ratio image. Three mice were analyzed independently, and the mean and one SD are indicated. (B) In vivo imaging of the lamina propria of the intestinal mucosa in Eisuke mice and a schematic view of this region. Images are cropped from Video 7. 0.25 mg/kg ONO-AE1-329 was injected intravenously at time 0. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. (C) Inhibition of the entry of neutrophils into the neutrophil recruitment cascade by ONO-AE1-329 treatment. The numbers of neutrophils on the endothelial cells in three mice were counted, and the mean values with one SD are plotted against time. Bars indicate the SD. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (D and E) ERK activity and migration velocity of neutrophils migrating in the connective tissue at the indicated time points. 20 neutrophils in each of two mice were analyzed. Black dots and red bars indicate the ERK activity (D) and migration velocity (E) in each neutrophil and the mean values, respectively. ***, P < 0.001 (D, Student’s t test; E, Mann–Whitney U test). (F) Cancellation of the effect of flurbiprofen axetil by ONO-AE1-329. FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. 7.5 mg/kg flurbiprofen axetil and 0.25 mg/kg ONO-AE1-329 were administered as indicated. Gamma, 1.14. Images are cropped from Video 8. The image is a representative view field of a mouse in three independent experiments. (G and H) ERK activities and migration velocities of neutrophils in the interstitial tissue at the indicated time points. 60 neutrophils from three mice were analyzed. Black dots and red bars indicate the ERK activity (G) and migration velocity (H) in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (I) PKA activity of intravascular and interstitial neutrophils 10 min before and 30 min after 5 mg/kg EP4 antagonist, ONO-AE3-208, injection. 60 neutrophils in and out of venules were randomly selected in the CFP images of three mice and examined for the PKA activity in the corresponding FRET/CFP ratio images. Red bars indicate mean values. Note that FRET values were normalized to the intravascular neutrophils in the absence of ONO-AE3-208. ***, P < 0.001 (Student’s t test). (J–L) Increase in ERK activity and migration velocity of neutrophils by ONO-AE3-208. (J) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 5 mg/kg ONO-AE3-208 at time 0. A CFP image and a scheme are also shown. Image is a representative view field of a mouse in three independent experiments. (B, F, and K) Bars, 50 µm. (K and L) The effect of ONO-AE3-208 on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −10 and 30 min, and ERK activity (K) and the migration velocity (L) of each neutrophil were measured. Three mice were used to collect the data. Dots indicate the ERK activity (K) and migration velocity (L) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Mann–Whitney U test). (M) C57BL/6 mice treated as indicated were sacrificed 24 h later to count the number of intestinal ulcers. Five mice were analyzed in each group. Error bars indicate the SD. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (N and O) Effect of an EP2 agonist, ONO-AE1-259-01, on PKA activity of intravascular and interstitial neutrophils and migration velocity of interstitial neutrophils. (N) Time courses of PKA activity of intravascular and interstitial neutrophils are plotted against time. An EP2 agonist, 0.5 mg/kg ONO-AE1-259-01, was injected intravenously at time 0. 30 neutrophils were randomly selected in the CFP images and examined for PKA activity in the corresponding FRET/CFP ratio image. Three independent experiments were performed, and the mean values with one SD are shown. (O) The effect of ONO-AE1-259-01 on the migration velocity of interstitial neutrophils. 30 neutrophils from three mice were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Dots and bars indicate migration velocities in each neutrophil and mean values, respectively. n.s., not significant (Student’s t test).

Inhibition of the recruitment to endothelial cells and the migration of neutrophils by an EP4 agonist. (A) Time courses of the PKA activity of intravascular and interstitial neutrophils are plotted against time. An EP4 agonist, ONO-AE1-329, was injected intravenously (0.25 mg/kg) at time 0. 10 neutrophils in each group were randomly selected in the CFP images and examined for PKA activity in the corresponding FRET/CFP ratio image. Three mice were analyzed independently, and the mean and one SD are indicated. (B) In vivo imaging of the lamina propria of the intestinal mucosa in Eisuke mice and a schematic view of this region. Images are cropped from Video 7. 0.25 mg/kg ONO-AE1-329 was injected intravenously at time 0. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. (C) Inhibition of the entry of neutrophils into the neutrophil recruitment cascade by ONO-AE1-329 treatment. The numbers of neutrophils on the endothelial cells in three mice were counted, and the mean values with one SD are plotted against time. Bars indicate the SD. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (D and E) ERK activity and migration velocity of neutrophils migrating in the connective tissue at the indicated time points. 20 neutrophils in each of two mice were analyzed. Black dots and red bars indicate the ERK activity (D) and migration velocity (E) in each neutrophil and the mean values, respectively. ***, P < 0.001 (D, Student’s t test; E, Mann–Whitney U test). (F) Cancellation of the effect of flurbiprofen axetil by ONO-AE1-329. FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. 7.5 mg/kg flurbiprofen axetil and 0.25 mg/kg ONO-AE1-329 were administered as indicated. Gamma, 1.14. Images are cropped from Video 8. The image is a representative view field of a mouse in three independent experiments. (G and H) ERK activities and migration velocities of neutrophils in the interstitial tissue at the indicated time points. 60 neutrophils from three mice were analyzed. Black dots and red bars indicate the ERK activity (G) and migration velocity (H) in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (I) PKA activity of intravascular and interstitial neutrophils 10 min before and 30 min after 5 mg/kg EP4 antagonist, ONO-AE3-208, injection. 60 neutrophils in and out of venules were randomly selected in the CFP images of three mice and examined for the PKA activity in the corresponding FRET/CFP ratio images. Red bars indicate mean values. Note that FRET values were normalized to the intravascular neutrophils in the absence of ONO-AE3-208. ***, P < 0.001 (Student’s t test). (J–L) Increase in ERK activity and migration velocity of neutrophils by ONO-AE3-208. (J) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 5 mg/kg ONO-AE3-208 at time 0. A CFP image and a scheme are also shown. Image is a representative view field of a mouse in three independent experiments. (B, F, and K) Bars, 50 µm. (K and L) The effect of ONO-AE3-208 on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −10 and 30 min, and ERK activity (K) and the migration velocity (L) of each neutrophil were measured. Three mice were used to collect the data. Dots indicate the ERK activity (K) and migration velocity (L) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Mann–Whitney U test). (M) C57BL/6 mice treated as indicated were sacrificed 24 h later to count the number of intestinal ulcers. Five mice were analyzed in each group. Error bars indicate the SD. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (N and O) Effect of an EP2 agonist, ONO-AE1-259-01, on PKA activity of intravascular and interstitial neutrophils and migration velocity of interstitial neutrophils. (N) Time courses of PKA activity of intravascular and interstitial neutrophils are plotted against time. An EP2 agonist, 0.5 mg/kg ONO-AE1-259-01, was injected intravenously at time 0. 30 neutrophils were randomly selected in the CFP images and examined for PKA activity in the corresponding FRET/CFP ratio image. Three independent experiments were performed, and the mean values with one SD are shown. (O) The effect of ONO-AE1-259-01 on the migration velocity of interstitial neutrophils. 30 neutrophils from three mice were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Dots and bars indicate migration velocities in each neutrophil and mean values, respectively. n.s., not significant (Student’s t test).

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