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In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.

Mizuno R, Kamioka Y, Kabashima K, Imajo M, Sumiyama K, Nakasho E, Ito T, Hamazaki Y, Okuchi Y, Sakai Y, Kiyokawa E, Matsuda M - J. Exp. Med. (2014)

Bottom Line: Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue.In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils.The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs).

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Affiliation: Department of Pathology and Biology of Diseases, Department of Gastrointestinal Surgery, Department of Dermatology, and Department of Immunology and Cell Biology, Graduate School of Medicine; Innovative Techno-Hub for Integrated Medical Bio-Imaging; and Laboratory of Bioimaging and Cell Signaling, Department of Molecular and System Biology, Graduate School of Biostudies; Kyoto University, Kyoto 606-8501, JapanDepartment of Pathology and Biology of Diseases, Department of Gastrointestinal Surgery, Department of Dermatology, and Department of Immunology and Cell Biology, Graduate School of Medicine; Innovative Techno-Hub for Integrated Medical Bio-Imaging; and Laboratory of Bioimaging and Cell Signaling, Department of Molecular and System Biology, Graduate School of Biostudies; Kyoto University, Kyoto 606-8501, Japan.

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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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fig6: 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).

Mentions: The accelerated migration and increased ERK activation of neutrophils in the NSAID-treated intestine led us to examine the role of PGE2 on neutrophil migration. We first intravenously administered an EP4 agonist, ONO-AE1-329, to examine the role of EP4 on neutrophils. The EP4 agonist ONO-AE1-329 strongly and rapidly increased PKA activity and decreased ERK activity in intravascular and interstitial neutrophils and in endothelial cells (Fig. 6, A and B; and Video 7). Intravenous injection of ONO-AE1-329 gradually reduced the number of neutrophils on the endothelial cells, in agreement with the role of ERK and PKA in the recruitment of neutrophils (Fig. 6 C). Both the ERK activity and migration velocity of interstitial neutrophils were gradually restored within 60 min after ONO-AE1-329 treatment, when PKA activity also returned to almost the basal level (Fig. 6, A, D, and E). The effect of EP4 activation was more clearly demonstrated when flurbiprofen axetil was injected before ONO-AE1-329 administration (Fig. 6 F). ONO-AE1-329 markedly inhibited ERK activity and migration of interstitial neutrophils (Fig. 6, F–H; and Video 8). The effect was lost within 80 min, probably as a result of inactivation in the lung. We next confirmed the role played by EP4 with an EP4 antagonist, ONO-AE3-208. As expected, intravenous injection of ONO-AE3-208 suppressed PKA, activated ERK, and promoted migration of neutrophils (Fig. 6, I–L). These results strongly suggest that PGE2-engaged EP4 inhibits ERK and thereby serves as an antiinflammatory signal. Because NSAIDs such as flurbiprofen axetil are known to cause ulcers in intestines, we examined the effect of ONO-AE1-329 and ONO-AE3-208 on the NSAID enteritis (Fig. 6 M). ONO-AE1-329 markedly decreased the number of flurbiprofen axetil–induced ulcers, whereas ONO-AE3-208 increased the number of ulcers, although ONO-AE3-208 alone could not cause ulcers. Thus, NSAID-mediated suppression of the EP4 signaling in the neutrophils seems to contribute at least partially to the development of NSAID enteritis. Of note, the EP2 agonist ONO-AE1-259-01 activated PKA only slightly and did not affect the migration velocity of the interstitial neutrophils (Fig. 6, N and O).


In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.

Mizuno R, Kamioka Y, Kabashima K, Imajo M, Sumiyama K, Nakasho E, Ito T, Hamazaki Y, Okuchi Y, Sakai Y, Kiyokawa E, Matsuda M - J. Exp. Med. (2014)

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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fig6: 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).
Mentions: The accelerated migration and increased ERK activation of neutrophils in the NSAID-treated intestine led us to examine the role of PGE2 on neutrophil migration. We first intravenously administered an EP4 agonist, ONO-AE1-329, to examine the role of EP4 on neutrophils. The EP4 agonist ONO-AE1-329 strongly and rapidly increased PKA activity and decreased ERK activity in intravascular and interstitial neutrophils and in endothelial cells (Fig. 6, A and B; and Video 7). Intravenous injection of ONO-AE1-329 gradually reduced the number of neutrophils on the endothelial cells, in agreement with the role of ERK and PKA in the recruitment of neutrophils (Fig. 6 C). Both the ERK activity and migration velocity of interstitial neutrophils were gradually restored within 60 min after ONO-AE1-329 treatment, when PKA activity also returned to almost the basal level (Fig. 6, A, D, and E). The effect of EP4 activation was more clearly demonstrated when flurbiprofen axetil was injected before ONO-AE1-329 administration (Fig. 6 F). ONO-AE1-329 markedly inhibited ERK activity and migration of interstitial neutrophils (Fig. 6, F–H; and Video 8). The effect was lost within 80 min, probably as a result of inactivation in the lung. We next confirmed the role played by EP4 with an EP4 antagonist, ONO-AE3-208. As expected, intravenous injection of ONO-AE3-208 suppressed PKA, activated ERK, and promoted migration of neutrophils (Fig. 6, I–L). These results strongly suggest that PGE2-engaged EP4 inhibits ERK and thereby serves as an antiinflammatory signal. Because NSAIDs such as flurbiprofen axetil are known to cause ulcers in intestines, we examined the effect of ONO-AE1-329 and ONO-AE3-208 on the NSAID enteritis (Fig. 6 M). ONO-AE1-329 markedly decreased the number of flurbiprofen axetil–induced ulcers, whereas ONO-AE3-208 increased the number of ulcers, although ONO-AE3-208 alone could not cause ulcers. Thus, NSAID-mediated suppression of the EP4 signaling in the neutrophils seems to contribute at least partially to the development of NSAID enteritis. Of note, the EP2 agonist ONO-AE1-259-01 activated PKA only slightly and did not affect the migration velocity of the interstitial neutrophils (Fig. 6, N and O).

Bottom Line: Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue.In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils.The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Biology of Diseases, Department of Gastrointestinal Surgery, Department of Dermatology, and Department of Immunology and Cell Biology, Graduate School of Medicine; Innovative Techno-Hub for Integrated Medical Bio-Imaging; and Laboratory of Bioimaging and Cell Signaling, Department of Molecular and System Biology, Graduate School of Biostudies; Kyoto University, Kyoto 606-8501, JapanDepartment of Pathology and Biology of Diseases, Department of Gastrointestinal Surgery, Department of Dermatology, and Department of Immunology and Cell Biology, Graduate School of Medicine; Innovative Techno-Hub for Integrated Medical Bio-Imaging; and Laboratory of Bioimaging and Cell Signaling, Department of Molecular and System Biology, Graduate School of Biostudies; Kyoto University, Kyoto 606-8501, Japan.

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