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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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Accelerated migration and activation of ERK in neutrophils treated with NSAID. (A) PKA activity of intravascular and interstitial neutrophils before and after 7.5 mg/kg flurbiprofen axetil injection. 40 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. ***, P < 0.001 (Student’s t test). (B) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 7.5 mg/kg flurbiprofen axetil at time 0. Images are cropped from Video 6. A CFP image and a scheme are also shown. Cr, crypt; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. Bars, 30 µm. (C) Increase in the crawling step and decrease in the rolling step in the neutrophil recruitment cascade by flurbiprofen axetil treatment. The neutrophils on the endothelial cells were categorized into the four steps of the neutrophil recruitment cascade. The fraction of each step was calculated after summing up three time points each for before and after flurbiprofen axetil treatment. The experiment was repeated three times, and 537 and 503 neutrophils in total were scored before and after flurbiprofen axetil injection, respectively. Error bars indicate the SD. *, P < 0.05 (Student’s t test). (D–G) Acceleration of neutrophil crawling and transmigration by flurbiprofen axetil injection. Eisuke mice pretreated with LPS and fMLP were time-lapse imaged before and after intravenous administration of flurbiprofen axetil. The images were acquired every 1.5 s for the detailed analysis. The flux of rolling neutrophils (D) and the number of adherent (E), crawling (F), or transmigrating neutrophils (G) were quantified with time-lapse essentially as described previously (Kubes et al., 2003). A neutrophil was defined as adherent to endothelial cells if it remained stationary for >30 s. The numbers of adherent, crawling, or transmigrating neutrophils were averaged per 10-min observation period. Four mice were analyzed independently, and mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (H and I) The effect of flurbiprofen axetil on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Three independent experiments were performed to collect the data. Dots indicate the ERK activity (H) and migration velocity (I) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Student’s t test and Mann–Whitney U test are used for the ERK activity [H] and migration velocity [I], respectively).
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fig5: Accelerated migration and activation of ERK in neutrophils treated with NSAID. (A) PKA activity of intravascular and interstitial neutrophils before and after 7.5 mg/kg flurbiprofen axetil injection. 40 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. ***, P < 0.001 (Student’s t test). (B) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 7.5 mg/kg flurbiprofen axetil at time 0. Images are cropped from Video 6. A CFP image and a scheme are also shown. Cr, crypt; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. Bars, 30 µm. (C) Increase in the crawling step and decrease in the rolling step in the neutrophil recruitment cascade by flurbiprofen axetil treatment. The neutrophils on the endothelial cells were categorized into the four steps of the neutrophil recruitment cascade. The fraction of each step was calculated after summing up three time points each for before and after flurbiprofen axetil treatment. The experiment was repeated three times, and 537 and 503 neutrophils in total were scored before and after flurbiprofen axetil injection, respectively. Error bars indicate the SD. *, P < 0.05 (Student’s t test). (D–G) Acceleration of neutrophil crawling and transmigration by flurbiprofen axetil injection. Eisuke mice pretreated with LPS and fMLP were time-lapse imaged before and after intravenous administration of flurbiprofen axetil. The images were acquired every 1.5 s for the detailed analysis. The flux of rolling neutrophils (D) and the number of adherent (E), crawling (F), or transmigrating neutrophils (G) were quantified with time-lapse essentially as described previously (Kubes et al., 2003). A neutrophil was defined as adherent to endothelial cells if it remained stationary for >30 s. The numbers of adherent, crawling, or transmigrating neutrophils were averaged per 10-min observation period. Four mice were analyzed independently, and mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (H and I) The effect of flurbiprofen axetil on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Three independent experiments were performed to collect the data. Dots indicate the ERK activity (H) and migration velocity (I) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Student’s t test and Mann–Whitney U test are used for the ERK activity [H] and migration velocity [I], respectively).

Mentions: We next examined the effect of an NSAID, flurbiprofen axetil, on the activities of ERK and PKA in the inflamed intestine. NSAIDs generally inhibit production of PGE2 and thereby suppress Gs-coupled receptors such as EP2 and EP4. In agreement with a previous study showing the expression of EP2 and EP4 in neutrophils (Yamane et al., 2000), PKA activity was decreased in both intravascular and interstitial neutrophils within 10 min after intravenous flurbiprofen axetil injection (Fig. 5 A). In contrast, flurbiprofen axetil injection caused a gradual increase in the ERK activity of neutrophils (Fig. 5 B and Video 6). In the venules, the major fraction of neutrophils was shifted from the rolling step to the crawling step by flurbiprofen axetil (Fig. 5 C). Detailed analysis of neutrophils on the endothelial cells revealed that flurbiprofen axetil enhanced progression from the adhesion step to the crawling step (Fig. 5, D–G). Meanwhile, in the interstitial tissue, both the ERK activity and migration velocity of interstitial neutrophils were significantly increased within 20 min after flurbiprofen axetil injection (Fig. 5, H and I). We obtained a similar result with another NSAID, indomethacin, suggesting that the observed effects arose mostly through the inhibition of COXs (not depicted).


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)

Accelerated migration and activation of ERK in neutrophils treated with NSAID. (A) PKA activity of intravascular and interstitial neutrophils before and after 7.5 mg/kg flurbiprofen axetil injection. 40 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. ***, P < 0.001 (Student’s t test). (B) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 7.5 mg/kg flurbiprofen axetil at time 0. Images are cropped from Video 6. A CFP image and a scheme are also shown. Cr, crypt; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. Bars, 30 µm. (C) Increase in the crawling step and decrease in the rolling step in the neutrophil recruitment cascade by flurbiprofen axetil treatment. The neutrophils on the endothelial cells were categorized into the four steps of the neutrophil recruitment cascade. The fraction of each step was calculated after summing up three time points each for before and after flurbiprofen axetil treatment. The experiment was repeated three times, and 537 and 503 neutrophils in total were scored before and after flurbiprofen axetil injection, respectively. Error bars indicate the SD. *, P < 0.05 (Student’s t test). (D–G) Acceleration of neutrophil crawling and transmigration by flurbiprofen axetil injection. Eisuke mice pretreated with LPS and fMLP were time-lapse imaged before and after intravenous administration of flurbiprofen axetil. The images were acquired every 1.5 s for the detailed analysis. The flux of rolling neutrophils (D) and the number of adherent (E), crawling (F), or transmigrating neutrophils (G) were quantified with time-lapse essentially as described previously (Kubes et al., 2003). A neutrophil was defined as adherent to endothelial cells if it remained stationary for >30 s. The numbers of adherent, crawling, or transmigrating neutrophils were averaged per 10-min observation period. Four mice were analyzed independently, and mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (H and I) The effect of flurbiprofen axetil on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Three independent experiments were performed to collect the data. Dots indicate the ERK activity (H) and migration velocity (I) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Student’s t test and Mann–Whitney U test are used for the ERK activity [H] and migration velocity [I], respectively).
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fig5: Accelerated migration and activation of ERK in neutrophils treated with NSAID. (A) PKA activity of intravascular and interstitial neutrophils before and after 7.5 mg/kg flurbiprofen axetil injection. 40 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. ***, P < 0.001 (Student’s t test). (B) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 7.5 mg/kg flurbiprofen axetil at time 0. Images are cropped from Video 6. A CFP image and a scheme are also shown. Cr, crypt; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. Bars, 30 µm. (C) Increase in the crawling step and decrease in the rolling step in the neutrophil recruitment cascade by flurbiprofen axetil treatment. The neutrophils on the endothelial cells were categorized into the four steps of the neutrophil recruitment cascade. The fraction of each step was calculated after summing up three time points each for before and after flurbiprofen axetil treatment. The experiment was repeated three times, and 537 and 503 neutrophils in total were scored before and after flurbiprofen axetil injection, respectively. Error bars indicate the SD. *, P < 0.05 (Student’s t test). (D–G) Acceleration of neutrophil crawling and transmigration by flurbiprofen axetil injection. Eisuke mice pretreated with LPS and fMLP were time-lapse imaged before and after intravenous administration of flurbiprofen axetil. The images were acquired every 1.5 s for the detailed analysis. The flux of rolling neutrophils (D) and the number of adherent (E), crawling (F), or transmigrating neutrophils (G) were quantified with time-lapse essentially as described previously (Kubes et al., 2003). A neutrophil was defined as adherent to endothelial cells if it remained stationary for >30 s. The numbers of adherent, crawling, or transmigrating neutrophils were averaged per 10-min observation period. Four mice were analyzed independently, and mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (H and I) The effect of flurbiprofen axetil on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Three independent experiments were performed to collect the data. Dots indicate the ERK activity (H) and migration velocity (I) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Student’s t test and Mann–Whitney U test are used for the ERK activity [H] and migration velocity [I], respectively).
Mentions: We next examined the effect of an NSAID, flurbiprofen axetil, on the activities of ERK and PKA in the inflamed intestine. NSAIDs generally inhibit production of PGE2 and thereby suppress Gs-coupled receptors such as EP2 and EP4. In agreement with a previous study showing the expression of EP2 and EP4 in neutrophils (Yamane et al., 2000), PKA activity was decreased in both intravascular and interstitial neutrophils within 10 min after intravenous flurbiprofen axetil injection (Fig. 5 A). In contrast, flurbiprofen axetil injection caused a gradual increase in the ERK activity of neutrophils (Fig. 5 B and Video 6). In the venules, the major fraction of neutrophils was shifted from the rolling step to the crawling step by flurbiprofen axetil (Fig. 5 C). Detailed analysis of neutrophils on the endothelial cells revealed that flurbiprofen axetil enhanced progression from the adhesion step to the crawling step (Fig. 5, D–G). Meanwhile, in the interstitial tissue, both the ERK activity and migration velocity of interstitial neutrophils were significantly increased within 20 min after flurbiprofen axetil injection (Fig. 5, H and I). We obtained a similar result with another NSAID, indomethacin, suggesting that the observed effects arose mostly through the inhibition of COXs (not depicted).

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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