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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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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.
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fig7: 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.

Mentions: Finally, we examined the role played by LTB4, one of the major chemoattractants in acute inflammation, on ERK activity and the neutrophil recruitment cascade. Neutrophils express the high-affinity receptor for LTB4, BLT1, which is coupled with the heterotrimeric Gi or Gq protein (Yokomizo et al., 1997). We could not observe any effect of either LTB4 or an LTB4 receptor antagonist, LY293111, on the PKA activity in neutrophils (not depicted), suggesting that the effect of BLT1-mediated PKA inhibition is weak in neutrophils. In contrast, upon intravenous injection of LTB4, we observed a surge in ERK activity followed by rapid suppression (Fig. 7, A and B). The ERK activity correlated positively with the migration velocity of the interstitial neutrophils (Fig. 7 C). Next, LTB4 signaling was abrogated by an LTB4 receptor antagonist, LY293111. Upon intravenous injection of LY293111, ERK activity was gradually decreased in the interstitial neutrophils (Fig. 7, D and E). LY293111 significantly decreased the fraction of neutrophils in the crawling step and thereafter (Fig. 7 F) and inhibited the migration velocity of the interstitial neutrophils (Fig. 7 G). These results suggest that LTB4 drives neutrophil emigration from the venules and migration in the interstitial tissues and that LTB4 was the canonical ERK activator of the neutrophils in the LPS-induced enteritis model. In conclusion, in vivo FRET imaging has revealed that Gi-coupled LTB4 activates ERK and transduces a “go” signal to neutrophils, whereas Gs-coupled EP4 activates PKA and thereby puts the brakes on neutrophil migration by suppressing ERK activity (Fig. 7 H).


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)

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.
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fig7: 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.
Mentions: Finally, we examined the role played by LTB4, one of the major chemoattractants in acute inflammation, on ERK activity and the neutrophil recruitment cascade. Neutrophils express the high-affinity receptor for LTB4, BLT1, which is coupled with the heterotrimeric Gi or Gq protein (Yokomizo et al., 1997). We could not observe any effect of either LTB4 or an LTB4 receptor antagonist, LY293111, on the PKA activity in neutrophils (not depicted), suggesting that the effect of BLT1-mediated PKA inhibition is weak in neutrophils. In contrast, upon intravenous injection of LTB4, we observed a surge in ERK activity followed by rapid suppression (Fig. 7, A and B). The ERK activity correlated positively with the migration velocity of the interstitial neutrophils (Fig. 7 C). Next, LTB4 signaling was abrogated by an LTB4 receptor antagonist, LY293111. Upon intravenous injection of LY293111, ERK activity was gradually decreased in the interstitial neutrophils (Fig. 7, D and E). LY293111 significantly decreased the fraction of neutrophils in the crawling step and thereafter (Fig. 7 F) and inhibited the migration velocity of the interstitial neutrophils (Fig. 7 G). These results suggest that LTB4 drives neutrophil emigration from the venules and migration in the interstitial tissues and that LTB4 was the canonical ERK activator of the neutrophils in the LPS-induced enteritis model. In conclusion, in vivo FRET imaging has revealed that Gi-coupled LTB4 activates ERK and transduces a “go” signal to neutrophils, whereas Gs-coupled EP4 activates PKA and thereby puts the brakes on neutrophil migration by suppressing ERK activity (Fig. 7 H).

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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