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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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Requirement of ERK activity for the neutrophil recruitment. (A) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice 2 h after LPS and fMLP treatment. At time 0, 5 mg/kg of the PD0325901 MEK inhibitor was injected intravenously. A CFP image and two FRET/CFP images are cropped from Video 2, with a schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. FRET/CFP images are prepared with the scale range shown at the bottom. Gamma, 1.7. The image is representative of a mouse in five independent experiments. Bar, 100 µm. (B) Time courses of the ERK activity of intravascular or interstitial neutrophils. In each of three mice, 10 neutrophils in and out of the venules were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. The mean and one SD of the average of each mouse are shown. (C) Neutrophils on the endothelial cells were classified into the four steps before and after PD0325901 treatment. We scored 152 and 147, 148 and 153, and 137 and 129 neutrophils before and after PD0325901 treatment in three mice. Error bars indicate the SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (D and E) Eisuke mice pretreated with LPS and fMLP were inoculated with the MEK inhibitor PD0325901 and imaged every 1.5 s. Rolling leukocyte flux (D) or number of adherent neutrophils (E) was quantified as described previously (Kubes et al., 2003). Three mice were analyzed independently, and the mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (F) The duration of neutrophil attachment to the endothelial cells was examined before and after PD0325901 treatment (n > 150 for each condition). Data were collected from four mice. Error 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. (G and H) Velocity of crawling over the endothelial cells (G) and the duration of transmigration (H) were measured for neutrophils that could be tracked during 30-min time-lapse imaging either before or after PD0325901 treatment. Data obtained from three mice were combined and plotted. Dots and bars indicate the crawling velocity (G) and duration of transmigration (H) of each neutrophil and the mean values, respectively. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (I) Migration velocity of interstitial neutrophils was measured before and after PD0325901 treatment. 50 neutrophils 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 < 0.001 (Mann–Whitney U test). (J–L) Inhibition of ERK activity by a p38 inhibitor, SB203580. Eisuke mice or PKAchu mice pretreated with LPS and fMLP were inoculated with 15 mg/kg SB203580. 60 neutrophils from three mice were randomly selected in the CFP images and examined for ERK activity (J), migration velocity during 5-min time-lapse imaging (K), and PKA activity (L). Dots and bars indicate the ERK activity (J), migration velocity (K), and PKA activity (L) of each neutrophil and the mean values, respectively. Data were acquired 10 min before and 20 min after SB203580 injection. ***, P < 0.001; n.s., not significant (J and L, Student’s t test; K, Mann–Whitney U test).
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fig2: Requirement of ERK activity for the neutrophil recruitment. (A) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice 2 h after LPS and fMLP treatment. At time 0, 5 mg/kg of the PD0325901 MEK inhibitor was injected intravenously. A CFP image and two FRET/CFP images are cropped from Video 2, with a schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. FRET/CFP images are prepared with the scale range shown at the bottom. Gamma, 1.7. The image is representative of a mouse in five independent experiments. Bar, 100 µm. (B) Time courses of the ERK activity of intravascular or interstitial neutrophils. In each of three mice, 10 neutrophils in and out of the venules were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. The mean and one SD of the average of each mouse are shown. (C) Neutrophils on the endothelial cells were classified into the four steps before and after PD0325901 treatment. We scored 152 and 147, 148 and 153, and 137 and 129 neutrophils before and after PD0325901 treatment in three mice. Error bars indicate the SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (D and E) Eisuke mice pretreated with LPS and fMLP were inoculated with the MEK inhibitor PD0325901 and imaged every 1.5 s. Rolling leukocyte flux (D) or number of adherent neutrophils (E) was quantified as described previously (Kubes et al., 2003). Three mice were analyzed independently, and the mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (F) The duration of neutrophil attachment to the endothelial cells was examined before and after PD0325901 treatment (n > 150 for each condition). Data were collected from four mice. Error 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. (G and H) Velocity of crawling over the endothelial cells (G) and the duration of transmigration (H) were measured for neutrophils that could be tracked during 30-min time-lapse imaging either before or after PD0325901 treatment. Data obtained from three mice were combined and plotted. Dots and bars indicate the crawling velocity (G) and duration of transmigration (H) of each neutrophil and the mean values, respectively. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (I) Migration velocity of interstitial neutrophils was measured before and after PD0325901 treatment. 50 neutrophils 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 < 0.001 (Mann–Whitney U test). (J–L) Inhibition of ERK activity by a p38 inhibitor, SB203580. Eisuke mice or PKAchu mice pretreated with LPS and fMLP were inoculated with 15 mg/kg SB203580. 60 neutrophils from three mice were randomly selected in the CFP images and examined for ERK activity (J), migration velocity during 5-min time-lapse imaging (K), and PKA activity (L). Dots and bars indicate the ERK activity (J), migration velocity (K), and PKA activity (L) of each neutrophil and the mean values, respectively. Data were acquired 10 min before and 20 min after SB203580 injection. ***, P < 0.001; n.s., not significant (J and L, Student’s t test; K, Mann–Whitney U test).

Mentions: The ERK activation during the adhesion of neutrophils prompted us to examine the effect of an MEK inhibitor, PD0325901, on the neutrophil recruitment cascade. PD0325901 was injected intravenously into Eisuke mice that had been subjected to LPS and fMLP injection. ERK activity in the interstitial neutrophils dropped within 20 min after PD0325901 administration to the level of the averaged ERK activity of neutrophils in the venules (Fig. 2, A and B; and Video 2). PD0325901 treatment did not significantly change the total number of neutrophils associated with the venules but decreased the number of neutrophils crawling over or transmigrating through the endothelial cells (Fig. 2 C). The proportion of neutrophils rolling over or adhering to the endothelial cells was increased accordingly. The effects on rolling and adhesion of neutrophils were further quantified as described previously (Fig. 2, D and E; Kubes et al., 2003). We found that PD0325901 profoundly increased the number of adherent neutrophils. These results agree with the finding that ERK activity increased before the initiation of crawling.


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)

Requirement of ERK activity for the neutrophil recruitment. (A) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice 2 h after LPS and fMLP treatment. At time 0, 5 mg/kg of the PD0325901 MEK inhibitor was injected intravenously. A CFP image and two FRET/CFP images are cropped from Video 2, with a schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. FRET/CFP images are prepared with the scale range shown at the bottom. Gamma, 1.7. The image is representative of a mouse in five independent experiments. Bar, 100 µm. (B) Time courses of the ERK activity of intravascular or interstitial neutrophils. In each of three mice, 10 neutrophils in and out of the venules were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. The mean and one SD of the average of each mouse are shown. (C) Neutrophils on the endothelial cells were classified into the four steps before and after PD0325901 treatment. We scored 152 and 147, 148 and 153, and 137 and 129 neutrophils before and after PD0325901 treatment in three mice. Error bars indicate the SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (D and E) Eisuke mice pretreated with LPS and fMLP were inoculated with the MEK inhibitor PD0325901 and imaged every 1.5 s. Rolling leukocyte flux (D) or number of adherent neutrophils (E) was quantified as described previously (Kubes et al., 2003). Three mice were analyzed independently, and the mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (F) The duration of neutrophil attachment to the endothelial cells was examined before and after PD0325901 treatment (n > 150 for each condition). Data were collected from four mice. Error 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. (G and H) Velocity of crawling over the endothelial cells (G) and the duration of transmigration (H) were measured for neutrophils that could be tracked during 30-min time-lapse imaging either before or after PD0325901 treatment. Data obtained from three mice were combined and plotted. Dots and bars indicate the crawling velocity (G) and duration of transmigration (H) of each neutrophil and the mean values, respectively. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (I) Migration velocity of interstitial neutrophils was measured before and after PD0325901 treatment. 50 neutrophils 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 < 0.001 (Mann–Whitney U test). (J–L) Inhibition of ERK activity by a p38 inhibitor, SB203580. Eisuke mice or PKAchu mice pretreated with LPS and fMLP were inoculated with 15 mg/kg SB203580. 60 neutrophils from three mice were randomly selected in the CFP images and examined for ERK activity (J), migration velocity during 5-min time-lapse imaging (K), and PKA activity (L). Dots and bars indicate the ERK activity (J), migration velocity (K), and PKA activity (L) of each neutrophil and the mean values, respectively. Data were acquired 10 min before and 20 min after SB203580 injection. ***, P < 0.001; n.s., not significant (J and L, Student’s t test; K, Mann–Whitney U test).
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fig2: Requirement of ERK activity for the neutrophil recruitment. (A) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice 2 h after LPS and fMLP treatment. At time 0, 5 mg/kg of the PD0325901 MEK inhibitor was injected intravenously. A CFP image and two FRET/CFP images are cropped from Video 2, with a schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. FRET/CFP images are prepared with the scale range shown at the bottom. Gamma, 1.7. The image is representative of a mouse in five independent experiments. Bar, 100 µm. (B) Time courses of the ERK activity of intravascular or interstitial neutrophils. In each of three mice, 10 neutrophils in and out of the venules were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. The mean and one SD of the average of each mouse are shown. (C) Neutrophils on the endothelial cells were classified into the four steps before and after PD0325901 treatment. We scored 152 and 147, 148 and 153, and 137 and 129 neutrophils before and after PD0325901 treatment in three mice. Error bars indicate the SD. *, P < 0.05; **, P < 0.01 (Student’s t test). (D and E) Eisuke mice pretreated with LPS and fMLP were inoculated with the MEK inhibitor PD0325901 and imaged every 1.5 s. Rolling leukocyte flux (D) or number of adherent neutrophils (E) was quantified as described previously (Kubes et al., 2003). Three mice were analyzed independently, and the mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (F) The duration of neutrophil attachment to the endothelial cells was examined before and after PD0325901 treatment (n > 150 for each condition). Data were collected from four mice. Error 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. (G and H) Velocity of crawling over the endothelial cells (G) and the duration of transmigration (H) were measured for neutrophils that could be tracked during 30-min time-lapse imaging either before or after PD0325901 treatment. Data obtained from three mice were combined and plotted. Dots and bars indicate the crawling velocity (G) and duration of transmigration (H) of each neutrophil and the mean values, respectively. **, P < 0.01; ***, P < 0.001 (Mann–Whitney U test). (I) Migration velocity of interstitial neutrophils was measured before and after PD0325901 treatment. 50 neutrophils 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 < 0.001 (Mann–Whitney U test). (J–L) Inhibition of ERK activity by a p38 inhibitor, SB203580. Eisuke mice or PKAchu mice pretreated with LPS and fMLP were inoculated with 15 mg/kg SB203580. 60 neutrophils from three mice were randomly selected in the CFP images and examined for ERK activity (J), migration velocity during 5-min time-lapse imaging (K), and PKA activity (L). Dots and bars indicate the ERK activity (J), migration velocity (K), and PKA activity (L) of each neutrophil and the mean values, respectively. Data were acquired 10 min before and 20 min after SB203580 injection. ***, P < 0.001; n.s., not significant (J and L, Student’s t test; K, Mann–Whitney U test).
Mentions: The ERK activation during the adhesion of neutrophils prompted us to examine the effect of an MEK inhibitor, PD0325901, on the neutrophil recruitment cascade. PD0325901 was injected intravenously into Eisuke mice that had been subjected to LPS and fMLP injection. ERK activity in the interstitial neutrophils dropped within 20 min after PD0325901 administration to the level of the averaged ERK activity of neutrophils in the venules (Fig. 2, A and B; and Video 2). PD0325901 treatment did not significantly change the total number of neutrophils associated with the venules but decreased the number of neutrophils crawling over or transmigrating through the endothelial cells (Fig. 2 C). The proportion of neutrophils rolling over or adhering to the endothelial cells was increased accordingly. The effects on rolling and adhesion of neutrophils were further quantified as described previously (Fig. 2, D and E; Kubes et al., 2003). We found that PD0325901 profoundly increased the number of adherent neutrophils. These results agree with the finding that ERK activity increased before the initiation of crawling.

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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