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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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Activation of PKA during the neutrophil recruitment cascade. (A) In vivo imaging of the lamina propria of the intestinal mucosa of PKAchu mice. A representative FRET/CFP ratio image from Video 3 and a scheme are shown. Cr, crypt; Ve, venule. Gamma, 1.14. The image is representative of a mouse in five independent experiments. (B) In vivo imaging of the lamina propria of the intestinal mucosa of a C57BL/6 mouse that was transplanted with bone marrow of PKAchu mice 7 wk before the experiment. Representative FRET/CFP ratio images and CFP/Qtracker 655 images from Video 4 and schemes during neutrophil extravasation are shown. Tracking of a neutrophil is shown by arrowheads. The same cells were also marked by yellow lines. Gamma, 1.44. The image is representative of a mouse in three independent experiments. Bars: (A) 30 µm; (B) 10 µm. (C) The PKA activity change was monitored in three representative neutrophils from three independently analyzed mice. The time of transition from the adhesion to crawling steps was set as zero. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (D) PKA activity of neutrophils in the four steps of extravasation is plotted. 30 neutrophils in each step were randomly chosen in the CFP images and examined for their PKA activity in the corresponding FRET/CFP ratio image. To accumulate the incidence, three independent experiments were performed. Dots and bars indicate the PKA activity in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (E) 90 neutrophils migrating in the interstitial tissue were randomly chosen in the CFP images and examined for their PKA activity and migration velocity during 5 min of time-lapse imaging. Results obtained from three mice were combined. The red line is an approximate curve showing the inverse correlation between PKA activity and migration velocity.
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fig3: Activation of PKA during the neutrophil recruitment cascade. (A) In vivo imaging of the lamina propria of the intestinal mucosa of PKAchu mice. A representative FRET/CFP ratio image from Video 3 and a scheme are shown. Cr, crypt; Ve, venule. Gamma, 1.14. The image is representative of a mouse in five independent experiments. (B) In vivo imaging of the lamina propria of the intestinal mucosa of a C57BL/6 mouse that was transplanted with bone marrow of PKAchu mice 7 wk before the experiment. Representative FRET/CFP ratio images and CFP/Qtracker 655 images from Video 4 and schemes during neutrophil extravasation are shown. Tracking of a neutrophil is shown by arrowheads. The same cells were also marked by yellow lines. Gamma, 1.44. The image is representative of a mouse in three independent experiments. Bars: (A) 30 µm; (B) 10 µm. (C) The PKA activity change was monitored in three representative neutrophils from three independently analyzed mice. The time of transition from the adhesion to crawling steps was set as zero. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (D) PKA activity of neutrophils in the four steps of extravasation is plotted. 30 neutrophils in each step were randomly chosen in the CFP images and examined for their PKA activity in the corresponding FRET/CFP ratio image. To accumulate the incidence, three independent experiments were performed. Dots and bars indicate the PKA activity in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (E) 90 neutrophils migrating in the interstitial tissue were randomly chosen in the CFP images and examined for their PKA activity and migration velocity during 5 min of time-lapse imaging. Results obtained from three mice were combined. The red line is an approximate curve showing the inverse correlation between PKA activity and migration velocity.

Mentions: Neutrophils at the inflammatory sites perceive signals from chemokines and prostaglandins, which transduce signals primarily via Gi- and Gs-coupled receptors, respectively. To examine which of the Gi- or Gs-mediated signaling cascades is dominant in the neutrophils during the neutrophil recruitment cascade, we examined PKA activity in the small intestines of PKAchu mice, which expressed a cytoplasmic FRET biosensor for PKA, AKAR3EV (Fig. 3 A and Video 3). Unlike the neutrophils of Eisuke mice, the expression of the FRET biosensor for PKA was relatively low in PKAchu mice. Therefore, in some experiments, C57BL/6 mice were transplanted with bone marrow of PKAchu mice 7 wk before analysis. In this way, we could eliminate the fluorescence from endothelial cells and mesenchymal cells and confirm the observations obtained with PKAchu mice (Fig. 3 B and Video 4). The PKA activity of neutrophils rolling on the endothelial cells was lower than that of the neutrophils that were adhering to, crawling over, or transmigrating through the endothelial cells (Fig. 3, C and D). In contrast to ERK activity, which showed a rapid increase before entering into the crawling step, PKA activity increased gradually during the neutrophil recruitment cascade (Fig. 3 C). After emigration to the interstitial tissue, the PKA activity in neutrophils varied cell by cell. Notably, the PKA activity was inversely correlated with the migration velocity of each neutrophil in the interstitial tissue (Fig. 3 E). These data suggested that PKA activity negatively regulated migration in the interstitial tissue.


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)

Activation of PKA during the neutrophil recruitment cascade. (A) In vivo imaging of the lamina propria of the intestinal mucosa of PKAchu mice. A representative FRET/CFP ratio image from Video 3 and a scheme are shown. Cr, crypt; Ve, venule. Gamma, 1.14. The image is representative of a mouse in five independent experiments. (B) In vivo imaging of the lamina propria of the intestinal mucosa of a C57BL/6 mouse that was transplanted with bone marrow of PKAchu mice 7 wk before the experiment. Representative FRET/CFP ratio images and CFP/Qtracker 655 images from Video 4 and schemes during neutrophil extravasation are shown. Tracking of a neutrophil is shown by arrowheads. The same cells were also marked by yellow lines. Gamma, 1.44. The image is representative of a mouse in three independent experiments. Bars: (A) 30 µm; (B) 10 µm. (C) The PKA activity change was monitored in three representative neutrophils from three independently analyzed mice. The time of transition from the adhesion to crawling steps was set as zero. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (D) PKA activity of neutrophils in the four steps of extravasation is plotted. 30 neutrophils in each step were randomly chosen in the CFP images and examined for their PKA activity in the corresponding FRET/CFP ratio image. To accumulate the incidence, three independent experiments were performed. Dots and bars indicate the PKA activity in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (E) 90 neutrophils migrating in the interstitial tissue were randomly chosen in the CFP images and examined for their PKA activity and migration velocity during 5 min of time-lapse imaging. Results obtained from three mice were combined. The red line is an approximate curve showing the inverse correlation between PKA activity and migration velocity.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: Activation of PKA during the neutrophil recruitment cascade. (A) In vivo imaging of the lamina propria of the intestinal mucosa of PKAchu mice. A representative FRET/CFP ratio image from Video 3 and a scheme are shown. Cr, crypt; Ve, venule. Gamma, 1.14. The image is representative of a mouse in five independent experiments. (B) In vivo imaging of the lamina propria of the intestinal mucosa of a C57BL/6 mouse that was transplanted with bone marrow of PKAchu mice 7 wk before the experiment. Representative FRET/CFP ratio images and CFP/Qtracker 655 images from Video 4 and schemes during neutrophil extravasation are shown. Tracking of a neutrophil is shown by arrowheads. The same cells were also marked by yellow lines. Gamma, 1.44. The image is representative of a mouse in three independent experiments. Bars: (A) 30 µm; (B) 10 µm. (C) The PKA activity change was monitored in three representative neutrophils from three independently analyzed mice. The time of transition from the adhesion to crawling steps was set as zero. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (D) PKA activity of neutrophils in the four steps of extravasation is plotted. 30 neutrophils in each step were randomly chosen in the CFP images and examined for their PKA activity in the corresponding FRET/CFP ratio image. To accumulate the incidence, three independent experiments were performed. Dots and bars indicate the PKA activity in each neutrophil and the mean values, respectively. ***, P < 0.001 (Student’s t test). (E) 90 neutrophils migrating in the interstitial tissue were randomly chosen in the CFP images and examined for their PKA activity and migration velocity during 5 min of time-lapse imaging. Results obtained from three mice were combined. The red line is an approximate curve showing the inverse correlation between PKA activity and migration velocity.
Mentions: Neutrophils at the inflammatory sites perceive signals from chemokines and prostaglandins, which transduce signals primarily via Gi- and Gs-coupled receptors, respectively. To examine which of the Gi- or Gs-mediated signaling cascades is dominant in the neutrophils during the neutrophil recruitment cascade, we examined PKA activity in the small intestines of PKAchu mice, which expressed a cytoplasmic FRET biosensor for PKA, AKAR3EV (Fig. 3 A and Video 3). Unlike the neutrophils of Eisuke mice, the expression of the FRET biosensor for PKA was relatively low in PKAchu mice. Therefore, in some experiments, C57BL/6 mice were transplanted with bone marrow of PKAchu mice 7 wk before analysis. In this way, we could eliminate the fluorescence from endothelial cells and mesenchymal cells and confirm the observations obtained with PKAchu mice (Fig. 3 B and Video 4). The PKA activity of neutrophils rolling on the endothelial cells was lower than that of the neutrophils that were adhering to, crawling over, or transmigrating through the endothelial cells (Fig. 3, C and D). In contrast to ERK activity, which showed a rapid increase before entering into the crawling step, PKA activity increased gradually during the neutrophil recruitment cascade (Fig. 3 C). After emigration to the interstitial tissue, the PKA activity in neutrophils varied cell by cell. Notably, the PKA activity was inversely correlated with the migration velocity of each neutrophil in the interstitial tissue (Fig. 3 E). These data suggested that PKA activity negatively regulated migration in the interstitial tissue.

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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Related in: MedlinePlus