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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).

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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Activation of ERK in neutrophils during adhesion to endothelial cells of the inflamed small intestine. (A) In vivo imaging of the lamina propria of the intestinal mucosa from an Eisuke mouse, which was subjected to LPS and fMLP 2 h before imaging. The representative FRET/CFP ratio image is shown in intensity-modulated display mode with 32-intensity in 8-ratio and a CFP image in grayscale from Video 1, with a schematic view of this region. Cr, crypt; Ve, venule. Gamma, 1.7. The image is representative of a mouse in five independent experiments. (B) ERK activities in three representative neutrophils from three independent experiments are plotted against time. The transition point from the adhesion to crawling steps was set as time 0. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (C) ERK activities from three representative neutrophils were recorded with a shorter interval than in B, at one image every 1.5 s, to discriminate arrest and spreading phases of the adhesion step. Neutrophils were defined as arrest when they stopped rolling for >30 s. The start of spreading was defined when neutrophils changed from a round to an amoeboid shape. (D) Schematic of the four steps of extravasation. Arrowheads indicate the same neutrophil at different time points. (E) Time-lapse FRET/CFP and CFP images of neutrophil extravasation. The boxed region in A was magnified and shown in a time series. The arrowheads indicate the same neutrophil traced in the video. Bars: (A) 100 µm; (E) 10 µm. (F) ERK activity of neutrophils during neutrophil recruitment to the inflamed tissue. 30 neutrophils in each step were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each neutrophil and the mean values, respectively. Difference from the rolling cells was evaluated by the Student’s t test: **, P < 0.01; ***, P < 0.001.
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fig1: Activation of ERK in neutrophils during adhesion to endothelial cells of the inflamed small intestine. (A) In vivo imaging of the lamina propria of the intestinal mucosa from an Eisuke mouse, which was subjected to LPS and fMLP 2 h before imaging. The representative FRET/CFP ratio image is shown in intensity-modulated display mode with 32-intensity in 8-ratio and a CFP image in grayscale from Video 1, with a schematic view of this region. Cr, crypt; Ve, venule. Gamma, 1.7. The image is representative of a mouse in five independent experiments. (B) ERK activities in three representative neutrophils from three independent experiments are plotted against time. The transition point from the adhesion to crawling steps was set as time 0. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (C) ERK activities from three representative neutrophils were recorded with a shorter interval than in B, at one image every 1.5 s, to discriminate arrest and spreading phases of the adhesion step. Neutrophils were defined as arrest when they stopped rolling for >30 s. The start of spreading was defined when neutrophils changed from a round to an amoeboid shape. (D) Schematic of the four steps of extravasation. Arrowheads indicate the same neutrophil at different time points. (E) Time-lapse FRET/CFP and CFP images of neutrophil extravasation. The boxed region in A was magnified and shown in a time series. The arrowheads indicate the same neutrophil traced in the video. Bars: (A) 100 µm; (E) 10 µm. (F) ERK activity of neutrophils during neutrophil recruitment to the inflamed tissue. 30 neutrophils in each step were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each neutrophil and the mean values, respectively. Difference from the rolling cells was evaluated by the Student’s t test: **, P < 0.01; ***, P < 0.001.

Mentions: We attempted to visualize ERK activity during the neutrophil recruitment cascade in the small intestines of mice after treating the intestinal tissue with LPS and fMLP to induce acute inflammation. For this purpose, we used Eisuke mice (C57BL/6-Tg(pT2A-3903NES)), which are transgenic mice expressing a cytoplasmic FRET biosensor for ERK and respond to LPS very similarly to C57BL/6 mice (not depicted). In Eisuke mice, the ERK activity can be represented by FRET/CFP ratio images (Fig. 1 A). Postcapillary venules in the lamina propria of the inflamed small intestine were live-imaged under an inverted two-photon excitation microscope. Neutrophils in the inflamed tissue could be easily distinguished by the segmented nuclei from lymphocytes or macrophages. Indeed, the cells with segmented nuclei could be stained with a neutrophil marker, anti-Gr1 antibody (not depicted). During the course of 2–5 h of live imaging (Video 1), neutrophils were observed to roll on, adhere to, crawl over, and transmigrate through the endothelial cells and then to migrate between the crypts (Fig. 1, A–E; and Video 1). Time-lapse videos with a shorter interval captured the precise timing of ERK activation during the neutrophil recruitment cascade (Fig. 1 C). At the beginning of the adhesion step when neutrophils arrested on the endothelial cells, ERK activity remained low and then increased rapidly when neutrophils spread over the endothelial cells. The high ERK activity was maintained during and after crawling, transmigration, and random migration between crypts. Fig. 1 E shows the activity change of ERK in a representative neutrophil, which incidentally migrated in the same XY plane, thereby allowing us to simultaneously trace the movement along with the activity change. To quantify these observations, neutrophils in each step were randomly selected in the CFP images and then analyzed for their ERK activity in the FRET/CFP ratio image. Clearly, the ERK activity was increased between the adhesion and crawling steps (Fig. 1 F).


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 ERK in neutrophils during adhesion to endothelial cells of the inflamed small intestine. (A) In vivo imaging of the lamina propria of the intestinal mucosa from an Eisuke mouse, which was subjected to LPS and fMLP 2 h before imaging. The representative FRET/CFP ratio image is shown in intensity-modulated display mode with 32-intensity in 8-ratio and a CFP image in grayscale from Video 1, with a schematic view of this region. Cr, crypt; Ve, venule. Gamma, 1.7. The image is representative of a mouse in five independent experiments. (B) ERK activities in three representative neutrophils from three independent experiments are plotted against time. The transition point from the adhesion to crawling steps was set as time 0. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (C) ERK activities from three representative neutrophils were recorded with a shorter interval than in B, at one image every 1.5 s, to discriminate arrest and spreading phases of the adhesion step. Neutrophils were defined as arrest when they stopped rolling for >30 s. The start of spreading was defined when neutrophils changed from a round to an amoeboid shape. (D) Schematic of the four steps of extravasation. Arrowheads indicate the same neutrophil at different time points. (E) Time-lapse FRET/CFP and CFP images of neutrophil extravasation. The boxed region in A was magnified and shown in a time series. The arrowheads indicate the same neutrophil traced in the video. Bars: (A) 100 µm; (E) 10 µm. (F) ERK activity of neutrophils during neutrophil recruitment to the inflamed tissue. 30 neutrophils in each step were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each neutrophil and the mean values, respectively. Difference from the rolling cells was evaluated by the Student’s t test: **, P < 0.01; ***, P < 0.001.
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fig1: Activation of ERK in neutrophils during adhesion to endothelial cells of the inflamed small intestine. (A) In vivo imaging of the lamina propria of the intestinal mucosa from an Eisuke mouse, which was subjected to LPS and fMLP 2 h before imaging. The representative FRET/CFP ratio image is shown in intensity-modulated display mode with 32-intensity in 8-ratio and a CFP image in grayscale from Video 1, with a schematic view of this region. Cr, crypt; Ve, venule. Gamma, 1.7. The image is representative of a mouse in five independent experiments. (B) ERK activities in three representative neutrophils from three independent experiments are plotted against time. The transition point from the adhesion to crawling steps was set as time 0. The rolling, adhesion, crawling, and transmigration steps are indicated by different colors. (C) ERK activities from three representative neutrophils were recorded with a shorter interval than in B, at one image every 1.5 s, to discriminate arrest and spreading phases of the adhesion step. Neutrophils were defined as arrest when they stopped rolling for >30 s. The start of spreading was defined when neutrophils changed from a round to an amoeboid shape. (D) Schematic of the four steps of extravasation. Arrowheads indicate the same neutrophil at different time points. (E) Time-lapse FRET/CFP and CFP images of neutrophil extravasation. The boxed region in A was magnified and shown in a time series. The arrowheads indicate the same neutrophil traced in the video. Bars: (A) 100 µm; (E) 10 µm. (F) ERK activity of neutrophils during neutrophil recruitment to the inflamed tissue. 30 neutrophils in each step were randomly selected in the CFP images and examined for their ERK activity in the corresponding FRET/CFP ratio image. Results obtained from three mice are combined. Black dots and red bars indicate the ERK activity in each neutrophil and the mean values, respectively. Difference from the rolling cells was evaluated by the Student’s t test: **, P < 0.01; ***, P < 0.001.
Mentions: We attempted to visualize ERK activity during the neutrophil recruitment cascade in the small intestines of mice after treating the intestinal tissue with LPS and fMLP to induce acute inflammation. For this purpose, we used Eisuke mice (C57BL/6-Tg(pT2A-3903NES)), which are transgenic mice expressing a cytoplasmic FRET biosensor for ERK and respond to LPS very similarly to C57BL/6 mice (not depicted). In Eisuke mice, the ERK activity can be represented by FRET/CFP ratio images (Fig. 1 A). Postcapillary venules in the lamina propria of the inflamed small intestine were live-imaged under an inverted two-photon excitation microscope. Neutrophils in the inflamed tissue could be easily distinguished by the segmented nuclei from lymphocytes or macrophages. Indeed, the cells with segmented nuclei could be stained with a neutrophil marker, anti-Gr1 antibody (not depicted). During the course of 2–5 h of live imaging (Video 1), neutrophils were observed to roll on, adhere to, crawl over, and transmigrate through the endothelial cells and then to migrate between the crypts (Fig. 1, A–E; and Video 1). Time-lapse videos with a shorter interval captured the precise timing of ERK activation during the neutrophil recruitment cascade (Fig. 1 C). At the beginning of the adhesion step when neutrophils arrested on the endothelial cells, ERK activity remained low and then increased rapidly when neutrophils spread over the endothelial cells. The high ERK activity was maintained during and after crawling, transmigration, and random migration between crypts. Fig. 1 E shows the activity change of ERK in a representative neutrophil, which incidentally migrated in the same XY plane, thereby allowing us to simultaneously trace the movement along with the activity change. To quantify these observations, neutrophils in each step were randomly selected in the CFP images and then analyzed for their ERK activity in the FRET/CFP ratio image. Clearly, the ERK activity was increased between the adhesion and crawling steps (Fig. 1 F).

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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