Limits...
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.

Show MeSH

Related in: MedlinePlus

PKA inhibition of ERK activity, recruitment to endothelial cells, and migration of neutrophils. (A) FRET images of the lamina propria of the intestinal mucosa in PKAchu mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously at 0.3 g/kg at time 0. The bottom left panel shows the schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.7. The image is representative of a mouse in three independent experiments. (B) Time courses of the PKA activity of intravascular and interstitial neutrophils and endothelial cells. In each of three mice, 10 neutrophils in and out of the venules and 10 endothelial cells were randomly selected in the CFP images and examined for PKA activity. Mean data of three mice are shown with one SD. (C) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously (0.3 g/kg) at time 0. Images are cropped from Video 5. Gamma, 1.2. The image is representative of a mouse in three independent experiments. (A and C) Bars, 30 µm. (D) Inhibition of the neutrophil attachment to the endothelial cells by dbcAMP treatment. The number of neutrophils on the endothelial cells was counted in three mice, and the mean value and one SD of the average of each mouse are plotted against time. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (E and F) Correlation of ERK activity and migration velocity of interstitial neutrophils before and after dbcAMP treatment. 60 neutrophils that were imaged in three mice were randomly selected in the CFP images before and after dbcAMP treatment and examined for their ERK activity (E) and migration velocity (F) during 5 min of time-lapse imaging. Red bars indicate the mean values. ***, P < 0.001 (E, Student’s t test; F, Mann–Whitney U test).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4042632&req=5

fig4: PKA inhibition of ERK activity, recruitment to endothelial cells, and migration of neutrophils. (A) FRET images of the lamina propria of the intestinal mucosa in PKAchu mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously at 0.3 g/kg at time 0. The bottom left panel shows the schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.7. The image is representative of a mouse in three independent experiments. (B) Time courses of the PKA activity of intravascular and interstitial neutrophils and endothelial cells. In each of three mice, 10 neutrophils in and out of the venules and 10 endothelial cells were randomly selected in the CFP images and examined for PKA activity. Mean data of three mice are shown with one SD. (C) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously (0.3 g/kg) at time 0. Images are cropped from Video 5. Gamma, 1.2. The image is representative of a mouse in three independent experiments. (A and C) Bars, 30 µm. (D) Inhibition of the neutrophil attachment to the endothelial cells by dbcAMP treatment. The number of neutrophils on the endothelial cells was counted in three mice, and the mean value and one SD of the average of each mouse are plotted against time. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (E and F) Correlation of ERK activity and migration velocity of interstitial neutrophils before and after dbcAMP treatment. 60 neutrophils that were imaged in three mice were randomly selected in the CFP images before and after dbcAMP treatment and examined for their ERK activity (E) and migration velocity (F) during 5 min of time-lapse imaging. Red bars indicate the mean values. ***, P < 0.001 (E, Student’s t test; F, Mann–Whitney U test).

Mentions: The role of PKA in the neutrophil recruitment cascade was further examined by intravenous injection of a cAMP analogue, dibutyryl cAMP (dbcAMP). PKA was rapidly activated in almost all cell types in the lamina propria of the small intestine, including neutrophils, endothelial cells, and epithelial cells (Fig. 4, A and B). In clear contrast, ERK activity was decreased in neutrophils with a slightly slower time course (Fig. 4 C and Video 5). Concomitant with the decrease in ERK activity, the number of neutrophils attached to the endothelial cells was also significantly decreased (Fig. 4 D). In the interstitial tissues, dbcAMP treatment also decreased the ERK activity and migration velocity of neutrophils (Fig. 4, E and F), strongly suggesting that PKA regulates neutrophils via negative regulation of ERK.


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)

PKA inhibition of ERK activity, recruitment to endothelial cells, and migration of neutrophils. (A) FRET images of the lamina propria of the intestinal mucosa in PKAchu mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously at 0.3 g/kg at time 0. The bottom left panel shows the schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.7. The image is representative of a mouse in three independent experiments. (B) Time courses of the PKA activity of intravascular and interstitial neutrophils and endothelial cells. In each of three mice, 10 neutrophils in and out of the venules and 10 endothelial cells were randomly selected in the CFP images and examined for PKA activity. Mean data of three mice are shown with one SD. (C) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously (0.3 g/kg) at time 0. Images are cropped from Video 5. Gamma, 1.2. The image is representative of a mouse in three independent experiments. (A and C) Bars, 30 µm. (D) Inhibition of the neutrophil attachment to the endothelial cells by dbcAMP treatment. The number of neutrophils on the endothelial cells was counted in three mice, and the mean value and one SD of the average of each mouse are plotted against time. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (E and F) Correlation of ERK activity and migration velocity of interstitial neutrophils before and after dbcAMP treatment. 60 neutrophils that were imaged in three mice were randomly selected in the CFP images before and after dbcAMP treatment and examined for their ERK activity (E) and migration velocity (F) during 5 min of time-lapse imaging. Red bars indicate the mean values. ***, P < 0.001 (E, Student’s t test; F, Mann–Whitney U test).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042632&req=5

fig4: PKA inhibition of ERK activity, recruitment to endothelial cells, and migration of neutrophils. (A) FRET images of the lamina propria of the intestinal mucosa in PKAchu mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously at 0.3 g/kg at time 0. The bottom left panel shows the schematic view of this region. Cr, crypt; Ly, lymphatic vessel; Ve, venule. Gamma, 1.7. The image is representative of a mouse in three independent experiments. (B) Time courses of the PKA activity of intravascular and interstitial neutrophils and endothelial cells. In each of three mice, 10 neutrophils in and out of the venules and 10 endothelial cells were randomly selected in the CFP images and examined for PKA activity. Mean data of three mice are shown with one SD. (C) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP. A cAMP analogue, dbcAMP, was injected intravenously (0.3 g/kg) at time 0. Images are cropped from Video 5. Gamma, 1.2. The image is representative of a mouse in three independent experiments. (A and C) Bars, 30 µm. (D) Inhibition of the neutrophil attachment to the endothelial cells by dbcAMP treatment. The number of neutrophils on the endothelial cells was counted in three mice, and the mean value and one SD of the average of each mouse are plotted against time. Asterisks indicate the result of the paired Student’s t test between each time point and time 0: *, P < 0.05; **, P < 0.01. (E and F) Correlation of ERK activity and migration velocity of interstitial neutrophils before and after dbcAMP treatment. 60 neutrophils that were imaged in three mice were randomly selected in the CFP images before and after dbcAMP treatment and examined for their ERK activity (E) and migration velocity (F) during 5 min of time-lapse imaging. Red bars indicate the mean values. ***, P < 0.001 (E, Student’s t test; F, Mann–Whitney U test).
Mentions: The role of PKA in the neutrophil recruitment cascade was further examined by intravenous injection of a cAMP analogue, dibutyryl cAMP (dbcAMP). PKA was rapidly activated in almost all cell types in the lamina propria of the small intestine, including neutrophils, endothelial cells, and epithelial cells (Fig. 4, A and B). In clear contrast, ERK activity was decreased in neutrophils with a slightly slower time course (Fig. 4 C and Video 5). Concomitant with the decrease in ERK activity, the number of neutrophils attached to the endothelial cells was also significantly decreased (Fig. 4 D). In the interstitial tissues, dbcAMP treatment also decreased the ERK activity and migration velocity of neutrophils (Fig. 4, E and F), strongly suggesting that PKA regulates neutrophils via negative regulation of ERK.

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.

Show MeSH
Related in: MedlinePlus