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. 2014 Jun 2;211(6):1123–1136. doi: 10.1084/jem.20132112

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© 2014 Mizuno et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — 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).