Accelerated migration and activation of ERK in neutrophils treated with NSAID. (A) PKA activity of intravascular and interstitial neutrophils before and after 7.5 mg/kg flurbiprofen axetil injection. 40 neutrophils in and out of venules were randomly selected in the CFP images of three mice and examined for the PKA activity in the corresponding FRET/CFP ratio images. Red bars indicate mean values. ***, P < 0.001 (Student’s t test). (B) FRET images of the lamina propria of the intestinal mucosa in Eisuke mice pretreated with LPS and fMLP and injected with 7.5 mg/kg flurbiprofen axetil at time 0. Images are cropped from Video 6. A CFP image and a scheme are also shown. Cr, crypt; Ve, venule. Gamma, 1.3. The image is a representative view field of a mouse in three independent experiments. Bars, 30 µm. (C) Increase in the crawling step and decrease in the rolling step in the neutrophil recruitment cascade by flurbiprofen axetil treatment. The neutrophils on the endothelial cells were categorized into the four steps of the neutrophil recruitment cascade. The fraction of each step was calculated after summing up three time points each for before and after flurbiprofen axetil treatment. The experiment was repeated three times, and 537 and 503 neutrophils in total were scored before and after flurbiprofen axetil injection, respectively. Error bars indicate the SD. *, P < 0.05 (Student’s t test). (D–G) Acceleration of neutrophil crawling and transmigration by flurbiprofen axetil injection. Eisuke mice pretreated with LPS and fMLP were time-lapse imaged before and after intravenous administration of flurbiprofen axetil. The images were acquired every 1.5 s for the detailed analysis. The flux of rolling neutrophils (D) and the number of adherent (E), crawling (F), or transmigrating neutrophils (G) were quantified with time-lapse essentially as described previously (Kubes et al., 2003). A neutrophil was defined as adherent to endothelial cells if it remained stationary for >30 s. The numbers of adherent, crawling, or transmigrating neutrophils were averaged per 10-min observation period. Four mice were analyzed independently, and mean values are indicated in red. *, P < 0.05; **, P < 0.01 (paired Student’s t test). (H and I) The effect of flurbiprofen axetil on the ERK activity and migration velocity of interstitial neutrophils. 60 neutrophils were randomly selected in the CFP images at −20 and 20 min, and the migration velocity of each neutrophil was measured. Three independent experiments were performed to collect the data. Dots indicate the ERK activity (H) and migration velocity (I) in each neutrophil, and red bars indicate the mean values. ***, P < 0.001 (Student’s t test and Mann–Whitney U test are used for the ERK activity [H] and migration velocity [I], respectively).