LTB4–BLT1 axis regulates Itgb2 dynamics during neutrophil extravasation in vivo. (A and B) Neutrophils purified from Itgb2−/− mice were labeled with CellTracker Green CMFDA, adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by 2P-IVM. Maximum-intensity projection of a Z-stack (A). Dashed tangerine lines indicate vessel boundaries. Scale bar = 50 µm. Quantitative analysis of the percentage of neutrophils that exhibit rolling, arrest, and extravasation response in Itgb2−/− neutrophils compared with WT counterpart (B). Values for WT neutrophils are the same as in Fig. 1 E–G. Data are plotted as mean ± SEM, and each dot represents the result from an individual animal. An unpaired t test with Welch’s correction was used to determine statistical significance. (C) Neutrophils purified from either WT or Itgb2−/− mice were labeled with CellTracker Green CMFDA and a fluorescently conjugated antibody against mouse Itgb2 (M18/2 clone, red), adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by ISMic (top panel). Maximum-intensity projections of Z-stacks are presented (bottom panels). Magenta arrowheads indicate neutrophils positive for Itgb2 staining. The results represent n = 3 mice per condition. Scale bars = 3 µm. (D–F) Neutrophils purified from either WT or Alox5−/− mice were labeled with CellTracker Green CMPTX and fluorescently conjugated M18/2 antibody (red), adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by ISMic. Additionally, WT neutrophils were pretreated with either vehicle (DMSO; D and F) or 40 µM Y27632 (F) or 10 µM nBleb (F) and transferred into the recipient. Still images represent maximum-intensity projections in x–y (large panels) and z–y (side panels) dimensions, derived from time-lapse sequences in Video 6 (min:s) for WT neutrophils (D) and Alox5−/− neutrophils (E). CellTracker is shown in magenta, blood vessels in blue, and anti-Itgb2 in white. Orange and green arrowheads indicate the back and protrusive front of arrested and extravasating neutrophils, respectively. Scale bars = 5 µm. The Itgb2 redistribution index was measured as described in the Materials and methods section and is presented as individual points for individual cells of WT (DMSO treated; 70 cells), WT + Y27632 (47 cells), WT + nBleb (54 cells), and Alox5−/− (59 cells) from n = 3 mice per condition (F). One-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
