PMNs require LTB4–BLT1 axis for sustained polarization, ITGB2 trafficking, and adhesion response in vitro. (A) PMNs were pretreated for 20 min with vehicle or 2 µM MK886 or 20 µM LY223982 and stimulated with 25 nM fNLFNYK on a fibrinogen-coated surface. Cells remained either unstimulated (30 min) or were stimulated for 30, 15, and 5 min independently before fixation. Adhesion was calculated as described in the Materials and methods section and is presented as fold change of adhered neutrophils compared with unstimulated controls. Data are plotted as mean ± SEM from n = 3 independent experiments. Two-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance. (B–D) PMNs were pretreated with vehicle or 2 µM MK886 or 20 µM LY223982 for 20 min, stimulated with 25 nM fNLFNYK for 15 min, fixed, and stained with rhodamine phalloidin (magenta) and anti-NMIIA (green). Representative confocal images for vehicle and LY223982 treated PMNs (B). Blue and orange arrowheads indicate the front and back of PMNs with polarized actomyosin distribution, respectively. Dashed white box indicates the region zoomed and presented on the right side for each condition, with individual channels in grayscale. Scale bars = 5 µm. Quantification of the percentage of neutrophils exhibiting cortical NMIIA (C) and polarized F-actin (D) in response to the above-mentioned treatments. Data are plotted as mean ± SEM from n = 3 independent experiments. One-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance. (E and F) PMNs were pretreated with either vehicle (E) or 20 µM LY223982 (F) for 20 min. They were incubated with an Alexa Fluor 488–conjugated antibody against human ITGB2 (αITGB2, CTB104 clone; white) in combination with CellMask Deep Red (magenta) for ∼1 min before stimulation with 100 nM fNLFNYK for 10 min on a fibrinogen-coated surface and acquired in 3D using time-lapse confocal microscopy. See Video 7. Left panel represents the 3D view of the cell as a function of time (min:s). Middle and right panels represent the middle and bottom slices, respectively, as indicated by the dashed lines in the 3D view. White arrows indicate the formation of ITGB2 ringlike structure. Blue arrowheads indicate ITGB2 vesicles. Images are representative of three independent experiments (see Fig. S2, G–I). (G) PMNs were labeled with CellMask Deep Red to visualize the PM (magenta) and with αITGB2 (green), pretreated with either the vehicle or 2 µM MK886 for 20 min, and stimulated with 25 nM fNLFNYK on a fibrinogen-coated surface. Neutrophils were fixed and either left untreated (− quenching; i and iii) or treated with an anti–Alexa Fluor 488 antibody (+ quenching; ii and iv). Images represent the maximum-intensity projections in x–y (large panels on the left) dimension for each condition. The cells in dashed white boxes are represented in the x–z dimension in small panels on the right. Blue and orange arrowheads indicate surface and internalized αITGB2 signals, respectively. Scale bars = 5 µm. Images are representative of n = 3 independent experiments. (H and I) Internalization and recycling of ITGB2. PMNs were treated with vehicle or 2 µM MK886 or 20 µM LY223982 or 20 µM Y27632 for 20 min and stimulated with 25 nM fNLFNYK for 10 min on a fibrinogen-coated surface in the presence of αITGB2 (H). Samples were processed and imaged as described in the Materials and methods section to determine the percentage of the internalized ITGB2. Data are plotted as mean ± SEM from n = 4 independent experiments. PMNs were treated with the vehicle or 2 µM MK886 or 20 µM LY223982 or 20 µM Y27632 for 20 min and stimulated with 25 nM fNLFNYK for 15 min on a fibrinogen-coated surface in the presence of αITGB2 (I). Samples were washed and either fixed or stimulated for an additional 45 min in the presence of fNLFNYK and inhibitors before fixation. Samples were processed and imaged as described in the Materials and methods section to determine the extent of recycling of internalized Itgb2. Data are plotted as mean ± SEM from n = 3 independent experiments. One-way ANOVA using Dunnett’s multiple comparisons test was performed to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
