Inhibitors of EV pathway impair neutrophil adhesion response in vitro. (A–C) A diagram of the 2P-IVM procedure in Alox5−/− mouse with inflamed footpad, where neutrophils from WT (green) were treated as indicated in Fig. 5 A for 30 min before they were coinjected with untreated Alox5−/− neutrophils into the recipient (A). An illustration of the possible autocrine/paracrine LTB4 signaling that occurs between WT and Alox5−/− neutrophils during extravasation response when coinjected into the recipient mice with inflamed footpads and how this mechanism can be blocked using the LTB4 inhibitor MK886 (B). An illustration of the possible EV-based autocrine/paracrine LTB4 signaling in the conditions mentioned above that occurs in the inflamed vasculature and how this mechanism can be blocked using inhibitors of N-SMase and Rab27 (C). See Fig. 5, A and B, as well as Videos 8 and 9. (D–G) PMNs were pretreated for 20 min with vehicle (DMSO) or 10 µM GW4869 or 1 µM Nexinhib20 or 10 µM Exo1 and stimulated in the absence or presence of αITGB2 for 15 min with 25 nM fNLFNYK, fixed, stained, and imaged by confocal microscopy. Quantification of the percentage of cells with cortical NMIIA distribution (D) and polarized F-actin (E) is presented. The extent of internalized ITGB2 (F) and cell adhesion (G) observed under the above-mentioned conditions was measured and represented as fold change and percentage with respect to vehicle control, respectively. 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 < 0.0001.
