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. 2022 Feb 8;7(3):e149870. doi: 10.1172/jci.insight.149870

Figure 1. Genetic deletion and pharmacological inhibition of Adam8 impair neutrophil motility in vivo.

Figure 1

(A) Schema for cremaster IVM after i.sc. TNF-α. (B) Number of cells adherent to endothelium, (C) rolling velocities, and (D) quantification of transmigrated Adam8−/− and Adam8+/+ neutrophils (right). Representative fields showing leukocyte extravasation (left); arrow indicates direction of emigration. Scale bar, 25 μm. Data for B–D are shown as mean ± SD. **, P < 0.005; Student’s t test. (E) Schema for ADAM8 inhibitor experiments after i.t. LPS. (F) BAL neutrophils and cytospins (left); scale bar, 25 μm. (G) BAL MPO ELISA. (H) Quantification of lung tissue and peripheral blood neutrophils. Data for F–H are mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. (I) Immunostaining of neutrophils (S100A8, green) in 100 μm lung sections. *, large airways. Scale bar, 2 mm. (J) Quantification and (K) representative images of intravascular (red) and total (green) neutrophils in lung sections at baseline and after i.t. LPS + BK1361 or CP. Ly6G antibody was injected i.v. 10 minutes prior to euthanasia to label intravascular neutrophils; total lung neutrophils were visualized by S100A8 staining (green) and nuclei by DAPI (blue). Neutrophils were quantified from 7 fields/animal (n = 5). Data in J are mean ± SD, 2-way ANOVA followed by Holm-Šídák multiple comparison test; LPS+CP vs. LPS+BK1361 (P = 0.0025); LPS+CP vs. PBS (P = 0.008); LPS+BK1361 vs. PBS (NS). Scale bar, 100 μm (K). (L) Two-photon lung IVM of MRP8-Cre mTmG mice + BK1361 or CP 3 hours after LPS challenge. Representative images of neutrophil influx (MRP8+, green). Arrowheads, neutrophil cluster. Scale bar, 50 μm. (M) Quantification of neutrophils circulating (track duration < 60 s, left graph) and migrating (track duration > 60 s, right graph) through the lung, n = 3. Blue, baseline; gray, LPS+BK1361; black, LPS+CP. **, P < 0.01; 2-way ANOVA. i.sc., intrascrotal injection.