Involvement of AnxA1 in Plg/Pla-induced apoptosis and efferocytosis. BALB/c mice were challenged with an i.pl. injection of Pla (2 µg per cavity) or PBS (vehicle). The cells that migrated to the pleural cavity were collected 3, 6, 24, and 48 hours after challenge and analyzed for AnxA1 mRNA expression by qRT-PCR (A), protein expression by western blot (B), and cell surface externalization by flow cytometry (C-F). Pleural cells obtained 48 hours after injection of Plg/Pla were surface stained and gated on F4/80-positive cells, and then analyzed for AnxA1. Data report the percentage (C) and the absolute number (D) of cells positive for AnxA1. Pleural cells were gated as in Figure 1, and then M2 (E) and Mres (F) subpopulations of macrophages were analyzed for AnxA1. Data are mean ± SEM. *P < .05; **P < .01; ***P < .001 when compared with mice injected with PBS. Neutrophils isolated from peripheral blood of healthy human donors were cultured in 96-well cell culture plates (10⁶ cells per well) with or without anti-AnxA1 antiserum (8 µg of hyperimmune serum per well) for 1 hour, and then treated with LPS (500 ng/ml) or LPS plus Plg (2 µg/ml) for an additional 5 hours. Cytospin slides of neutrophils were counted for apoptosis. **P < .01 when compared with untreated (UT) neutrophils; ^##P < .01 when compared with the LPS-treated group. The experiments were performed twice with different donors in biological triplicates (G). WT and AnxA1 KO mice received an i.p. injection of zymosan, and were injected i.p. 62 hours later with Plg (2 µg per cavity) or vehicle. Seven hours after treatment, mice were injected i.p. with 10⁶ apoptotic neutrophils. Mice were killed 3 hours after injecting prey neutrophils (see the schematic representation of experimental protocol in Figure 5). Efferocytosis was assessed on cytospin preparations of cells harvested from peritoneal lavage. Results are presented as the mean efferocytosis index ± SEM of 5 mice. **P < .01 comparing Plg-treated WT mice vs Plg-treated Anx1 KO mice (H). qRT-PCR results are presented as the fold increase of mRNA expression relative to the amount present in control samples. Analyses of gene expression were performed with 2 replicates with samples of all groups run on 1 plate. Blots were normalized with β-actin and are representative of 3 independent experiments using pooled cells from at least 5 animals. All experiments were performed at least 3 times with similar results.