Figure 5. Macrophages recruited to the pleura post–Ang-(1-7) injection present a regulatory phenotype.

Briefly, BALB/c mice received an i.pl. injection of Ang-(1-7) (100 ng/cavity) or PBS (controls), and the macrophages recruited to the cavity were harvested at 48 hours for phenotyping by flow cytometry as shown in gating strategy (A). (B) Graphs present the absolute numbers of M1 (F4/80^loLy6C⁺CD11b^med), M2 (F4/80^hiLy6C^–CD11b^hi), and Mres (F4/80^medCD11b^lo) recruited into the pleura. TGF-β levels were assessed in the pleural lavage supernatant from Ang-(1-7)–injected mice at different time points postinjection (C). Leukocytes recruited into the pleural cavity were processed for Western blot analysis of Arg-1 and Ym1 levels (D). β-Actin was used as a loading control. During in vitro settings, the kinetics of production of IL-10 and TGF-β by BMDMs were evaluated (E). Data are presented as mean ± SEM of 8 mice per group (in vivo) or are representative results of 3 independent experiments with BMDMs performed in biological quadruplicates (n = 4). Western blot quantification was performed using ImageJ software from the representative blots shown in D, which used whole cell extracts from 3 mice. * for P < 0.05 and *** for P < 0.001 when compared with the control group (PBS) by t test (B) or 1-way ANOVA (C and E).