Angiotensin-(1-7) Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

Figure 1. Angiotensin-(1-7) [Ang-(1-7)] promotes resolution of the inflammatory response by inducing neutrophils apoptosis in a model of antigen-induced arthritis (AIA) in mice. (A) Experimental protocol: on day 0, mice were immunized by injecting 10 µg of mBSA into each knee joint. Fourteen days later, arthritis was induced by an additional injection of 100 µg of mBSA into each joint (Challenge). At the peak of inflammation (12 h post-mBSA challenge, arrow), 100 ng of Ang-(1-7) was injected in mice knee joints. Some animals also received 1 mg/kg of the caspase inhibitor, zVAD-FMK 10 min before Ang-(1-7) treatment. Control mice were injected with 10 µL of PBS. At 24 h post-challenge (12 h after treatment), the mice joints were washed; (A) the number of neutrophils measured at different time points 12, 24, 36, 48, and 72 h post-arthritis induction and the calculated kinetics parameters and resolution index; (B) number of neutrophils present in the synovial cavity; (C) relative number of neutrophils present in the periarticular tissue, as determined by the activity of myeloperoxidase (MPO); (D) percentage of apoptotic neutrophils 24 h post-arthritis induction; (E) the increase in the…

Figure 2. The Mas antagonist, A779, reverses the pro-resolution effect of angiotensin-(1-7) [Ang-(1-7)] in a model of arthritis in mice. (A,B) The number of neutrophils and the percentage of apoptotic neutrophils in the synovial cavity, respectively, in antigen-induced arthritis (AIA) mice 12 h after treatment with Ang-(1-7) alone or Ang-(1-7) and A779. (C,D) The number of neutrophils and the percentage of apoptotic neutrophils in the synovial cavity, respectively, in AIA mice 48 and 72 h after challenge treated with A779. Bars show the mean ± SEM from eight mice per group. *p ≤ 0.05 as compared with PBS-control group; #p ≤ 0.05 as compared with AIA group (one-way ANOVA followed by Newman–Keuls test).

Figure 3. Angiotensin-(1-7) [Ang-(1-7)] induces apoptosis of human neutrophils. Ang-(1-7) (30, 100, and 300 nM/well) or PBS (control, C) was incubated for 4 h with isolated human neutrophils. A779 (300 nM/well) was added 15 min before Ang-(1-7). (A) Percentage of apoptotic human neutrophils in response to increasing concentration of Ang-(1-7); (B) percentage of cells expressing Annexin V after 4 h incubation with Ang-(1-7) (100 nM/well); (C) effect of the Mas antagonist, A779, on neutrophil apoptosis induced by Ang-(1-7); (D) images of neutrophils stained for Mas receptor (green) and propidium iodide (PI, red). *p ≤ 0.05 when compared with PBS control (one-way ANOVA followed by Newman–Keuls test).

Figure 4. Angiotensin-(1-7) [Ang-(1-7)] decreases NF-κB activation. (A,B) NF-κB expression by neutrophils obtained from the knees of antigen-induced arthritis (AIA) mice treated or not with Ang-(1-7); (C,D) NF-κB expression by human neutrophils treated or not with Ang-(1-7). Bars show the mean ± SEM from eight mice per group. *p ≤ 0.05 when compared with PBS control (one-way ANOVA followed by Newman–Keuls test).

Figure 5. Angiotensin-(1-7) [Ang-(1-7)] induces efferocytosis of apoptotic human neutrophils. In panel (A), human neutrophils (PMNs) were isolated from peripheral venous blood and apoptosis induced by staurosporine (10 µM). Apoptotic PMNs were injected intraperitoneally into mice bearing a 72-h peritonitis elicited by 0.1 mg of zymosan; these mice were injected previously (30 min before apoptotic cells) with Ang-(1-7) (0.1, 0.3, 1.0, and 3.0 μg/mice). Mice were killed after 90 min, and exudates were collected for morphologic analysis; in panel (B), an image showing an example of efferocytosis of apoptotic neutrophils by macrophages. In panel (C), human PMNs were treated with Ang-(1-7), staurosporine, or both before injection intraperitoneally into mice bearing a 72-h peritonitis elicited by 0.1 mg of zymosan. Mice were killed after 90 min, and exudates were collected for morphologic analysis. In panels (A,C), human neutrophils were obtained from a pool of three donors, and there were five mice in each group.