Activation of the alpha7 nAChR reduces acid-induced acute lung injury in mice and rats

Abstract

New evidence indicates that neural mechanisms can down-regulate acute inflammation. In these studies, we tested the potential role of the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) in a rodent model of acid-induced acute lung injury. We first determined that the alpha7 nAChR was expressed by alveolar macrophages and lung epithelial cells. Then, using an acid-induced acute lung injury mouse model, we found that nicotine, choline, and PNU-282,987 (a specific alpha7 nAChR agonist) decreased excess lung water and lung vascular permeability, and reduced protein concentration in the bronchoalveolar lavage (BAL). Deficiency of alpha7 nAChR resulted in a 2-fold increase in excess lung water and lung vascular permeability. The reduction of proinflammatory cytokines (macrophage inflammatory protein-2 and TNF-alpha) in the BAL with nicotine probably resulted from the suppression of NF-kappaB activation in alveolar macrophages. The beneficial effect of nicotine was also tested in rat model of acid-induced acute lung injury in which BAL protein and receptor for advanced glycation end products (RAGE), a marker of type I cell injury, were reduced by nicotine treatment. These results indicate that activation of alpha7 nAChR may provide a new therapeutic pathway for the treatment of acute lung injury.

Figure 1.

Expression of α7 nAChR in mouse airway epithelial cells, human alveolar type II cells, mouse alveolar macrophages, and neutrophils. (A and B) Representative immunohistochemistry of α7 nAChR in the lung section in mice. (A) Without H-302 antibody; (B) With H-302 antibody. In the inset, red-brownish staining indicates α7 nAChR–positive bronchial epithelial cells. (C and D) Immunofluorescence of α7 nAChR in isolated alveolar epithelial type II cells in human. (C) Without H-302 antibody; (D) With H-302 antibody (the second antibody labeled with FITC). (E and F) Immunofluorescence of α7 nAChR in alveolar macrophages in mice (the second antibody labeled with Cy3). (E) Without H-302 antibody; (F) With H-302 antibody. (G and H) Immunohistochemistry of α7 nAChR in BAL proinflammatory cells. (G) The control group was instilled with saline. (H) The acid-instilled group. Cells were treated with H-302 antibody and Histostain SP rabbit primary (AEC) kit (Zymed Laboratories). Green arrows indicate α7 nAChR–positive alveolar macrophages. Red arrows indicate α7 nAChR–positive neutrophils. (I) The intensity of immunostaining of α7 nAChR in G and H measured by NIH Image-J software.

Figure 2.

(A and B) Nicotine reduced pulmonary edema and lung vascular permeability in mice. (A) Excess lung water. (B) Extravascular plasma equivalents. The mice were pretreated with nicotine 5–10 min before intratracheal instillation of acid and killed at 4 h. **P < 0.01 for saline + acid (n = 6) versus the saline + saline group (n = 5); *P < 0.05 for nicotine + acid (n = 6) versus the saline + acid group (n = 6). (C and D). Representative histologic change (objective magnification: ×20; scale bar: 50 μm) (H&E staining). (C) Saline + acid. (D) Nicotine + acid (n = 3, each). (E and F). Effects of nicotine on acid-induced acute lung injury in ventilated rats. (E) The protein concentration in the BAL. (F) Soluble RAGE levels in the BAL. n = 3, in the saline + acid group; n = 4 in the nicotine +acid group. (G and H). Specific blockade of α7 nAChR counteracted the protective effect of nicotine in acid-induced acute lung injury in mice. To antagonize the effects of nicotine, MLA was also pretreated 5–10 min before acid. (G) Excess lung water. (H) Extravascular plasma equivalents. *P < 0.05 versus acid only; ^#P < 0.05 for MLA + nicotine versus nicotine treatment only (n = 5 in each group). Data are mean ± SD.

Figure 3.

Effects of nicotine on protein concentration, neutrophil counts, and cytokine levels in the BAL and activation of NF-κB in the BAL proinflammatory cells in mice. The mice were pretreated with nicotine 5–10 min before intratracheal instillation of acid. (A) Protein concentration. (B) Leukocyte counts. (C) Neutrophil counts. **P < 0.01 for saline +acid (n = 5) versus the saline + saline group (n = 4); ^#P < 0.05 or ^##P < 0.01 nicotine + acid (n = 5) versus the saline + acid group (n = 5). (D and E). Cytokine levels in the BAL. (D) TNF-α. (E) MIP-2. *P < 0.05 for nicotine + acid (n = 5) versus the saline + acid (n = 4) group. Data are mean ± SD. (F) Western blot for NF-κB p65 in the cytoplasm and nucleus in the BAL proinflammatory cells (n = 3 in each group).

Figure 4.

Effects of choline on excess lung water, extravascular plasma equivalents, and neutrophil differentiation in the BAL in mice. The mice were pretreated with choline 5–10 min before intratracheal instillation of saline or acid. (A) Excess lung water. (B) Extravascular plasma equivalents. **P < 0.01 for the saline + acid (n = 5) versus the saline + saline group (n = 4); ^#P < 0.05 for the nicotine + acid (n = 5) versus the saline + acid group. (C) Neutrophil differentiation in the BAL. **P < 0.01 versus the saline + saline group; ^##P < 0.01 versus the saline + acid group (n = 3 in each group). (D and E). BAL cytological change (Wright-Giemsa staining). (D) The saline + acid group (n = 4). (E) The choline + acid group (n = 5). Arrows indicate neutrophils. Data are mean ± SD.

Figure 5.

Effects of PNU-282987 (a specific α7 nAChR agonist) on excess lung water, extravascular plasma equivalents, neutrophil accumulation, protein concentration, and cytokine levels in the BAL in the acid-induced acute lung injury in mice. (A) Excess lung water. (B) Extravascular plasma equivalents. **P < 0.01 for PNU282987 versus the saline group (n = 5 in each group). (C) Neutrophil counts in the BAL. (D) Protein concentration in the BAL. (E and F). TNF-α and MIP-2 levels in the BAL. *P < 0.05, **P < 0.01 for PNU282987 versus the saline group (n = 5 in each group). Data are mean ± SD.

Figure 6.

Deficiency of α7 nAChR worsened acid induced acute lung injury in mice. (A) Excess lung water. (B) Extravascular plasma equivalents. *P < 0.05,**P < 0.01 for wild-type versus knockout mice (n = 5 wild-type, n = 4 α7 nAChR knockout). Data are mean ± SD.