Requisite role of the cholinergic alpha7 nicotinic acetylcholine receptor pathway in suppressing Gram-negative sepsis-induced acute lung inflammatory injury

Abstract

Although activation of the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) modulates the response to sepsis, the role of this pathway in the development of sepsis-induced acute lung injury (ALI) is not known. In this study, we addressed the contribution of alpha7 nAChR in mediating endotoxin- and live Escherichia coli-induced ALI in mice. Because we found that alpha7 nAChR(+) alveolar macrophages and neutrophils were present in bronchoalveolar lavage and injured lungs of mice, we tested whether acetylcholine released by lung vagal innervation stimulated these effector cells and thereby down-regulated proinflammatory chemokine/cytokine generation. Administration of alpha7 nAChR agonists reduced bronchoalveolar lavage MIP-2 production and transalveolar neutrophil migration and reduced mortality in E. coli pneumonia mice, whereas vagal denervation increased MIP-2 production and airway neutrophil accumulation and increased mortality. In addition, alpha7 nAChR(-/-) mice developed severe lung injury and had higher mortality compared with alpha7 nAChR(+/+) mice. The immunomodulatory cholinergic alpha7 nAChR pathway of alveolar macrophages and neutrophils blocked LPS- and E. coli-induced ALI by reducing chemokine production and transalveolar neutrophil migration, suggesting that activation of alpha7 nAChR may be a promising strategy for treatment of sepsis-induced ALI.

FIGURE 1

Alveolar macrophages express α7 nAChR. A, Morphology of normal BAL cells. B and C, α7 nAChR immunofluorescence in normal BAL cells (mainly alveolar macrophages). B, Control α7 nAChR Ab. C, Rabbit anti-mouse α7 nAChR Ab (× 200; second Ab labeled with Alex Fluor-488). D, Western blot for α7 nAChR in normal BAL cells. PC12 cells and rat brain extract were used as positive controls. E and F, α7 nAChR and CD11b are coexpressed in normal BAL cells identified by flow cytometry. E, Control Abs. F, CD11b and α7 nAChR Abs (repeated 4 times). G–J. α7 nAChR and CD11b coexpression in the lung sections from E. coli pneumonia at 12 h. G, Control Abs followed by DAPI staining. H–J, Anti-CD11b (H) and anti-α7 nAChR (I) Abs staining followed by DAPI (× 200; second Ab labeled with Alexa Fluor-594 and Alexa Fluor-488). K and L, Inhibitory effects of nicotine on proinflammatory cytokines in LPS-stimulated alveolar macrophages (isolated from normal BAL cells), which is reversed by MLA (an α7 nAChR antagonist). K, MIP-2. L, TNF-α. Cells were treated with PBS, nicotine, or nicotine + MLA, then stimulated with LPS. *p < 0.05 versus LPS only; n = 3–5 in each group. M and N, HMGB1 levels in the media of cultured alveolar macrophages, which were treated with nicotine or ACh and then stimulated with LPS for 20 h. M, Effect of nicotine. N, Effect of ACh. *p < 0.05, **p < 0.01 versus LPS only; n = 3–5 in each group. Data are mean ± SD.

FIGURE 2

Neutrophils express α7 nAChR. A, Cytologic change of BAL cells (enriched in neutrophils) from E. coli pneumonia mice at 12 h. B, Western blot for α7 nAChR in BAL cells from normal and E. coli pneumonia at 4 h (equal quantity loading in protein). C and D, α7 nAChR immunofluorescence in pneumonia BAL cells (segmented neutrophils, arrow heads). C, Control α7 nAChR Ab. D, Rabbit anti-mouse α7 nAChR Ab (× 200; second Ab labeled with Alex Fluor-488). E and F, Neutrophils (Gr1⁺ cells) coexpress α7 nAChR in BAL cells from pneumonia at 12 h confirmed by flow cytometry (repeated 3 times). E, Control Abs. F, BAL cells were labeled with anti-α7 nAChR and Gr1 Abs. G–J. α7 nAChR and Gr1 (a neutrophil marker) coexpression in the lung sections from E. coli pneumonia at 12 h. G, Control Abs followed by DAPI staining. H-J. Anti-Gr1 (H) and anti-α7 nAChR (I) Abs staining followed by DAPI (× 200; second Ab labeled with Alexa Fluor-594 and Fluor-488). K–M. Effect of nicotine, DMAB, and PNU (specific α7 nAChR agonist) on production of MIP-2 in LPS-stimulated neutrophils. K, Nicotine. L, DMAB. M, PNU. *p < 0.05, **p < 0.01 versus LPS only; n = 5–6 in each group. N and O, Deficiency of α7 nAChR in neutrophils increases production of proinflammatory cytokines under LPS-stimulation. N, TNF-α. O, MIP-2. **p < 0.01 versus WT; n = 8 in each group. Data are mean ± SD.

FIGURE 3

Activation of α7 nAChR by nicotine protects against LPS-induced ALI at 24 and 48 h. At 24 h, nicotine therapy reduced BAL protein (A), HMGB1 in the lung homogenate (B), and plasma MIP-2 (C). *p < 0.05 versus saline; n = 5 in each group. Data were pooled from 2 different experiments. D–F. Activation of α7 nAChR by its agonists nicotine, DMAB, or PNU attenuates LPS-induced ALI. Body weight loss at 24 h (D); *p < 0.05 versus saline + LPS. Lung myeloperoxidase activity at 48 h (E); **p < 0.01 versus saline only; ^#p < 0.05 versus saline + LPS. ELW at 48 h (F); *p < 0.05 versus saline + LPS; n = 5 in each group. Data were pooled from 4 different experiments. Data are mean ± SD. G–K. Representative photomicrographs of lung histology in LPS-induced ALI at 24 h treated with α7 nAChR agonists nicotine, DMAB, or PNU. Magnification ×400; scale bar = 50 μm; n = 2–3 in each group.

FIGURE 4

α7 nAChR is a key regulator of lung inflammation and injury in E. coli pneumonia. Administration of nicotine reduces pulmonary edema (A) and lung vascular permeability (B) in E. coli pneumonia at 4 h. Mice were pretreated with either saline or nicotine (3.5 mg/kg, i.v.), then instilled IT with E. coli (10⁷ CFU), and killed at 4 h. **p < 0.01 versus saline + saline and nicotine + saline; ^#p < 0.05 versus saline + E. coli; n = 4–6 in each group. Data were pooled from three different experiments. Administration of nicotine reduces protein concentration TNF-α (C), MIP-2 (D), and neutrophil counts (E) in the BAL in E.coli pneumonia at 4 h. *p < 0.05; **p < 0.01 versus saline controls; n = 4–6 in each group. Data were pooled from three different experiments. F and G, Activation of α7 nAChR by nicotine attenuates E. coli pneumonia at 24 h. F, ELW. G, Levels of HMGB1 in the lung homogenate. *p < 0.05; n = 4–6 in each group. H, Deficiency of α7 nAChR worsens pulmonary edema in E. coli pneumonia. *p < 0.05 for α7 nAChR^+/+ versus α7 nAChR^−/− mice; n = 3 in each group. Data are mean ± SD. I and J, Changes of AChE and choline in the BAL at 12 h after E. coli pneumonia. I, AChE activity in BAL cells from normal and E. coli pneumonia mice. J, Choline levels in the BAL. **p < 0.01 versus normal mice; n = 6 in each group. Data are mean ± SD.

FIGURE 5

Activation of α7 nAChR reduces BAL proinflammatory cells and protein in E. coli pneumonia. A–D, Effect of nicotine. A, BAL leukocytes. B, BAL neutrophils. C, BAL monocytes. D, Protein levels. *p < 0.05 for nicotine versus saline; n = 3–5 in each group. Data were pooled from three different experiments. E–H, Effect of PHA 568487. E, BAL leukocytes. F, BAL neutrophils. G, BAL monocytes. H, Protein levels. *p < 0.05 for PHA 568487 versus saline; n = 3–5 in each group. Data were pooled from three different experiments. I–L, Unilateral cervical vagotomy (right side) increases BAL proinflammatory cells and protein levels in E. coli pneumonia. I, BAL leukocytes. J, BAL neutrophils. K, BAL monocytes. L, Protein levels. *p < 0.05 for vagotomy versus sham group at 4, 12, or 24 h after IT E. coli challenge; n = 4–5 in each group. Data were pooled from three different experiments. Data are mean ± SD.

FIGURE 6

Activation of α7 nAChR reduces MIP-2 generation in E. coli pneumonia. A and B, Effect of nicotine. A, BAL MIP-2. B, Plasma MIP-2. *p < 0.05 for nicotine versus saline; n = 3–5 in each group. Data were pooled from three different experiments. C and D, Effect of PHA 568487. C, BAL MIP-2. D, Plasma MIP-2. *p < 0.05 for PHA 568487 versus saline; n = 3–5 in each group. Data were pooled from three different experiments. E and F, Unilateral cervical vagotomy (right side) increases MIP-2 levels in BAL from isolateral lung and plasma in E. coli pneumonia. E, BAL MIP-2. F, Plasma MIP-2. *p < 0.05 versus sham group at 4 and 12 h after IT E. coli challenge; n = 4–5 in each group. Data were pooled from three different experiments. Data are mean ± SD.

FIGURE 7

Activation of α7 nAChR enhances survival in E. coli pneumonia. A, Effects of nicotine. Mice were treated with saline or nicotine (2.4 mg/kg, delivered by an osmotic pump) and followed up for 24 h. *p < 0.05 for nicotine versus saline treated mice; n = 12 in each group. Data were pooled from two different experiments. B, Antagonism of α7 w E. coli pneumonia. Mice were treated with nicotine or nicotine + MLA (2.4 mg/kg, delivered by an osmotic pump; MLA was given i.p.) and followed up for 24 h. *p < 0.05 for nicotine (n = 9) versus nicotine + MLA (n = 5) treated mice. Data were pooled from two different experiments. C, Unilateral vagotomy reduces survival in E. coli pneumonia. Sham and vagotomized mice were challenged with E. coli and followed for 48 h. *p < 0.05 for vagotomy (n = 30) versus sham (n = 30). Data were pooled from 5 different experiments. D, Deficiency of α7 nAChR worsens reduces survival in E. coli pneumonia; n =5 α7 nAChR^+/+ mice; n =4 α7 nAChR^−/− mice; *p < 0.05 for α7 nAChR^+/+ versus α7 nAChR^−/− mice.