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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2877486
NIHMSID: NIHMS203396

Requisite Role of the Cholinergic α7 Nicotinic Acetylcholine Receptor Pathway in Suppressing Gram-Negative Sepsis-Induced Acute Lung Inflammatory Injury

Abstract

Although activation of the α7 nicotinic acetylcholine receptor (α7 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 α7 nAChR in mediating endotoxin- and live Escherichia coli–induced ALI in mice. Because we found that α7 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 α7 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, α7 nAChR−/− mice developed severe lung injury and had higher mortality compared with α7 nAChR+/+ mice. The immunomodulatory cholinergic α7 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 α7 nAChR may be a promising strategy for treatment of sepsis-induced ALI.

The cholinergic antiinflammatory pathway (1) has been described in a series of experiments showing that vagus nerve stimulation attenuated the systemic inflammatory response to endotoxin (13). The α7 nicotinic acetylcholine receptor (α7 nAChR) expressed in macrophages regulates this pathway during inflammation (4) such that cholinergic activation of these cells dampens the inflammatory response. The studies showed that activation of α7 nAChR by agonists (e.g., nicotine) downregulated expression of HMGB1 protein (a late mediator of sepsis) and improved survival (5, 6). Stimulation of the cholinergic antiinflammatory pathway also appears to play a protective role in other inflammatory models, peritonitis (7, 8), and renal ischemia and reperfusion injury (9).

Activation of α7 nAChR in macrophages and monocytes may downregulate production of proinflammatory cytokines and attenuate the inflammatory response by several possible but poorly understood mechanisms: 1) suppression of NF-κB translocation and I-κB phosphorylation (5, 10), 2) activation of Jak2-STAT3 signaling (11), and 3) inhibition of expression of LPS receptors and binding proteins CD14 and TLR4 (12). Some studies have also indicated a possible role of specific α7 nAChR agonists GTS-21 (13) and choline (14) or cholinesterase inhibitors (15) for improving outcome in experimental sepsis.

Sepsis-induced acute lung injury (ALI) causes acute respiratory failure in critically ill patients and has a mortality rate of 40% (16, 17). The most common causes of ALI are pneumonia and sepsis (17, 18). Although activation of the α7 nAChR modulates the response to sepsis, the role of this pathway in the development of sepsis-induced ALI is not known. Our objectives here are to determine the role of activation of alveolar macrophages and neutrophils expressing α7 nAChR on the production of proinflammatory cytokines and chemokines, whether α7 nAChR affects the ALI response induced by endotoxin and Escherichia coli pneumonia, and whether antagonism or deficiency of α7 nAChR activity and vagus denervation has the opposite effects of lung inflammation and injury and survival in mouse models.

Materials and Methods

Reagents

(−)-nicotine, acetylcholine (ACh), methyllycaconitine (MLA), and LPS were purchased from Sigma-Aldrich (St. Louis, MO). Dimethylaminobenzaldehyde (DMAB), PNU 282987 (PNU), and PHA 568487 (specific agonists of α7 nAChR) were purchased from Tocris Bioscience (Ellisville, MO) and dissolved in 0.9% saline before each experiment. H-302, an anti-α7 nAChR Ab used to detect α7 nAChR of mouse and human origin, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE anti-mouse CD11, Ly-6G (Gr-1, Gr1), and corresponding isotype Abs were purchased from eBioscience (San Diego, CA).

Animals

Most experiments were done with CD1 mice (purchased from Charles River Laboratories, Wilmington, MA). α7 nAChR–deficient mice (C57BL/6 background, B6.129S7-Chrna7tm1Bay, number 003232) and wild type (WT) littermates (C57BL/6J, 8 wk old) were purchased from The Jackson Laboratory (Bar Harbor, ME) (4). Anesthesia was induced with an i.p. injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg). The Committees on Animal Research of the University of Illinois at Chicago and the University of California, San Francisco approved the protocols.

Isolation and culture of alveolar macrophages

Alveolar macrophages were isolated by a bronchoalveolar lavage (BAL) (19, 20). Greater than 90% alveolar macrophage purity was confirmed with a cytospin preparation (Cytospin 3, Thermo Electron, Milford, MA) and Hema 3 staining (Fisher Scientific, Kalamazoo, MI). Alveolar macrophages were cultured in RPMI 1640 medium (2.5 × 105 /ml). α7 nAChR agonists or an antagonist (MLA) were added 20 min before LPS stimulation (3 mM). PBS was used as a negative control. The media was collected after 12 h incubation for TNF-α and MIP-2, and after 20 h for HMGB1 measurements.

Neutrophil isolation and culture

As described (20), mice were euthanized by cervical dislocation, and the bone marrow from the femurs and tibias was flushed with PBS using a 25-gauge needle. The whole bone marrow was centrifuged and washed in PBS, and red blood cells were hypotonically lysed with 0.2% NaCl. This solution was resumed to isotonicity with 1.2% NaCl and then filtered over a 70-μm nylon cell strainer (BD Discovery Labware, Bedford, MA). The solution was centrifuged and resuspended in PBS and then delicately applied over a 62% Percoll gradient. The Percoll solution was centrifuged for 30 min at 1500 × g. The neutrophil pellet was then isolated, washed, and centrifuged twice, and counted with a Coulter counter (Z1 series; Beckman Coulter, Fullerton, CA). Greater than 90% neutrophil purity was confirmed with the cytospin preparation and Hema 3 staining. Neutrophils were suspended in RPMI 1640 medium (106/ml). α7 nAChR agonists or an antagonist (MLA) were added 20 min before LPS stimulation (3 μM). PBS was used as a negative control. The media was collected after 12 h incubation for measuring MIP-2 or TNF-α concentration.

LPS-induced ALI mouse model

As described, mice were intratracheally (IT) instilled with LPS (5 mg/kg) by a direct visualized instillation method (21). Mice were monitored for 24 and 48 h, and killed to perform BAL or measure extravascular lung water (ELW).

Acute E. coli pneumonia mouse model and survival study

Live E. coli were obtained from American Type Culture Collection, Manassas, VA (ATCC 25992) (22). E. coli (107 CFU) was instilled into the air spaces of lungs. Immediately before exposure to E. coli, mice received 0.05 μCi [125I]-albumin via the right jugular vein. Mice were monitored for 4 h and killed to measure ELW and lung endothelial permeability to protein or to perform BAL.

We had found a correlation between the degree of lung injury and mortality and the dose of IT E. coli. Therefore, different doses were used depending on the experimental objectives: 1) E. coli (107 CFU) for early experiments at 4 h to ensure that the lungs were substantially injured; 2) E. coli (2.5 × 106 CFU) for longer experiments (24 h) to ensure that there was no death in both control and treated groups; 3) E. coli (5 × 106 CFU) for the survival study, to be certain that some mice died of substantial lung injury within 24 h, which facilitated observing the difference between the control and treated groups; and 4) E. coli (4 × 106 CFU) for the survival study with vagotomized or α7 nAChR−/− mice, because they were more susceptible to the E. coli infection compared with the WT mice.

ELW and lung extravascular plasma equivalent

As described (19), homogenate and supernatant of lung, and blood were weighed and then desiccated in an oven (60°C for 24 h). ELW was calculated by standard formula:

ELW=[(QWexpQdexp×Qdexp)(QWcontrolQdcontrol×Qdcontrol)]×1000(μ1)

where QW exp equals water volume of the lung in the experimental group; Qd exp equals dry weight of lung in the experimental group. The controls were the normal mice with the same age as the experimental group. Lung extravascular plasma equivalents (EPEs; index of lung vascular permeability to protein) were calculated as the counts of [125I]-albumin in the blood free lung tissue divided by the counts of [125I]-albumin in the plasma.

Unilateral vagotomy

Right or sham cervical vagotomy was performed with the animals under anesthesia. The procedure involved a longitudinal midline incision in the ventral region of the neck. Using blunt dissection, the overlying muscles and fascia were separated until the right vagus and carotid artery were visible. The vagus was carefully stripped away from carotid artery and lightly cut off in the vagotomy group. The vagus was kept intact in sham group. The wound was closed and sutured. The respiration rhythm was not affected by unilateral vagotomy.

BAL and plasma cytokine and protein concentration measurements

BAL and plasma samples were obtained from mice at designated time points. Supernatant was used to measure protein and cytokines. TNF-α and MIP-2 were measured with ELISA kits (R&D Systems, Minneapolis, MN). Protein concentration was measured by a Bio-Rad protein assay kit (Hercules, CA).

Measurement of leukocyte, neutrophil, and monocyte counts in BAL

BAL leukocytes, neutrophils, and monocytes were measured by Hemavet 950FS (Drew Scientific, Dallas, TX). Morphology of BAL cells was determined by BAL cytospin and Hema 3 staining.

Determination of lung myeloperoxidase activity

As described (20), supernatants of lung homogenate were mixed with o-Dianisidine HCl (Sigma-Aldrich) and H2O2 to measure optical density by a spectrophotometer at 405 nm.

HMGB1 enzyme-linked immunosorbent assay

To measure HMGB1 using ELISA in this study, we used a capture Ab (clone HAP46.5, anti-HMGB1 monoclonal Ab; Abcam, Cambridge, MA), which was diluted in coating buffer and added to a 96-well plate overnight at 4°C. After blocking and washing, standard curve was made by adding recombinant human HMGB1 (H4652; Sigma-Aldrich; 500, 250, 125, 62.5, 31, 16, and 8 ng/ml) to each well and incubated at room temperature for 2 h. Detection Ab (anti-mouse RAGE Ab labeled with HRP; SureLINK HRP Conjugation Kit; Gaithersburg, MD) was added and incubated at room temperature for 2 h. Color was developed by adding substrate A and B mixture (R&D Systems, Minneapolis, MN), and the plate was read at 450 nm in a spectrophotometer.

Measurement of acetylcholinesterase activity and choline levels

Acetylcholinesterase (AChE) activity of BAL cells was measured as previously described (23). Briefly, 105 BAL cells (sonicated after removal of red blood cells) were mixed with 5,5′-dithiobis-2-nitrobenzoic acid (Sigma-Aldrich) to measure OD405nm[A], and then acetylthiocholine iodide (Sigma-Aldrich) to measure OD405nm[B] at 30 min. AChE activity (ΔOD405nm) was calculated by OD405[B] − OD405[A]. Choline in BAL was measured by a Choline/Acetylcholine Quantification kit (BioVision Research Products, Mountain View, CA).

Detection of α7 nAChR expression in alveolar macrophages and neutrophils by immunofluorescence

As described (19), using cytospin, alveolar macrophages, or neutrophils were prepared on slides and fixed in 4% paraformaldehyde. The smear was permeabilized in 0.25% triton and incubated with 1:67 rabbit anti-mouse α7 nAChR Ab (H-302). Then, goat-anti-rabbit fluorescein isothiocyanate (FITC)-labeled secondary Ab (Santa Cruz Biotechnology) was added on the smear and incubated for 1 h. After washing with hypertonic PBS (2.7% NaCl), the slides were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). For immunofluorescence in frozen lung sections, lungs were collected at endpoints of experiments and embedded in OCT (Sakura FineTek, Torrance, CA), and frozen on dry ice. Frozen sections (5 μm) were prepared in a cryostat (Microm, Walldorf, Germany). The lung sections was permeablized in 0.2% triton and incubated with the following Abs: 1:50 rabbit anti-mouse α7 nicotinic ACh receptor Ab (H302; Santa Cruz Biotechnology); 1:50 rat anti-mouse CD11b monoclonal Ab (M1/70; BD Pharmingen, San Diego, CA), or 1:50 rat anti-mouse Gr1 Ab (LEW; BD Pharmingen). Then, anti-rabbit or rat Alexa Fluor 594 or 488 labeled secondary Abs (Invitrogen, Carlsbad, CA) was added on the smear and incubated for 1 h. After washing with hypertonic PBS (2.7% NaCl), the slides were mounted with ProLong Gold antifade reagent (Molecular Probes). The fluorescence was visualized and analyzed using a digitized fluorescence microscope (Carl Zeiss, Thornton, NY).

Flow cytometric analysis

Monoclonal Abs PE anti-mouse CD11b (M1/70), Gr1 (RB6-8C5), and IgG isotype controls were obtained from eBioscience. Rabbit anti-mouse α7 nAChR and control Abs were obtained from Santa Cruz Biotechnology. All samples were pretreated with Fc receptor blocking reagent to prevent nonspecific binding. After BAL, red blood cells were lysed. The BAL cell pellets were washed with PBS (2.5% FCS), fixed in 2% paraformaldehyde, permeabilized with 0.2% Triton, and then labeled with corresponding Abs. Fluorescent cells were analyzed after exclusion of debris and aggregates with CyAn ADP or MoFlo (DakoCytomation, Carpinteria, CA). Data were analyzed by Summit 4.3 software (DakoCytomation).

α7 nAChR Western blotting of BAL samples

As described (19), denatured proteins (20 μg) from BAL cells were loaded and run on a 4–12% gradient Bis-Tris gel (Invitrogen, Carlsbad, CA). The proteins were then transferred to a nitrocellulose membrane and incubated with anti-α7 nAChR Ab (H-302). Membranes were exposed to HRP-labeled Ab and developed with an ECL kit (Amersham, Piscataway, NJ).

Statistical analysis

Statistical analysis was done with SPSS software (SPSS, Chicago, IL). An unpaired t test was used unless there were multiple comparisons, in which case we used ANOVA with post hoc Bonferoni test (significance level was set at p < 0.05). The log-rank test was used for comparing survival data by GraphPad Prism software (GraphPad, San Diego, CA). The results are shown as mean ± SD.

Results

Alveolar macrophages express α7 nAChR

In normal BAL cells, alveolar macrophages are dormant or resting cells (Fig. 1A) α7 nAChR immunoreactivity was found in the cell membrane and cytoplasm (Fig. 1B, 1C). Western blotting demonstrated that normal BAL cells (mainly alveolar macrophages) expressed α7 nAChR with a band at 55 kDA, using PC12 cells and rat brain extract as positive controls (Fig. 1D). To study alveolar macrophage cell surface expression of α7 nAChR, normal BAL cell pellets were double stained with anti-mouse CD11b (Mac-1, a surface marker of macrophages) and α7 nAChR Abs to perform flow cytometry. We observed that 14.7 ± 4.8% of normal BAL cells coexpressed CD11b and α7 nAChR (Fig. 1E, 1F). To investigate coexpression of α7 nAChR and CD11b in E. coli– injured lungs, lung sections were obtained from pneumonia mice at 12 h after IT challenge of E. coli for immunostaining. Coexpression of α7 nAChR and CD11b was found in injured lung sections (Fig. 1GJ).

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 ...

Effects of nicotine and MLA on TNF-α and MIP-2 production in alveolar macrophages

To test whether activation of α7 nAChR by nicotine in alveolar macrophages alters proinflammatory cytokine production and whether MLA (a specific α7 nAChR antagonist) reverses the effects of nicotine, alveolar macrophages were pretreated separately with PBS, 10−26 M nicotine, or 10−26 M + MLA 10−26 M nicotine. After 20 min, cells from these three groups were stimulated with LPS (3 μM) for 12 h to measure TNF-α and MIP-2 in the media. Untreated cells with PBS challenge were used as controls. LPS stimulation increased MIP-2 (545 ± 24 versus 31 ± 18 pg/ml in PBS group) and TNF-α (704 ± 6 versus 16 ± 7 pg/ml in PBS group) production in alveolar macrophages. Nicotine reduced LPS-induced MIP-2 and TNF-α production. MLA counteracted this effect of nicotine on TNF-α and MIP-2 production (Fig. 1K, 1L).

Effects of nicotine and ACh on HMGB1 production in alveolar macrophages

To test whether activation of α7 nAChR agonists by nicotine and ACh affects HMGB1 production in alveolar macrophages, alveolar macrophages were pretreated separately with PBS, 10−7 M nicotine or ACh, 10−6 M nicotine or ACh, or 10−5 M nicotine or ACh. At 20 min, the cells from these four groups were stimulated with LPS (3 μM) for 20 h to measure HMGB1 in the media. Untreated cells with PBS challenge were used as controls. LPS stimulation increased HMGB1 production in alveolar macrophages (3998 ± 204 versus 28 ± 6 pg/ml in the PBS stimulated group. Nicotine reduced HMGB1 by 25 and 32% at 10−6 and 10−5 M, respectively (Fig. 1M). ACh suppressed HMGB1 by 30% at 10−7 to 10−5 M (Fig. 1N).

Neutrophils express α7 nAChR

To determine whether neutrophils express α7 nAChR, we collected BAL cells from E. coli pneumonia (2.5 ×10−6 CFU, IT, sacrificed at 12 h) to perform immunofluorescence and Western blotting. The smear of BAL cells by Hema 3 staining demonstrated that the number of neutrophils (Fig. 2A) was increased in BAL from E. coli pneumonia compared with normal BAL cells, which are mainly alveolar macrophages (Fig. 1A). By Western blotting, we observed that the expression of α7 nAChR in BAL cells from E. coli pneumonia (alveolar macrophages mixed with neutrophils) was markedly increased compared with normal BAL cells (Fig. 2B). Immunofluorescence demonstrated that BAL neutrophils from pneumonia expressed α7 nAChR in the cell membrane and cytoplasm (Fig. 2C, 2D, segmented neutrophils). To confirm that neutrophils expressed α7 nAChR, BAL cell pellets (from pneumonia mice at 12 h) were double stained with anti-mouse Gr1 (a surface marker of neutrophils) and α7 nAChR Abs to perform flow cytometry. We observed that 43.6 ± 1.8% of BAL cells coexpressed Gr1 and α7 nAChR (Fig. 2E, 2F), confirming the finding that neutrophils express α7 nAChR. To investigate coexpression of α7 nAChR and Gr1 in E. coli–injured lungs, lung sections were obtained from pneumonia mice at 12 h after IT challenge of E. coli to perform immunostaining. Coexpression of α7 nAChR and Gr1 was found in injured lung sections (Fig. 2GJ).

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 ...

α7 nAChR activation downregulates TNF-α and MIP-2 production in neutrophils

To investigate whether activation of α7 nAChR agonists by nicotine, DMAB (a novel partial α7 nAChR agonist), or PNU (a highly specific α7 nAChR agonist, Ki 27 nM) affect MIP-2 production in neutrophils, neutrophils were pretreated separately with PBS; 10−7 M nicotine, DMAB or PNU; 10−6 M nicotine, DMAB or PNU; or 10−5 M nicotine, DMAB or PNU. At 20 min, cells from these four groups were stimulated with LPS (3 μM) for 12 h to measure MIP-2 concentration in the media. Untreated cells with PBS challenge were used as controls. The MIP-2 concentration in LPS-stimulated neutrophils was increased (1124 ± 84 pg/ml) compared with 11 ± 3 pg/ml in PBS group. Nicotine reduced MIP-2 by 23–40% at 10−7 to 10−6 M, and 50% at 10−5 M (Fig. 2K). DMAB inhibited MIP-2 by 51–53% at 10−7 to 10−5 M (Fig. 2L), and PNU suppressed MIP-2 by 49% at 10−7 to 10−6 M and 30% at 10−5 M (Fig. 2M).

α7 nAChR-deficient neutrophils propagate proinflammatory cytokine production

To test whether deficiency of α7 nAChR in neutrophils facilitates proinflammatory cytokine production, the isolated bone marrow neutrophils from α7 nAChR+/+ and α7 nAChR−/− mice were stimulated with LPS (3 μM). The media was collected at 12 h. After LPS stimulation, TNF-α and MIP-2 concentrations in the media from α7 nAChR−/− neutrophils were increased compared with α7 nAChR+/+ neutrophils (Fig. 2N,2O).

α7 nAChR activation attenuates LPS-induced ALI

To study whether administration of nicotine suppresses LPS-induced ALI, mice were administrated nicotine (0.4 mg/kg, i.p., every 6 h). The first dose was given 15 min before LPS (5 mg/kg, IT), and the control group received the same volume of saline (5) for 24 h. Lungs were lavaged to measure protein, and then homogenized to measure HMGB1 and blood was collected to measure plasma MIP-2. The BAL protein (an index of permeability of the lung vascular and epithelial barriers) was decreased in the nicotine-treated group (Fig. 3A). HMGB1 levels in lung homogenate were also reduced by nicotine therapy (Fig. 3B). MIP-2 generation in the plasma (a surrogate of the systemic inflammatory response) was abolished by nicotine treatment (Fig. 3C).

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. ...

To compare the effects of three different α7 nAChR agonists (nicotine, DMAB, and PNU) on LPS-induced ALI, three agonists were separately given (0.4 mg/kg, i.p., every 6 h). The first dose was given 15 min before IT challenge with LPS (5 mg/kg). The control group received the same volume of saline. At 24 h, body weight loss in the agonist-treated group was reduced compared with the saline group (Fig. 3D). At 48 h, myeloperoxidase activity in lung homogenates in the nicotine, DMAB, and PNU groups was lower than in the saline group (Fig. 3E). ELW in the agonist-treated groups was also reduced compared with the saline-treated group (Fig. 3F).

To test for histologic differences, LPS-challenged mice (5 mg/kg, IT) weregiven saline, nicotine, DMAB, or PNU (0.4 mg/kg, i.p., every 6 h) separately and then euthanized after 24 h. The control mice received saline IT and saline i.p. In the LPS + saline group, more consolidation, hemorrhage, neutrophil infiltration, and interstitial thickness (Fig. 3H) were found compared with the control group (Fig. 3G). Administration of nicotine, DMAB, or PNU markedly reduced the consolidation, hemorrhage, neutrophil infiltration, and interstitial thickness compared with the LPS + saline group (Fig. 3IK).

α7 nAChR modulates lung inflammation and edema by suppressing inflammatory mediator production in local milieu

To test whether activation of α7 nAChR reduces ELWand lung EPE in E. coli pneumonia, we divided mice into four groups: 1) saline + saline group, in which the mice were pretreated with saline and then IT saline; 2) nicotine + saline group, in which the mice were treated with nicotine (3.5 mg/kg, i.v.) (19) 5–10 min before IT saline; 3) saline + E. coli (107 CFU; the lungs were substantially injured at 4 h at this dose) group, in which the mice were pretreated with saline and then IT E. coli; and 4) nicotine + E. coli (107 CFU) group, in which mice were pretreated with nicotine and then IT E. coli. At 4 h, blood was withdrawn and the lungs were removed to measure ELW and lung EPE. ELW and lung EPE in the saline + E. coli group were increased eightfold to ninefold compared with the saline + saline group. ELW and lung EPE in the nicotine + E. coli group were reduced compared with the saline + E. coli group at 4 h (Fig. 4A, 4B). We also used the same experimental design to determine BALTNF-α and MIP-2 levels and neutrophil numbers. Nicotine reduced the BAL neutrophil counts and TNF-α and MIP-2 levels compared with the saline group at 4 h (Fig. 4CE).

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 ...

To address whether activation of α7 nAChR by nicotine affects E. coli pneumonia at a 24-h time frame, mice were treated with either nicotine (0.4 mg/kg, i.p.) or saline 15 min before IT challenge with 2.5 × 106 CFU E. coli (at this dose, there was no death in both control and treated groups within 24 h of the experiment). The same therapy was repeated every 6 h (the total dose 2.4 mg/kg within 24 h) (5). At 24 h, ELW in pneumonia treated with nicotine was decreased compared with pneumonia treated with saline (Fig. 4F). Because HMGB1 is a later mediator of sepsis and inflammation, lung HMGB1 levels were measured. HMGB1 levels in lung homogenates after nicotine therapy were reduced compared with saline therapy (Fig. 4G), indicating that activation of α7 nAChR attenuates lung inflammation and injury by reducing HMGB1 locally, also in the late time frame. To address whether a lack of α7 nAChR worsens pulmonary edema, α7 nAChR+/+ and α7 nAChR−/− mice were instilled IT with E. coli (2.5 × 106 CFU) and sacrificed at 12 h. ELW in α7 nAChR−/− mice was 2.3-fold higher compared with α7 nAChR+/+ mice (Fig. 4H).

AChE activity and choline concentration in BAL in E. coli pneumonia

To study AChE activity in alveolar proinflammatory cells and BAL choline concentrations in E. coli pneumonia, mice were challenged IT with E. coli (2.5 × 106 CFU) and sacrificed at 12 h with BAL collected. BAL from normal mice was used as a control. AChE activity in BAL cells and choline levels in the BAL were increased in E. coli pneumonia mice at 12 h (Fig. 4I, 4J) compared with normal mice. There was no difference in BAL ACh levels between control (2.1 ± 2.4 nmol/ml) and E. coli pneumonia groups (3.2 ± 2.1 nmol/ml) at 12 h.

α7 nAChR pathway regulates proinflammatory cell transmigration and transalveolar permeability

To study the effects of nicotine (a nonselective agonist of α7 nAChR) on transmigration of proinflammatory cells into the airspaces as well as lung epithelial and endothelial permeability, mice were pretreated with either nicotine (0.4 mg/kg, i.p.) or saline 15 min before IT challenge with E. coli (2.5 × 106 CFU). The therapy was repeated every 6 h. The mice were killed at 4, 12, and 24 h. Normal mice were used as controls. BAL was collected to measure numbers of leukocytes, neutrophils, and monocytes. BAL protein (an index of lung epithelial and endothelial permeability) was also analyzed. At 4 h, BAL leukocyte (1.15 ± 0.34 k/μl versus 0.62 ± 0.14 k/μl) and neutrophil counts (0.125 ± 0.017 versus 0.065 ± 0.017 k/μl) were reduced (Fig. 5A, 5B) with nicotine therapy. At 12 h, BAL leukocyte, neutrophil, and monocyte counts showed a trend in reduction in the nicotine-treated group (Fig. 5AC). At 24 h, there were no differences in leukocyte, neutrophil, and monocyte counts (Fig. 5AC) compared with nicotine- and saline-treated groups. BAL protein levels were diminished at 4, 12, and 24 h compared with nicotine-and saline-treated groups (Fig. 5D).

FIGURE 5
Activation of α7 nAChR reduces BAL proinflammatory cells and protein in E. coli pneumonia. AD, Effect of nicotine. A, BAL leukocytes. B, BAL neutrophils. C, BAL monocytes. D, Protein levels. *p < 0.05 for nicotine versus saline; ...

To test the effects of PHA 568487 (a specific agonist of α7 nAChR) on transmigration of proinflammatory cells into the airspace and lung epithelial and endothelial permeability, mice were pretreated with either PHA 568487 (0.4 mg/kg, i.p.) or saline 15 min before IT challenge with E. coli (2.5 × 106 CFU). The therapy was repeated every 6 h. The mice were killed at 4, 12, and 24 h. Normal mice were used as controls. BAL was collected to measure numbers of leukocytes, neutrophils, and monocytes, and BAL protein levels. At 4 h, BAL leukocyte (0.32 ± 0.02 versus 0.16 ± 0.04 k/μl) and neutrophil counts (0.09 ± 0.05 verus 0.01 ± 0 k/μl) were reduced (Fig. 5E, 5F) with PHA 568487 therapy. At 12 h, BAL neutrophil and monocyte counts showed a trend in reduction in the PHA 568487-treated group (Fig. 5F, 5G). At 24 h, BAL neutrophil count was lower in the PHA 568487-treated group (Fig. 5F). BAL protein levels were lower at 4, 12, and 24 h in the PHA 568487-treated groups (Fig. 5H).

Based on the above data, we hypothesized that the vagus nerve- mediated ACh-α7 nAChR pathway regulated transendothelial or epithelial migration of proinflammatory cells and lung epithelial and vascular permeability. Therefore, mice with denervation of vagus were used to test this hypothesis. Mice were first surgically prepared with a unilateral cervical vagotomy (right side) or sham operation and then instilled IT with E. coli (2.5 × 106 CFU). The mice were killed at 4, 12, and 24 h. The right lungs were lavaged. Normal mice were used as controls. BAL was collected to measure the numbers of leukocytes, neutrophils, and monocytes, and BAL protein levels. BAL leukocyte, neutrophil, and monocyte counts were increased at 12 and 24 h in the vagotomized group (Fig. 5IK). BAL protein levels in vagotomized group were also increased compared with the sham group at 4 and 12 h (Fig. 5F).

α7 nAChR pathway negatively regulates MIP-2 production

To study the effects of nicotine (a nonselective agonist of α7 nAChR) on MIP-2 production in BAL and circulation, mice were pretreated with either nicotine (0.4 mg/kg, i.p.) or saline 15 min before IT challenge with E. coli (2.5 × 106 CFU). The therapy was repeated every 6 h. The mice were killed at 4, 12, and 24 h. Normal mice were used as controls. BAL and plasma were collected to measure MIP-2 levels by ELISA. The MIP-2 levels in the BAL were reduced at 4 and 12 h in the nicotine-treated group compared with the saline group (Fig. 6A). MIP-2 levels in the plasma in the nicotine-treated group also showed a lower trend at 4 h (Fig. 6B).

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 ...

To study the effects of PHA 568487 (a specific agonist of α7 nAChR) on MIP-2 production in BAL and circulation, mice were pretreated with either PHA 568487 (0.4 mg/kg, i.p.) or saline 15 min before IT challenge of E. coli 2.5 × 106 CFU, and then the same therapy was repeated every 6 h. The mice were killed at 4, 12, and 24 h. Normal mice were used as controls. BAL and plasma were collected to measure MIP-2 levels by ELISA. MIP-2 levels in the BAL were reduced at 4 h and showed a lower trend at 12 and 24 h in the PHA 568487-treated group (Fig. 6C). The MIP-2 levels in the plasma in the PHA 568487-treated group were also reduced at 4 h (Fig. 6D). Because vagus nerve signaling via the ACh-α7 nAChR pathway may limit MIP-2 production, we studied the effects of denervation of vagus on the response. First, unilateral cervical vagotomy (right side) was done and then mice were instilled IT with E. coli (2.5 × 106 CFU). The mice were killed at 4, 12, and 24 h, and the right lungs were lavaged. Normal sham-operated mice were used as controls. BAL and plasma were collected to measure MIP-2 levels by ELISA. MIP-2 levels in the BAL were increased at 4 and 12 h in the vagotomized group (Fig. 6E), and MIP-2 levels in the plasma in vagotomized group were also increased compared with the sham group at 4 and 12 h (Fig. 6F).

Nicotine activation of α7 nAChR increases survival in E. coli pneumonia

To test whether activation of α7 nAChR by nicotine affects survival in E. coli pneumonia, mice were challenged IT with E. coli (5 × 106 CFU) and treated with either nicotine (2.4 mg/kg, delivered by a ALZET [Cupertino, CA] osmotic pump implanted under skin) or saline. Mice were followed for 24 h, and survival was increased in the nicotine therapy group compared with saline (Fig. 7A). To address whether MLA, a specific α7 nAChR antagonist, counteracts the effect of nicotine, mice were treated with either nicotine (2.4 mg/kg, via osmotic pump) or nicotine + MLA (4 mg/kg, i.p. at 8 and 16 h). At 24 h, survival in MLA treated mice was reduced compared with the nicotine-only group (Fig. 7B).

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 ...

Cervical vagotomy and α7 nAChR deficiency reduces survival in E. coli pneumonia

To study whether vagus denervation affects survival in E. coli pneumonia, unilateral cervical vagotomy in mice (right side) or a sham operation was performed; E. coli (4 × 106 CFU) was instilled IT, and mice were followed for 48 h. Survival in vagotomized mice was reduced compared with the sham group (Fig. 7C). To determine whether the deficiency of α7 nAChR affects survival from E. coli pneumonia, α7 nAChR+/+ and α7 nAChR−/− mice were instilled IT with E. coli (4 × 106 CFU) and followed for 12 h. Survival in α7 nAChR−/− mice was reduced compared with α7 nAChR+/+ mice (Fig. 7D).

Discussion

The current study demonstrates for the first time that activation of α7 nAChR in alveolar macrophages and neutrophils is a critical mechanism that decreases the lung inflammatory response to either E. coli pneumonia or LPS challenge in mice. Activation of α7 nAChR in these cells reduced the production of proinflammatory cytokines and chemokines (especially MIP-2) and neutrophil transmigration, and thereby reduced lung injury and mortality. There was a causal relationship between α7 nAChR activation and the reduction in lung inflammation, because lung MIP-2 production, lung neutrophil infiltration, and mortality were increased in α7 nAChR−/− and vagus-denervated mice.

α7 nAChR+CD11b+ alveolar macrophages and α7 nAChR+ Gr1+ neutrophils activated by α7 nAChR agonists induced a decreased production of MIP-2, TNF-α, and HMGB1 consistent with an important role of α7 nAChR activation in mediating the inactivation of inflammatory mediator production. The question arises as to the mechanisms of vagal innervation-induced protection mediated by α7 nAChR receptors in alveolar macrophages and neutrophils. It is known that airway epithelia and periepithelial tissue are vagally innervated (6), thus release of ACh in airways may activate α7 nAChR+ inflammatory cells accumulating in the airways. The ACh concentration, however, did not increase in BAL in our models consistent with rapid hydrolysis of ACh (half-life, 2 min) by AChE occurring after ACh release from nerve endings (24). This possibility was assessed by measuring AChE activity in proinflammatory cells and BAL choline concentration (50 nmol/ml); thus, it is likely that α7 nAChR+CD11b+ and α7 nAChR+Gr1+ cells are activated in the local milieu at sites of vagal innervation. Vagus denervation failed to activate α7 nAChR+CD11b+ and α7 nAChR+Gr1+, resulting in persistent MIP-2 production and neutrophil infiltration, and worsened lung inflammation and injury compared with control animals.

In LPS and E. coli pneumonia ALI mouse models, alveolar macrophages are the central effector cells in the production of proinflammatory cytokines (25, 26), which initiate and amplify neutrophil transmigration into the lungs to mediate inflammation and injury (2729). Infiltrated neutrophils may function in a feed-forward manner to generate MIP-2 and promote further neutrophil transmigration (30, 31). Using exogenous α7 nAChR agonists to stimulate α7 nAChR+CD11b+ and α7 nAChR+Gr1+ cells in injured lungs disrupted this feed-forward inflammatory loop. In this sense, activation of α7 nAChR by endogenous ACh represents a homeostatic negative feedback mechanism that probably fine-tunes the lung-host defense system by dampening neutrophil transmigration, and thereby mitigating lung inflammation and injury.

E. coli pneumonia shares similar pathogenic mechanisms with LPS-induced ALI, including activation of alveolar macrophages, increased production of early and late proinflammatory mediators, and neutrophil transmigration (17, 24, 29). Recently, studies have shown that nicotine (nonspecific), DMAB (partial), PNU (specific), and PHA 568487 (specific) are α7 nAChR agonists, but these chemical preparations are different in molecular structure and specificity. To compare the effects of α7 nAChR agonists on proinflammatory cytokine production and lung inflammation, we administrated α7 nAChR agonists (nicotine, DMAB, PNU, or PHA 568487) to disrupt the propagation of inflammation by reducing MIP-2 production in α7 nAChR+CD11b+ and α7 nAChR+Gr1+ cells, decreasing pulmonary edema and neutrophil infiltration in the early time frame(4 and 12h),and thereby enhancing the survival of pneumonia in the nicotine treated mice. The role of vagal signaling was established by the vagus denervation study, which increased MIP-2 production in BAL and lung neutrophil infiltration at these early time points. These findings demonstrate that regulation of proinflammatory responses (e. g., MIP-2 production) by α7 nAChR-cholinergic antiinflammatory pathway plays an important role in the early stage of lung inflammation induced by E. coli pneumonia or LPS challenge in mice.

Studies of Streptococcus pneumoniae–induced pneumonia showed that nicotine treatment increased bacterial burden and worsened lung inflammation at 24 h (32), results that are different from the lung antiinflammatory effects of activating α7 nAChR and the previous observations that activation of α7 nAChR protected against Gram-negative sepsis (1, 5, 1215, 33). A reason for this apparent difference in results with live bacteria may be that TLR4 recognizes the Gram-negative product LPS, whereas TLR2 recognizes Gram-positive components (34); therefore, the protective effects by α7 nAChR may occur downstream of TLR4 activation. In the sepsis models (endotoxemia and cecal ligation and puncture), the protective effects of α7 nAChR activation were mediated by reducing HMGB1, a late mediator of inflammation (5). In this study, we found that acute lung inflammation and injury induced by Gram-negative bacteria can be regulated by the cholinergic antiinflammatory pathway. α7 nAChR+CD11b+ and α7 nAChR+Gr1+ cells might be important players in this pathway, which deserves further study.

In summary, activation of α7 nAChR inhibited production of proinflammatory cytokines (MIP-2, TNF-α, HMGB1) in alveolar macrophages and neutrophils. α7 nAChR agonists reduced pulmonary edema and lung inflammation in both LPS-induced ALI and E. coli pneumonia; it also increased survival from E. coli pneumonia. Thus, activation of α7 nAChR may be useful as a novel antiinflammatory strategy to treat ALI.

Acknowledgments

We thank Jae Woo Lee, Sandra Brady, and Xiaopei Gao for suggestions and technical assistance.

This work was supported by National Heart, Lung, and Blood Institute HL-51854 and HL-51856 (to M.M.); R01-HL-045638 (to A.M.); and a Parker B. Francis Award (to X.S.).

Abbreviations used in this paper

ACh
acetylcholine
AChE
acetylcholinesterase
ALI
acute lung injury
BAL
bronchoalveolar lavage
DMAB
dimethylaminobenzaldehyde
ELW
extravascular lung water
EPE
extravascular plasma equivalent
IT
intratracheal
MLA
methyllycaconitine
α7 nAChR
α7 nicotinic acetylcholine receptor
PNU
PNU 282987
WT
wild type

Footnotes

Disclosures The authors have no financial conflicts of interest.

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