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Requisite Role of the Cholinergic α7 Nicotinic
Acetylcholine Receptor Pathway in
Suppressing Gram-Negative Sepsis-Induced
Acute Lung Inflammatory Injury
Xiao Su, Michael A. Matthay and Asrar B. Malik
J Immunol 2010; 184:401-410; Prepublished online 30
November 2009;
doi: 10.4049/jimmunol.0901808
http://www.jimmunol.org/content/184/1/401
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References
The Journal of Immunology
Requisite Role of the Cholinergic a7 Nicotinic Acetylcholine
Receptor Pathway in Suppressing Gram-Negative
Sepsis-Induced Acute Lung Inflammatory Injury
Xiao Su,*,† Michael A. Matthay,† and Asrar B. Malik*
T
he cholinergic antiinflammatory pathway (1) has been
described in a series of experiments showing that vagus
nerve stimulation attenuated the systemic inflammatory
response to endotoxin (1–3). The a7 nicotinic acetylcholine receptor (a7 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 a7 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 a7 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-kB translocation
and I-kB 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
*Center for Lung and Vascular Biology and Department of Pharmacology, University
of Illinois at Chicago, Chicago, IL 60612; and †Cardiovascular Research Institute,
University of California, San Francisco, CA 94143
Received for publication June 8, 2009. Accepted for publication October 23, 2009.
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.).
Address correspondence and reprint requests to Dr. Xiao Su, Center for Lung
and Vascular Biology, Department of Pharmacology, University of Illinois at
Chicago, E403 MSB, 835 S. Wolcott Ave, Chicago, IL 60612. E-mail address:
[email protected]
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; a7 nAChR, a7 nicotinic acetylcholine receptor; PNU, PNU 282987; WT, wild type.
Copyright Ó 2009 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901808
also indicated a possible role of specific a7 nAChR agonists GTS21 (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 a7 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 a7 nAChR on the production of proinflammatory cytokines and chemokines, whether a7 nAChR affects
the ALI response induced by endotoxin and Escherichia coli
pneumonia, and whether antagonism or deficiency of a7 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 a7 nAChR) were purchased from Tocris Bioscience (Ellisville,
MO) and dissolved in 0.9% saline before each experiment. H-302, an antia7 nAChR Ab used to detect a7 nAChR of mouse and human origin, was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE antimouse 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). a7 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.
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Although activation of the a7 nicotinic acetylcholine receptor (a7 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
a7 nAChR in mediating endotoxin- and live Escherichia coli–induced ALI in mice. Because we found that a7 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 a7 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, a7 nAChR2/2 mice developed severe lung injury and
had higher mortality compared with a7 nAChR+/+ mice. The immunomodulatory cholinergic a7 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 a7 nAChR may be a promising strategy for treatment of sepsis-induced
ALI. The Journal of Immunology, 2010, 184: 401–410.
ACTIVATION OF a7 nAChR PREVENTS ACUTE LUNG INJURY
402
Isolation and culture of alveolar macrophages
Unilateral vagotomy
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 3 105 /ml). a7 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-a and MIP-2, and after 20 h for
HMGB1 measurements.
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. Thewound was closed
and sutured. The respiration rhythm was not affected by unilateral vagotomy.
Neutrophil isolation and culture
BAL and plasma samples were obtained from mice at designated time
points. Supernatant was used to measure protein and cytokines. TNF-a 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 oDianisidine HCl (Sigma-Aldrich) and H2O2 to measure optical density by
a spectrophotometer at 405 nm.
HMGB1 enzyme-linked immunosorbent assay
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 mCi [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 3 106 CFU) for longer experiments (24 h) to ensure that there
was no death in both control and treated groups; 3) E. coli (5 3 106 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 3 106 CFU) for the survival
study with vagotomized or a7 nAChR 2/2 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 ¼ QW exp =Qd exp 3 Qd exp
− QW control =Qd control 3 Qd control
3 1000 ml
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.
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 5,59-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
(DOD405nm) 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 a7 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 a7
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 mm) 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 a7 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).
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As described (20), mice were euthanized by cervical dislocation, and the
bone marrow from the femurs and tibias was flushed with PBS using a 25gauge 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-mm 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 3 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). a7 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 measuring MIP-2 or TNF-a concentration.
BAL and plasma cytokine and protein concentration
measurements
The Journal of Immunology
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 a7
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).
403
olar macrophages were pretreated separately with PBS, 1027 M
nicotine or ACh, 1026 M nicotine or ACh, or 1025 M nicotine or
ACh. At 20 min, the cells from these four groups were stimulated
with LPS (3 mM) 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 6 204 versus 28 6 6 pg/ml in the PBS stimulated group.
Nicotine reduced HMGB1 by 25 and 32% at 1026 and 1025 M,
respectively (Fig. 1M). ACh suppressed HMGB1 by 30% at 1027 to
1025 M (Fig. 1N).
a7 nAChR Western blotting of BAL samples
As described (19), denatured proteins (20 mg) 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-a7 nAChR Ab (H-302). Membranes were exposed to HRPlabeled Ab and developed with an ECL kit (Amersham, Piscataway, NJ).
Statistical analysis
Results
Alveolar macrophages express a7 nAChR
In normal BAL cells, alveolar macrophages are dormant or resting
cells (Fig. 1A). a7 nAChR immunoreactivity was found in the cell
membrane and cytoplasm (Fig. 1B, 1C). Western blotting demonstrated that normal BAL cells (mainly alveolar macrophages)
expressed a7 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 a7 nAChR, normal BAL
cell pellets were double stained with anti-mouse CD11b (Mac-1,
a surface marker of macrophages) and a7 nAChR Abs to perform
flow cytometry. We observed that 14.7 6 4.8% of normal BAL
cells coexpressed CD11b and a7 nAChR (Fig. 1E, 1F). To investigate coexpression of a7 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 a7 nAChR and CD11b was found in injured lung
sections (Fig. 1G–J).
Effects of nicotine and MLA on TNF-a and MIP-2 production
in alveolar macrophages
To test whether activation of a7 nAChR by nicotine in alveolar
macrophages alters proinflammatory cytokine production and
whether MLA (a specific a7 nAChR antagonist) reverses the
effects of nicotine, alveolar macrophages were pretreated separately with PBS, 1026 M nicotine, or 1026 M + MLA 1026 M
nicotine. After 20 min, cells from these three groups were stimulated with LPS (3 mM) for 12 h to measure TNF-a and MIP-2 in
the media. Untreated cells with PBS challenge were used
as controls. LPS stimulation increased MIP-2 (545 6 24 versus
31 6 18 pg/ml in PBS group) and TNF-a (704 6 6 versus 16 6
7 pg/ml in PBS group) production in alveolar macrophages.
Nicotine reduced LPS-induced MIP-2 and TNF-a production.
MLA counteracted this effect of nicotine on TNF-a and MIP-2
production (Fig. 1K, 1L).
Effects of nicotine and ACh on HMGB1 production in alveolar
macrophages
To test whether activation of a7 nAChR agonists by nicotine and
ACh affects HMGB1 production in alveolar macrophages, alve-
To determine whether neutrophils express a7 nAChR, we collected BAL cells from E. coli pneumonia (2.5 3106 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 a7 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 a7 nAChR in the cell
membrane and cytoplasm (Fig. 2C, 2D, segmented neutrophils).
To confirm that neutrophils expressed a7 nAChR, BAL cell pellets (from pneumonia mice at 12 h) were double stained with antimouse Gr1 (a surface marker of neutrophils) and a7 nAChR Abs
to perform flow cytometry. We observed that 43.6 6 1.8% of BAL
cells coexpressed Gr1 and a7 nAChR (Fig. 2E, 2F), confirming
the finding that neutrophils express a7 nAChR. To investigate
coexpression of a7 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
a7 nAChR and Gr1 was found in injured lung sections (Fig. 2G–J).
a7 nAChR activation downregulates TNF-a and MIP-2
production in neutrophils
To investigate whether activation of a7 nAChR agonists by nicotine, DMAB (a novel partial a7 nAChR agonist), or PNU (a
highly specific a7 nAChR agonist, Ki 27 nM) affect MIP-2 production in neutrophils, neutrophils were pretreated separately with
PBS; 1027 M nicotine, DMAB or PNU; 1026 M nicotine, DMAB
or PNU; or 1025 M nicotine, DMAB or PNU. At 20 min, cells
from these four groups were stimulated with LPS (3 mM) 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 6 84 pg/ml)
compared with 11 6 3 pg/ml in PBS group. Nicotine reduced
MIP-2 by 23–40% at 1027 to 1026 M, and 50% at 1025 M (Fig.
2K). DMAB inhibited MIP-2 by 51–53% at 1027 to 1025 M (Fig.
2L), and PNU suppressed MIP-2 by 49% at 1027 to 1026 M and
30% at 1025 M (Fig. 2M).
a7 nAChR-deficient neutrophils propagate proinflammatory
cytokine production
To test whether deficiency of a7 nAChR in neutrophils facilitates
proinflammatory cytokine production, the isolated bone marrow
neutrophils from a7 nAChR+/+ and a7 nAChR2/2 mice were
stimulated with LPS (3 mM). The media was collected at 12 h.
After LPS stimulation, TNF-a and MIP-2 concentrations in the
media from a7 nAChR2/2 neutrophils were increased compared
with a7 nAChR+/+ neutrophils (Fig. 2N, 2O).
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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 6 SD.
Neutrophils express a7 nAChR
404
ACTIVATION OF a7 nAChR PREVENTS ACUTE LUNG INJURY
a7 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).
To compare the effects of three different a7 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) were given saline, nicotine, DMAB, or PNU (0.4 mg/kg, i.p., every
6 h) separately and then euthanized after 24 h. The control mice re-
ceived 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. 3I–K).
a7 nAChR modulates lung inflammation and edema by
suppressing inflammatory mediator production in local milieu
To test whether activation of a7 nAChR reduces ELW and 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 BAL TNFa and MIP-2 levels and neutrophil numbers. Nicotine reduced the
BAL neutrophil counts and TNF-a and MIP-2 levels compared with
the saline group at 4 h (Fig. 4C–E).
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FIGURE 1. Alveolar macrophages express a7 nAChR. A, Morphology of normal BAL cells. B and C, a7 nAChR immunofluorescence in normal BAL
cells (mainly alveolar macrophages). B, Control a7 nAChR Ab. C, Rabbit anti-mouse a7 nAChR Ab (3 200; second Ab labeled with Alex Fluor-488). D,
Western blot for a7 nAChR in normal BAL cells. PC12 cells and rat brain extract were used as positive controls. E and F, a7 nAChR and CD11b are
coexpressed in normal BAL cells identified by flow cytometry. E, Control Abs. F, CD11b and a7 nAChR Abs (repeated 4 times). G–J. a7 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-a7
nAChR (I) Abs staining followed by DAPI (3 200; 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 a7 nAChR
antagonist). K, MIP-2. L, TNF-a. Cells were treated with PBS, nicotine, or nicotine + MLA, then stimulated with LPS. pp , 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. pp , 0.05, ppp , 0.01 versus LPS only; n = 3–5 in each group. Data are mean 6 SD.
The Journal of Immunology
405
To address whether activation of a7 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 3 106 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 a7
nAChR attenuates lung inflammation and injury by reducing
HMGB1 locally, also in the late time frame. To address whether
a lack of a7 nAChR worsens pulmonary edema, a7 nAChR+/+ and
a7 nAChR2/2 mice were instilled IT with E. coli (2.5 3 106 CFU)
and sacrificed at 12 h. ELW in a7 nAChR2/2 mice was 2.3-fold
higher compared with a7 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 3 106 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 6 2.4 nmol/ml) and E. coli pneumonia
groups (3.2 6 2.1 nmol/ml) at 12 h.
a7 nAChR pathway regulates proinflammatory cell
transmigration and transalveolar permeability
To study the effects of nicotine (a nonselective agonist of a7 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 3 106 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 6 0.34 k/ml versus 0.62 6 0.14 k/ml) and
neutrophil counts (0.125 6 0.017 versus 0.065 6 0.017 k/ml) 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. 5A–C). At 24 h, there were no differences in leukocyte, neutrophil, and monocyte counts (Fig. 5A–C)
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FIGURE 2. Neutrophils express a7 nAChR. A, Cytologic change of BAL cells (enriched in neutrophils) from E. coli pneumonia mice at 12 h. B, Western
blot for a7 nAChR in BAL cells from normal and E. coli pneumonia at 4 h (equal quantity loading in protein). C and D, a7 nAChR immunofluorescence in
pneumonia BAL cells (segmented neutrophils, arrow heads). C, Control a7 nAChR Ab. D, Rabbit anti-mouse a7 nAChR Ab (3 200; second Ab labeled
with Alex Fluor-488). E and F, Neutrophils (Gr1+ cells) coexpress a7 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-a7 nAChR and Gr1 Abs. G–J. a7 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-a7 nAChR (I) Abs staining followed
by DAPI (3 200; second Ab labeled with Alexa Fluor-594 and Fluor-488). K–M. Effect of nicotine, DMAB, and PNU (specific a7 nAChR agonist) on
production of MIP-2 in LPS-stimulated neutrophils. K, Nicotine. L, DMAB. M, PNU. pp , 0.05, ppp , 0.01 versus LPS only; n = 5–6 in each group. N and
O, Deficiency of a7 nAChR in neutrophils increases production of proinflammatory cytokines under LPS-stimulation. N, TNF-a. O, MIP-2. ppp , 0.01
versus WT; n = 8 in each group. Data are mean 6 SD.
406
ACTIVATION OF a7 nAChR PREVENTS ACUTE LUNG INJURY
compared with nicotine- and saline-treated groups. BAL protein
levels were diminished at 4, 12, and 24 h compared with nicotineand saline-treated groups (Fig. 5D).
To test the effects of PHA 568487 (a specific agonist of a7
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 3 106 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 6 0.02 versus 0.16
6 0.04 k/ml) and neutrophil counts (0.09 6 0.05 verus 0.01 6 0 k/
ml) 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 568487treated groups (Fig. 5H).
Based on the above data, we hypothesized that the vagus nerve–
mediated ACh-a7 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 3 106 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. 5I–K). BAL protein levels in vagotomized group were also
increased compared with the sham group at 4 and 12 h (Fig. 5F).
a7 nAChR pathway negatively regulates MIP-2 production
To study the effects of nicotine (a nonselective agonist of a7
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 3 106 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
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FIGURE 3. Activation of a7 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). pp , 0.05 versus saline; n = 5 in each group. Data were pooled from 2 different experiments.
D–F. Activation of a7 nAChR by its agonists nicotine, DMAB, or PNU attenuates LPS-induced ALI. Body weight loss at 24 h (D); pp , 0.05 versus saline
+ LPS. Lung myeloperoxidase activity at 48 h (E); ppp , 0.01 versus saline only; #p , 0.05 versus saline + LPS. ELW at 48 h (F); pp , 0.05 versus saline
+ LPS; n = 5 in each group. Data were pooled from 4 different experiments. Data are mean 6 SD. G–K. Representative photomicrographs of lung histology
in LPS-induced ALI at 24 h treated with a7 nAChR agonists nicotine, DMAB, or PNU. Magnification 3400; scale bar = 50 mm; n = 2–3 in each group.
The Journal of Immunology
407
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 nicotinetreated group also showed a lower trend at 4 h (Fig. 6B).
To study the effects of PHA 568487 (a specific agonist of a7 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 3 106 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 568487treated group were also reduced at 4 h (Fig. 6D). Because vagus nerve
signaling via the ACh–a7 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 ITwith E. coli (2.5 3 106 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 a7 nAChR increases survival in E. coli
pneumonia
To test whether activation of a7 nAChR by nicotine affects survival
in E. coli pneumonia, mice were challenged IT with E. coli (5 3 106
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 a7 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).
Cervical vagotomy and a7 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 3 106 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 a7 nAChR affects
survival from E. coli pneumonia, a7 nAChR+/+ and a7 nAChR2/2
mice were instilled IT with E. coli (4 3 106 CFU) and followed for
12 h. Survival in a7 nAChR2/2 mice was reduced compared with
a7 nAChR+/+ mice (Fig. 7D).
Discussion
The current study demonstrates for the first time that activation of
a7 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 a7
nAChR in these cells reduced the production of proinflammatory
cytokines and chemokines (especially MIP-2) and neutrophil
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FIGURE 4. a7 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. ppp , 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-a (C), MIP-2 (D), and neutrophil counts (E) in the
BAL in E.coli pneumonia at 4 h. pp , 0.05; ppp , 0.01 versus saline controls; n = 4–6 in each group. Data were pooled from three different experiments. F
and G, Activation of a7 nAChR by nicotine attenuates E. coli pneumonia at 24 h. F, ELW. G, Levels of HMGB1 in the lung homogenate. pp , 0.05; n = 4–
6 in each group. H, Deficiency of a7 nAChR worsens pulmonary edema in E. coli pneumonia. pp , 0.05 for a7 nAChR+/+ versus a7 nAChR2/2 mice; n =
3 in each group. Data are mean 6 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. ppp , 0.01 versus normal mice; n = 6 in each group. Data are mean 6 SD.
408
ACTIVATION OF a7 nAChR PREVENTS ACUTE LUNG INJURY
transmigration, and thereby reduced lung injury and mortality.
There was a causal relationship between a7 nAChR activation and
the reduction in lung inflammation, because lung MIP-2 production, lung neutrophil infiltration, and mortality were increased
in a7 nAChR2/2 and vagus-denervated mice.
a7 nAChR+CD11b+ alveolar macrophages and a7 nAChR+
Gr1+ neutrophils activated by a7 nAChR agonists induced a decreased production of MIP-2, TNF-a, and HMGB1 consistent
with an important role of a7 nAChR activation in mediating the
inactivation of inflammatory mediator production. The question
arises as to the mechanisms of vagal innervation-induced protection mediated by a7 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 a7 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 a7 nAChR+CD11b+
and a7 nAChR+Gr1+ cells are activated in the local milieu at sites
of vagal innervation. Vagus denervation failed to activate a7
nAChR+CD11b+ and a7 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 (27–29). Infiltrated neutrophils may function in a feedforward manner to generate MIP-2 and promote further neutrophil
transmigration (30, 31). Using exogenous a7 nAChR agonists to
stimulate a7 nAChR+CD11b+ and a7 nAChR+Gr1+ cells in injured
lungs disrupted this feed-forward inflammatory loop. In this sense,
activation of a7 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,
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FIGURE 5. Activation of a7 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. pp , 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. pp , 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.
pp , 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 6 SD.
The Journal of Immunology
and neutrophil transmigration (17, 24, 29). Recently, studies have
shown that nicotine (nonspecific), DMAB (partial), PNU (specific), and PHA 568487 (specific) are a7 nAChR agonists, but
these chemical preparations are different in molecular structure
and specificity. To compare the effects of a7 nAChR agonists on
proinflammatory cytokine production and lung inflammation, we administrated a7 nAChR agonists (nicotine, DMAB, PNU, or PHA
568487) to disrupt the propagation of inflammation by reducing MIP-2
production in a7 nAChR+CD11b+ and a7 nAChR+Gr1+ cells, decreasing pulmonary edema and neutrophil infiltration in the early time
frame (4 and 12 h), 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 a7 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 a7 nAChR
FIGURE 7. Activation of a7 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. pp , 0.05
for nicotine versus saline treated mice; n = 12 in each group. Data were
pooled from two different experiments. B, Antagonism of a7 nAChR by its
specific antagonist MLA counteracts the protective effects of nicotine on
survival in 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. pp , 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. pp ,
0.05 for vagotomy (n = 30) versus sham (n = 30). Data were pooled from 5
different experiments. D, Deficiency of a7 nAChR worsens reduces survival
in E. coli pneumonia; n = 5 a7 nAChR+/+ mice; n = 4 a7 nAChR2/2 mice; pp
, 0.05 for a7 nAChR+/+ versus a7 nAChR2/2 mice.
and the previous observations that activation of a7 nAChR protected against Gram-negative sepsis (1, 5, 12–15, 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 a7 nAChR may occur downstream of TLR4 activation. In
the sepsis models (endotoxemia and cecal ligation and puncture), the
protective effects of a7 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. a7 nAChR+CD11b+ and a7 nAChR+Gr1+ cells might be important players in this pathway, which deserves further study.
In summary, activation of a7 nAChR inhibited production of
proinflammatory cytokines (MIP-2, TNF-a, HMGB1) in alveolar
macrophages and neutrophils. a7 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 a7 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.
Disclosures
The authors have no financial conflicts of interest.
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ACTIVATION OF a7 nAChR PREVENTS ACUTE LUNG INJURY