Pulmonary, gastrointestinal and urogenital pharmacology

Cigarette smoke alters non-neuronal cholinergic system componentsinducing MUC5AC production in the H292 cell line

Angela Marina Montalbano a, Giusy Daniela Albano a,b, Giulia Anzalone a, Anna Bonanno a,Loredana Riccobono a, Caterina Di Sano a, Rosalia Gagliardo a, Liboria Siena a,Michael Paul Pieper c, Mark Gjomarkaj a, Mirella Profita a,n

a Institute of Biomedicine and Molecular Immunology “A. Monroy” (IBIM), Italian National Research Council (CNR), Palermo, Italyb Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S.), Sezione di Pneumologia, University of Palermo, Palermo, Italyc Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

a r t i c l e i n f o

Article history:Received 29 November 2013Received in revised form11 April 2014Accepted 16 April 2014Available online 2 May 2014

Keywords:Cigarette smokeCholine Acetyl-TransferaseAnticholinergic drugsMucin production.

a b s t r a c t

Cigarette smoke extract (CSE) affects the expression of Choline Acetyl-Transferase (ChAT), muscarinicacetylcholine receptors, and mucin production in bronchial epithelial cells. Mucin 5AC (MUC5AC),muscarinic acetylcholine receptor M3, ChAT expression, acetylcholine levels and acetylcholine bindingwere measured in a human pulmonary mucoepidermoid carcinoma cell line (H292) stimulated with CSE.We performed ChAT/RNA interference experiments in H292 cells stimulated with CSE to study the roleof ChAT/acetylcholine in MUC5AC production. The effects of Hemicholinium-3 (HCh-3) (50 μM)(a potent and selective choline uptake blocker) and Tiotropium bromide (Spirivas) (100 nM), alone orin combination with Salmeterol (SL) and Fluticasone propionate (FP), were tested in this model. MUC5AC,muscarinic acetylcholine receptor M3, ChAT, acetylcholine expression and acetylcholine bindingsignificantly increased in H292 cells stimulated with CSE (5%) compared to untreated cells. HCh-3reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. ChAT/RNAinterference eliminated the effect of CSE on MUC5AC production. FP reduced ChAT and acetylcholinebinding in unstimulated cells, while showing a partial effect in CSE stimulated cells. SL increased theChAT expression and acetylcholine binding in H292 cells stimulated with or without CSE. Tiotropium,alone or together with FP and SL, reduced acetylcholine binding and MUC5AC production in H292 cellsstimulated with CSE. CSE affects the ChAT/acetylcholine expression, increasing MUC5AC production inH292 cells. Pharmacological treatment with anticholinergic drugs reduces the secretion of MUC5ACgenerated by autocrine acetylcholine activity in airway epithelial cells.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Mucus protects the underlying airway epithelium from dehydra-tion, pathogens, and chemical and particulate irritants. The airwaysurface is covered with mucus secretions produced by goblet cellsand submucosal glands. Mucins are divided into the following twogroups: membrane-associated and secreted mucins. Mucins foundin secretions include MUC2, MUC5AC, MUC5B, MUC7, and MUC8(Caramori et al., 2009; Davies et al., 1996, Thornton et al., 1996).

MUC5AC is the mucin most expressed in the airway epithelium,and cigarette smoke, a source of oxidative stress within theairways, is able to induce mucin synthesis in airway epithelialcells and subsequent goblet cell production (Takeyama et al.,2001). The mucociliary dysfunction component of ChronicObstructive Pulmonary Disease (COPD) is due to mucus hyperse-cretion coupled with a decrease in mucus transport, and it is animportant pathophysiological feature requiring appropriate treat-ment (Rogers, 2005). Acetylcholine, synthesized from choline byCholine Acetyl-Transferase (ChAT), can exert its pathophysiologicaleffects in the airway epithelial cells by activating muscarinicacetylcholine receptors which contribute to the inflammatoryprocesses of COPD (Gosens et al., 2004; Profita et al., 2005;Bateman et al., 2009; Profita et al., 2009). Muscarinic acetylcholinereceptors have a potentially important role in the control ofneuronally mediated airway mucus secretion, with the mainreceptor type responsible for this action being the muscarinic

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/ejphar

European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.04.0220014-2999/& 2014 Elsevier B.V. All rights reserved.

Abbreviations: ChAT, Choline Acetyl-Transferase; COPD, Chronic ObstructivePulmonary Disease; CS, cigarette smoke; CSE, cigarette smoke extract; FBS, fetalbovine serum; FP, fluticasone propionate; H292 cells, human pulmonarymucoepidermoid carcinoma cell line; HCh-3, hemicholinium-3; LABAs, long-actingß2-agonists; MUC5AC, mucin 5 AC; SL, salmeterol

n Corresponding author.E-mail address: profita@ibim.cnr.it (M. Profita).

European Journal of Pharmacology 736 (2014) 35–43

acetylcholine receptor M3 (Rogers, 2002). CSE increases the pro-inflammatory activity of human bronchial epithelial cells andpromotes the cellular response to acetylcholine affecting theexpression of ChAT and muscarinic acetylcholine receptor M3.Tiotropium may prevent the pro-inflammatory events generatedby acetylcholine together with CSE (Profita et al., 2011). Only a fewstudies, conducted in vitro, have tried to explain the relationshipbetween the production of MUC5AC and the non-neuronal com-ponents of the cholinergic system, and although cigarette smokeis thought to be involved in mucus hypersecretion in COPD,the mechanisms underlining this phenomenon are not known(Takeyama et al., 2001).

Current international guidelines recommend using bronchodi-lators in the symptomatic management of COPD and includeβ2-adrenoceptor agonists, anti-cholinergics and methylxanthinesin the treatment of stable COPD (Rogers, 2005). Corticosteroids areoften used in combination with β2-agonist and anticholinergicbronchodilators in the treatment of COPD (Johnson, 2005). A rangeof studies have shown that treatment with inhaled long-actingβ2-agonists (LABAs) has beneficial effects on lung function (Mahleret al., 1999; Jones and Bosh, 1997; Aalbers et al., 2002). However,it has been observed that β2-agonists stimulate airway mucinoutput in experimental animal models rather than in humantissue (Phipps et al., 1982). On the other hand, it is unlikelythat bronchodilatation alone could cause this phenomenon aslong-acting β2-agonist therapy is, by itself, unable to reduceexacerbation rates and severity to a clinically meaningful extent(Calverley et al., 2007). Anticholinergic drugs, including Tiotro-pium, are currently used for the treatment of COPD, and preclinicalstudies have demonstrated that stimulation with cholinergicagonists increases mucociliary clearance (Cammer et al., 1974;Gatto, 1993; Berger et al., 1978). Anticholinergics block muscarinicreceptors on airway secretory cells and smooth muscle and so,theoretically, may reduce vagal tone and mucus secretion in COPDfacilitating cough-induced mucus clearance (Abad et al., 2003,Coulson and Fryer, 2003). Consistent with this suggestion is theobservation that Tiotropium reduces levels of mucus secretion inpatients with COPD (Tamaoki et al., 1994).

The aim of the present study was to characterize the potentialrole of non-neuronal components of the cholinergic system onMUC5AC production in bronchial epithelial cells stimulated withCSE. In addition, we investigated the effects of the combination ofcorticosteroids, β2-agonist and anticholinergic bronchodilators onmucin production in the same model.

2. Materials and methods

2.1. Cell cultures

The human pulmonary mucoepidermoid carcinoma cell line(H292) used for these studies is a valid in vitro model for evaluatingthe functional properties of bronchial epithelial cells in inflamma-tion and repair processes (Newland and Richter, 2008). H292cells were cultured as adherent monolayer in RPMI 1640 medium(Euroclone, Milan, Italy), supplemented with HEPES (25 mM),1% MEM (non-essential aminoacids, Euroclone), 2 mM L-glutamine,penicillin (100 U/ml) and streptomycin (100 μg/ml, Euroclone), and10% heat-inactivated (56 1C, 30 min) fetal bovine serum (FBS)at 37 1C in a humidified 5% CO2 water jacketed incubator.

2.2. CSE preparation

Commercially available cigarettes (Marlboro Red Label, PhilipMorris International, Switzerland) were used in this study. CSEwas prepared and further diluted to the required concentrations in

fresh culture medium as previously described (Profita et al., 2011).The viability of the cells exposed to CSE was evaluated by trypanblue exclusion dye assay.

2.3. H292 cell stimulation with CSE

H292 cells (1.0�105 cells/ml) were seeded in 6-well cell cultureplates (Falcon, Becton Dickinson, CA, USA) and grown in 1 ml ofRPMI 1640 (10% FBS for 72 h) until 70–80% confluence. The mediumwas then removed and replaced with FBS-free complete RPMI 1640for an additional 24 h (5% CO2 at 37 1C) to make the cells quiescent.The cells were treated for 18 h with CSE (0.5–10%) or medium aloneto evaluate the expression of MUC5AC, ChAT, and muscarinicacetylcholine receptor M3. The cells were treated for 18 h withCSE (5%) or medium alone to evaluate acetylcholine concentrationand binding. To clarify the role of ChAT and autocrine/paracrineacetylcholine, we tested the effect of Hemicholinium-3 (HCh-3), apotent and selective choline uptake blocker (50 μM) (Sigma-Aldrich,Milan, Italy), on MUC5AC production and endogenous acetylcholinebinding in H292 cells stimulated in the presence and absenceof CSE 5%.

2.4. H292 cell stimulation with acetylcholine

H292 cells were plated and grown, as described above, andstimulated with acetylcholine (100 nM) (Sigma-Aldrich, Milan,Italy) in the presence or absence of CSE 5% for 18 h to evaluateMUC5AC expression by flow-cytometry.

2.5. H292 cell stimulation with fluticasone propionateand Salmeterol

Cultured H292 cells were stimulated with Fluticasone Propio-nate (FP) (10�9–10�6 M) or the long-acting β2-agonist (LABA)Salmeterol (SL) (10�9–10�6 M) in 1 ml of RPMI 1640 FBS-free for18 h to evaluate the dose–response, and cells were collected forChAT expression by flow-cytometry analysis. Furthermore, thecells were stimulated with and without CSE (5%) under FBS-freeconditions (5% CO2, 37 1C) in the presence or absence of theselected dose of FP (10�8 M) and SL (10�8 M), alone or incombination with Tiotropium bromide (Spirivas) (100 nM) (Boeh-ringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany).

2.6. Flow-cytometry analyses of ChAT, muscarinic acetylcholinereceptor M3, and MUC5AC

The expression of ChAT, muscarinic acetylcholine receptor M3,and MUC5AC in H292 cells was determined by flow-cytometryanalyses using indirect label immunofluorescence by a FACSCali-bur™ flow cytometer (Becton Dickinson, Mountain View, CA, USA)supported by Cell Quest acquisition and data analysis soft-ware (Becton Dickinson, Mountain View, CA, USA), as previouslydescribed (Profita et al., 2009). Details about the primary andsecondary antibodies used are shown in Table 1. 5�105 cells werepreviously fixed with 4% paraformaldehyde in PBS, washed, andpermeabilized with PBS (containing 0.1% saponin, 1% sodium azideand 10% FBS) to evaluate MUC5AC expression. The cells werestained with 4 μl of the mouse monoclonal anti-MUC5AC antibodyclone 45M1 (200 μg/ml) (Neomarkers, CA, USA) for 30 min at 4 1C,washed twice, and then incubated with a FITC-conjugated rabbitpolyclonal anti-mouse IgG F(abʹ)2 (Dako, Glostrup, Denmark) for30 min at 4 1C. The unbound antibody was washed with cold PBS,and cells were analyzed by flow-cytometer. Cells were consideredMUC5AC positive if their fluorescence 1 (FL1) was greater thana gate set to exclude all cells in the FL1 peak from an isotype-matched control antibody (Kim et al., 2005).

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–4336

2.7. Acetylcholine measurement

We quantified acetylcholine in H292 cell protein extractsstimulated with and without CSE 5% for 18 h by a fluorimetricmethod using a commercial kit (BioVision Research Products, CA,USA). The kit can detect choline (Ch) and total choline (TCh)(by adding acetylcholinesterase to the reaction, convertingacetylcholine to Ch) with a sensitivity of 50 pmol/well by plottingfluorescence readings (Ex/Em 535/587 nm) against the standardcurve. This sensitivity corresponds to the concentration of 1 μM ofTCh or Ch. Acetylcholine was evaluated as the difference betweenTCh and Ch. Fluorescence intensity was read using a Wallac 1420Victor2 multilabel counter (Perkin-Elmer Life Sciences, Turku,Finland). Results were expressed as pmol/μg protein.

2.8. Acetylcholine binding

The cultured cells were collected after trypsin treatment, andapproximately 5�105 cells were washed in cold PBS and incu-bated for 1 h at 4 1C with or without acetylcholine 100 nM. Afterwashing, the cells were incubated with a rabbit polyclonal anti-acetylcholine antibody (Abcam, Cambridge, UK) to test the bindingof endogenous and exogenous acetylcholine. This antibody speci-fically recognizes the acetylcholine bound by glutaric acid and is atool to visualize the acetylcholine bound to biological structuressuch as receptors. An anti-rabbit IgG was used as an isotypenegative control antibody (Dako, Glostrup, Denmark). After wash-ing with cold PBS, a FITC-conjugated polyclonal swine anti-rabbitIgG (Dako, Glostrup, Denmark) was added to the cells for 30 min at4 1C. Fluorescence-positive cells were analyzed using a FACSCali-bur™ flow cytometer (Becton Dickinson, Mountain View, CA, USA).The percentage of positive cells was determined from forward (FS)and sideways (SS) scatter patterns. Non-specific binding andbackground fluorescence were detected by analyzing negativecontrol samples.

2.9. RNA interference in the ChAT pathway

H292 cells were plated in 6-well tissue culture plates andgrown in medium containing 10% FBS until 60–80% confluence.Cell transfection was performed according to the manufacturer'sinstructions (72 h at 37 1C) in siPORT Amine Transfection Agent(Ambion Inc., Texas, USA) diluted in OPTI-MEM (Gibco Invitrogen,Milan, Italy), and ChAT siRNA (20 μM; s-2969 Ambion Inc., Texas,USA) diluted in OPTI-MEM for a final concentration of 30 nM.A siRNA (20 μM; Ambion Inc., Texas, USA), containing a scrambledsequence without significant homology to the human genome,was used as negative control. Finally, cells were stimulated withCSE (5%) for 18 h, and MUC5AC expression was evaluated by flow-cytometry. The silencing efficacy of the RNA interference for ChATwas checked by Western blot analysis and flow-cytometry.

2.10. Statistical analysis

Data are expressed as mean7standard deviation (S.D.). Statis-tical analysis was calculated using one-way analysis of variance(ANOVA) test followed by Fisher's PLDS multiple comparison testto compare three or more experimental conditions obtained fromin vitro experiments, while Student's t test was used to comparetwo different experimental conditions. A P valueo0.05 wasconsidered statistically significant.

3. Results

3.1. CSE increased the expression of MUC5AC, ChAT, and muscarinicacetylcholine receptor M3

H292 cells stimulated for 18 h with CSE 1% and 5% showedsignificantly higher levels of MUC5AC expression in comparison tobaseline conditions (Po0.016; Po0.006 respectively) (Fig. 1A).CSE 1%, 5%, and 10% significantly increased the levels of ChAT(Fig. 1B) and muscarinic acetylcholine receptor M3 expression(Fig. 1C) in H292 cells compared to the baseline levels.

3.2. Stimulation of H292 cells with acetylcholine and CSE increasedacetylcholine binding and MUC5AC protein expression

The stimulation of H292 cells with acetylcholine (100 nM) andCSE 5% for 24 h, alone or in combination, significantly increasedacetylcholine binding when compared to unstimulated cells.Acetylcholine binding was significantly higher in the cells stimu-lated with both acetylcholine and CSE compared to the cellsstimulated with acetylcholine alone (Fig. 2A). The differencesbetween the conditions are underwhelming statistically signifi-cant since we stimulated the H292 with exogenous acetylcholine100 nM in the presence of an increased production of endogenousacetylcholine in the cells stimulated with CSE. Furthermore,stimulation with acetylcholine (100 nM) and CSE 5% for 24 h,alone or in combination, significantly increased MUC5AC expres-sion (Fig. 2B) compared to unstimulated cells. The levels ofMUC5AC were significantly higher in the cells stimulated withboth acetylcholine and CSE compared to the cells stimulated withacetylcholine or CSE. Finally, MUC5AC was significantly increasedin the cells stimulated with CSE than in the cells stimulated withacetylcholine (Fig. 2B). These effects may be associated with theincreased expression of muscarinic acetylcholine receptor M3 dueto stimulation with CSE.

3.3. CSE increased intracellular acetylcholine level and binding

H292 cell stimulationwith CSE 5% for 18 h significantly increasedintracellular levels of acetylcholine (Fig. 3A) and endogenouslyproduced acetylcholine binding (Fig. 3B and C), compared to

Table 1Reagent list for flow-cytometry analyses of ChAT, muscarinic receptor M3, and MUC5AC.

Instrument: FACS Calibur™ flow cytometer (Becton Dickinson)

Type Antibody Fluorochrome Vendor/ Cat. no./ Clone

PrimaryPolyclonal Rabbitanti-human muscarinicreceptorM3 (H-210) Santa Cruz Biotech., Inc/ # sc-9108Monoclonal Mouse anti-ChAT Chemicon (Millipore) # MAB5270/ 1.B3.9B3Monoclonal Mouse anti-MUC5AC Neomarkers/ # MS-145-P/ 45M1

SecondaryPolyclonal Anti-rabbit IgG swine F(abʹ)2 FITC Dako, Glostrup, Denmark/ # F 0054Polyclonal Anti-mouse IgG rabbit F(abʹ)2 FITC Dako, Glostrup, Denmark/ # F 0313

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–43 37

unstimulated cells. These findings suggest that CSE mediated ChATactivity.

3.4. Effect of HCh-3 on MUC5AC expression and acetylcholinebinding in cells stimulated with CSE

The treatment of H292 cells with HCh-3 (50 μM) significantlyreduced the binding of endogenous acetylcholine (Fig. 4A) and theproduction of MUC5AC (Fig. 4B) in cells stimulated with CSE 5%,compared to untreated cells. These results suggest that endogenous

acetylcholine, generated by CSE-mediated ChAT activity, is able toregulate the production of MUC5AC in H292 cells stimulatedwith CSE.

3.5. Effect of ChAT silencing on MUC5AC production in H292 cells

The temporary transfection of H292 cells, stimulated with CSE5%, with ChAT siRNA, generated a significant decrease in MUC5ACproduction compared to unsilenced and scrambled cells (Fig. 5).These results further confirm that ChAT and its related acetylcholine

BaselineCSE 5% CSE 5%

BaselineBaselineCSE 5%

Baseline 0.5 1 5 10CSE (%)

)IFM(

CA5

CU

M

p<0.016p<0.006

ChA

T (M

FI)

0

200

400

600

800

1000

1200

Baseline 0.5 1 5 10CSE (%)

p<0.016

p<0.001p<0.001

0

20

40

60

80

100

120

0.5 1 5 10

Mus

carin

ic re

cept

or M

3 (M

FI)

p<0.018

p<0.001p<0.001

BaselineCSE (%)

0

35

70

105

140

Fig. 1. Dose dependent effect of CSE on MUC5AC, ChAT, and muscarinic acetylcholine receptor M3 protein expression in H292 cell line. Cells were cultured, stimulated (seedetails in “Section 2”) and analyzed by flow-cytometry to evaluate (A) MUC5AC, (B) ChAT, and (C) muscarinic acetylcholine receptor M3 protein expression. Bars representmean7S.D. fluorescence intensity (MFI) of six different experiments. Representative flow-cytometry of the data is shown. Statistical analysis was performed by ANOVAfollowed by Fisher's PLSD correction. Significance was at Po0.05.

p<0.001p<0.001

MU

C5A

C (

MFI

)

0

35

70

105

140

gnidnibenilohclyte

CA

(MFI

)

0

35

70

p<0.001

p<0.003

p<0.033 p<0.017

Baseline ACh100 nM

CSE 5% CSE 5%+

ACh100 nM

ACh100 nM

p<0.001

p<0.001p<0.001

p<0.001

Baseline ACh100 nM

CSE 5% CSE 5%+

Fig. 2. Effect of acetylcholine and CSE stimulation on acetylcholine binding and MUC5AC expression in H292 cell line. Cells were cultured, stimulated (see details in“Section 2”) and analyzed by flow-cytometry to evaluate (A) acetylcholine binding and (B) MUC5AC expression. Bars represent mean7S.D. fluorescence intensity (MFI) ofpositive cells of six separate experiments. Statistical analysis was performed by ANOVA followed by Fisher's PLSD correction. Significance was at Po0.05.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–4338

production are involved in the synthesis of MUC5AC in H292 cellsstimulated with CSE 5%.

3.6. Effect of Fluticason and salbutamol in H292 cells

The treatment of H292 cells with FP (from 10�9–10�6 M)significantly reduced ChAT levels (Fig. 6A). The treatment ofH292 cells with SL (from 10�9 M to 10�6 M) significantlyincreased ChAT expression, reaching higher levels at the concen-tration of 10�8 M (Fig. 6B). Subsequently, we tested the combinedeffect of FP 10�8 M and SL 10�8 M on ChAT and muscarinicacetylcholine receptor M3 expression in H292 cells stimulatedwith and without CSE 5%. Furthermore, FP alone significantlyreduced ChAT expression and the effect of SL on ChAT expressionin H292 cells stimulated with CSE 5% (Fig. 7A). Finally, weobserved that FP and SL, alone or in combination, did not affectthe expression of muscarinic acetylcholine receptor M3 (Fig. 7B) inH292 cells whether or not they were stimulated with CSE.

3.7. Effect of Fluticason, salbutamol and Tiotropium on MUC5ACexpression and acetylcholine binding in H292 cells

Treatment with SL alone generated significantly higher levels ofMUC5AC expression in H292 cells compared to untreated cells.The use of Tiotropium reduced MUC5AC expression in H292 cellstreated with SL. FP, alone or in combination with SL, decreasedMUC5AC expression in H292 cells stimulated with CSE 5%.Furthermore, Tiotropium is able to reduce MUC5AC expression inH292 cells stimulated with CSE in the presence or absence of bothFL and SL (Fig. 8A).

Treatment with SL alone generated significantly increasedlevels of acetylcholine binding in H292 cells compared tountreated cells. Treatment with FP, alone or in combination withSL, decreased endogenous acetylcholine binding in H292 cellscompared to untreated cells. Tiotropium, alone or in combinationwith FL and SL, reduced the binding of acetylcholine in H292cells compared to untreated cells and to cells treated with SL.

p<0.015

0

0.1

0.2

0.3

0.4

0.5

CSE (5%)Baseline

Ace

tylc

holin

e bi

ndin

g (M

FI)

0

10

20

30

40

50

60

70 p<0.002

CSE (5%)Baseline

/lomp(

enilohcly teC

A) nieto rp

gµ

Fig. 3. Effect of CSE 5% on the production of acetylcholine and endogenous acetylcholine binding in H292 cell line. Cells were cultured, stimulated (see details in “Section 2”)and analyzed to evaluate (A) acetylcholine level in protein extract by a fluorimetric method using a commercial kit and expressed as pmol/μg protein; (B) endogenousacetylcholine binding by flow-cytometry. Bars represent mean7S.D. fluorescence intensity (MFI) of six different experiments; (C) a representative flow-cytometry ofacetylcholine binding is shown. Statistical analysis was performed by Student's t test. Significance was at Po0.05.

p<0.001

gnidnibenil ohclytec

A(M

FI)

p<0.030

Baseline CSE 5%0

20

40

60p<0.001

MU

C5A

C (

MFI

)

Baseline CSE 5%0

35

70

105

140p<0.001

HCh-3 (50 M)

Untreated

µ

Fig. 4. Effect of Hemicholinium-3 (HCh-3) on endogenous acetylcholine binding and MUC5AC expression in H292 cell line. H292 cells were cultured, stimulated (see detailsin “Section 2”) and analyzed by flow-cytometry to evaluate (A) acetylcholine binding and (B) MUC5AC expression. Bars represent mean7S.D. fluorescence intensity (MFI) ofpositive cells of six separate experiments. Statistical analysis was performed by Student's t test. Significance was at Po0.05.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–43 39

Stimulation with CSE (5%) increased levels of endogenous acet-ylcholine binding in H292 cells, and treatment with FP, alone or incombination with SL, significantly reduced this effect. Tiotropium,alone or in combination with FL and SL, reduced the binding ofacetylcholine in H292 cells stimulated with CSE (Fig. 8B).

3.8. Discussion

In this study, using an in vitro model of cigarette smoke inflam-mation, we demonstrated for the first time that CSE increasesacetylcholine production and that acetylcholine is able to increase

MU

C5A

C (M

FI)

70

105

140 p<0.001

p<0.001

CSE (5%)Baseline

p<0.001

0

35

Unsilenced cell

Silenced cell

Scrambled cell

Fig. 5. Effect of silencing of ChAT mRNA on MUC5AC expression in H292 cell line. H292 cells were cultured, stimulated (see details in “Section 2”) and analyzed by flow-cytometry to evaluate (A) MUC5AC expression in unsilenced cells and in cells transfected with scrambled siRNA or siRNA for ChAT. Bars represent mean7S.D. fluorescenceintensity (MFI) of positive cells of six independent experiments; (B) a representative flow-cytometry of MUC5AC is shown. Statistical analysis was performed by ANOVAfollowed by Fisher's PLSD correction. Significance was at Po0.05.

BaselineFPBaseline

SL

0

400

800

1200

ChA

T (M

FI)

p<0.001

p<0.001p<0.001

p<0.001

0

400

800

1200 p<0.027

p<0.015p<0.007

p<0.001

FP

Baseline 10-9 M 10-8 M 10-7 M 10-6 M

SL

Baseline 10-9 M 10-8 M 10-7 M 10-6 M

200

600

1000

200

600

1000

ChA

T (M

FI)

Fig. 6. Dose response of Fluticasone propionate (FP) and Salmeterol (SL) on ChAT expression in H292 cell line. The cells were treated with different concentrations of (A) FP(10�9–10�6 M) and (B) SL (10�9–10�6 M). ChAT protein expression was evaluated by flow-cytometry. Bars represent mean7S.D. fluorescence intensity (MFI) of fivedifferent experiments. Representative flow-cytometry of the data is shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD correction. Significancewas at Po0.05.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–4340

MUC5AC release in the H292 cell line. This effect is due to theinduction of ChAT and muscarinic acetylcholine receptor M3 expres-sion by CSE, promoting increased acetylcholine production and

binding to the cells. Furthermore, we showed that Tiotropium is ableto control the MUC5AC production generated by CSE. Mucus hyper-production causes cough and wheezing and can impair mucociliary

CSE (5%)0

200

400

600

800

1000C

hAT

(MFI

)p<0.001

p<0.003

p<0.001

p<0.040

p<0.038 p<0.022

Baseline

p<0.001

p<0.001

p<0.001

p<0.001

Mus

carin

ic re

cept

or M

3

(MFI

)

0

20

40

60

80

100

CSE (5%)Baseline

FP (10-8M)+SL (10-8M)

SL (10-8M)

FP (10-8M)

Untreated cells

Fig. 7. Effect of FP and SL on ChAT and muscarinic acetylcholine receptor M3 protein expression in H292 cell line stimulated with CSE. Cells were cultured, stimulated (seedetails in “Section 2”) and analyzed by flow-cytometry to evaluate (A) ChAT and (B) muscarinic acetylcholine receptor M3 protein expression. Bars represent mean7S.D.fluorescence intensity (MFI) of six separate experiments. Statistical analysis was performed by ANOVA followed by Fisher's PLSD correction. Significance was at Po0.05.

Tiotropium+FP+SLTiotropium+SLTiotropium+FPTiotropiumFP+SLSLFPUntreated cells

Ace

tylc

holin

e bi

ndin

g (f

old

chan

ge)

0.5

0.9

1.3

1.7

2.1

2.5

CSE (5%)Baseline0.5

0.7

0.9

1.1

1.3

1.5

MU

C5A

C (

fold

cha

nge )

CSE (5%)Baseline

+ ++*

+*

* * +*

+*+*

+*+*

+*

§°

§°

§° §

°§°

§°

§°

§° §°§°

Fig. 8. Effect of Tiotropium on MUC5AC expression and acetylcholine binding in H292 cell line. Cells were cultured, stimulated (see details in “Section 2”) and analyzed byflow-cytometry to evaluate (A) MUC5AC protein expression and (B) acetylcholine binding. Bars represent mean7S.D. fluorescence intensity (MFI) of positive cells of sixseparate experiments and were plotted as fold-change compared to untreated cells, which were chosen as the reference sample. Statistical analysis was performed byANOVA followed by Fisher's PLSD correction. þPo0.001 vs. untreated cells; nPo0.001 vs. SL treated cells; 1Po0.001 vs. untreated cells; §Po0.01 vs. SL treated cells.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–43 41

clearance through the retention of pathogens and toxic particles,generating airway obstruction which can lead to ventilation perfusionmismatching. COPD is associated with a mucus hypersecretory phe-notype, which contributes significantly to the morbidity and mortalityrelated to these conditions. The blockade of mucus in COPD exacer-bations may affect morbidity (Curran and Cohn, 2010).

Anticholinergics block muscarinic receptors on airway secre-tory cells and smooth muscle, reducing vagal tone and mucussecretion and facilitating cough-induced mucus clearance in COPDpatients (Abad et al., 2003; Coulson and Fryer, 2003). Consistentwith this concept is the observation that Tiotropium reducesmucus secretion (Tamaoki et al., 1994) and exacerbation frequency(Powrie et al., 2007), improving lung function and the naturalcourse of airway disease in COPD patients (Dusser et al. 2006,Tashkin et al., 2008). The underlying mechanism by which long-term treatment with Tiotropium prevents exacerbations is unclear,since the drug is a bronchodilator apparently lacking anti-inflammatory capabilities in vivo in COPD patients (Powrie et al.,2007; Perng et al., 2009). Furthermore, a number of preclinicalstudies have demonstrated that stimulation with cholinergicagonists increases mucociliary clearance (Cammer et al., 1974;Gatto, 1993). These effects were exerted via the activation ofmuscarinic acetylcholine receptors M1, M2, and M3 (Gosenset al., 2006; Profita et al., 2008).

We have previously demonstrated that CSE increases the pro-inflammatory activity of human bronchial epithelial cells, affecting theexpression of ChAT and muscarinic acetylcholine receptor M3 (Profitaet al., 2011). The use of anticholinergic drugs is able to prevent the pro-inflammatory events generated by acetylcholine and CSE (Profita et al.,2011) and to decrease carbachol and cigarette smoke-induced over-expression of MUC5AC in human airway epithelial cells (Cortijo et al.,2011). All together, this evidence suggests a strong link between CSE,MUC5AC and non-neuronal components of the cholinergic system.Accordingly, we show here that CSE is able to increase MUC5AC, ChAT,and muscarinic acetylcholine receptor M3 expression in the H292 cellline, demonstrating for the first time the relationship betweencigarette smoke, mucin production and non-neuronal cholinergicsystem components in an in vitro model of COPD.

Acetylcholine contributes to controlling airway tone and bronchialsecretions concurrent to airway obstruction in inflammatory diseases(Barnes et al., 1989). In the last century, morphological evidence of aneuronal acetylcholine supply was reported, and a cholinergic inner-vationwas demonstrated, in the airways of various species (Anderssonand Grundstrom, 1987). It was recently demonstrated that acetylcho-line is increased in the induced sputum of COPD patients (Profita et al.,2012). During COPD, the released acetylcholine may diffuse into thealveolar spaces and bronchial lumens exerting inflammatory effects,and it may be able, directly or indirectly, to produce MUC5AC or othermarkers involved in the pathogenesis of airway disease.

Acetylcholine is synthesized by ChAT in various cell types (macro-phages, T-lymphocytes, fibroblasts, and epithelial cells) (Wessler et al.,1999) and increased ChAT expression levels are often associated withthe increased production and activity of acetylcholine, which isconsidered an autocrine or paracrine hormone of the airway epithelialcells (Proskocil et al., 2004). CSE increases the pro-inflammatoryactivity of human bronchial epithelial cells and promotes cellularresponse to lower concentrations of acetylcholine (Profita et al., 2011).Accordingly, we observed that H292 cells stimulated with CSE showedan increase in acetylcholine production, together with an increase ofacetylcholine binding (exogenous and endogenous) to the cells. Theeffect involving MUC5AC production may be linked to increasedmuscarinic acetylcholine receptor M3 expression in H292 cells stimu-lated with CSE. This observationwas supported by the fact that HCh-3,a potent choline uptake blocker, decreased the binding of endogen-ously produced acetylcholine and MUC5AC production in theH292 cells stimulated with CSE. Furthermore, ChAT enzyme RNA

interference experiments, which showed the reduction of MUC5AC inthe presence of CSE in silenced H292 cells compared with unsilencedcells, clearly demonstrated the potential involvement of the ChAT/acetylcholine pathway in the regulation of mucin production duringairway inflammation in COPD.

Corticosteroids are often used in combination with β2-agonistand anticholinergic bronchodilators in the treatment of COPD(Johnson, 2005). COPD exacerbations are, at the cellular level,characterized by increasing systemic and bronchial inflammationthat, at least in part, can be influenced by long-term treatmentwith inhaled corticosteroids (Groenewegen et al., 2008; Burge etal., 2000; Wouters et al., 2005; Jones et al., 2003; Vestbo et al.,1999). Long-acting β2-agonist therapy alone is unable to reduceexacerbation rates and severity to a clinically meaningful extent(Calverley et al., 2007). We studied the effect of corticosteroids,β2-agonists, and anticholinergic bronchodilators on MUC5AC pro-duction generated by CSE and regulated by ChAT/acetylcholineand muscarinic acetylcholine receptor M3. We showed that FPalone is able to reduce the basal levels of ChAT and the levels ofChAT induced by cigarette smoke and SL. Furthermore, weobserved that FP and SL did not affect the levels of muscarinicacetylcholine receptor M3 generated by CSE in H292 cells. Ourfindings suggest that the use of muscarinic receptor antagonistssuch as Tiotropium is able to control the MUC5AC production byblocking the activity of acetylcholine bound to muscarinic acet-ylcholine receptor M3 in epithelial cells. Accordingly, we showedthat the use of Tiotropium is able to reduce acetylcholine bindingin H292 cells stimulated with CSE. Our in vitro model suggests thepotentially relevant contribution of Tiotropium in the blockade ofmucus secretion, controlling the ChAT/acetylcholine pathwayduring COPD exacerbations. Our observations were supported bydata showing the inhibitory effect of Tiotropium on NE-inducedglobet cell metaplasia, on mucin production in a mouse model ofCOPD (Arai et al., 2010) and on inflammation in a cigarette smokemouse model of COPD (Wollin and Pieper, 2010). Accordingly, wesuggest that cigarette smoke might have an effect on multipleinflammatory events in the airways producing mucus, and thatanticholinergic drugs might be able to exert their effect directly orindirectly on epithelial cells. However, little is known about theantinflammatory activity of Tiotropium in COPD patients.

In conclusion, our findings suggest that anticholinergic bronchodi-lators are a useful tool for the treatment of COPD patients who smokecigarettes, helping to reduce mucus secretion in airway epithelial cells.Although it has the limitation of being an in vitro study, this workopens up new perspectives on the use of Tiotropium as a mucolytic inthe treatment of COPD. in vivo studies and appropriate clinical trialsare necessary to confirm the usefulness of Tiotropium, alone or incombination with corticosteroids or long-acting β2-agonists, for con-trolling mucus secretion in COPD.

Authorship contribution

A. M. Montalbano was involved in the study design, dataanalysis and paper preparation. A. Bonanno, L. Riccobono,R. Gagliardo, C. Di Sano, G. D. Albano, G. Anzalone and L. Sienawere involved in the technical approach. M. Gjomarkaj andM. P. Pieper gave us suggestions regarding data analysis. M. Profitadefined the study and discussed the results.

Acknowledgment

The authors thank the Italian National Research Council andBoehringer Ingelheim Pharma GmbH & Co. KG (4303029) for theirfinancial support.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–4342

References

Aalbers, R., Ayres, J., Decramer, M., Lier, P.A., Magyar, P., Malolepszy, J., Ruffin, R.,Sybrecht, G.W., 2002. Formoterol in patients with chronic obstructive pulmon-ary disease: a randomized, controlled, 3-month trial. Eur. Respir. J. 19, 936–943.

Abad, S.F., Novalbos, J., Gallego, S.S., Galvez Mugica, M.A., 2003. [Regulation ofbronchial tone in chronic obstructive pulmonary disease (COPD): role ofmuscarinic receptors] Regulacion del tono bronquial en la enfermedad pulmo-nar obstructiva cronica (EPOC): papel de los receptores muscarinicos. An. Med.Interna 20, 201–205.

Andersson, R.G., Grundstrom, N., 1987. Innervation of airway smooth muscle.Efferent mechanisms. Pharmacol. Ther. 32, 107–130.

Arai, N., Kondo, M., Izumo, T., Tamaoki, J., Nagai, A., 2010. Inhibition of neutrophilelastase-induced goblet cell metaplasia by tiotropium in mice. Eur. Respir. J. 35,1164–1171.

Barnes, N.C., Evans, J., Zakrzewski, J.T., Piper, P.J., Costello, J.F., 1989. A criticalevaluation of the clinical relevance of leukotrienes in bronchial asthma. AgentsActions Suppl. 28, 123–133.

Bateman, E.D., Rennard, S., Barnes, P.J., Dicpinigaitis, P.V., Gosens, R., Gross, N.J.,Nadel, J.A., Pfeifer, M., Racke, K., Rabe, K.F., Rubin, B.K., Welte, T., Wessler, I.,2009. Alternative mechanisms for Tiotropium. Pulm. Pharmacol. Ther. 22 (6),533–542.

Berger, J., Albert, R.E., Sanborn, K., Lippmann, M., 1978. Effects of atropine andmethacholine on deposition and clearance of inhaled particles in the donkey.J. Toxicol. Environ. Health 4, 587–604.

Burge, P.S., Calverley, P.M.A., Jones, P.W., Anderson, J.A., Maslen, T.K., 2000.Randomised, double blind, placebo controlled study of fluticasone propionatein patients with moderate to severe chronic obstructiv pulmonary disease: theISOLDE trial. Br. Med. J. 320, 1297–1303.

Calverley, P.M.A., Anderson, J.A., Celli, B.R., Ferguson, G.T., Jenkins, C., Jones, P.W.,Yates, J., Vestbo, J., 2007. Salmeterol and fluticasone propionate and survival inchronic obstructive pulmonary disease. N. Engl. J. Med. 356, 775–789.

Cammer, P., Strandberg, K., Philipson, K., 1974. Increased mucociliary transport bycholinergic stimulation. Arch. Environ. Health 29, 220–224.

Caramori, G., Casolari, P., Di Gregorio, C., Saetta, M., Baraldo, S., Boschetto, P., Ito, K.,Fabbri, M.L., Barnes, P.J., Adcock, I.M., Cavallesco, G., Chung, K.F., Papi, A., 2009.MUC5AC expression is increased in bronchial submucosal glands of stableCOPD patients. Histopathology 55, 321–331.

Cortijo, J., Mata, M., Milara, J.E., Donet, A., Gavalda, M., Miralpeix, E.J., 2011. MorcilloAclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in humanairways. Eur. Respir. J. 37, 244–254.

Coulson, F.R., Fryer, A.D., 2003. Muscarinic acetylcholine receptors and airwaydiseases. Pharmacol. Ther. 98, 59–69.

Curran, D.R., Cohn, L., 2010. Advances in mucous cell metaplasia: a plug for mucusas a therapeutic focus in chronic airway disease. Am. J. Respir. Cell Mol. Biol.42 (3), 268–275.

Davies, J.R., Hovenberg, H.W., Lindén, C.J., Howard, R., Richardson, P.S., Sheehan, J.K.,Carlstedt, I., 1996. Mucins in airway secretions from healthy and chronicbronchitic subjects. Biochem. J. 313, 431–439.

Dusser, D., Bravo, M.L., Iacono, P., 2006. The effect of Tiotropium on exacerbationsand airflow in patients with COPD. Eur. Respir. J. 27, 547–555.

Gatto, L.A., 1993. Cholinergic and adrenergic stimulation of mucociliary transport inthe rat trachea. Respir. Physiol. 92, 209–217.

Gosens, R., Zaagsma, J., Grootte Bromhaar, M., Nelemans, A., Meurs, H., 2004.Acetylcholine: a novel regulator of airway smooth muscle remodelling? Eur.J. Pharmacol. 500 (1–3), 193–201.

Gosens, R., Zaagsma, J., Meurs, H., Halayko, A.J., 2006. Muscarinic receptor signalingin the pathophysiology of asthma and COPD. Respir. Res. 7, 73.

Groenewegen, K.H., Postma, D.S., Hop, W.C., Wielders, P.L.M.L, Schlösser, N.J.J,Wouters, E.F.M., 2008. Increased systemic inflammation is a risk factor forCOPD exacerbations. Chest 133, 350–357.

Johnson, M., 2005. Corticosteroids: potential β2-agonist and anticholinergic inter-actions in chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 2 (4),320–325.

Jones, P.W., Bosh, T.K., 1997. Quality of life changes in COPD patients treated withsalmeterol. Am. J. Respir. Crit. Care Med. 155, 1283–1289.

Jones, P.W., Willits, L.R., Burge, P.S., Calverley, P.M.A., 2003. Disease severity and theeffect of fluticasone propionate on chronic obstructive pulmonary diasaseexacerbations. Eur. Respir. J. 21, 68–73.

Kim, S., Schein, A.J., Nadel, J.A., 2005. E-cadherin promotes EGFR-mediated celldifferentiation and MUC-5AC mucin expression in cultured human airwayepithelial cells. Am. J. Physiol. – Lung Cell. Mol. Physiol. 289, L1049–L1060.

Mahler, D.A., Donohue, J.F., Barbee, R.A., Goldman, M.D., Gross, N.J., Wisniewski, M.E.,Yancey, S.W., Zakes, B.A., Rickard, K.A., Anderson, W.H., 1999. Efficacy of salmeterolxinafoate in the treatment of COPD. Chest 115, 957–965.

Newland, N., Richter, A., 2008. Agents associated with lung inflammation inducesimilar responses in H292 lung epithelial cells. Toxicol. in vitro 22 (7),1782–1788.

Perng, D.W., Tao, C.W., Su, K.C., Tsai, C.C., Liu, L.Y., Lee, Y.C., 2009. Anti-inflammatoryeffects of salmeterol/fluticasone, Tiotropium/fluticasone or Tiotropium inCOPD. Eur. Respir. J. 38, 778–784.

Phipps, R.J., Williams, I.P., Richardson, P.S., Pell, J., Pack, R.J., Wright, N., 1982.Sympathomimetic drugs stimulate the output of secretory glycoproteins fromhuman bronchi in vitro. Clin. Sci. 63, 23–28.

Powrie, D.J., Wilkinson, T.M.A., Donaldson, G.C., Jones, P., Scrine, K., Viel, K., Kesten, S.,Wedzicha, J.A., 2007. Effect of Tiotropium on sputum and serum inflammatorymarkers and exacerbations in COPD. Eur. Respir. J. 30, 472–478.

Profita, M., Di Giorgi, R., Sala, A., Bonanno, A., Riccobono, L., Mirabella, F., Gjomarkaj,M., Bonsignore, G., Bousquet, J., Vignola, A.M., 2005. Muscarinic receptors,leukotriene B4 production and neutrophilic inflammation in COPD patients.Allergy 60 (11), 1361–1369.

Profita, M., Bonanno, A., Siena, L., Ferraro, M., Montalbano, A.M., Pompeo, F.,Riccobono, L., Pieper, M.P., Gjomarkaj, M., 2008. Acetylcholine mediates therelease of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependentmechanism. Eur. J. Pharmacol. 582 (1–3), 145–153.

Profita, M., Bonanno, A., Siena, L., Bruno, A., Ferraro, M., Montalbano, A.M., Albano,G.D., Riccobono, L., Casarosa, P., Pieper, M.P., Gjomarkaj, M., 2009. Smoke,choline acetyltransferase, muscarinic receptors, and fibroblast proliferation inchronic obstructive pulmonary disease. J. Pharmacol. Exp. Ther. 329 (2),753–763.

Profita, M., Bonanno, A., Montalbano, A.M., Ferraro, M., Siena, L., Bruno, A., Girbino, S.,Albano, G.D., Casarosa, P., Pieper, M.P., Gjomarkaj, M., 2011. Cigarette smoke extractactivates human bronchial epithelial cells affecting non-neuronal cholinergicsystem signalling in vitro. Life Sci. 89 (1–2), 36–43.

Profita, M., Bonanno, A., Montalbano, A.M., Albano, G.D., Riccobono, L., Siena, L.,Ferraro, M., Casarosa, P., Pieper, M.P., Gjomarkaj, M., 2012. β2long-acting andanticholinergic drugs control TGF-β1-mediated neutrophilic inflammation inCOPD. Biochim. Biophys. Acta 1822 (7), 1079–1089.

Proskocil, B.J., Sekhon, H.S., Jia, Y., Savchenko, V., Blakely, R.D., Lindstrom, J.,Spindel, E.R., 2004. Acetylcholine is an autocrine or paracrine hormonesynthesized and secreted by airway bronchial epithelial cells. Endocrinology145 (756), 2498–2506.

Rogers, D.F., 2002. Pharmacological regulation of the neuronal control of airwaymucus secretion. Curr. Opin. Pharmacol. 2, 249–255.

Rogers, D.F., 2005. Mucociliary dysfunction in COPD: effect of current pharma-cotherapeutic options. Pulm. Pharmacol. Ther. 18 (1), 1–8.

Takeyama, K., Jung, B., Shim, J.J., Burgel, P.R., Dao-Pick, T., Ueki, I.F., Protin, U.,Kroschel, P., Nadel, J.A., 2001. Activation of epidermal growth factor receptors isresponsible for mucin synthesis induced by cigarette smoke. Am. J. Physiol.Lung Cell. Mol. Physiol. 280, L165–L172.

Tamaoki, J., Chiyotani, A., Tagaya, E., Sakai, N., Konno, K., 1994. Effect of long termtreatment with oxitropium bromide on airway secretion in chronic bronchitisand diffuse panbronchiolitis. Thorax 49, 545–548.

Tashkin, D.P., Celli, B.R., Senn, S., Burkhart, D., Kesten, S., Menjoge, S.S., Decramer,M., 2008. A 4-year trial of Tiotropium in chronic obstructive pulmonary disease.N. Engl. J. Med. 359, 1543–1554.

Thornton, D.J., Carlstedt, I., Howard, M., Devine, P.L., Price, M.R., Sheehan, J.K., 1996.Respiratory mucins: identification of core proteins and glycoforms. Biochem. J.316 (967), 75.

Vestbo, J., Sørensen, T., Lange, P., Brix, A., Torre, P., Viskum, K., 1999. Long-termeffect of inhaled budesonide in mild and moderate chronic obstructivepulmonary disease: a randomised controlled trial. Lancet 355, 1819–1823.

Wessler, I., Kirkpatrick, C.J., Racke, K., 1999. The cholinergic ‘pitfall’: acetylcholine, auniversal cell molecule in biological systems, including humans. Clin. Exp.Pharmacol. Physiol. 26, 198–205.

Wollin, L., Pieper, M.P., 2010. Tiotropium bromide exerts anti-inflammatory activityin a cigarette smoke mouse model of COPD. Pulm. Pharmacol. Ther. 23,345–354.

Wouters, E.F., Postma, D.S., Fokkens, B., Hop, W.C., Prins, J., Kuipers, A.F., Pasma, H.R.,Hensing, C.A., Creutzberg, E.C., 2005. Withdrawal of fluticasone propionatefrom combined salmeterol/fluticasone treatment in patients with COPD causeimmediate and sustained disease deterioation: a randomised controlled trial.Thorax 60, 480–487.

A.M. Montalbano et al. / European Journal of Pharmacology 736 (2014) 35–43 43