ORIGINAL ARTICLE

Effects of Cigarette Smoke Extract

and Nicotine on Bronchial Tone and
Acetylcholine-Induced Airway Contraction
in Mouse Lung Slices
E Streck,1 RA Jörres,2 RM Huber,1 A Bergner1
1
Department of Thoracic Medicine, Medizinische Klinik-Innenstadt, Ludwig-Maximilians-University, Munich,
Germany
2
Institute and Outpatient Clinic for Occupational, Social and Environmental Medicine, Ludwig-Maximilians-
University, Munich, Germany

QAbstract
Background: Tobacco smoke is a key risk factor for chronic obstructive pulmonary disease, but it may also alter the pathophysiology of
asthma. In the present study, we analyzed whether tobacco smoke has acute or chronic effects on bronchial tone and whether it alters
bronchial reactivity in vitro.
Methods: Airways in murine lung slices were digitally recorded and the change in cross-sectional area with time was quantified. T-bet KO mice
served as a model for bronchial hyperreactivity. T-bet KO mice show a shift towards type 2 helper T lymphocytes and display histological as
well as functional characteristics of asthma. Cigarette smoke extract (CSE) was obtained using commercially available cigarettes (Gauloise
Blondes) by drawing cigarette smoke slowly through a water pump into a tube containing 10 mL of DMEM culture medium.
Results: Acute exposure to CSE led to relaxation of the airway. Acute exposure to nicotine resulted in a minor relaxation of the airway in
Balb/C mice and in nonsignificant relaxation of the airway in T-bet KO mice. The nicotinic acetylcholine-receptor hexamethonium partially
inhibited CSE-induced airway relaxation. Airway contraction in response to acetylcholine was stronger in T-bet KO mice than in Balb/C
mice. After exposure to CSE or nicotine for 48 hours, acetylcholine-induced airway contraction was no longer different between the 2
types of mice.
Conclusions: Our data indicate that acute exposure to CSE leads to airway relaxation, which is partially mediated by nicotine. Chronic exposure
to CSE reverses bronchial hyperreactivity in the airways of T-bet KO mice; this effect can be mimicked by chronic exposure to nicotine.
Key words: Tobacco smoke. Nicotine. Bronchial tone. Hyperreactivity. Asthma.

QResumen
Antecedentes: El humo del tabaco es un factor de riesgo clave de la enfermedad pulmonar obstructiva crónica (EPOC), si bien también
puede alterar la fisiopatología del asma. En este estudio se analizó si el humo del tabaco tiene efectos agudos o crónicos sobre el tono
bronquial y si altera la reactividad bronquial in vitro.
Métodos: Se registraron digitalmente las vías respiratorias en secciones pulmonares de ratón y se cuantificó el cambio del área transversal
con el tiempo. Se emplearon ratones KO T-bet como modelo de hiperreactividad bronquial. Los ratones KO T-bet muestran un cambio a
linfocitos T cooperadores de tipo 2 y presentan las características histológicas y funcionales del asma. Se obtuvo extracto de humo de
tabaco (EHT) a partir de cigarrillos comercialmente disponibles (Gauloise Blondes) extrayendo lentamente el humo de tabaco a través de
una bomba de agua y transfiriéndolo a un tubo con 10 ml de medio de cultivo DMEM.
Resultados: La exposición aguda al EHT provocó una relajación de las vías respiratorias. La exposición aguda a la nicotina provocó una
leve relajación de las vías respiratorias en los ratones Balb/C y una relajación no significativa de las vías respiratorias en los ratones KO
T-bet. El hexametonio del receptor nicotínico de acetilcolina inhibió parcialmente la relajación de las vías respiratorias inducida por el
EHT. La contracción de las vías respiratorias en respuesta a la acetilcolina fue mayor en los ratones KO T-bet que en los ratones Balb/C.
Tras la exposición a EHT o a nicotina durante 48 horas, la contracción de las vías respiratorias inducida por acetilcolina ya no presenta
diferencias entre ambos tipos de ratones.

© 2010 Esmon Publicidad J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330
325 E Streck, et al

Conclusiones: Los datos indican que una exposición aguda al EHT provoca una relajación de las vías respiratorias, mediada parcialmente
por la nicotina. La exposición crónica al EHT invierte la hiperreactividad bronquial de las vías respiratorias de los ratones KO T-bet; este
mismo efecto también puede producirse por una exposición crónica a la nicotina.
Palabras clave: Humo de tabaco. Nicotina. Tono bronquial. Hiperreactividad. Asma.

Introduction Subsequently, 0.1 to 0.2 mL of air was injected to flush the

agarose-sHBSS out of the airways and the agarose was gelled
Tobacco smoke is a key risk factor for chronic obstructive by keeping the mouse preparation at 4°C. The lungs were
pulmonary disease, lung cancer, and coronary heart disease, removed and slices of approximately 200 µm were cut with an
and it may also play a role in asthma. Lazarus et al [1] showed EMS-4000 Tissue Slicer (Electron Microscopy Sciences, Fort
that the response to inhaled corticosteroids, which are the Washington, Pennsylvania, USA). The slices were maintained
backbone of asthma therapy today, is diminished by smoking. by floating them in DMEM supplemented with antibiotics and
Furthermore, children with asthma show worse symptoms if antimycotics at 37°C in 5% CO2 for up to 5 days. In culture,
their parents smoke [2]. Nicotine, which is one of the most lung slices maintain their contractile properties for up to 1 week
active pharmacological compounds in tobacco smoke, has anti- [7]. Experiments were therefore performed on days 2 to 5 of
inflammatory effects in ulcerative colitis [3] and has been shown cultivation and each slice was used for 1 experiment only. For
to decrease the release of interleukin 1ß and tumor necrosis factor each experimental group, slices from at least 3 different mice
in alveolar macrophages [4]. We performed an in vitro study to were used. To measure airway cross-sectional area, lung slices
determine whether tobacco smoke has acute or chronic effects were placed in culture dishes, immersed in sHBSS, and held in
on bronchial tone and whether it alters bronchial reactivity to position by a custom-made gold grid. Bright field images were
acetylcholine (ACH). We also evaluated whether possible effects recorded using a digital CCD camera (AxioCam MRm, Carl
are mediated by nicotine. Zeiss Vision, Munich, Germany). Frames were captured in time-
T-bet is a type 1 helper T cell (TH1)–specific transcription lapse (1 frame/s) and the cross-sectional area of the airway was
factor that has the ability to convert TH2 cells into TH1 cells. measured by pixel summing using the image analysis software
Mice in whom the T-bet gene has been deleted (T-bet KO mice) “Scion” (Scion Corporation, Frederick, Maryland, USA).
spontaneously develop airway remodeling similar to that seen in Cigarette smoke extract (CSE) was obtained using
asthma and have several functional and inflammatory features commercially available cigarettes (Gauloise Blondes, nominal
that are characteristic of this disease [5]. We recently showed that nicotine 0.8 mg) as described previously [8]. Briefly, the smoke
airways in lung slices from T-bet KO mice maintain bronchial of 1 cigarette was drawn slowly through a water pump into a tube
hyperreactivity in vitro in that they have an elevated contractile containing 10 mL of DMEM culture medium. Each cigarette was
response to ACH [6]. In the present study, we used this model to completely burned after 5 minutes. The CSE was then sterile
test the effects of tobacco smoke on bronchial hyperreactivity. filtered and kept at –32°C. The medium nicotine concentration
Acute exposure to cigarette smoke extract (CSE) leads in the CSE was found to be 168 µg/mL.
to airway relaxation, which is partially mediated by nicotine. The Mann-Whitney rank sum test or analysis of variance
Chronic exposure to CSE reverses bronchial hyperreactivity in by ranks (combined with pairwise multiple comparisons)
the airways of T-bet KO mice. This effect can be mimicked by was performed using Sigma Stat software (Jandel Scientific,
chronic exposure to nicotine. Chicago, Illinois, USA). A P value of less than .05 was
considered statistically significant.

Methods
Results
Cell culture reagents were obtained from Life Technologies
(Eggenstein, Germany); other reagents were from Sigma Airways in lung slices (Figure 1) were monitored using
(Deisenhofen, Germany). Balb/C mice were purchased from phase-contrast microscopy and recorded in time-lapse at 1
Harlan-Winkelmann (Borchen, Germany) and T-bet KO mice frame/s. For each frame, the area of the airway was calculated
on a Balb/C background were purchased from Charles River and the change in airway area in response to agonists was
(Charles River Breeding Labs, Needham, Massachusetts, USA). monitored (Figure 2). Airways in lung slices from Balb/C
All procedures were approved by the Ethics Committee of the and T-bet KO mice were exposed to 1 µM ACH with no prior
Ludwig-Maximilians-University in Munich, Germany. incubation with CSE or nicotine. Contractions in lung slices from
Lung slices were prepared as described elsewhere [6]. T-bet KO mice were significantly stronger (7±3% of starting
Briefly, 42 to 77-day-old mice were sacrificed by intraperitoneal value for Balb/C vs 45±6% for T-bet KO; P<.001; n=15 airways
injection of pentobarbital and the chest wall was removed. from 3 different mice; Figure 3).
The trachea was cannulated using an intravenous catheter Next, the acute effects of 5% CSE or 5% nicotine
and the lungs were inflated with 2% agarose-sHBSS at 37°C. (10 µg/mL) on airway tone were investigated. CSE induced

J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330 © 2010 Esmon Publicidad
 Cigarette Smoke and Bronchial Tone 326

ACH
200

Airway Area, % Starting Value

CSE

100

Figure 1. Phase-contrast image of an airway in a lung slice. The airway

is cut in cross-section and has a clearly visible lumen. The scale bar
represents 50 µm.
0 50 100 150 200 250 300

Figure 2. Airways were monitored using phase-contrast microscopy

and recorded in time-lapse at 1 frame/s in response to cigarette
smoke extract (CSE, 5%) and 1 µM acetylcholine. CSE led to relaxation
(increase in airway area) while ACH induced contraction of the airway
(decrease in airway area). ACH indicates acetylcholine; CSE, cigarette
smoke extract.

+10

–10
Change in Area, % Starting Value

–20

–30

–40

–50

–60
Balb/C T-bet KO

Figure 3. Airways in lung slices from Balb/C mice and T-bet KO mice–serving as an asthma model–were exposed to ACH and the change in airway area
was quantified. Contractions in the airways of T-bet KO mice were significantly stronger (*P<.001, n=15 airways from 3 different mice). ACH indicates
acetylcholine.

© 2010 Esmon Publicidad J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330
327 E Streck, et al

50

40
Balb/C

T-bet KO
Change in Area, % Starting Value

30

20

10

Control CSE Nicotine

-10

Figure 4. Airways in lung slices from Balb/C and T-bet KO mice were exposed to 5% CSE or 5% nicotine (10 µg/ml) and the change in airway area was
quantified. Control slices were exposed to medium. In lung slices from Balb/C mice CSE and nicotine led to relaxation of the airways, with the relaxation
being stronger for CSE (*P<.05 vs control and nicotine, **P<.05 vs control, n=8-12 lung slices from 3 different mice). In lung slices from T-bet KO mice,
only CSE caused significant airway relaxation (***P<.05 vs control and nicotine, n=10 lung slices from 3 different mice). CSE indicates cigarette smoke
extract.

A 40
Balb/C

30
Change in Area, % Starting Value

20

10

0
HM HM
CSE Nicotine

J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330 © 2010 Esmon Publicidad
 Cigarette Smoke and Bronchial Tone 328

B 40
T-bet KO

30
Change in Area, % Starting Value

20

10

0
HM HM
CSE Nicotine

Figure 5. Airways in lung slices from Balb/C and T-bet KO mice were exposed to 5% CSE or 5% nicotine with or without 10 µM of hexamethonium to
block nicotinic ACH-receptors. A, In lung slices from Balb/C mice, HM significantly reduced CSE-induced airway relaxation but did not alter the response
to nicotine (*P<.01, n=11 lung slices from 3 different mice). B, In lung slices from T-bet KO mice, HM also significantly reduced CSE-induced relaxation of
the airway. Interestingly, HM increased nicotine-induced airway relaxation (*P<.05, n=11 lung slices from 3 different mice). ACH indicates acetylcholine;
CSE, cigarette smoke extract; HM, hexamethonium.

strong relaxation of the airway, which was comparable in significantly reduced CSE-induced airway relaxation (33±6%
Balb/C and T-bet KO lung slices (31±6% of starting value for without hexamethonium vs 9±8% with hexamethonium;
Balb/C versus 33±6% for T-bet KO; n=8-12 lung slices from P<.01, Figure 5B). Interestingly, hexamethonium increased
3 different mice; P<.05 vs control and nicotine, Figure 4). nicotine-induced relaxation of the airway (5±2% without
Nicotine induced minor relaxation of the airway in lung slices hexamethonium vs 1±7% with hexamethonium; P<.05; n=11
from Balb/C mice, while relaxation in lung slices from T-bet lung slices from 3 different mice).
KO mice was not statistically significant (9±2% of starting To test the long-term effects of CSE and nicotine on
value for Balb/C; P<.05 vs control, 5±2% for T-bet KO; n=10 ACH-induced airway contraction, lung slices were cultured
lung slices from 3 different mice; Figure 4). for 48 hours in medium containing 5% CSE or 5% nicotine
To investigate whether the acute effects of CSE and nicotine and subsequently exposed to 1 µM ACH. In the control groups
were due to nicotinic ACH-receptor activation, lung slices were (cultivation in medium alone), contractions in the airways
exposed to 5% CSE or 5% nicotine with or without 10 µM of T-bet KO mice were significantly stronger (7±3% of the
of the nicotinic ACH-receptor inhibitor hexamethonium. In starting value for Balb/C vs 45±6% for T-bet KO, P<.001;
lung slices from Balb/C mice, hexamethonium significantly n=15 airways from 3 different mice, Figure 6). However,
reduced CSE-induced airway relaxation (31±6% without after culture with CSE or nicotine, no difference was observed
hexamethonium vs 10±2% with hexamethonium; P<.01, Figure between airways from Balb/C or T-bet KO mice (CSE, 20±3%
5A) but did not alter the response to nicotine (9±2% without vs for Balb/C vs 22±4% for T-bet KO; nicotine, 17±2% for
5±3 % with hexamethonium; n=11 lung slices from 3 different Balb/C versus 17±2% for T-bet KO; n=15 airways from 3
mice). In lung slices from T-bet KO mice, hexamethonium also different mice).

© 2010 Esmon Publicidad J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330
329 E Streck, et al

Balb/C

-10

-20
Change in Area, % Starting Value

-30

Balb/C
-40
T-bet KO

-50

Control CSE 48h Nicotine 48h

-60

Figure 6. Lung slices from Balb/C mice and T-bet KO mice were cultured for 48 hours in medium containing 5% CSE or 5% nicotine and subsequently
exposed to 1 µM ACH. In the control groups (culture in medium alone), contractions in the airways of T-bet KO mice were significantly stronger (*P<.001,
n=15 airways from 3 different mice). However, after cultivation with CSE or nicotine no differences between airways from Balb/C or T-bet KO mice could
be observed. ACH indicates acetylcholine; CSE, cigarette smoke extract.

Discussion channels and open to Na+, K+, and Ca2+ upon the binding of
nicotine [10]. Chronic exposure to nicotine leads not only
We show that acute exposure to CSE led to relaxation of the to inactivation but also to upregulation of nicotinic ACH
airway. Acute exposure to nicotine resulted in minor relaxation receptors [11]. Hexamethonium unselectively blocks nicotinic
in Balb/C and in nonsignificant relaxation in T-bet KO mice. ACH receptors.
The nicotinic ACH-receptor hexamethonium partially inhibited We found that acute exposure to CSE led to relaxation of the
CSE-induced relaxation. Exposure of airways in lung slices airway. This was not because of toxic effects damaging airway
from T-bet KO mice to CSE or nicotine for 48 hours completely smooth muscle cells, given that subsequent exposure to ACH
reversed bronchial hyperreactivity. resulted in strong airway contraction (Figure 2). Furthermore,
Tobacco smoke is a mixture of solid and liquid components hexamethonium partially reversed CSE-induced relaxation,
with a particle diameter of 0.1-1 µm and a particle concentration thus indicating a specific effect via nicotinic ACH-receptors.
of 107-108/mL. Over 4000 different chemical compounds have However, even in the presence of hexamethonium, the airways
been identified, and nicotine is the most pharmacologically active still relaxed in response to CSE, and exposure to nicotine
one [9]. For incubation with nicotine alone, we used 10 µg/mL of alone led to relaxation that was considerably smaller than that
nicotine because this was the concentration we measured in 5% induced by CSE. Given the enormous number of chemical
CSE. Therefore, the dissimilar effects of CSE and nicotine we compounds in tobacco smoke, identification of the specific
observed were not due to different concentrations of nicotine, agents in CSE responsible for nicotine-independent relaxation
but to the complex composition of tobacco smoke. seems unfeasible. In fact, it may be the complex composition
Muscarinic ACH receptors are G protein–coupled and can of tobacco smoke, rather than specific compounds, which leads
be activated by muscarine and deactivated by atropine. To to nicotine-independent airway relaxation.
date, 5 subtypes (M1-M5) have been identified. Nicotinic ACH Addition of nicotine to lung slices from T-bet KO mice
receptors are integral 252-kDa membrane proteins consisting did not induce significant relaxation of the airway. However,
of 5 subunits (2α, ß, γ, δ, binding site on α-subunits) and can in the presence of hexamethonium, the airways relaxed in
be activated by nicotine. These receptors form unspecific cation response to nicotine. Although seemingly conflicting, these

J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330 © 2010 Esmon Publicidad
 Cigarette Smoke and Bronchial Tone 330

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© 2010 Esmon Publicidad J Investig Allergol Clin Immunol 2010; Vol. 20(4): 324-330