Spontaneous airway hyperresponsiveness in estrogen receptor-α α deficient mice
Michelle A. Carey,1 Jeffrey W. Card,1 J. Alyce Bradbury,1 Michael P. Moorman,1 Najwa
Haykal-Coates,2 Stephen H. Gavett,2 Joan P. Graves,1 Vickie R. Walker,1 Gordon P. Flake,1
James W. Voltz,1 Daling Zhu,3 Elizabeth R. Jacobs,3 Azzeddine Dakhama,4 Gary L. Larsen,4
Joan E. Loader,4 Erwin W. Gelfand,4 Dori R. Germolec,1 Kenneth S. Korach1 and Darryl C.
1Division of Intramural Research, National Institute of Environmental Health Sciences, National
Institutes of Health, Research Triangle Park, North Carolina 27709; 2Experimental Toxicology
Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, North Carolina 27711; 3Departments of Medicine
and Physiology, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee,
Wisconsin 53226; 4Division of Cell Biology, Department of Pediatrics, National Jewish Medical
and Research Center, Denver, Colorado 80206.
Address correspondence and reprint requests to:
Darryl C. Zeldin, M.D.
NIH/NIEHS, 111 T.W. Alexander Drive, Building 101, Room D236
Research Triangle Park, NC 27709
AJRCCM Articles in Press. Published on November 9, 2006 as doi:10.1164/rccm.200509-1493OC
Copyright (C) 2006 by the American Thoracic Society.
Funding: This research was supported by the Intramural Research Program of the NIEHS, NIH.
J.W. Card was supported by a Research Fellowship Award from the Davies Charitable
Foundation and by a Senior Research Training Fellowship from the American Lung Association
of North Carolina.
Running head: ERα and airway hyperresponsiveness
Word Count: 5,294
Descriptor number: 60
This article has an online data supplement which is accessible from this issues table of content
online at www.atsjournals.org
This paper has been reviewed and approved for release by the National Health and
Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. Approval
does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor
does mention of trade names or commercial products constitute endorsement or recommendation
Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic
evidence supports a role for female sex hormones in modulating lung function and airway
hyperresponsiveness in humans. Objectives: To examine the role of estrogen receptors in
modulating lung function and airway responsiveness using estrogen receptor deficient mice.
Methods: Lung function was assessed by a combination of whole body barometric
plethysmography, invasive measurement of airway resistance and isometric force measurements in
isolated bronchial rings. M2 muscarinic receptor expression was assessed by western blotting and
function was assessed by electrical field stimulation of tracheas in the presence/absence of
gallamine. Allergic airway disease was examined following ovalbumin sensitization and exposure.
Measurements and main results: Estrogen receptor-α knockout mice exhibit a variety of lung
function abnormalities and have enhanced airway responsiveness to inhaled methacholine and
serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor
expression and function in the lungs. Absence of estrogen receptor-α also leads to increased airway
responsiveness without increased inflammation following allergen sensitization and challenge.
Conclusions: These data suggest that estrogen receptor-α is a critical regulator of airway
hyperresponsiveness in mice.
Word count: 185
Keywords: lung function, asthma, hyperreactivity, M2 muscarinic receptor, estrogen receptor
A compelling body of evidence supports a role for female sex hormones in modulating lung
function, airway hyperresponsiveness and asthma in humans. Asthma prevalence rates are higher in
women than in men between the ages of puberty and menopause (1, 2). Menstrual cycle variations
in pulmonary function and airway hyperresponsiveness have been well documented (3, 4). Females
also appear to exhibit more severe airway hyperresponsiveness and more severe asthma than males
(5, 6). The effect of estrogens in asthma is highly controversial and the results of published studies
are contradictory. Both beneficial and detrimental effects have been reported (7, 8). For example,
long-term and/or high doses of postmenopausal estrogen therapy have been reported to increase
subsequent risk of asthma (7). In contrast, another study reported that supplemental estrogens could
be used as steroid-sparing agents in asthmatic women (8).
Airway hyperresponsiveness is one of the main features of asthma and is also a major risk
factor for accelerated lung function decline, and hence the development of chronic obstructive
pulmonary disease. The dominant autonomic control of airway smooth muscle in the lungs is
provided by the parasympathetic nervous system (9, 10). The parasympathetic nerves release
acetylcholine which stimulates muscarinic M3 receptors on the smooth muscle to cause contraction.
Concurrently, acetylcholine also stimulates M2 muscarinic receptors located on the nerves to limit
further acetylcholine release (9). Loss of M2 receptor function increases acetylcholine release and
potentiates vagally mediated bronchoconstriction (11). There is substantial evidence that loss of M2
muscarinic receptor expression and/or function on parasympathetic nerves is responsible for the
development of airway hyperresponsiveness (9). Indeed, M2 muscarinic receptors are dysfunctional
in asthmatics (12, 13) and in animal models of allergic airway disease (14, 15).
Estrogens mediate both transcriptional and non-genomic effects via alpha (α) or beta (β)
estrogen receptors (ERs). Both nuclear receptors are expressed in the lung with ERβ being more
abundant than ERα (16), but their functions in this organ are largely unknown. Mice lacking either
ERα (αERKO) or ERβ (βERKO) have been developed using gene targeting strategies (17, 18). The
objective of the present study was to examine the role of ERs in modulating lung function and
airway hyperresponsiveness. Our results show that ERα is a critical regulator of airway
hyperresponsiveness and that αERKO mice have reduced M2 muscarinic receptor expression and
function in the lung. Hence ERs could represent a novel therapeutic target for asthma and other
diseases associated with reactive airways. Parts of this work have been published in abstract form
Methods (Word count: 376)
All procedures were performed under an approved animal study protocol in accordance with
the NIH Guide for the Care and Use of Laboratory Animals. Mice (αERKO, βERKO and wild type
littermate controls on a pure C57BL/6 background, 12-16 weeks of age) were obtained from Taconic
Farms. Further details about animals and treatments are provided in the online supplement.
Whole body barometric plethysmography
Basal lung function was measured in unrestrained mice using whole body barometric
plethysmography (Buxco Electronics). Greater detail about these measurements is provided in the
Invasive analysis of lung function was performed on anesthetized mice using the Flexivent
system (Scireq). Further detail is provided in the online supplement.
Acetylcholine release assay
Acetylcholine (ACh) release was determined by spectrofluorometry using the Amplex® Red
acetylcholine/acetylcholinesterase assay kit following the manufacturer’s instructions (Molecular
Probes). This assay is described in more detail in the online supplement.
Lungs homogenates were analyzed by western blotting for M2 muscarinic receptor
expression which was then normalized to actin expression by analyzing the M2 muscarinic
receptor/actin band density ratio in each sample. Details are provided in the online supplement.
Assessment of M2 muscarinic receptor function by electrical field stimulation
Airway responsiveness to electrical field stimulation in presence or absence of the M2
muscarinic receptor antagonist gallamine was assessed ex vivo as previously described (20), with
some modifications which are described in detail in the online supplement.
Isometric force measurements in isolated bronchial rings
The tension response in isolated bronchial rings to incrementally increasing concentrations of
carbachol (10-7 M to 10-3 M) was examined. Details are provided in the online supplement.
Allergic airway disease model
Allergic airway disease was induced as previously described (21). Invasive lung function
measurements were performed as described in the “Respiratory Mechanics” section.
Bronchoalveolar lavage was performed and various endpoints examined as described in detail in the
Values for all measurements are expressed as mean ± SEM. ANOVA was used to determine
the levels of difference between all groups. Comparisons for all pairs were performed by unpaired
two-tailed Student’s t test. Statistics were performed using GraphPad Prism (version 4) statistical
software (GraphPad Software Inc., San Diego, CA) and Microsoft Excel 2002 software.
Significance levels were set at a p value of 0.05.
Baseline lung function and airway hyperresponsiveness
Whole body barometric plethysmography
Whole body barometric plethysmography was used to non-invasively assess baseline lung
function in αERKO and βERKO mice. Breathing frequency was significantly reduced in male and
female αERKO mice relative to wild type controls (Table 1). Male wild type mice were found to
have a significantly higher tidal volume than female wild type mice; however, this pattern was
reversed in αERKO mice (Table 1). Similarly, minute ventilation, peak inspiratory flow and peak
expiratory flow were higher in male versus female wild type but not in αERKO mice (Table 1). In
contrast, disruption of ERβ had no influence on gender differences in tidal volume, minute
ventilation, peak inspiratory flow and peak expiratory flow (Table 1). However, breathing frequency
was significantly lower and peak inspiratory flow was significantly higher in female βERKO mice
relative to female wild type mice. Moreover, tidal volume was higher in both male and female
βERKO mice relative to their gender-matched wild type controls (Table 1). Together, these data
suggest that both ERα and ERβ play a role in the regulation of breathing with ERα having the more
αERKO females exhibited substantially enhanced bronchial responsiveness to inhaled
methacholine compared to wild type females (Figure 1A). Similarly, male αERKO mice were
hyperresponsive to methacholine compared to their male wild type counterparts, although the
differences were less pronounced than in females (Figure 1B). In contrast, there were no significant
differences in methacholine responsiveness between male or female βERKO mice and their gender-
matched wild type controls (Figure 1, A and B). As Penh is not a universally accepted measure of
bronchoconstriction in mice (22), we also examined airway hyperresponsiveness using invasive
Invasive measurement of lung function and airway responsiveness
We focused our attention on the female αERKO mice with the more robust lung phenotype.
Lung function and methacholine responsiveness were measured in anesthetized, intubated and
mechanically ventilated mice. Under basal conditions, there were no significant differences between
αERKO and wild type females with respect to total elastance (E), Newtonian resistance (Rn) or
tissue elastance (H) (Table 2). However, total respiratory resistance (R), tissue resistance (G) and
hysteresivity (η) were significantly reduced in αERKO female mice compared to wild type females
(Table 2). The reductions in total respiratory resistance and in tissue resistance at baseline in the
αERKO females are consistent with the reduced baseline Penh as assessed by barometric
plethysmography (Table 1). Consistent with the non-invasive barometric plethysmography results,
invasive measurement of lung function confirmed that αERKO females exhibit hyperresponsiveness
to inhaled methacholine (Figure 2). Specifically, PC200R, PC50E and PC200G were significantly
reduced in αERKO female mice relative to wild type females indicating that the lung periphery
plays a role in the enhanced bronchoconstriction to inhaled methacholine in the αERKO mice.
αERKO females also tended to have reduced PC200Rn and reduced PC50H, although these
differences were not statistically significant. Together, these data confirm, via an alternative
method, that lack of ERα leads to basal lung function abnormalities and hyperresponsiveness to
Role of circulating estrogen in the α αERKO phenotype
Female αERKO mice have elevated circulating levels of estrogen and androgen (23). To
address the possibility that altered sex hormone levels in the female αERKO mice were responsible
for the methacholine hyperresponsiveness, wild type and αERKO female mice were ovariectomized
and lung function was assessed 3 weeks later using whole body barometric plethysmography.
Ovariectomy reduced absolute responsiveness to methacholine in the αERKO mice (compare
αERKO mice in Figure 1A to αERKO ovariectomized mice in Figure 3). However, removal of the
ovaries failed to completely abolish differences in methacholine responsiveness between αERKO
and wild type female mice as ovariectomized αERKO mice were still hyperresponsive relative to
ovariectomized wild type mice (Figure 3). Interestingly, ovariectomy did not alter the response of
wild type mice to methacholine. Ovariectomy abolished most of the differences between wild type
and αERKO mice with respect to basal lung function parameters (Table E1, online supplement).
Following ovariectomy, breathing frequency in αERKO mice was still reduced relative wild type
mice, but this did not reach statistical significance (p = 0.06) (Table E1, online supplement).
We next investigated the role of estrogen in modulating airway hyperresponsiveness using
invasive measurements of lung function. Similar to the whole body plethysmography experiments,
we examined airway hyperresponsiveness in ovariectomized mice. In addition, to determine
whether high levels of estradiol alone could recapitulate the hyperresponsive phenotype in wild type
mice, we administered estradiol using implantable, sustained-release pellets, which have been shown
to produce circulating, steady-state estradiol levels comparable to those found in αERKO female
mice. In wild type mice, neither ovariectomy nor estradiol treatment had any effect on airway
responsiveness to methacholine (Figure 4A). In contrast, ovariectomy reduced responsiveness to
methacholine in αERKO mice as evidenced by increased values for PC200R, PC200G and PC50H
(Figure 4B). Estradiol treatment had no potentiating effect on airway hyperresponsiveness in
αERKO mice. Collectively, these data suggest that the high circulating levels of estrogen in the
αERKO mice may contribute to the hyperresponsive phenotype; however, the data also suggest that
high circulating estrogen levels alone are not sufficient for the phenotype to occur – the ERα must
also be absent.
Role of nerve and muscle in airway hyperresponsiveness
Acetylcholine release following electrical field stimulation of tracheas
In order to assess the involvement of neural pathways in the hyperresponsive phenotype of
αERKO mice, we measured release of acetylcholine from isolated tracheas stimulated by electrical
field stimulation. As shown in Figure 5, there was enhanced release of acetylcholine from tracheas
of αERKO female mice relative to wild type controls. As acetylcholine release is controlled by
prejunctional inhibitory M2 muscarinic autoreceptors (9), the increased release of acetylcholine
strongly suggests that these receptors are dysfunctional in the αERKO mice.
M2 muscarinic autoreceptor function
We next investigated the function of the M2 muscarinic receptors. Tracheas were subjected
to electrical field stimulation in the presence or absence of the specific M2 muscarinic receptor
antagonist, gallamine. Electrical field stimulation of tracheas from both genotypes resulted in
frequency-dependent contractile responses. As predicted, pre-treatment with gallamine potentiated
the contractile response in tissues from wild type mice (Figure 6A) demonstrating the presence of
functional muscarinic M2 muscarinic receptors. In contrast, pre-treatment of αERKO mouse tissues
with gallamine had no effect on contractile response to electrical field stimulation (Figure 6B). The
lack of effect of gallamine on contractile response in αERKO mouse tissues indicates that αERKO
mice have dysfunctional M2 muscarinic receptors. One possible mechanism for M2 muscarinic
receptor dysfunction is downregulation of M2 receptor expression (9). To examine this possibility,
we measured protein levels of this receptor in lung homogenates using a specific M2 muscarinic
receptor antibody. We found significantly reduced M2 muscarinic receptor expression in lungs from
αERKO female mice relative to wild type controls (Figure 7A). Densitometric analyses normalized
to actin expression revealed an approximate 50% reduction in M2 muscarinic receptor expression in
αERKO females (Figure 7B). Together, these data suggest that dysfunctional M2 muscarinic
receptors may contribute to the hyperresponsive phenotype in αERKO mice.
Assessment of airway smooth muscle responsiveness.
In order to assess the involvement of airway smooth muscle in the hyperresponsive
phenotype of αERKO mice, isometric force measurements using isolated bronchial ring preparations
were employed. Carbachol induced a significantly greater increase in isometric tension in female
αERKO compared to female wild type bronchial rings ex vivo (Figure 8). These data suggest that
the hyperresponsive phenotype observed in αERKO female mice may be due, at least in part, to
alterations in airway smooth muscle contractile function.
Airway responsiveness to serotonin
In order to determine whether the hyperresponsive phenotype of αERKO mice was specific
to cholinergic agonists, we examined responsivity to serotonin. Similar to the results for
methacholine, αERKO mice were hyperresponsive to inhaled serotonin with significantly reduced
PC200R, PC50E, PC200Rn, PC200G and PC50H relative to wild type mice (Figure 9). These data
suggest that αERKO mice are also hyperresponsive to other bronchoconstrictors.
Role of ERα α in allergic airway disease
Airway responsiveness following allergen challenge
Our next objective was to assess the role of ERα in a clinically relevant lung disease model.
Airway responsiveness to methacholine was assessed in wild type and αERKO mice in an
established model of allergic airway disease involving initial sensitization and subsequent exposure
to ovalbumin. Disruption of ERα had profound effects on the degree of airway hyperresponsiveness
following allergen challenge (Figure 10). Thus, compared to allergic wild type mice and non-
allergic mice of both genotypes, allergic αERKO mice exhibited significantly reduced PC200R,
PC50E, PC200Rn, PC200G and PC50H. It should be noted that, in contrast to naïve αERKO mice,
allergic αERKO mice exhibited enhanced responsiveness to methacholine in the central airways as
evidenced by significantly reduced PC200Rn. These results indicate that absence of ERα leads to
greatly enhanced airway responsiveness following allergen challenge.
Inflammation and cytokine release following allergen challenge
Allergen sensitization and exposure resulted in an influx of inflammatory cells into the
airways. Interestingly, there were no differences between allergic wild type and allergic αERKO
mice with respect to numbers of total cells, eosinophils, lymphocytes and macrophages recovered in
the bronchoalveolar lavage (BAL) fluid (Figure 11A). There was a small reduction in the number of
neutrophils in the airways of allergic αERKO relative to allergic wild type mice. There were no
differences between allergic wild type and allergic αERKO mice with respect to tissue inflammation
as assessed histologically (Figure E1, online supplement). There were no differences between
allergic wild type and allergic αERKO mice in BAL fluid levels of IL-4, IL-5, IL-12 or TNF-
α (Figure 11B). BAL fluid levels of total protein, a marker of alveolar epithelial permeability, were
also similar in allergic wild type and αERKO mice (Figure 11C). These results suggest that lack of
ERα does not appreciably alter the inflammatory response in the allergic airway despite having
profound effects on the development of allergen-induced airway hyperresponsiveness.
The physiological roles of ERs in the lung are largely unknown, hence we examined lung
function and airway hyperresponsiveness in ER deficient mice. The results described herein
implicate a major role for ERα in modulating lung function and airway hyperresponsiveness, and
describe a potential mechanism by which ERα mediates airway responsiveness.
There is considerable evidence supporting a role for sex hormones in the neural control of
breathing (24). Breathing disorders such as obstructive sleep apnea have been linked to sex hormone
levels (24). There is an increase in sleep disordered breathing after menopause which can be
alleviated by hormone replacement therapy (25, 26). Respiratory rhythm is generated by medullary
neurons in the brainstem (27), a site where ERα has been shown to be abundantly expressed (27-29).
Interestingly, we found a marked reduction in breathing frequency in male and female αERKO mice
relative to wild type controls. Male wild type mice have a significantly higher tidal volume than
female wild type mice; however, this pattern is reversed in αERKO mice. Similarly, minute
ventilation, peak inspiratory flow, and peak expiratory flow are higher in male versus female wild
type but not αERKO mice. Together, these data indicate that functional disruption of ERα leads to
changes in a variety of respiratory parameters and suggest that this nuclear receptor may be a critical
regulator of breathing and respiratory rhythmogenesis in mice.
ERβ disruption has no influence on gender differences in tidal volume, minute ventilation,
peak inspiratory flow and peak expiratory flow. However, breathing frequency is significantly lower
and peak inspiratory flow is significantly higher in female βERKO relative to female wild type mice.
Tidal volume is higher in both male and female βERKO mice relative to their respective wild type
controls. Consistent with this observation, Massaro and Massaro recently reported that βERKO
mice have a higher body mass-specific lung volume relative to wild type mice (30). These data
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suggest that ERβ does play a role in the regulation of breathing, albeit a much less dominant role
Airway hyperresponsiveness to cholinergic stimuli is a cardinal feature of asthma and a
major risk factor for accelerated decline of lung function and development of chronic obstructive
pulmonary disease (COPD) in humans (31, 32). The exact mechanism(s) underlying the
development of airway hyperresponsiveness in chronic lung diseases such as asthma remains
unknown. Several studies of risk factors associated with airway hyperresponsiveness have reported
higher responsiveness in females compared to males (32-34) suggesting the involvement of sex
hormones in the pathogenesis. Herein we demonstrate that in the absence of immunologic
stimulation, αERKO female mice exhibit substantially enhanced airway responsiveness to inhaled
methacholine compared to wild type females, suggesting that ERα is a critical regulator of this
Traditionally, airway hyperresponsiveness has been presumed to mainly involve the central
airways and not the periphery. However, physiologic and pathologic evidence has emerged in recent
years to support the role of the lung parenchyma and distal airways in the pathogenesis of airway
hyperresponsiveness (35-38). Airway hyperresponsiveness is influenced by properties of the central
airways and the surrounding pulmonary parenchyma which is tethered to the airways, and by
interactions between these two compartments (39). The exact location and precise mechanism for
changes in tissue resistance are controversial, but many hypotheses have been proposed including
contraction of parenchymal interstitial cells, contraction of smooth muscle cells within alveolar ducts
and changes in the architecture of the alveoli and alveolar ducts (40-42). It has also been suggested
that parenchymal changes could be secondary to airway narrowing, either by direct interaction
between the airways and parenchyma or indirectly by altering lung volume (39). Invasive