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Published ahead of print on November 9, 2006, doi:10.1164/rccm.200509-1493OC
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American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 126-135, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200509-1493OC

Original Article

Spontaneous Airway Hyperresponsiveness in Estrogen Receptor-{alpha}–deficient Mice

Michelle A. Carey, Jeffrey W. Card, J. Alyce Bradbury, Michael P. Moorman, Najwa Haykal-Coates, Stephen H. Gavett, Joan P. Graves, Vickie R. Walker, Gordon P. Flake, James W. Voltz, Daling Zhu, Elizabeth R. Jacobs, Azzeddine Dakhama, Gary L. Larsen, Joan E. Loader, Erwin W. Gelfand, Dori R. Germolec, Kenneth S. Korach and Darryl C. Zeldin

Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health; Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; Departments of Medicine and Physiology, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin; and Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado

Correspondence and requests for reprints should be addressed to Darryl C. Zeldin, M.D., NIH/NIEHS, 111 T.W. Alexander Drive, Building 101, Room D236 Research Triangle Park, NC 27709. E-mail: zeldin{at}niehs.nih.gov


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 after ovalbumin sensitization and exposure.

Measurements and Main Results: Estrogen receptor-{alpha} 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-{alpha} also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.

Conclusions: These data suggest that estrogen receptor-{alpha} is a critical regulator of airway hyperresponsiveness in mice.

Key Words: lung function • asthma • hyperreactivity • M2 muscarinic receptor • estrogen receptor



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
The effect of estrogens in the lung is highly controversial and the results of published studies are contradictory.

What This Study Adds to the Field
This study suggests that estrogen receptor-{alpha} is involved in airway hyperresponsiveness.

 
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 women with asthma (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 (COPD). 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 (ACh), 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 ACh release (9). Loss of M2 receptor function increases ACh 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 subjects with asthma (12, 13) and in animal models of allergic airway disease (14, 15).

Estrogens mediate both transcriptional and nongenomic effects via {alpha} or beta estrogen receptors (ERs). Both nuclear receptors are expressed in the lung, with ERbeta being more abundant than ER{alpha} (16), but their functions in this organ are largely unknown. Mice lacking either ER{alpha} (ER{alpha} knockout [{alpha}ERKO]) or ERbeta (betaERKO) 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 ER{alpha} is a critical regulator of airway hyperresponsiveness and that {alpha}ERKO 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 (19).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
All procedures were performed under an approved animal study protocol in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice ({alpha}ERKO, betaERKO, and wild-type littermate controls on a pure C57BL/6 background; 12–16 wk of age) were obtained from Taconic Farms (Germantown, NY). 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, Wilmington, NC). Greater detail about these measurements is provided in the online supplement.

Respiratory Mechanics
Invasive analysis of lung function was performed on anesthetized mice using the Flexivent system (Scireq, Montreal, QC, Canada). Further detail is provided in the online supplement.

ACh Release Assay
ACh release was determined by spectrofluorometry using the Amplex Red ACh/acetylcholinesterase assay kit following the manufacturer's instructions (Molecular Probes, Carlsbad, CA). This assay is described in more detail in the online supplement.

Immunoblotting
Lung 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 the presence or absence of the M2 muscarinic receptor antagonist gallamine was assessed ex vivo as previously described (20), with some modifications that 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 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 previously. Bronchoalveolar lavage (BAL) was performed and various endpoints examined as described in detail in the online supplement.

Statistical Analysis
Values for all measurements are expressed as mean ± SEM. Analysis of variance 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 statistical software (version 4; GraphPad Software, Inc., San Diego, CA) and Microsoft Excel 2002 software. Significance levels were set at a p value of 0.05.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline Lung Function and Airway Hyperresponsiveness
Whole-body barometric plethysmography.
Whole-body barometric plethysmography was used to noninvasively assess baseline lung function in {alpha}ERKO and betaERKO mice. Breathing frequency was significantly reduced in male and female {alpha}ERKO mice relative to wild-type control animals (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 {alpha}ERKO mice (Table 1). Similarly, minute ventilation, peak inspiratory flow, and peak expiratory flow were higher in male versus female wild-type but not in {alpha}ERKO mice (Table 1). In contrast, disruption of ERbeta had no influence on sex 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 betaERKO mice relative to female wild-type mice. Moreover, tidal volume was higher in both male and female betaERKO mice relative to their sex-matched wild-type controls (Table 1). Together, these data suggest that both ER{alpha} and ERbeta play a role in the regulation of breathing, with ER{alpha} having the more dominant role.


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  		TABLE 1. BASAL LUNG FUNCTION IN WILD-TYPE, {alpha}ERKO, AND betaERKO MICE

 
{alpha}ERKO females exhibited substantially enhanced bronchial responsiveness to inhaled methacholine compared with wild-type females (Figure 1A). Similarly, male {alpha}ERKO mice were hyperresponsive to methacholine compared with 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 betaERKO mice and their sex-matched wild-type controls (Figures 1A and 1B). Because Penh (enhanced pause) is not a universally accepted measure of bronchoconstriction in mice (22), we also examined airway hyperresponsiveness using invasive techniques.


Figure 1
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  		Figure 1. Airway responsiveness to inhaled methacholine in wild-type, estrogen receptor-{alpha} knockout ({alpha}ERKO) and betaERKO mice. Responsiveness to inhaled methacholine was measured using whole-body barometric plethysmography in awake, unrestrained (A) female and (B) male mice. Penh, an index of bronchoconstriction, was measured at baseline and after sequential delivery of increasing concentrations of methacholine (6.25–100 mg/ml). Results are reported as % increase in Penh over baseline values. Data represent means ± SEM of at least eight mice per group; *p < 0.05 versus wild type; ^p < 0.05 versus betaERKO.

 
Invasive measurement of lung function and airway responsiveness.
We focused our attention on the female {alpha}ERKO 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 {alpha}ERKO 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 ({eta}) were significantly reduced in {alpha}ERKO female mice compared with wild-type females (Table 2). The reductions in total respiratory resistance and in tissue resistance at baseline in the {alpha}ERKO females are consistent with the reduced baseline Penh as assessed by barometric plethysmography (Table 1). Consistent with the noninvasive barometric plethysmography results, invasive measurement of lung function confirmed that {alpha}ERKO females exhibit hyperresponsiveness to inhaled methacholine (Figure 2). Specifically, PC200(provocative concentration of methacholine aerosol which produces a 200% increase over baseline values)R, PC50(provocative concentration of methacholine aerosol which produces a 50% increase over baseline values)E, and PC200G were significantly reduced in {alpha}ERKO female mice relative to wild-type females, indicating that the lung periphery plays a role in the enhanced bronchoconstriction to inhaled methacholine in the {alpha}ERKO mice. {alpha}ERKO 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 ER{alpha} leads to basal lung function abnormalities and hyperresponsiveness to inhaled methacholine.


Figure 2
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  		Figure 2. Invasive measurement of airway responsiveness in female wild-type and {alpha}ERKO mice. After acquisition of baseline data, airway responsiveness to inhaled methacholine (0–25 mg/ml) was assessed using the forced oscillation technique in anesthetized, intubated, and mechanically ventilated mice. Linear interpolation was used to determine the provocative concentration of methacholine aerosol at which a 200% increase (PC200) over baseline values was observed for total respiratory resistance (R), Newtonian resistance (Rn), and tissue resistance (G), and at which a 50% increase (PC50) over baseline values was observed for total elastance (E) and tissue elastance (H). Data represent the means ± SEM of at least six mice per group; *p < 0.05 versus wild type.

 

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  		TABLE 2. BASAL LUNG FUNCTION IN WILD-TYPE AND {alpha}ERKO MICE MEASURED INVASIVELY

 
Role of Circulating Estrogen in the {alpha}ERKO Phenotype
Female {alpha}ERKO mice have elevated circulating levels of estrogen and androgen (23). To address the possibility that altered sex hormone levels in the female {alpha}ERKO mice were responsible for the methacholine hyperresponsiveness, wild-type and {alpha}ERKO female mice underwent ovariectomy and lung function was assessed 3 wk later using whole-body barometric plethysmography. Ovariectomy reduced absolute responsiveness to methacholine in the {alpha}ERKO mice (compare {alpha}ERKO mice in Figure 1A with {alpha}ERKO ovariectomized mice in Figure 3). However, removal of the ovaries failed to completely abolish differences in methacholine responsiveness between {alpha}ERKO and wild-type female mice because ovariectomized {alpha}ERKO 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 {alpha}ERKO mice with respect to basal lung function parameters (Table E1 of the online supplement). After ovariectomy, breathing frequency in {alpha}ERKO mice was still reduced relative wild-type mice, but this did not reach statistical significance (p = 0.06) (Table E1).


Figure 3
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  		Figure 3. Airway responsiveness to inhaled methacholine in ovariectomized female wild-type and {alpha}ERKO mice. Responsiveness to inhaled methacholine was measured using whole-body barometric plethysmography in awake, unrestrained female mice. Penh, an index of bronchoconstriction, was measured at baseline and after sequential delivery of increasing concentrations of methacholine (6.25–100 mg/ml). Results are reported as % increase in Penh over baseline values. ovex = ovariectomized. Data represent means ± SEM of eight mice per group; *p < 0.05 versus wild-type ovex.

 
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 {alpha}ERKO 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 {alpha}ERKO mice as evidenced by increased values for PC200R, PC200G, and PC50H (Figure 4B). Estradiol treatment had no potentiating effect on airway hyperresponsiveness in {alpha}ERKO mice. Collectively, these data suggest that the high circulating levels of estrogen in the {alpha}ERKO 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 ER{alpha} must also be absent.


Figure 4
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  		Figure 4. Invasive measurement of airway responsiveness to methacholine in female (A) wild-type and (B) {alpha}ERKO mice after ovariectomy (ovex) or implantation of sustained-release pellets containing estradiol. Control animals were sham ovariectomized or received placebo pellets and combined into one control group. After acquisition of baseline data, airway responsiveness to inhaled methacholine (0–25 mg/ml) was assessed using the forced oscillation technique in anesthetized, intubated, and mechanically ventilated mice. Linear interpolation was used to determine the provocative concentration of methacholine aerosol at which a 200% increase (PC200) over baseline values was observed for R, Rn, and G, and at which a 50% increase (PC50) over baseline values was observed for E and H. Data represent the means ± SEM of at least six mice per group; *p < 0.05 versus {alpha}ERKO sham/placebo.

 
Role of Nerve and Muscle in Airway Hyperresponsiveness
ACh release after electrical field stimulation of tracheas.
To assess the involvement of neural pathways in the hyperresponsive phenotype of {alpha}ERKO mice, we measured release of ACh from isolated tracheas subjected to electrical field stimulation. As shown in Figure 5, there was enhanced release of ACh from tracheas of {alpha}ERKO female mice relative to wild-type control animals. Because ACh release is controlled by prejunctional inhibitory M2 muscarinic autoreceptors (9), the increased release of ACh strongly suggests that these receptors are dysfunctional in the {alpha}ERKO mice.


Figure 5
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  		Figure 5. Acetylcholine release from wild-type and {alpha}ERKO tracheas in response to electrical field stimulation. Data represent the means ± SEM of at least four mice per group; *p < 0.05 versus wild type.

 
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, pretreatment with gallamine potentiated the contractile response in tissues from wild-type mice (Figure 6A), demonstrating the presence of functional M2 muscarinic receptors. In contrast, pretreatment of {alpha}ERKO 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 {alpha}ERKO mouse tissues indicates that {alpha}ERKO mice have dysfunctional M2 muscarinic receptors. One possible mechanism for M2 muscarinic receptor dysfunction is down-regulation 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 {alpha}ERKO female mice relative to wild-type control animals (Figure 7A). Densitometric analyses normalized to actin expression revealed an approximate 50% reduction in M2 muscarinic receptor expression in {alpha}ERKO females (Figure 7B). Together, these data suggest that dysfunctional M2 muscarinic receptors may contribute to the hyperresponsive phenotype in {alpha}ERKO mice.


Figure 6
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  		Figure 6. M2 muscarinic receptor function in wild-type and {alpha}ERKO tracheas. The ex vivo response of tracheal smooth muscle from (A) wild-type female and (B) {alpha}ERKO female mice to electrical field stimulation in the presence and absence of the M2 muscarinic receptor antagonist gallamine. Data represent the means ± SEM of at least eight mice per group; *p < 0.05 versus wild type.

 

Figure 7
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  		Figure 7. Pulmonary M2 muscarinic receptor expression in female wild-type and {alpha}ERKO mice. (A) M2 muscarinic receptor and actin expression by immunoblotting in lungs from female wild-type and {alpha}ERKO mice. (B) Density of M2 muscarinic receptor expression was normalized to actin expression in each sample. Data represent the means ± SEM of four mice per group; *p < 0.05 versus wild type.

 
Assessment of airway smooth muscle responsiveness.
To assess the involvement of airway smooth muscle in the hyperresponsive phenotype of {alpha}ERKO mice, isometric force measurements using isolated bronchial ring preparations were performed. Carbachol induced a significantly greater increase in isometric tension in female {alpha}ERKO compared with female wild-type bronchial rings ex vivo (Figure 8). These data suggest that the hyperresponsive phenotype observed in {alpha}ERKO female mice may be due, at least in part, to alterations in airway smooth muscle contractile function.


Figure 8
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  		Figure 8. Isometric force measurements in isolated bronchial rings from female wild-type and {alpha}ERKO mice. The tension response of rings to incrementally increasing concentrations of carbachol (10–7 to 10–3 M) was examined. Data represent the means ± SEM of at least eight mice per group; *p < 0.05 versus wild type.

 
Airway responsiveness to serotonin.
To determine whether the hyperresponsive phenotype of {alpha}ERKO mice was specific to cholinergic agonists, we examined responsivity to serotonin. Similar to the results for methacholine, {alpha}ERKO 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 {alpha}ERKO mice are also hyperresponsive to other bronchoconstrictors.


Figure 9
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  		Figure 9. Invasive measurement of airway responsiveness to inhaled serotonin in female wild-type and {alpha}ERKO mice. After acquisition of baseline data, airway responsiveness to inhaled serotonin (0–20 mg/ml) was assessed using the forced oscillation technique in anesthetized, intubated, and mechanically ventilated mice. Linear interpolation was used to determine the provocative concentration of serotonin aerosol at which a 200% increase (PC200) over baseline values was observed for R, Rn, and G, and at which a 50% increase (PC50) over baseline values was observed for E and H. Data represent the means ± SEM of at least 10 mice per group; *p < 0.05 versus wild type.

 
Role of ER{alpha} in Allergic Airway Disease
Airway responsiveness after allergen challenge.
Our next objective was to assess the role of ER{alpha} in a clinically relevant lung disease model. Airway responsiveness to methacholine was assessed in wild-type and {alpha}ERKO mice in an established model of allergic airway disease involving initial sensitization and subsequent exposure to ovalbumin. Disruption of ER{alpha} had profound effects on the degree of airway hyperresponsiveness after allergen challenge (Figure 10). Thus, compared with allergic wild-type mice and nonallergic mice of both genotypes, allergic {alpha}ERKO mice exhibited significantly reduced PC200R, PC50E, PC200Rn, PC200G, and PC50H. It should be noted that, in contrast to naive {alpha}ERKO mice, allergic {alpha}ERKO mice exhibited enhanced responsiveness to methacholine in the central airways as evidenced by significantly reduced PC200Rn. These results indicate that absence of ER{alpha} leads to greatly enhanced airway responsiveness after allergen challenge.


Figure 10
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  		Figure 10. Airway responsiveness to inhaled methacholine after ovalbumin (OVA) sensitization and exposure in female wild-type and {alpha}ERKO mice. After acquisition of baseline data, airway responsiveness to inhaled methacholine (0–25 mg/ml) was assessed using the forced oscillation technique in anesthetized, intubated, and mechanically ventilated mice. Linear interpolation was used to determine the provocative concentration of methacholine aerosol at which a 200% increase (PC200) over baseline values was observed for R, Rn, and G, and at which a 50% increase (PC50) over baseline values was observed for E and H. Data represent the means ± SEM of at least 4 nonallergic and 12 allergic mice per group; *p < 0.05 versus wild-type vehicle; ^p < 0.05 versus {alpha}ERKO vehicle; #p < 0.05 versus wild-type OVA.

 
Inflammation and cytokine release after 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 {alpha}ERKO mice with respect to numbers of total cells, eosinophils, lymphocytes, and macrophages recovered in the BAL fluid (Figure 11A). There was a small reduction in the number of neutrophils in the airways of allergic {alpha}ERKO relative to allergic wild-type mice. There were no differences between allergic wild-type and allergic {alpha}ERKO mice with respect to tissue inflammation as assessed histologically (Figure E1). There were no differences between allergic wild-type and allergic {alpha}ERKO mice in BAL fluid levels of IL-4, IL-5, IL-12, or tumor necrosis factor-{alpha} (Figure 11B). BAL fluid levels of total protein, a marker of alveolar epithelial permeability, were also similar in allergic wild-type and {alpha}ERKO mice (Figure 11C). These results suggest that lack of ER{alpha} does not appreciably alter the inflammatory response in the allergic airway despite having profound effects on the development of allergen-induced airway hyperresponsiveness.


Figure 11
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  		Figure 11. (A) Bronchoalveolar lavage (BAL) fluid cells, (B) cytokines, (C) and protein after OVA sensitization and exposure in female wild-type and {alpha}ERKO mice. BAL fluid was collected 24 h after the fifth daily OVA aerosol challenge. Data represent the means ± SEM of at least 3 nonallergic and 12 allergic mice per group; * p < 0.05 versus wild-type OVA.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The physiologic 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 ER{alpha} in modulating lung function and airway hyperresponsiveness, and describe a potential mechanism by which ER{alpha} 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 in which ER{alpha} has been shown to be abundantly expressed (27–29). Interestingly, we found a marked reduction in breathing frequency in male and female {alpha}ERKO mice relative to wild-type control animals. Male wild-type mice have a significantly higher tidal volume than female wild-type mice; however, this pattern is reversed in {alpha}ERKO mice. Similarly, minute ventilation, peak inspiratory flow, and peak expiratory flow are higher in male versus female wild-type but not {alpha}ERKO mice. Together, these data indicate that functional disruption of ER{alpha} 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.

ERbeta disruption has no influence on sex 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 betaERKO relative to female wild-type mice. Tidal volume is higher in both male and female betaERKO mice relative to their respective wild-type controls. Consistent with this observation, Massaro and Massaro recently reported that betaERKO mice have a higher body mass–specific lung volume relative to wild-type mice (30). These data suggest that ERbeta does play a role in the regulation of breathing, albeit a much less dominant role than ER{alpha}.

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 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 with males (32–34), suggesting the involvement of sex hormones in the pathogenesis. Herein we demonstrate that in the absence of immunologic stimulation, {alpha}ERKO female mice exhibit substantially enhanced airway responsiveness to inhaled methacholine compared with wild-type females, suggesting that ER{alpha} is a critical regulator of this process.

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 measurement of lung function in the {alpha}ERKO mice at baseline revealed hyperresponsiveness primarily in the periphery. After allergen challenge, there was marked hyperresponsiveness in both the central and peripheral airways. Interestingly, Massaro and Massaro recently reported that ERs are required for the formation of a full complement of alveoli in female mice (30). Thus, it is possible that structural abnormalities related to formation and size of alveoli may play a role in the abnormal hyperresponsive phenotype observed in these mice. Alternatively, the parenchymal defect in the {alpha}ERKO mice could be secondary to airway narrowing.

The reduced airway responsiveness to methacholine after ovariectomy of the {alpha}ERKO mice suggests that ovarian products may play a role in the hyperresponsive phenotype. However, the lack of an effect of estrogen supplementation or ovariectomy on airway responsiveness in wild-type mice suggests that estrogen alone is not the only culprit. One possible explanation for these findings may be that the absence of ER{alpha} allows ERbeta to predominate in this model. ER{alpha} and ERbeta have distinct expression patterns, with some organs having a predominance of one receptor over the other and other tissues having comparable expression of both receptors (16, 43, 44). Studies with {alpha}ERKO and betaERKO mice have revealed that these receptors have both overlapping and unique (and sometimes opposite) roles in mediating estrogen-dependent action in vivo. Both receptors are expressed in the lung, with ERbeta levels being higher than ER{alpha} levels (16). Studies have revealed that there is a complex interplay between ER{alpha} and ERbeta in the regulation and autoregulation of their respective promoters (44) and that some of the biological functions of one receptor may be dependent on the presence of the other receptor (16, 44, 45). In the present study, estrogen may produce different effects via ERbeta, and the observed hyperresponsive phenotype in {alpha}ERKO mice may be due to an altered estrogen response rather than an absent one, as has been postulated with other phenotypes displayed by these mice (46–48). An alternative possibility could be that the hyperresponsive phenotype is driven by an ovarian product other than estrogen, such as an androgen. In this regard, the ovaries of the {alpha}ERKO mice produce 17beta-hydroxysteroid dehydrogenase type III, an enzyme normally found only in testes, which converts androstenedione to testosterone. Indeed, {alpha}ERKO females have elevated plasma levels of androgens, similar to those seen in wild-type males (23).

Estrogen has been shown to modulate the density of muscarinic receptors in vivo in extrapulmonary tissues (49, 50), but there are no reports of estrogen modulation of muscarinic receptor expression or function in the lung. Importantly, expression of the M2 muscarinic receptor is markedly reduced in {alpha}ERKO female mice relative to wild-type control mice. Consistent with this finding, tracheas from {alpha}ERKO female mice release more ACh in response to electrical field stimulation than tracheas from wild-type control mice. Furthermore, the lack of effect of gallamine, a selective M2 muscarinic receptor antagonist, on the contractile response of {alpha}ERKO tracheas to electrical field stimulation conclusively demonstrates M2 muscarinic receptor dysfunction in these mice. Together, these data indicate that one potential mechanism for airway hyperresponsiveness in {alpha}ERKO female mice could be down-regulation of M2 muscarinic receptor expression and function leading to increased ACh in the neuromuscular junction and resulting in enhanced bronchoconstriction after cholinergic agonist stimulation.

In light of our finding that {alpha}ERKO mice are also hyperresponsive to inhaled serotonin, it is possible that a reflex mechanism may be contributing to the generalized airway hyperresponsiveness in these mice. It is generally assumed that the response to methacholine reflects only a direct effect of the agonist on airway smooth muscle. However, studies suggest that a substantial component of the airway smooth muscle response to cholinergic agonists depends on a vagally mediated reflex (51). For example, administering cholinergic agonists increases the firing of sensory nerves in the lung, and vagotomy decreases the bronchoconstrictive response to methacholine (52, 53). Studies suggest that, in the mouse, the respiratory response to serotonin also involves a vagal reflex (54). Blockade of muscarinic M2 receptors with the subtype selective antagonist gallamine potentiates vagally induced bronchoconstriction by increasing ACh release (55). In addition, dysfunctional muscarinic M2 receptors have been shown to enhance reflex bronchoconstriction (56). Thus, if reflex bronchoconstriction is contributing to the enhanced airway hyperresponsiveness to methacholine and serotonin, then dysfunctional M2 receptors could be playing a major role by leading to enhanced ACh release after vagal stimulation. The in vitro response to carbachol may reflect an additional smooth muscle defect and this may or may not contribute to the airway hyperresponsiveness observed in the intact animal. The combined defects at the level of smooth muscle and nerve may explain why the hyperresponsiveness is so prominent in the {alpha}ERKO female mice.

Although deletion of ER{alpha} has minimal effects on airway inflammation after allergen challenge, it has a profound effect on the development of allergen-induced airway hyperresponsiveness. Indeed, the differences in airway responsiveness between wild-type and {alpha}ERKO mice are even more pronounced after exposure to ovalbumin. Although airway inflammation is frequently correlated with airway hyperresponsiveness to methacholine, and treatment of inflammation often improves hyperresponsiveness, dissociation between airway inflammation and hyperresponsiveness has been observed in other murine models of allergic airway disease (57, 58). Of note, allergen challenge induced minimal changes in airway responsiveness in wild-type mice in our study. This is not surprising because other investigators have shown that the C57BL/6 strain is one of the least responsive strains in terms of the development of airway responsiveness after ovalbumin sensitization and exposure (59, 60). Indeed, the fact that allergen challenge induced such a profound degree of airway hyperresponsiveness in {alpha}ERKO mice on a C57BL/6 background indicates that ER{alpha} is a potent regulator of this process.

Although targeted disruption of specific genes in mice offers a powerful tool to investigators, caution must be exercised in extrapolating from these studies to physiologic effects in humans. Knockout of a gene in mice may produce different effects than inactivation of the same gene in humans. In addition, targeted disruption in mice may lead to compensatory changes in expression of other genes, which may hamper interpretation of the results. Nevertheless, it is of interest to speculate on the potential clinical significance of our findings. There are several polymorphic sites in the human ER{alpha} locus and some of these polymorphisms have been associated with diseases such as cancer and osteoporosis (61, 62). Interestingly, in some instances, the phenotype associated with the polymorphism is also dependent on high levels of estrogen (62). The role of hormone replacement therapy in the treatment and prevention of cardiovascular disease has been highly controversial due to conflicting findings. It has been suggested that variation in estrogen effects on the cardiovascular system may be related, at least in part, to common variants in the ER{alpha} gene, which can significantly alter the way a person responds to endogenous and exogenous estrogen (63). It is possible that the conflicting human data on estrogen effects in the lung could also be due to genetic variability in ER{alpha}. Indeed, genetic variability in ER{alpha} with resultant effects on estrogen sensitivity could underlie the greater incidence and severity of asthma and airway hyperresponsiveness in females between the ages of puberty and menopause when circulating estrogen levels are high, and could also underlie the phenomenon of premenstrual asthma as estrogen levels fluctuate during the menstrual cycle. Of interest, the relevance of our findings to humans is supported by a recent publication by Dijkstra and coworkers who found that variations in ER{alpha} were associated with airway hyperresponsiveness and more rapid lung function decline in subjects with asthma (64).

In conclusion, our data suggest that, in the mouse, lack of ER{alpha} leads to airway hyperresponsiveness via defects at the level of airway smooth muscle and nerves, and may involve regulation of M2 muscarinic receptor expression and function. Our data also suggest that the {alpha}ERKO mouse may prove to be a useful model to study the mechanisms of sex hormone modulation of airway responsiveness in humans.


   Acknowledgments
 
The authors thank Drs. Anton Jetten, William Schrader, and Steven Kleeberger for helpful suggestions during preparation of this manuscript. They also thank Laura Miller DeGraff for assistance with pellet implantations and surgeries, and Sandy Ward for help with cell differentials.


   FOOTNOTES
 
Supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health. J.W.C. 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.

This article has been reviewed and approved for release by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency (EPA). Approval does not signify that the contents necessarily reflect the views and policies of the EPA, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200509-1493OC on November 9, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 23, 2005; accepted in final form November 3, 2006


   REFERENCES
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 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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