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TRANSLATIONAL PHYSIOLOGY
1Department of Medicine, Baylor College of Medicine, Houston, Texas; 2Department of Physiology, Vallabhbhai Patel Chest Institute, Delhi, India; 3Molecular Immunogenetics Laboratory, Institute of Genomic and Integrative Biology, Delhi, India; 4Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina; and 5Department of Pulmonary Medicine, MD Anderson Cancer Center, Houston, Texas
Submitted 6 June 2006 ; accepted in final form 18 August 2006
ABSTRACT
Allergic asthma is associated with airway epithelial cell mucous metaplasia and mucin hypersecretion, but the consequences of mucin hypersecretion on airway function are unclear. Recently, a peptide derived from the myristoylated alanine-rich C kinase substrate protein NH2-terminal sequence (MANS) was shown to inhibit methacholine (MCh)-induced mucin secretion from airway mucous cells by >90%. We studied the effect of intranasal pretreatment with this peptide on specific airway conductance (sGaw) during challenge with MCh in mice with allergen-induced mucous cell metaplasia. sGaw was noninvasively measured in spontaneously breathing restrained mice, using a double-chamber plethysmograph. Pretreatment with MANS peptide, but not a control peptide [random NH2-terminal sequence (RNS)], resulted in partial inhibition of the fall in sGaw induced by 60 mM MCh (mean ± SE; baseline 1.15 ± 0.06; MANS/MCh 0.82 ± 0.05; RNS/MCh 0.55 ± 0.05 cmH2O/s). The protective effect of MANS was also seen in mice challenged with allergen for 3 consecutive days to increase airway hyperresponsiveness, although the degree of protection was less (baseline 1.1 ± 0.08; MANS/MCh, 0.65 ± 0.06; RNS/MCh 0.47 ± 0.03 cmH2O/s). Because routine sGaw measurement in mice includes nasal airways, the effectiveness of MANS was also confirmed in mice breathing through their mouths after nasal occlusion (baseline 0.92 ± 0.05; MANS/MCh 0.83 ± 0.06; RNS/MCh 0.61 ± 0.03 cmH2O/s). In all instances, sGaw in the MANS-pretreated group was 
35% higher than in RNS-treated controls, and mucous obstruction accounted for 50% of the MCh-induced fall in sGaw. In summary, mucin secretion has a significant role in airway obstruction in a mouse model of allergic asthma, and strategies to inhibit mucin secretion merit further investigation.
myristoylated alanine-rich C kinase substrate NH2-terminus sequence; specific airway conductance; goblet cells
An alternative approach to dissecting the pathophysiological role of mucus in airflow obstruction is selective inhibition of mucin secretion. This was not possible until recently, when a 24-amino acid peptide [myristoylated alanine-rich C kinase substrate (MARCKS) NH2-terminus sequence, (MANS)] corresponding to the NH2-terminal domain of the MARCKS protein, was found to inhibit mucin release from mouse airway epithelial cells by 90% (18). We used the MANS peptide to selectively block methacholine (MCh)-induced mucin hypersecretion in mice with allergen-induced airway epithelial cell mucous metaplasia. This allowed us to quantitatively resolve the contribution of mucin hypersecretion to pulmonary airflow obstruction and test the possible efficacy of specific inhibition of mucin secretion by the MANS peptide as a therapeutic approach in allergic asthma.
METHODS
This study was conducted in conformity with the American Physiological Society’s Guiding Principles in the Care and Use of Animals. Experimental protocols and study design were approved by the designated institutional review boards at the Vallabhbhai Patel Chest Institute and Institute of Genomic and Integrative Biology.
Induction of airway mucous metaplasia. Six- to eight-week-old BALB/c mice between 16 and 20 g were purchased from the Center of Cellular and Molecular Biology, India, and housed in accordance with institutional guidelines. To induce mucous metaplasia, mice were sensitized to ovalbumin (20 µg ovalbumin grade V, 2.25 mg alum in saline, pH 7.4; Sigma, St. Louis, MO) administered by weekly intraperitoneal injection for 4 wk. Sensitized mice were then challenged by exposure for 30 min to an aerosol of either 0.9% (wt/vol) saline (Ova/Sal) or 2.5% (wt/vol) ovalbumin in 0.9% saline (Ova/Ova), as described (4). Twenty mice were subjected to daily ovalbumin challenge for 3 days (Ova/Ova3) to increase airway inflammation and airway hyperresponsiveness (AHR). Experiments were conducted 7 days after challenge because intracellular mucin reaches a maximum at that time (4).
Quantification of airflow obstruction. Specific airway conductance (sGaw) was measured as the time lag between flows recorded from the head and thoracoabdominal chambers in consciously breathing, restrained mice, using the two-chambered body plethysmograph system devised by Buxco (PLY-3351, Biosystem XA software) (5, 15). Briefly, this equipment consists of two plastic cylinders used as the thoracoabdominal and head compartments that can be attached to one another with an orifice, such that the head and neck of a mouse can pass through but not its shoulders. Mice were acclimatized to being restrained in the plethysmograph during the sensitization and challenge phase. To measure sGaw, mice were placed in the thoracoabdominal chamber, maneuvered to pass their head through the orifice, and gently wedged between the orifice and a piston posteriorly. Before attaching the head chamber, a suitably cut latex sheet was passed snugly over the neck and interposed between the two chambers to ensure that the chambers would be airtight. Phase lag between the airflow signals recorded from the two chambers was approximated using the time lag at zero flow, and sGaw was calculated (5, 15). This technique has been extensively used in guinea pigs (15) and more recently mice (5). It is very sensitive to changes in airflow resistance in upper and central airways, but insensitive to peripheral and tissue resistance changes.
Experimental protocol. Following the sensitization, challenge, and an additional 7 days to allow for epithelial cell mucous metaplasia, baseline sGaw was recorded for 10 min. The head chamber was then removed, and each mouse was administered 50 µl of either 100 µM MANS or a control random NH2-terminal sequence (RNS) peptide as small volumes into each nostril alternately over 2 min. After 15 min, either a dose-response or a single challenge experiment was performed as follows. To obtain a dose-response curve, stepwise increasing concentrations of MCh [0–200 mM solution in normal saline (NS)] were administered during 1.5-min aerosolizations using an aerogen micropump nebulizer (Buxco) connected to the plethysmograph and were followed by measurement of sGaw for 3 min. In single-challenge experiments, a single 3-min MCh aerosol challenge (60 mM solution in NS) was administered to mice pretreated with MANS or RNS, with or without an additional 3-min ATP aerosol challenge (100 mM solution in NS) immediately afterwards. This was followed by continuous measurement of sGaw. Stable sGaw values at the end of 3-min windows were recorded for analysis. In some experiments, the nostrils of mice were occluded with adhesive tape before a single MCh challenge, thereby eliminating the effect of changes in nasal resistance on measured sGaw.
Histological analysis of mucous secretion. At the end of the experiment, mice were killed, and intracellular mucin remaining in the airways was assessed by routine and fluorescent periodic acid Schiff (PAS) staining (4). Serial sections of the main axial bronchus along with minor daughter branches that we refer to as penetrating bronchi were used for semiquantitative scoring. We scored each airway from 1 to 4 using a retained mucin score as follows: 1, near complete mucin secretion with many ghost cells (almost completely depleted of mucin granules, creating an empty appearance with faint outlines, see Fig. 1C) and most cells partially empty; 2, semicomplete mucin secretion with many cells partially empty, few ghost cells, and few cells densely populated with mucin granules (full cells); 3, limited mucin secretion with full cells and partially empty cells being equally predominant; 4, minimal mucus secretion with mostly full cells. To eliminate bias, scoring was done while blinded to the treatment. Two to three sections were scored per lung, and specimens from at least three different experiments were used per group.
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RESULTS
MANS therapy is safe. Intranasal administration of 50 µl MANS or RNS peptide solutions in 104 mice was associated with no ill effects other than a transient fall in sGaw with recovery to baseline during the 15-min observation period.
Intranasal MANS inhibits mucus secretion from the tracheobronchial airways. To determine whether intranasal MANS is effective in inhibiting tracheobronchial mucin secretion induced by MCh and/or ATP, retained intracellular mucin in axial and penetrating airways of MANS or RNS-pretreated mice was compared. Representative sections showing mucin secretion in response to MCh and/or ATP challenge after MANS or RNS treatment are shown in Fig. 1. Fluorescent PAS staining was found to be superior to conventional PAS staining for assessment of retained mucin, since the traditional method has greater background staining, which limits visualization of mucin granules (4). Pretreatment with MANS, but not RNS, blocked mucin release. Almost all airways (10 of 11) from RNS-pretreated mice were scored as 1, and nine of these had nearly complete mucin secretion with almost no retained mucin (Fig. 1C). In contrast, 14 of 20 airways from MANS-pretreated mice were scored as 4, of which eight had negligible mucus secretion (Fig. 1D). Thus mucin secretion was significantly inhibited in MANS-pretreated mice (Fig. 1, bottom, retained mucin score: MANS/MCh, 3.4 ± 0.2; RNS/MCh, 1.1 ± 0.08; P < 0.05).
MANS therapy inhibits airflow obstruction induced by escalating doses of MCh. To determine whether inhibition of mucin secretion by MANS can attenuate MCh-induced airflow obstruction, we studied the effect of MANS pretreatment on the MCh dose-response curve in mice. AHR to MCh was observed in both Ova/Ova and Ova/Ova3 mice compared with control (Fig. 2). As expected, repeated allergen challenge increased AHR, as seen by a leftward shift of the dose-response curve, i.e., lower doses of MCh were required for similar degree of fall in sGaw. In mice with mucous metaplasia, pretreatment with MANS significantly improved sGaw in the midrange of the dose-response curve and caused a rightward shift (Fig. 2). No significant effect of MANS was found in control mice that lack mucous metaplasia, indicating that the mechanism of MANS action is by inhibiting mucus secretion. The MCh dose associated with a 35% fall in sGaw from baseline was >200 mM in Ova/Sal mice, 110 mM in Ova/Ova mice, and 75 mM in Ova/Ova3 mice. MANS pretreatment increased this to 165 mM in Ova/Ova mice and 100 mM in Ova/Ova3 mice. Thus MANS inhibits MCh-induced airway obstruction in mice with mucous metaplasia. However, it was not possible to precisely separate the effect of mucus and bronchoconstriction in an escalating MCh dose-response experiment, since the former is likely to get depleted, while the latter increases progressively. Thus, to precisely study the role of mucus in MCh-induced airflow obstruction, single-challenge experiments were performed.
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In this study, we found that intranasal instillation of a MARCKS-related peptide, MANS, inhibited mucin secretion in the lung and improved airflow obstruction after MCh challenge. This extends the previous work by Singer et al. (18) describing the inhibitory effect of intratracheal MANS on mucin secretion in vivo and provides a functional basis for potential use of MARCKS-related peptides as therapy in diseases characterized by mucus-related obstruction.
While we histologically confirmed the inhibition of mucin secretion from bronchial mucous cells, we did not directly measure the amount of mucus secreted into the lumen. Singer et al. have previously found that MANS therapy caused near complete inhibition of secreted mucin (Muc5AC). Importantly, this was by ELISA-based measurements in bronchoalveolar lavage specimens and was different from histological bronchial mucin measurements. In their study, post-MCh intracellular bronchial mucin content was reduced by 20% in MANS-treated mice and 97% in RNS-treated mice, i.e., MANS treatment resulted in 80% inhibition of secretion. That is comparable to the changes seen by us.
To determine the functional consequences of MANS treatment, we utilized restrained double-chamber plethysmography that has been shown to be more sensitive to MCh-induced airflow obstruction than classical invasive mechanics (5). However, noninvasive methods are unable to adequately resolve changes in peripheral airway resistance (Raw) and lung parenchymal mechanics. Additional investigations based on partitioning of respiratory system impedance may, therefore, be useful in future studies.
We found that mucus-related obstruction accounted for about one-half of the total airflow obstruction induced by an intermediate dose of MCh that induced maximal mucous secretion but not maximal bronchoconstriction (Fig. 2). Mucus-related obstruction was a significant component of MCh-induced obstruction seen in dose-response experiments used to quantify AHR (Fig. 3). It is, therefore, important that changes in AHR to MCh should not be directly equated with changes in airway smooth muscle contractility, especially when using noninvasive methods.
Since changes in nasal resistance can affect sGaw, it was necessary to eliminate the possibility that MANS was effecting changes by altering the nasal airway architecture. We occluded the nostrils of spontaneously breathing mice, forcing them to breathe from their mouths, thereby nullifying the effect of any changes in nasal resistance. We found that MANS-induced improvement in sGaw was not significantly affected by nasal occlusion (open, 0.27 ± 0.07; occluded, 0.22 ± 0.08) and is thus unrelated to changes in nasal resistance. It was not possible to quantitatively isolate the nasal component of the MCh or MANS response in our experiments because oropharyngeal resistance in orally breathing mice was an unknown additional variable. The limited effect of MANS on the nasal airways is probably related to differences in mucin secretion between the lung and the nose. The nose contains mucin- secreting submucosal glands, connected to the lumen by ducts (2, 12). In contrast, the respiratory epithelium lining the tracheobronchial airways of mice lacks such glands or mucous cells, but is rich in Clara cells, similar to the peripheral small airways of humans that are of approximately the same size. During mucous metaplasia, Clara cells increase mucin production, resulting in the development of a mucous cell phenotype. Since the mechanism of action of MANS involves contact with the target cell membrane, it is much more likely to affect mucous cells lining the airway lumen than submucosal glands. Newer peptides related to MANS that are more soluble and suitable for aerosol delivery may be more effective and should be studied further.
The mechanisms of mucus-related airflow obstruction are likely to be primarily related to changes in airway caliber. This can be divided into two components. First, airway epithelial height increases by up to 5 µm during mucous metaplasia (4). This should slightly reduce the diameter of the proximal airways and increase Raw that varies to the fourth power of the radius during laminar flow. In our experiments, development of mucous metaplasia (Ova/Ova and Ova/Ova3 mice) was associated with a trend toward increased Raw (15% rise in Raw assuming constant lung volume) that failed to reach statistical significance (P = 0.065). Second, during triggered mucin secretion, intracellular mucin that is stored in a condensed phase is secreted into the lumen, where it gets hydrated and expands up to 500 times (20). Since this expansion is far greater than the associated loss of epithelial height during mucin secretion, it is likely to cause airway narrowing and consequent airway obstruction. From the differences in post-MCh sGaw in MANS- and RNS-treated mice that were breathing orally, it can be estimated that tracheobronchial Raw increased by about one-third (assuming constant lung volume) due to mucin secretion. This requires an airway narrowing of slightly less than 10% on average or 15–20% in the proximal and upper airways that are the sites of mucous production and the major contributors to resistance to airflow (cross-sectional area increases with increasing generations). The potential mucous volume [conservatively assuming hundredfold expansion (20)] stored in the mucin granules of mucous cells is 0.1 µl/mm2 {10 nl/mm2 [mucin volume density (4)] x 100}. Thus a 1-mm segment of a large proximal airway with mucous metaplasia (0.5–1 mm diameter) has potential mucous content of 0.18–0.35 µl and a volume of 0.2–0.8 µl. Even if one-half were secreted, the airway would narrow by >20%. These numbers are only representative of the potential for mucous-related narrowing, since the actual dynamics of mucous accumulation into the lumen are complex and include the function of the mucociliary escalator that clears mucus. However, it can be seen that the magnitude of changes in Raw reported by us are well within the potential for mucus-related airway narrowing.
The time course of mucus-related obstruction in our study was similar to the kinetics of mucin secretion. In recent studies, mucin exocytosis was 100% complete at 3 min when measured by capacitance changes during granule fusion (3); and 50% complete by 3.7 min, when measured as morphometric apical volume loss (8). Differences between MANS- and RNS-treated mice were seen within the first set of stable measurements (6 min from start of MCh exposure) and lasted for 10–15 min, by which time both recovered to baseline. This correlates well with the expected time frame of mucous secretion and implies that mucus is being effectively cleared, since no lasting effect is seen.
The mouse model of mucous hypersecretion we used was designed to mimic mucus-related obstruction in human asthma. While the model provides an excellent means of analyzing the secretory event, there are some differences between this model and human asthma. Unlike severe human asthma where extensive mucus plugging can be seen, the effect of mucous secretion on airway conductance in our model was modest and transient (13). The reasons for this could be manifold. First, the airways of mice constitute a large percentage of the lung and have a relatively large airway lumen compared with humans. This large airway caliber is speculated to reduce the flow-resistive load that would otherwise result from the rapid respiratory rate (250–350 breaths/min) required by the mouse to maintain body temperature (11). Second, occlusion of airways by mucus probably requires either excessive mucus and/or extremely narrow airways. Since murine airways have relatively large caliber and mucous metaplasia is limited to the first five generations of airways (5), it is likely that significant airway plugging is uncommon in murine models. While there were no plugged airways in our histological specimens, displacement during inflation with formalin cannot be excluded. Third, marked airway inflammation as in severe asthma is associated with compromised epithelial integrity and reduced mucociliary clearance. Exuded plasma proteins and cellular debris may promote the production of viscous mucus and the formation of luminal mucus plugs (7). These features may not be sufficiently recapitulated in our model, since our experiments were performed 1 wk after allergen challenge, by which time mucous metaplasia is maximal, but inflammation may have partially subsided. Airway closure has been detected in response to MCh challenge in allergically inflamed mice lungs shortly after allergen exposure (21). Thus further evaluation of the MANS peptide in alternative models of chronic or severe asthma would be helpful (19).
In summary, mucin secretion has a significant role in airway obstruction. Administration of a MARCKS-related peptide blocks mucin hypersecretion and improves mucus obstruction.
ACKNOWLEDGMENTS
This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-072984 (B. F. Dickey) and R37 HL-36982 (K. B. Adler), and Council of Scientific and Industrial Research (India) Asthma Task Force Project SMM0006 (B. Ghosh).
FOOTNOTES
Address for reprint requests and other correspondence: A. Agrawal, Dept. of Physiology, Vallabhbhai Patel Chest Institute, Delhi Univ., Delhi 110017, India (e-mail: aagrawal{at}bcm.tmc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
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