Characterisation of an ovalbumin-allergic mouse model of airway mucus secretion

Katherine Choy1, Kevin Coote1, Henry Danahay2. 1Novartis Institutes of Biomedical Research, Horsham, West Sussex, United Kingdom, 2Novartis Institutes of Biomedical Research, Cambridge, MA, United States.

Secretory mucins are stored within intracellular secretory granules and are released by regulated exocytosis in response to secretagogues including muscarinic and purinergic agonists (Williams et al, 2006). Our aim was to characterize an ovalbumin (OVA)-allergic mouse model first described by Evans et al (2004), by testing the effects of purinergic and muscarinic secretagogues on epithelial mucus load (EML) as an inverse measurement of mucin secretion.

An airway mucus hypersecretory phenotype was induced in female Balb/C mice (5-6 weeks old) by 4 once-weekly sensitisations (20μg OVA + 2.25mg Al(OH)3 in 0.9% saline, 200μL i.p.) followed a week later by a 30 minute aerosol (Sidestream, Respironics, UK) challenge of either 0.9% saline or 2.5% (w/v) OVA in 0.9% saline. Vehicle (0.9% saline) or secretagogues were administered as aerosols 3 days post-challenge. Animals were terminated by overdose of barbiturate anesthesia 10 minutes post-exposure. Lungs were removed, fixed in 4% paraformaldehyde and EML quantified by image analysis (ImagePro, Media Cybernetics, UK) of Periodic Acid Fluorescent Schiff -stained lung sections. Data are expressed as mean % ± s.e.mean mucus/μm2 epithelium, and tested for significance using ANOVA with Newman-Keuls post-hoc test. All groups were n = 8.

OVA-challenged mice had significantly increased EML compared to saline-challenged mice (0.9% ± 0.2 vs 17.5% ± 2.1, P<0.01). As Evans et al observed a significant decrease in EML after 5 minutes exposure to 100mM ATP, we attempted to increase the maximum achievable window of secretion by testing the effect of increased exposure time (Table 1).

Table1. Effect of increasing time of exposure to vehicle or 100mM ATP on EML

After both 5- and 10-minute exposure to ATP, there was a significant decrease in EML(* = P<0.05) consistent with mucus secretion. However after 20 minutes the decrease was not significant. The maximum achievable window was not significantly different between any of the exposures.

We then tested alternative purinergic agonist UTP at 100mM for 5 minutes, the muscarinic agonist methacholine (0.51M) for 90 seconds or 100mM ATP for 5 minutes immediately followed by methacholine (Table 2).

Table 2. Effect of alternative secretagogues on EML.

All treatments caused significant decreases in EML compared to vehicle (* = P<0.05). However, there were no significant differences between the treatments.

We have demonstrated murine mucus secretion in response to both purinergic agonists (ATP and UTP), and to methacholine. The maximum decrease in epithelial mucus load appeared to be limited to approximately 50%, even when two classes of secretagogues were combined.

Evans CM et al Am J Respir Cell Mol Biol, 2004, 31(4): 382-394

Williams OW et al Am J Respir Cell Mol Biol 2006, 34: 527-536