Evidence that Mast Cells Regulate the Cough Hypersensitivity Associated with Eosinophilic Bronchitis

Abstract

Purpose: Eosinophils have been implicated as key effectors in the emergence of chronic cough. But causality has not been firmly established, and many patients with cough secondary to eosinophilic bronchitis (EB) are likely misdiagnosed as asthmatics based on successful empiric trials using inhaled corticosteroids. Better diagnostics and more precise therapeutic strategies for EB and other diseases that lead to chronic cough remain important but unmet clinical needs.

Methods: With the goal of better defining the mechanisms of cough in disease, we established a model of EB in guinea pigs using allergen challenges that induce a cough hypersensitivity responsive to steroid therapy.

Results: The heightened cough responsiveness associated with the eosinophilic inflammation was mimicked by LTD₄ inhalation and prevented by cysLT₁ receptor blockade with pranlukast or montelukast. But cysLT₁ receptor antagonism failed to prevent the eosinophilic infiltration of the airways evoked by allergen challenge. Additionally, inhalation of the mast cell selective stimulant Compound 48/80 mimicked the effects of allergen challenge on cough but failed to significantly increase eosinophilic infiltration of the airways. We also observed that thromboxane A2, through TP receptor engagement, acts downstream from and simultaneously with the leukotrienes to promote cough hypersensitivity in EB.

Conclusions: These results suggest that mast cells and not eosinophils may be essential to the emergence of cough hypersensitivity in EB. We speculate that therapeutic strategies targeting mast cells, cysLT₁ receptors, and TP receptors may represent endotype-specific treatments for chronic cough.

Keywords: Cough; Leukotrienes; MrgPRX2; Thromboxane.

Fig. 1

These representative traces (5-min duration) reveal the enhanced cough responses to 0.1 M citric acid challenge after induction of allergic inflammation in actively sensitized guinea pigs. Sensitized guinea pigs were challenged with aerosols of A vehicle (saline; n = 14) or B ovalbumin (1–1000 µg/mL; n = 15). An eosinophilic infiltrate to the airways 24 h after allergen challenge was documented (see Fig. 2), with a coincident cough hypersensitivity. As described in the Methods section, coughs (*) were defined by visual observation of a cough like effort, a sound recording (not shown; see supplemental Fig. 3) coinciding with a pressure recording (measured in arbitrary units) within the single chamber plethysmograph depicting an enhanced inspiratory effort (downward deflections in the traces above) followed by a forceful expiratory efforts (upward deflection) producing peak pressures > 5 times the pressures of tidal expiration. Augmented breaths/sighs (arrows) produced nearly symmetric inspiratory and expiratory pressure changes that were only modestly larger than those produced during tidal breathing. The expiratory pressures in the traces above were cropped for presentation

Fig. 2

Ovalbumin challenge in actively sensitized guinea pigs produces an eosinophilic bronchitis with associated cough hypersensitivity that is prevented by corticosteroid treatment. Eosinophilic infiltrates into the A airways (as assessed by BAL) and B bronchial mucosa were evoked by allergen challenge. C The eosinophilia is prevented by dexamethasone pretreatment (20 mg/kg ip twice before allergen challenge and again just prior to 0.1 M citric acid challenge). D A cough hypersensitivity accompanies the eosinophilic bronchitis that is also reversed by dexamethasone. The data are presented as medians and interquartile ranges of 6–15 separate experiments. Asterisks (**) indicate statistically significant differences between endpoints measured in challenged and unchallenged or steroid-treated guinea pigs (p < 0.001)

Fig. 3

Inhalation of LTD₄ acutely enhanced cough responses evoked by 0.1 M citric acid inhalation. A Enhancement of the cough responses was observed after inhalation of either 10 or 30 nM LTD₄. B The effects of 30 nM LTD₄ inhalation on cough were prevented by pretreatment with the cysLT₁ receptor antagonists pranlukast (30 µM inhaled) or montelukast (0.5 mg/kg ip). The data are presented as the medians and interquartile ranges of 5–15 experiments. An asterisk (*) indicates statistically significant differences between cough responses in the various treatment groups studied (p < 0.02); β: not different from responses in unchallenged animals (p > 0.1)

Fig. 4

The allergen-induced enhancement of cough responsiveness in guinea pigs is prevented by prior cysLT₁ receptor blockade with either pranlukast (30 µM inhaled) or montelukast (0.5 mg/kg IP). The data are presented as the medians and interquartile ranges of 5–15 experiments. An asterisk (*) indicates statistically significant differences in the cough responses in the various treatment groups studied (p < 0.05)

Fig. 5

Despite preventing allergen inhalation-induced cough hypersensitivity (Fig. 4), cysLT₁ receptor blockade with pranlukast (30 µM inhaled) did not prevent allergen-induced eosinophilia as assessed by A counting cells recovered by BAL or by B counting eosinophils in the mucosa of thin sections of guinea pig bronchi (ns: not significant (p > 0.05)). Each symbol denotes the results recorded in a single animal (horizontal bars: medians). An asterisk (*) indicates that a statistically significant increase in eosinophilia was observed following allergen challenge with or without prior pretreatment with pranlukast (p < 0.05)

Fig. 6

The effects of both allergen and LTD₄ inhalation on cough responsiveness are mimicked by inhalational challenge with the mast cell and MrgPRX2 receptor activator Compound 48/80. We have shown previously that Compound 48/80 evoked contractions of guinea pig airways that, like the contractions evoked by allergen, are partially inhibited by cysLT₁ receptor blockade [38]. A Compound 48/80 challenges potentiated 0.1 citric acid evoked coughing measured 1 day after inhalation of the MrgPRX2 receptor agonist. B Unlike allergen, however, compound 48/80 failed to significantly increase eosinophil influx into the airways as assessed by bronchoalveolar lavage (ns: not significant (p > 0.05). The data are presented as the medians and interquartile ranges of 5–8 experiments. An asterisk (*) indicates that a statistically significant increase in citric acid evoked cough responses was observed 24 h after compound 48/80 inhalation (p < 0.05)

Fig. 7

The thromboxane/TP receptor antagonist ICI 192605 (10 µM inhaled) prevents the heightened cough responses produced following A inhalation of the TP receptor agonist U46619 (0.1 µM), B 30 nM LTD₄ inhalation, and C aerosolized ovalbumin challenge in actively sensitized guinea pigs. Each symbol denotes the results recorded in a single animal (horizontal bars: medians). We have shown previously that ICI 192605 has little or no affinity for histamine H1 receptors, cysLT1 receptors, prostanoid EP receptors, or bradykinin B2 receptors [38, 49]. An asterisk (*) indicates statistically significant differences in cough responses among the treatment groups (p < 0.05; n = 7–15); β: not different from responses in unchallenged animals (p > 0.1)

Fig. 8

The eicosanoids A LTC₄, LTD₄, and/or LTE₄ and B TxB₂ are elevated in bronchoalveolar lavage fluid recovered from the lungs of actively sensitized guinea pigs 24 h after allergen/ovalbumin challenge. The release of TxB₂-IR was largely abolished by pretreatment with the cysLT₁ receptor antagonist Pranlukast (30 µM inhaled) or the steroid dexamethasone (20 mg/kg ip 24 and 1 h prior to allergen challenge and 1 h prior to citric acid challenge). The data are presented as the medians and interquartile ranges of 5–8 experiments. An asterisk (*) indicates statistically significant differences between treatment groups (p < 0.02)