Monensin inhibits mast cell mediated airway contractions in human and guinea pig asthma models

Abstract

Asthma is a common respiratory disease associated with airway hyperresponsiveness (AHR), airway inflammation and mast cell (MC) accumulation in the lung. Monensin, an ionophoric antibiotic, has been shown to induce apoptosis of human MCs. The aim of this study was to define the effect of monensin on MC responses, e.g., antigen induced bronchoconstriction, and on asthmatic features in models of allergic asthma. Tracheal segments from house dust mite (HDM) extract sensitized guinea pigs were isolated and exposed to monensin, followed by histological staining to quantify MCs. Both guinea pig tracheal and human bronchi were used for pharmacological studies in tissue bath systems to investigate the monensin effect on tissue viability and antigen induced bronchoconstriction. Further, an HDM-induced guinea pig asthma model was utilized to investigate the effect of monensin on AHR and airway inflammation. Monensin decreased MC number, caused MC death, and blocked the HDM or anti-IgE induced bronchoconstriction in guinea pig and human airways. In the guinea pig asthma model, HDM-induced AHR, airway inflammation and MC hyperplasia could be inhibited by repeated administration of monensin. This study indicates that monensin is an effective tool to reduce MC number and MCs are crucial for the development of asthma-like features.

Figure 1

Astra blue staining of guinea pig trachea after 24 and 72 h exposure to monensin. The staining intensity score of mast cells (1: pale, 2: medium, 3: strong) after (A) 24 h and (C)72 h culture with 1 µM (M1) or 10 µM (M10) monensin, or vehicle (1% ethanol, Ctrl). The mast cell numbers after culturing for (B) 24 h and (D) 72 h. Representative pictures were listed on the right (n = 6–8 per group). Arrows denote mast cells. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Astra blue-Hematoxylin staining of guinea pig trachea after 2 to 72 h exposure to monensin. Percentage of mast cells that have detectable nuclei after (A) 2 h, (B) 24 h and (C)72 h exposure to 1 µM (M1) or 10 µM (M10) monensin, or vehicle (1% ethanol; Ctrl) with representative pictures on the right (n = 3–8 per group). Arrows denote the magnified mast cells. *p < 0.05 and **** p < 0.0001.

Figure 3

Responses of guinea pig trachea and human bronchi to monensin. (A) Smooth muscle contraction of tracheal segments and (B) human bronchi to 1 µM (M1) or 10 µM (M10) monensin with or without the presence of mepyramine (1 µM; Mepy) (n = 5–6 per group).

Figure 4

Responses of guinea pig and human airways to carbachol and histamine after monensin exposure. Guinea pig tracheal responses to carbachol after (A) 24 h and (C) 72 h, and histamine after (B) 24 h and (D) 72 h exposure to 1 µM (M1) or 10 µM (M10) monensin, or vehicle (1% ethanol; Ctrl). The responses of human bronchi to (E) carbachol and (F) histamine after 24 h incubation (n = 6 to 14 per group). ** p < 0.01.

Figure 5

Responses of guinea pig trachea to HDM and human bronchi to anti-IgE after monensin exposure. The responses of guinea pig tracheal segments to HDM after culturing with 1 µM (M1) or 10 µM (M10) monensin, or vehicle (1% ethanol; Ctrl) for (A) 2 h, (B) 24 h and (C) 72 h (n = 3–8 per group). The responses of human bronchi to anti-IgE after (D) 2 h and (E) 24 h incubation (n = 5–13 per group).

Figure 6

Effect of monensin on airway hyperresponsiveness. (A) Schematic diagram of the guinea pig asthma model. Animals were sensitized with a single i.p. injection of HDM + adjuvant followed by intranasal HDM challenges for three or five weeks. Monensin (0.5 mg/mL in 200 µL 12.5%EtOH/PBS) was given 24 h before each challenge. Airway responsiveness to methacholine (MCh) was assessed in a flexiVent system one day after (B–D) the third or (E–G) the fifth challenge. Differences of resistance in conducting airways (Rn; B and E), tissue damping (G; C and F) and tissue elastance (H; D and F) were compared between the animals exposed to HDM with or without monensin treatment (n = 4–7 per group). Control: animals exposed to PBS, received vehicle (12.5% EtOH) or monensin as pretreatment. HDM: animals exposed to HDM and received vehicle as pretreatment. HMH: animals exposed to HDM and received monensin as pretreatment *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 7

Inflammation and mast cells in airways of guinea pigs. (A) Inflammatory areas were compared in Hematoxylin–Eosin-stained slides. Mast cell number was counted in Astra blue stained sections after (B) three weeks and (C) five weeks challenges. Control: animals exposed to PBS, received vehicle (12.5% EtOH/PBS) or monensin as pretreatment. HDM: animals exposed to HDM and received vehicle as pretreatment. HMH: animals exposed to HDM and received monensin as pretreatment (n = 6–7 per group). *p < 0.05 and *** p < 0.001, ****p < 0.0001.

Figure 8

Weight curves of guinea pigs. The weights of animals from the first to the fifth challenge. Control: animals exposed to PBS, received vehicle (12.5% EtOH/PBS) or monensin as pretreatment. HDM: animals exposed to HDM and received vehicle as pretreatment. HMH: animals exposed to HDM and received monensin as pretreatment (n = 6–7 in each group).