Chronic inflammation of the airway causes structural abnormalities and the development of airway remodeling. Among these changes, the hyperplasia of goblet cells and structural alterations of the subbasement membrane induce functional impairment resulting in the appearance of the typical clinical symptoms from bronchial hyperreactivity and airway constriction (18
). Clinically, although early prevention or treatment of airway remodeling may facilitate improvements in asthma symptoms, specific interventions that improve airway remodeling are scarce at present (20
). Thus, in order to develop the therapeutics that can treat the airway remodeling morphology, characterization of the mechanism mediating airway remodeling is required. The development of a murine airway-remodeling model would help reach this goal. Here, we present a murine experimental system with repeated exposure to the common allergen, HDM, for 12 weeks, and present findings of airway remodeling factors, hypertrophy of smooth muscles, increase in the degree of fibrosis in mucous membranes, and hyperplasia of goblet cells. Regarding the airway responsiveness and the bronchoalveolar lavage fluid, HDM-exposed mice demonstrated that the number of eosinophils and other allergic inflammatory reactions were enhanced and severe airway hyperreactivity was observed in response to methacholine.
Some experiments, however, have reported that inhalation of ovalbumin for 12 weeks weakens the degree of airway remodeling and observations of airway responsiveness were lost (21
). Clinically, ovalbumin is a rare allergen. It cannot induce chronic airway inflammation readily by itself, and induces tolerance rather than priming in most cases (14
). In this study, allergic bronchial asthma induced by the repeated or continuous exposure to aeroallergens resulted in a chronic inflammatory condition, using a common clinical allergen, HDM. This exposure resulted in the maintenance of the specific characteristic of bronchial asthma, airway remodeling.
Airway remodeling is a repair phenomenon in chronic airway inflammation and damage and involves various mediators. The mechanism to maintain the normal conditions is disturbed resulting in the appearance of hypertrophy of smooth muscles, the fibrosis of the subbasement membrane, and hyperplasia of goblet cells to name a few of the altered factors. In addition, the cytokines that are involved in the immune response of the type 2 helper cells, such as IL-13, IL-4 and TGF-β, mediate their effects on the epithelial cells in the airway. These immune mediators result in hyperplasia of goblet cells, and simultaneously induce fibrosis in the subbasement membrane, causing alteration of the components of the extracellular matrix (5
). The evaluation of cytokines in the bronchoalveolar lavage fluid performed to assess the effect of such mediators showed that after 7 weeks and 12 weeks, the concentrations of IL-4, IL-13, and TGF-β increased continuously (). These findings are consistent with prior reports demonstrating that among growth factors, TGF-β secreted by the epithelial cells in the airway induces hypertrophy of smooth muscle cells and fibrosis in the subbasement membrane by altering myofibroblasts. In addition, the type 2 helper immune response cytokines IL-4 and IL-13 are produced. The interaction between TGF-β and the cytokines results in chronic inflammation and the acceleration of airway remodeling (6
Concerning the proportion of inflammatory cells in the bronchoalveolar lavage fluid, in CFA 7 and 12 groups, the total cell numbers as well as eosinophils were increased. As the exposure to HDM continued, in the CFA 12 group, an increase in neutrophils was detected. Simultaneous assessment of the degree of airway remodeling showed that hyperplasia of goblet cells, subepithelial fibrosis, and the hypertrophy of the peribronchial smooth muscle were observed until 12 weeks, suggesting that eosinophils play an important role in airway remodeling.
Eosinophils released by TGF-β primarily, increase the synthesis of fibronectin, collagen I, and III, and thus facilitate the progression of airway remodeling (23
). The interaction of the immune response of the Th2 helper cells and the bronchial epithelial cells stimulates the release of various cytokines resulting in airway remodeling (6
). Thus, it is believed that the increase of eosinophils maintains the Th2 immune response and simultaneously, through TGF-β, facilitates the progression, or at least the maintenance, of the airway remodeling.
In addition, in the airway or blood of bronchial asthma patients, neutrophils account for over 50% of cells that are involved in the thickening of the subbasement membrane and cells positive for TGF-β (24
). Neutrophils have been reported to play an important role in the degranulation of goblet cells (25
), which suggests an important role for neutrophils in airway remodeling. However, in animal studies, from 2 hr to 48 hr after a challenge with allergens, neutrophils have been reported to be temporarily increased in the bronchoavleolar lavage fluid (26
It has been reported that such a response is the result of a challenge with a large allergen bolus in the laboratory; these observations were not reported with chronic exposure to a small amount of allergen where neutrophils were increased continuously (27
). In addition, it has been reported that neutrophils induce hyperplasia of goblet cells and the progression of subepithelial fibrosis by stimulating the airway epithelial cells through the release of oxidants resulting in the release of epidermal growth factor and TGF-β, which stimulates the infiltration of neutrophils in the airway by the release of IL-8 (27
). In our study, in the 12 week group, the number of neutrophils in the CFA 12 group was increased even though the number of eosinophils in the CFA-12 group was reduced more than in the CFA 7 group. IL-13 was increased in the supernatant of the bronchoalveolar lavage fluid, and the high level of IL-4 and TGF-β was maintained. This suggests the involvement of neutrophils in airway remodeling with increased exposure time.
It was observed that the total serum IgE antibody response induced by the type two helper cell responses was increased in the CFA group until 6 weeks and continued for 12 weeks, and the D. farinae specific IgG1 antibody response, which is influenced by IL-4, increased with the increase of the exposure time to allergens.
IFA is a surfactant composed of paraffin oil containing mannide mono-oleate. It has been reported that CFA is a potent adjuvant as it contains heat-inactivated mycobacteria, lengthens the period of the presence of allergens when administered together, and delivers them efficiently to the immune system. IFA induces the type 2 helper cell response and thus increases antibody production (28
). Here, our data demonstrate that the application of such an adjuvant together with HDM allows maintenance of the type 2 helper response continuously.
In conclusion, our data demonstrate that repeated exposure to HDM maintains continuously airway-remodeling factors, hypertrophy of smooth muscles, increase of fibrosis in the subbasement membrane, hyperplasia of goblet cells, and the allergic inflammatory response such as airway hyperreactivity. In addition, in the bronchoalveolar lavage fluid, the increase of IL-4, IL-13, and TGF-β was maintained, and this may play an important role in the airway remodeling process. In addition, neutrophils increased as the exposure time increased, which suggests that neutrophils may also play an important role in the etiology of chronic bronchial asthma.