In the present study, we tried to develop a mouse model of chronic airway inflammation and remodeling induced by chronic exposure to antigen. Furthermore, the expressions of MMPs-9 and -12 were also investigated. BALB/c mice were sensitized and then repeatedly challenged with OVA every 3 days for 54 days. At the following day after the last challenge, of days 24, 39, and 54, histological changes of the airways were studied by hematoxylin-eosin and Masson's trichrome stains. The expressions of MMPs-9 and -12 were also measured by western blot. Persistent inflammatory cells infiltration and collagen deposition in the lung tissue were observed in repeatedly challenged mice. Furthermore, the expressions of MMPs-9 and -12 were increased in the airways after repeated antigen challenges. The severest inflammation was observed in the day-54 challenged group. These results suggest that MMPs-9 and -12 might be involved in the pathogenesis of chronic airway inflammation and remodeling induced by antigen exposure in mice.
Airway inflammation and remodeling are important features of chronic allergic airway diseases such as asthma [
Matrix metalloproteinases (MMPs) are a family of proteinases with zinc-dependent proteolysis, which play important roles in matrix turnover [
In particular, MMP-9 (gelatinase B) and MMP-12 (macrophage elastase), both of which are reportedly increased in the airways of asthmatic patients [
In the present study, a mouse model of chronic airway inflammation and remodeling was developed by repeated antigen exposure. By using this animal model of chronic airway inflammation and remodeling, the changes in the expressions of MMPs-9 and -12 were also investigated.
Male BALB/c mice (7-week old, specific-pathogen-free) were used. All experiments were approved by the Animal Care Committee at the Hoshi University (Tokyo, Japan). Mice were sensitized by intraperitoneal injection of 8
Schematic representation of the sensitization and challenge protocol. BALB/c mice (7-week old) were sensitized at days 0 and 5 and received repeated aerosol ovalbumin (OVA) challenge every 3 days from day 12 to day 54.
Twenty-four hr after the last antigen challenge, mice were anesthetized with urethane (1.6 g/kg, i.p.). One mL of phosphate-buffered saline (PBS) was instilled into the lungs and lavaged 3 times. The BAL fluid (BALF) was collected and then centrifuged (1,000 g, 10 min at room temperature). Then the supernatants were collected and stored at −80°C for the following experiments.
Lungs were fixed in 10% formaldehyde and embedded in Paraplast X-TRATM paraffin (Fisher Healthcare, Houston, TX). Four
Twenty-five
Inflammatory cells infiltration into the lung tissue was investigated with HE staining. Inflammatory cells infiltration in the peripheral bronchial and subepithelium was observed from day 24 (Figure
Inflammatory cells infiltration into the lungs of the repeatedly antigen challenged mice. Lung tissue sections from mice at days 24 ((a) and (b)), 39 ((c) and (d)), and 54 ((e) and (f)) were studied by HE stain (×40). Inflammatory cells infiltration in the peripheral bronchial and subepithelium was observed from day 24 (b) and most seriously at day 54 (f) when mice were repeatedly challenged with OVA. The inflammation scores in peripheral bronchial and subepithelium were graded as mentioned in Section
The distribution of collagen fiber in lung tissue was studied with Masson’s trichrome staining. Collagen fibers were found in the endothelium of vessel, the subepithelium and smooth muscle layer in the lung tissues of sensitized and challenged mice (Figures
Distribution of collagen fiber in lungs of the repeatedly antigen challenged mice. Lung tissue sections from mice at days 24 ((a) and (b)), 39 ((c) and (d)), and 54 ((e) and (f)) were studied by Masson’s trichrome staining (×40). Collagen fibers were found in the endothelium of vessel, the airway subepithelium and smooth muscle layer both in sensitized and challenged mice ((a) and (b)). Increased deposition of collagen in the airway subepithelium and smooth muscle layer was found in repeatedly challenged mice at day 54 (f), which was not observed in the airways of sensitized mice (e).
The bands corresponding to the proenzyme of MMP-9 (98 kD) (Figure
Upregulations of MMPs-9 and -12 in BALFs of the repeatedly antigen challenged mice. The bands corresponding to the proenzyme of MMP-9 (98 kD) (a), the proenzyme (54 kD) and intermediate form (45 kD) of MMP-12 (b) were detected by western blot. The expressions of MMPs-9 and -12 were persistently increased after repeated antigen challenges from day 24 to day 54, and significantly upregulated at day 54. Each column represents the mean with SEM from 3 to 4 independent experiments.
Allergic airway diseases are characterized by chronic airway inflammation and remodeling. A number of animal models that imitate much of the pathophysiology have been developed [
In the current mouse model, inflammation in lung tissue was observed at day 24 after antigen exposure till day 54 (Figures
MMPs-9 and -12, which degrade ECM components such as collagen, are thought to be deeply involved in the pathogenesis of chronic airway diseases [
Upregulated expressions of MMPs are thought to mainly contribute to the abnormal degradation of ECM in the airways, which leads to the physiological structural changes of the airways and is also thought to be one of the main reasons of airway hyperresponsiveness [
Besides ECM, MMPs are known for their proteolysis on nonmatrix such as cell surface receptors [
In summary, our results suggested that the processes of airway inflammation and remodeling are coordinated, and MMPs-9 and -12 are cooperative in these pathophysiological changes of chronic allergic airway diseases. These findings might provide a better understanding of the regulation of airway inflammation and remodeling in allergic airway disease.