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Induction of airway remodeling of nasal mucosa by repetitive allergen challenge in a murine model of allergic rhinitis
Induction of airway remodeling of nasal mucosa by repetitive allergen challenge in a murine model of allergic rhinitis Yune Sung Lim, MD*; Tae-Bin Won, MD*; Woo Sub Shim, MD*; Yong Min Kim, MD*; Jeong-Whun Kim, MD*; Chul Hee Lee, MD*†; Yang-Gi Min, MD*†; and Chae-Seo Rhee, MD*†
Background: Although many studies regarding airway remodeling in asthma have been reported, only a few studies have investigated airway remodeling in allergic rhinitis. Objectives: To determine whether repetitive allergen challenge could induce airway remodeling in the nose and evaluate the effect of steroids using a murine model of allergic rhinitis. Methods: To develop a mouse model of airway remodeling, ovalbumin-sensitized mice were repeatedly exposed to inhaled ovalbumin administration twice a week for 1 month and 3 months. Matched control mice were challenged with phosphatebuffered saline, and the treatment group received intraperitoneal dexamethasone injection. Trichrome, periodic acid–Schiff, hematoxylin-eosin, and immunohistochemical staining against matrix metalloproteinase 9 and tissue inhibitors of metalloproteinase 1 were performed to nasal and lung tissues, and the level of transforming growth factor  in the nasal lavage fluid was analyzed. Results: Repetitive ovalbumin challenge for 3 months induced circumferential peribronchial fibrosis in the lung. In the nose, subepithelial fibrosis, increased matrix metalloproteinase 9 and tissue inhibitors of metalloproteinase 1 expression, goblet cell hyperplasia, and submucous gland hypertrophy were observed compared with the control group. Features of airway remodeling were more prominent in the lung tissue. Administration of dexamethasone significantly inhibited these histologic changes. Conclusion: Airway remodeling associated with long-term allergen challenge can occur in the nasal mucosa and the lung. Steroid treatment prevents airway inflammation in response to acute allergen challenge, as well as airway remodeling by long-term allergen challenge. Ann Allergy Asthma Immunol. 2007;98:22–31.
INTRODUCTION Allergic rhinitis and bronchial asthma are representative allergic diseases of the upper and lower airways, sharing common pathophysiologic features such as infiltration of inflammatory cells, including eosinophils and lymphocytes, degranulation of mast cells, and expression of various cytokines in response to allergens. Thus, once regarded as 2 separate disease entities, they are now thought to be a common disease with different clinical manifestations.1 However, differences between the 2 tissues exist. The nose and bronchial airways differ in embryologic origin, the nose being of ectodermal and the lower airway of endodermal origin. Except for those smooth muscles associated with vessels, there is no smooth muscle in the nasal cavity compared with the bronchus, and although bronchial asthma is characterized by disruption and desquamation of bronchial epithelial cells by continuous exposure to allergen, epithelial cells in the nasal cavity are relatively well preserved in allergic rhinitis.2
* Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Korea. † Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Center, Seoul, Korea. Received for publication April 28, 2006. Accepted for publication in revised form September 12, 2006.
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It is well known that the airway remodeling process develops in asthmatic patients.3 These manifestations include epithelial disruption, goblet cell hyperplasia, mucous gland hypertrophy, increased collagen and matrix protein deposition, and smooth muscle hypertrophy and hyperplasia. However, studies on the airway remodeling process of the upper airway in allergic rhinitis are sparse, with previous reports showing conflicting results.4 In this study, to better understand the development of nasal airway remodeling in response to allergic inflammation, we investigated the features of airway remodeling in the nose and lung associated with repetitive allergen challenge in ovalbumin-sensitized mice and evaluated the effects of steroid administration. MATERIALS AND METHODS Induction of the Murine Model of Allergic Rhinitis and Airway Remodeling Animals. Specific pathogen-free BALB-c mice were used as the experimental animals. Each mouse weighed 20 to 30 g and was 3 to 5 weeks of age. The studies herein followed the principles for laboratory animal research as outlined in the Animal Welfare Act and Department of Health, Education, and Welfare (National Institutes of Health) guidelines for the experimental use of animals, and these experimental protocols were approved by our institution’s animal subjects committees.
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Figure 1. Mouse ovalbumin experimental protocol (0, 1, and 3 months). Mice were immunized by intraperitoneal injection of ovalbumin on days 0, 7, 14, and 21. Intranasal challenges were administered via daily ovalbumin inhalation from days 28 to 35, and then repeated ovalbumin challenges were performed twice a week for 1 or 3 months. Control mice were immunized but challenged with phosphate-buffered saline instead of ovalbumin twice a week during the 1- or 3-month study. Mice were killed 24 hours after the final ovalbumin challenge. Systemic dexamethasone was administered intraperitoneally on days 27, 36, and 50. Upward arrow indicates intraperitoneal ovalbumin injection; triangle, intranasal ovalbumin challenge; rectangle, intranasal ovalbumin or phosphate-buffered saline challenge; and circle, intraperitoneal dexamethasone injection.
Reagents. Ovalbumin (grade V) and aluminum hydroxide were purchased from Sigma Chemical Co (St Louis, MO). Dexamethasone was purchased form Merck & Co (West Point, PA). Sensitization and allergen challenge. Allergen sensitization and challenge for the development of the allergic rhinitis murine model was performed as described previously5 and is summarized in Figure 1. Briefly, on days 0, 7, 14, and 21, mice were immunized by intraperitoneal injection of 25 g of ovalbumin and 1 mg of aluminum hydroxide in 300 L of phosphate-buffered saline (PBS). One week after last immunization (day 28), mice received a series of 7 daily 1% ovalbumin challenges via a nebulizer (PulmoAide, Somerset, PA). The aerosolized ovalbumin (particle size, 0.5–5.0 m) was generated into a closed chamber of 8,800 cm3 (20 ⫻ 22 ⫻ 20 cm) made of acrylic for 30 minutes. The treatment group received a single intraperitoneal injection of 5 mg/kg of dexamethasone in PBS 1 day before the first ovalbumin challenge. Twentyfour hours after the final ovalbumin challenge, mice were killed for analysis (0-month group). In selected groups, 1% ovalbumin was repeatedly inhaled twice a week for 1 or 3 months for the development of airway remodeling (1- and 3-month groups, respectively). The control group received PBS inhalation instead of allergen. For the treatment group, additional doses of dexamethasone (5 mg/kg) were injected intraperitoneally 24 hours before ovalbumin challenge of the first and third weeks of ovalbumin challenge. Mice were killed 24 hours after the final ovalbumin challenge, and nasal and lung tissues and nasal lavage fluid were obtained for analysis. Mice were divided into 0-, 1-, and 3-month groups, depending on the duration of local allergen challenge, and each of these experimental groups had control and treatment groups with 10 mice each, making a total of 90 mice. Nasal lavage and histologic analysis. After the mice were killed, nasal lavages were performed after partial tracheal resection using 22-gauge catheters. The catheter was inserted into the tracheal opening in the direction of the upper airway into the nasopharynx. The nasal passages were gently perfused with 1 mL of PBS from the choana to the nostril, and
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nasal lavage fluid was collected from the nares. The nasal lavage fluid was cytospun and the supernatant stored for further analysis. For histologic analysis of the nasal cavity, the heads of the mice were obtained and fixed with 10% formaldehyde solution, decalcified with hydrochloric acid, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E). Respiratory bronchiole with a diameter of 150 to 200 m was observed for lung tissues, and the middle turbinate was chosen for observation in the nasal cavity. Two sections of the middle turbinate, 4 m apart, were made 5 mm posterior to the nasal vestibule. Trichrome staining, matrix metalloproteinase 9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) immunohistochemical staining, and periodic acid–Schiff (PAS) staining were performed for nasal and lung tissues. Eosinophil Quantification Under light microscopic vision (⫻400 magnification; Laborlux K, Lekca, Wetzlar, Germany), eosinophils were counted in the submucosal area of the whole nasal septum using an eyepiece reticule. A single observer, not knowing the experimental conditions, counted all of the slides. Quantification of Airway Remodeling Peribronchial trichrome staining. All slides were read by a histopathologist blinded to the experimental conditions. The degree of subepithelial fibrosis was examined by measuring the trichrome-stained area per unit length. For a quantitative measurement, the areas of interest were measured by using an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD) with an attached light microscope (BX51; Olympus, Tokyo, Japan). The positively stained area was selected and the area of stained tissue in micrometers squared per micrometers of length was calculated. Immunohistochemical staining for MMP-9 and TIMP-1. After deparaffinization, 3% hydrogen peroxide and 10% fetal bovine serum were applied, and antibodies (Santa Cruz, San Diego, CA) for either MMP-9 or TIMP-1 were diluted at 1:200 and reacted in 4°C. Then biotinylated secondary antimouse antibody and streptavidin horseradish peroxidase (labeled streptavidin-biotinkit, Dako, Glostrup, Denmark) were
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Figure 2. Airway remodeling in lung. A, Peribronchial trichrome staining. B, Peribronchial matrix metalloproteinase 9 (MMP-9) immunostaining. C, Peribronchial tissue inhibitors of metalloproteinase 1 (TIMP-1) immunostaining. Control bronchi from mice challenged with phosphate-buffered saline (PBS) exhibited minimal peribronchial inflammation and fibrosis (a). In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced circumferential inflammation, resulting in peribronchial fibrosis and tissue remodeling (b), which was significantly inhibited by systemic administration of dexamethasone (DEXA) (c). Representative pictures with arrows showing positive staining for trichrome (A), MMP-9 (B), and TIMP-1 (C) in peribronchial lung airway.
added for 1 hour and 30 minutes, respectively. Then the section was reacted with 3,3-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. PAS staining for goblet cell hyperplasia. PAS staining was performed to observe the development of goblet cell hyperplasia. Sections of the middle turbinate were used, and the result was presented as the number of goblet cells expressed per unit length (1 m) in the middle nasal turbinate. H&E staining for submucosal gland hypertrophy. Quantification of submucosal gland hypertrophy in the middle turbinate was performed using an image analysis system. Sections of the middle turbinate were stained with H&E. The
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results were presented as the maximal height of epithelial cells in the submucosal glands. Measurement of TGF- in nasal lavage. The expression of transforming growth factor  (TGF-) was measured in the nasal lavage fluid by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). The sensitivity of TGF- concentration that was measurable through this method was 61 pg/mL. Statistical Analysis Results in the different groups of mice were compared by analysis of variance using the nonparametric Kruskal-Wallis
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Table 1. Parameters of Airway Remodeling in the Lung* Parameter Trichrome stained area, m /m PBS Ovalbumin Ovalbumin and dexamethasone MMP-9 stained area, m2/m PBS Ovalbumin Ovalbumin and dexamethasone TIMP-1 stained area, m2/m PBS Ovalbumin Ovalbumin and dexamethasone
0 mo
1 mo
3 mo
3.40 ⫾ 0.77 4.44 ⫾ 1.54 3.18 ⫾ 0.09
3.38 ⫾ 0.37 2.86 ⫾ 0.51 3.24 ⫾ 0.13
2.99 ⫾ 0.48 18.11 ⫾ 1.84† 2.95 ⫾ 0.29†
5.17 ⫾ 1.71 4.84 ⫾ 0.75 4.03 ⫾ 1.37
3.41 ⫾ 0.85 4.64 ⫾ 1.05 2.78 ⫾ 0.85
3.70 ⫾ 0.57 21.17 ⫾ 9.63† 2.84 ⫾ 1.58†
5.99 ⫾ 1.79 2.77 ⫾ 1.25 4.99 ⫾ 0.40
7.97 ⫾ 1.41 7.26 ⫾ 1.46 5.21 ⫾ 0.35
6.42 ⫾ 0.61 12.97 ⫾ 3.88† 5.30 ⫾ 0.41†
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Abbreviation: MMP-9, matrix metalloproteinase 9; PBS, phosphate-buffered saline; TIMP-1, tissue inhibitors of metalloproteinase 1. * Data are presented as mean ⫾ SD. † P ⬍ .05.
no statistically significant difference was found among the 0-month, 1-month, control, and treatment groups. Immunohistochemical staining for MMP-9 and TIMP-1 showed similar results as those found in the trichrome staining. Expression was significantly increased in the 3-month group compared with the control group (P ⫽ .002), and these findings were significantly inhibited by steroid administration (P ⫽ .02) (Figs 2B and C). The parameters of airway remodeling in the lung are summarized in Table 1.
Figure 3. Eosinophil counts in the nasal mucosa. The mean ⫾ SD eosinophil count in the positive control group was 84.2 ⫾ 15.6, 34.6 ⫾ 10.6, and 35.5 ⫾ 15.7 in the 0-, 1-, and 3-month groups, respectively, and these were significantly different from that of negative control group (P ⫽ .001, P ⫽ .008, and P ⫽ .03, respectively) and the steroid treatment group (P ⫽ .002, P ⫽ .008, and P ⫽ .02, respectively).
test followed by posttesting using the Dunn multiple comparison of means. Statistical analysis was performed using SPSS statistical software for Windows, version 11.5 (SPSS Inc, Chicago, IL), and P ⬍ .05 was considered statistically significant. RESULTS Airway Remodeling in the Lung Subepithelial fibrosis significantly increased in the 3-month group compared with the control group (P ⫽ .03), and this was inhibited by steroid administration (P ⫽ .02) (Fig 2A). Also, subepithelial fibrosis observed in the 3-month group was significantly increased compared with the 0- and 1-month groups, respectively (P ⫽ .03 for both). However,
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Eosinophil Quantification The number of eosinophils in the submucosal area in the whole nasal septum was counted under a light microscope (⫻400 magnification; Fig 3). The mean ⫾ SD eosinophil number in the positive control group was 84.2 ⫾ 15.6, 34.6 ⫾ 10.6, and 35.5 ⫾ 15.7 in the 0-, 1-, and 3-month group, respectively, and it was significantly different from that of the negative control group (P ⫽ .001, P ⫽ .008, and P ⫽ .03, respectively) and the steroid treatment group (P ⫽ .002, P ⫽ .008, and P ⫽ .02, respectively) (Fig 3). Airway Remodeling in the Nose Expression of subepithelial fibrosis. Subepithelial fibrosis significantly increased in the 3-month group compared with the control group (P ⫽ .04), and this was inhibited by steroid administration (P ⫽ .003) (Fig 4). Also, subepithelial fibrosis in the 3-month group showed a significant increase compared with the 0- and 1-month groups, respectively (P ⫽ .001 and P ⫽ .002). However, no significant difference was found among the 0-month, 1-month, control, and treatment groups. Expression of MMP-9 and TIMP-1. The expressions of MMP-9 and TIMP-1 were significantly increased in the 3-month group compared with the control group (P ⫽ .005 and P ⫽ .001), and this was inhibited by steroid administration (P ⫽ .001 and P ⫽ .002) (Fig 5). However, no significant difference was found among the 0-month, 1-month, and control groups. Expression of MMP-9 and TIMP-1 was also significantly increased in the 3-month group compared with
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Figure 4. Subepithelial fibrosis in the nose. A, Subepithelial trichrome staining. Control middle turbinate derived from mice challenged with phosphatebuffered saline (PBS) (a) exhibited minimal subepithelial trichrome staining. In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced subepithelial trichrome staining (arrow) (b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Subepithelial trichrome stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .04, OVA vs PBS) but not 0 or 1 month developed increased subepithelial trichrome staining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced levels of subepithelial trichrome staining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .003, DEXA and OVA vs OVA). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
the 0- and 1-month groups, respectively (MMP-9 and TIMP-1: vs 0 month, P ⫽ .001 and P ⫽ .003, respectively; MMP-9 and TIMP-1: vs 1 month, P ⫽ .002 and P ⫽ .002, respectively). Interestingly, the expression of MMP-9 and TIMP-1 in the 3-month control group was significantly increased compared with the 0- and 1-month control groups, respectively (MMP-9: vs 0- and 1-month control groups, P ⫽ .003 and P ⫽ .02; TIMP-1: vs 0- and 1-month control groups). However, the expression of MMP-9 and TIMP-1 was not significantly different among other groups. Goblet cell hyperplasia. PAS staining performed in the nasal cavity showed that goblet cells were significantly increased in the 3-month group compared with the 3-month control group (P ⫽ .001), and this was significantly inhibited by steroid administration (P ⫽ .002) (Fig 6). However, no significant differences were found among the 0-month, 1-month, control, and treatment groups. Goblet cell was significantly increased in the 3-month group compared with the 0- and 1-month groups, respectively (P ⫽ .001 and P ⫽
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.002). The 3-month control group showed a significant increase in goblet cell when compared with the 0- and 1-month control groups, respectively (P ⫽ .01 and P ⫽ .02). Submucous gland hypertrophy. Submucous gland hypertrophy was significantly increased in the 3-month group compared with the control group (P ⫽ .002), and this was significantly inhibited by steroid administration (P ⫽ .001) (Fig 7). However, no significant differences were found among the 0-month, 1-month, control, and treatment groups. Submucous gland hypertrophy was significantly increased in the 3-month group compared with the 0- and 1-month groups (P ⫽ .001 and P ⫽ .001). However, these findings were not observed in control or treatment groups. Levels of TGF- in the nasal lavage fluid. Mice repetitively challenged with ovalbumin for 3 months did not show significant change of TGF- expression in the nasal lavage fluid compared with control PBS-challenged mice. Mice challenged with ovalbumin and treated with dexamethasone did not exhibit any significant change. Also, no significant
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Figure 5. Expression of matrix metalloproteinase 9 (MMP-9) (A) and tissue inhibitors of metalloproteinase 1 (TIMP-1) (B) in the nose. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited MMP-9 and TIMP-1 immunostaining (yellow). Repetitive ovalbumin (OVA) challenge for 3 months induced an increase in the area of MMP-9 and TIMP-1 immunostaining (arrows, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). C, Subepithelial MMP-9 stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .005, OVA vs PBS) but not 0 or 1 month developed increased MMP-9 immunostaining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the area of subepithelial MMP-9 immunostaining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .001, DEXA and OVA vs OVA). There was increased MMP-9 immunostaining in the subepithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 or 1 month (P ⫽ .003 and P ⫽ .02, respectively). D, Subepithelial TIMP-1 stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .001, OVA vs PBS) but not 0 or 1 month developed increased TIMP-1 immunostaining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the area of subepithelial TIMP-1 immunostaining in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .002, DEXA and OVA vs OVA). There was increased TIMP-1 immunostaining in the subepithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 month (P ⫽ .02). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
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Figure 6. Goblet cell hyperplasia in the nose. A, Periodic acid–Schiff (PAS) staining. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited minimal epithelial PAS staining (red). In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced epithelial PAS staining (arrow, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Goblet cell count. Mice repetitively challenged with OVA for 3 months (P ⫽ .001, OVA vs PBS) but not 0 or 1 month developed increased PAS staining of epithelium compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the PAS staining in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .002, DEXA and OVA vs OVA). There was increased PAS staining in the epithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 or 1 month (P ⫽ .001 and P ⫽ .02, respectively). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
differences were found in the level of TGF- expression among the 0-, 1-, and 3-month experimental groups. DISCUSSION In this study we demonstrated that repetitive ovalbumin challenge for 3 months in a murine model of allergic rhinitis could induce features of airway remodeling in the nose and the lung. These features include increased subepithelial fibrosis, increased MMP-9 and TIMP-1 expression, goblet cell hyperplasia, and submucous gland hypertrophy. Subepithelial fibrosis is an important feature of the airway remodeling process and can be evaluated through tissue staining with trichrome. Jeffery6 showed that this change in the bronchial mucosa is caused by the deposition of fibrous tissues and extracellular matrix (ECM) under the basement membrane. It has been reported that type I and type III collagens were significantly increased in subepithelial fibro-
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sis of bronchial asthma patients compared with healthy counterparts.7 However, studies of subepithelial fibrosis in the nasal cavity of allergic patients have rarely been performed. It was reported that the amount of type I and type III collagen deposition was increased in the basement membrane of allergic rhinitis patients and thickness of the basement membrane was also increased compared with healthy individuals.8 In this study we have demonstrated that mice repetitively challenged with ovalbumin for 3 months developed increased subepithelial trichrome staining compared with control PBSchallenged mice, strongly suggesting subepithelial fibrosis occurring in the nose in response to long-term allergen challenge. MMP is a zinc- and calcium-dependent substrate-specific endopeptidase, which is synthesized mainly from structural and inflammatory cells. MMP is one of the major proteinases involved in the turnover of ECM and basement membrane
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Figure 7. Submucous gland hypertrophy in the nose. A, Hematoxylin-eosin staining. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited minimal submucosal gland hypertrophy. In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced submucosal gland hypertrophy (arrow, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Width of submucosal gland. Mice repetitively challenged with OVA for 3 months (P ⫽ .002, OVA vs PBS) but not 0 or 1 month developed submucosal gland hypertrophy compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the submucosal gland hypertrophy in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .001, DEXA and OVA vs OVA). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
proteins in the respiratory epithelium. Therefore, it is considered to be involved in the remodeling process of the tissue after inflammations. Previous studies have reported that MMP-9 and TIMP-1 are abundant in the sputum and bronchial tissues of patients with bronchial asthma, and the expression of MMP-9 significantly correlates with the amount of collagen deposition in the basement membrane.9,11 Furthermore, the concentration of TIMP-1 is increased in the bronchial alveolar fluid of untreated bronchial asthma patients, suggesting that high concentration of TIMP-1 is related to airway remodeling.10 It has been suggested that imbalance between MMP-9 and TIMP-1 causes structural changes in the airway, leading to airway remodeling and obstruction in bronchial asthma and bronchitis.11 The expression of MMP-9 and TIMP-1 in the bronchial tissue in our study is consistent with previous reports. However, previous studies performed in the nasal cavity show inconsistent results. Some reported
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that expression of MMP-9 did not change in allergic rhinitis,12 whereas others reported increased eosinophil cationic proteins and MMP-9 after nasal allergen challenge.13 In this study there was an increase in MMP-9 and TIMP-1 after repetitive allergen challenge. Inflammation caused by repetitive exposure to allergens can lead to epithelial damage, which is subsequently followed by a healing process. We can speculate that a series of these injuries and healing processes can be the mechanism for airway remodeling in the nasal cavity. Normally, goblet cells are not present in terminal bronchioles; however, goblet cell hyperplasia can be observed in bronchial asthma patients. Previous animal experiments have shown findings of goblet cell hyperplasia and mucous hypersecretion in response to repetitive allergen inhalation.9 Contradictory reports exist in the nasal cavity; some report no change in the number of goblet cells,14 whereas some show
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goblet cell hyperplasia and increased number of submucosal glands in patients with allergic rhinitis.15,16 In this study, we have demonstrated an increase in goblet cells and hypertrophy of submucosal glands in response to repetitive allergen stimulation for 3 months. TGF- is produced and secreted from macrophages, fibroblasts, epithelial cells, and eosinophils. It is a common profibrotic cytokine that mediates the synthesis and degradation of ECM and is thought to be the major cytokine involved in the fibrosis process of the lower airway. In vitro experiments have proven that TGF- is involved in the synthesis and secretion of abundant ECM proteins such as collagen type I and III, fibronectin, tenascin, and proteoglycans by stimulating fibroblasts. In asthma patients, the concentration of TGF- in the bronchoalveolar fluid was elevated, and the concentration was higher when exposed to allergen.17 Also, it has been reported that a close correlation exists among expression of TGF-, clinical symptoms, and the degree of subepithelial fibrosis.18 However, the expression of TGF- in allergic rhinitis is not well defined. Some show no change,19 whereas others report an increase of TGF-, suggesting its role in the activation of epithelial cells in response to tissue injury caused by allergic inflammation.20 In this study, the level of TGF- in the nasal lavage fluid in mice repetitively challenged with ovalbumin for 3 months was slightly increased compared with the control group; however, this finding was not statistically significant. Considering the fact that TGF- is a cytokine expressed in cells such as macrophages, fibroblasts, epithelial cells, and eosinophils, the relatively well-maintained integrity of the epithelial cell layer in the nasal cavity can account for this nonsignificant difference. We therefore speculate that although features of airway remodeling in the nose with allergic inflammation resemble the changes in the lung airway, the role of TGF- may not be as significant as in the lung. Further studies to determine the factors that contribute to airway remodeling in the nose are warranted. Interestingly, mice challenged with PBS instead of ovalbumin for 3 months showed a significant increase in MMP-9 and TIMP-1 expression and goblet cell hyperplasia compared with the 0- and 1-month group. This finding was not as remarkable as in the ovalbumin-challenged group, and these findings were not observed in the lung tissue. This result could imply that, though minimal, inflammation and airway remodeling in the nasal mucosa of an already established allergic rhinitis may also occur after nonspecific environmental stimuli, which in this case was PBS. We think that PBS, even though it does not induce direct inflammation, can act as a nonspecific stimulant. The nasal cavity mucosa is constantly exposed to various harmful substances and pathogens in the air and therefore is prone to developing inflammations. As a result of these constant stimuli, although not remarkable, thickening of the basement membrane may occur. This is evidenced by findings of basement membrane thickening found not only in allergic rhinitis patients but also in healthy individuals.21 The absence of airway remodeling in the lung
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can be attributable to the elimination to a certain extent of this nonspecific stimulus by the nasal cavity. However, the possibility that these changes observed in the 3-month group could be the result of a physiologic aging process should be considered. Steroids are commonly used for the treatment of bronchial asthma and allergic rhinitis. They cause reduction in the density of inflammatory cells such as eosinophils, mast cells and lymphocytes, increase of normal ciliated epithelium, reduction of basement membrane thickness, and decrease of tenascin in the basement membrane.22 Steroid administration also decreases subepithelial collagen deposition through regulation of MMP-9 and TIMP-1 expression23 and has also been known to reduce allergen-induced late reactions23,24 in both the lung and nasal cavity. In this study, although airway remodeling occurred in the 3-month and control groups, steroid administration inhibited this process, suggesting that steroids can effectively inhibit airway remodeling caused by allergic reactions in the lung and nasal cavity. In summary, this study demonstrates that long-term, repetitive allergen challenge induces airway remodeling in the nasal cavity and the lung. In addition, long-term nonspecific stimulation of the allergic nasal mucosa may also induce minimal airway remodeling. Steroids can prevent not only the allergic inflammation but also the airway remodeling process in response to long-term allergic challenge in the upper and lower airways. ACKNOWLEDGMENTS This study was partly supported by grant 11 to 2003– 008 from the Samsung Research Fund. REFERENCES 1. Rowe-Jones JM. The link between the nose and lung, perennial rhinitis and asthma—Is it the same disease? Allergy 1997;52(36 suppl):20 –28. 2. Calderon MA, Lozewicz S, Prior A, Jordan S, Trigg CJ, Davies RJ. Lymphocyte infiltration and thickness of the nasal mucous membrane in perennial and seasonal allergic rhinitis. J Allergy Clin Immunol. 1994;93:635– 643. 3. Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A, Olivieri D. Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest. 1997;111: 852– 857. 4. Frieri M. Clinical and basic science research on allergic rhinitis and asthma from Nassau University Medical Center. Allergy Asthma Proc. 2001;22:167–172. 5. Rhee CS, Libet L, Chisholm D, et al. Allergen-independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis. Immunology. 2004;113: 106 –113. 6. Jeffery PK. Pathology of asthma. Br Med Bull. 1992;48:23–39. 7. Chakir J, Laviolette M, Boutet M, Laliberte´ R, Dube´ J, Boulet L-P. Lower airways remodeling in nonasthmatic subjects with allergic rhinitis. Lab Invest. 1996;75:735–744. 8. Sanai A, Nagata H, Konno A. Extensive interstitial collagen deposition on the basement membrane zone in allergic nasal mucosa. Acta Otolaryngol. 1999;119:473– 478.
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9. Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol. 1998;102:783–788. 10. Mautino G, Henriquet C, Jaffuel D, Bousquet J, Capony F. Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects. Am J Respir Crit Care Med. 1999;160:324 –330. 11. Vignola AM, Riccobono L, Mirabella A, et al. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med. 1998;158:1945–1950. 12. Shaida A, Kenyon G, Devalia J, Davies RJ, MacDonald TT, Pender SL. Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis. J Allergy Clin Immunol. 2001;108:791–796. 13. van Toorenenbergen AW, Gerth van Wijk R, Vermeulen AM. Allergen induced matrix metalloproteinase-9 in nasal lavage fluid. Allergy. 1999;54:293–294. 14. Berger G, Marom Z, Ophir D. Goblet cell density of the inferior turbinates in patients with perennial allergic and nonallergic rhinitis. Am J Rhinol. 1997;11:233–236. 15. Gluck U, Gebbers J. Epithelial changes in seasonal allergic rhinitis throughout the year: evidence of coexistent air pollution and local secretory IgA deficiency? ORL J Otorhinolaryngol Relat Spec. 2000;62:68 –75. 16. Malekzadeh S, Hamburger MD, Whelan PJ, Biedlingmaier JF, Baraniuk JN. Density of middle turbinate subepithelial mucous glands in patients with chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2002;127:190 –195. 17. Redington AE, Madden J, Frew AJ, et al. Transforming growth factor-[beta]1 in asthma. Measurement in bronchoalveolar lavage fluid. Am J Respir Crit Care Med. 1997;156:642– 647. 18. Minshall EM, Leung DY, Martin RJ, et al. Eosinophilassociated TGF-[beta]1 mRNA expression and airways fibrosis
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19. 20. 21.
22. 23.
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in bronchial asthma. Am J Respir Cell Mol Biol. 1997;17: 326 –333. Bousquet J, Jacot W, Vignola AM, Bachert C, Van Cauwenberge P. Allergic rhinitis: a disease remodeling the upper airways? J Allergy Clin Immunol. 2004;113:43– 49. Salib RJ, Howarth PH. Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clin Exp Allergy. 2003;33:1629 –1633. Braunstahl GJ, Fokkens WJ, Overbeek SE, KleinJan A, Hoogsteden HC, Prins JB. Mucosal and systemic inflammatory changes in allergic rhinitis and asthma: a comparison between upper and lower airways. Clin Exp Allergy. 2003;33:579 –587. Fish JE, Peters SP. Airway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol. 1999;104: 509 –516. Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol. 1999;104:356 –363. Cockcroft D, Murdock K. Comparative effects of inhaled salbutamol, sodium cromoglycate, and beclomethasone dipropionate on allergen-induced early asthmatic responses, late asthmatic responses, and increased bronchial responsiveness to histamine. J Allergy Clin Immunol. 1987;79:734 –740.
Requests for reprints should be addressed to: Chae-Seo Rhee, MD Department of Otorhinolaryngology-Head and Neck Surgery Seoul National University Hospital 28 Yongon-dong Chongno-gu Seoul 110-744, Korea E-mail: [email protected]
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Background: Although many studies regarding airway remodeling in asthma have been reported, only a few studies have investigated airway remodeling in allergic rhinitis. Objectives: To determine whether repetitive allergen challenge could induce airway remodeling in the nose and evaluate the effect of steroids using a murine model of allergic rhinitis. Methods: To develop a mouse model of airway remodeling, ovalbumin-sensitized mice were repeatedly exposed to inhaled ovalbumin administration twice a week for 1 month and 3 months. Matched control mice were challenged with phosphatebuffered saline, and the treatment group received intraperitoneal dexamethasone injection. Trichrome, periodic acid–Schiff, hematoxylin-eosin, and immunohistochemical staining against matrix metalloproteinase 9 and tissue inhibitors of metalloproteinase 1 were performed to nasal and lung tissues, and the level of transforming growth factor  in the nasal lavage fluid was analyzed. Results: Repetitive ovalbumin challenge for 3 months induced circumferential peribronchial fibrosis in the lung. In the nose, subepithelial fibrosis, increased matrix metalloproteinase 9 and tissue inhibitors of metalloproteinase 1 expression, goblet cell hyperplasia, and submucous gland hypertrophy were observed compared with the control group. Features of airway remodeling were more prominent in the lung tissue. Administration of dexamethasone significantly inhibited these histologic changes. Conclusion: Airway remodeling associated with long-term allergen challenge can occur in the nasal mucosa and the lung. Steroid treatment prevents airway inflammation in response to acute allergen challenge, as well as airway remodeling by long-term allergen challenge. Ann Allergy Asthma Immunol. 2007;98:22–31.
INTRODUCTION Allergic rhinitis and bronchial asthma are representative allergic diseases of the upper and lower airways, sharing common pathophysiologic features such as infiltration of inflammatory cells, including eosinophils and lymphocytes, degranulation of mast cells, and expression of various cytokines in response to allergens. Thus, once regarded as 2 separate disease entities, they are now thought to be a common disease with different clinical manifestations.1 However, differences between the 2 tissues exist. The nose and bronchial airways differ in embryologic origin, the nose being of ectodermal and the lower airway of endodermal origin. Except for those smooth muscles associated with vessels, there is no smooth muscle in the nasal cavity compared with the bronchus, and although bronchial asthma is characterized by disruption and desquamation of bronchial epithelial cells by continuous exposure to allergen, epithelial cells in the nasal cavity are relatively well preserved in allergic rhinitis.2
* Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, Seoul, Korea. † Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Center, Seoul, Korea. Received for publication April 28, 2006. Accepted for publication in revised form September 12, 2006.
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It is well known that the airway remodeling process develops in asthmatic patients.3 These manifestations include epithelial disruption, goblet cell hyperplasia, mucous gland hypertrophy, increased collagen and matrix protein deposition, and smooth muscle hypertrophy and hyperplasia. However, studies on the airway remodeling process of the upper airway in allergic rhinitis are sparse, with previous reports showing conflicting results.4 In this study, to better understand the development of nasal airway remodeling in response to allergic inflammation, we investigated the features of airway remodeling in the nose and lung associated with repetitive allergen challenge in ovalbumin-sensitized mice and evaluated the effects of steroid administration. MATERIALS AND METHODS Induction of the Murine Model of Allergic Rhinitis and Airway Remodeling Animals. Specific pathogen-free BALB-c mice were used as the experimental animals. Each mouse weighed 20 to 30 g and was 3 to 5 weeks of age. The studies herein followed the principles for laboratory animal research as outlined in the Animal Welfare Act and Department of Health, Education, and Welfare (National Institutes of Health) guidelines for the experimental use of animals, and these experimental protocols were approved by our institution’s animal subjects committees.
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Figure 1. Mouse ovalbumin experimental protocol (0, 1, and 3 months). Mice were immunized by intraperitoneal injection of ovalbumin on days 0, 7, 14, and 21. Intranasal challenges were administered via daily ovalbumin inhalation from days 28 to 35, and then repeated ovalbumin challenges were performed twice a week for 1 or 3 months. Control mice were immunized but challenged with phosphate-buffered saline instead of ovalbumin twice a week during the 1- or 3-month study. Mice were killed 24 hours after the final ovalbumin challenge. Systemic dexamethasone was administered intraperitoneally on days 27, 36, and 50. Upward arrow indicates intraperitoneal ovalbumin injection; triangle, intranasal ovalbumin challenge; rectangle, intranasal ovalbumin or phosphate-buffered saline challenge; and circle, intraperitoneal dexamethasone injection.
Reagents. Ovalbumin (grade V) and aluminum hydroxide were purchased from Sigma Chemical Co (St Louis, MO). Dexamethasone was purchased form Merck & Co (West Point, PA). Sensitization and allergen challenge. Allergen sensitization and challenge for the development of the allergic rhinitis murine model was performed as described previously5 and is summarized in Figure 1. Briefly, on days 0, 7, 14, and 21, mice were immunized by intraperitoneal injection of 25 g of ovalbumin and 1 mg of aluminum hydroxide in 300 L of phosphate-buffered saline (PBS). One week after last immunization (day 28), mice received a series of 7 daily 1% ovalbumin challenges via a nebulizer (PulmoAide, Somerset, PA). The aerosolized ovalbumin (particle size, 0.5–5.0 m) was generated into a closed chamber of 8,800 cm3 (20 ⫻ 22 ⫻ 20 cm) made of acrylic for 30 minutes. The treatment group received a single intraperitoneal injection of 5 mg/kg of dexamethasone in PBS 1 day before the first ovalbumin challenge. Twentyfour hours after the final ovalbumin challenge, mice were killed for analysis (0-month group). In selected groups, 1% ovalbumin was repeatedly inhaled twice a week for 1 or 3 months for the development of airway remodeling (1- and 3-month groups, respectively). The control group received PBS inhalation instead of allergen. For the treatment group, additional doses of dexamethasone (5 mg/kg) were injected intraperitoneally 24 hours before ovalbumin challenge of the first and third weeks of ovalbumin challenge. Mice were killed 24 hours after the final ovalbumin challenge, and nasal and lung tissues and nasal lavage fluid were obtained for analysis. Mice were divided into 0-, 1-, and 3-month groups, depending on the duration of local allergen challenge, and each of these experimental groups had control and treatment groups with 10 mice each, making a total of 90 mice. Nasal lavage and histologic analysis. After the mice were killed, nasal lavages were performed after partial tracheal resection using 22-gauge catheters. The catheter was inserted into the tracheal opening in the direction of the upper airway into the nasopharynx. The nasal passages were gently perfused with 1 mL of PBS from the choana to the nostril, and
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nasal lavage fluid was collected from the nares. The nasal lavage fluid was cytospun and the supernatant stored for further analysis. For histologic analysis of the nasal cavity, the heads of the mice were obtained and fixed with 10% formaldehyde solution, decalcified with hydrochloric acid, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E). Respiratory bronchiole with a diameter of 150 to 200 m was observed for lung tissues, and the middle turbinate was chosen for observation in the nasal cavity. Two sections of the middle turbinate, 4 m apart, were made 5 mm posterior to the nasal vestibule. Trichrome staining, matrix metalloproteinase 9 (MMP-9) and tissue inhibitors of metalloproteinase 1 (TIMP-1) immunohistochemical staining, and periodic acid–Schiff (PAS) staining were performed for nasal and lung tissues. Eosinophil Quantification Under light microscopic vision (⫻400 magnification; Laborlux K, Lekca, Wetzlar, Germany), eosinophils were counted in the submucosal area of the whole nasal septum using an eyepiece reticule. A single observer, not knowing the experimental conditions, counted all of the slides. Quantification of Airway Remodeling Peribronchial trichrome staining. All slides were read by a histopathologist blinded to the experimental conditions. The degree of subepithelial fibrosis was examined by measuring the trichrome-stained area per unit length. For a quantitative measurement, the areas of interest were measured by using an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD) with an attached light microscope (BX51; Olympus, Tokyo, Japan). The positively stained area was selected and the area of stained tissue in micrometers squared per micrometers of length was calculated. Immunohistochemical staining for MMP-9 and TIMP-1. After deparaffinization, 3% hydrogen peroxide and 10% fetal bovine serum were applied, and antibodies (Santa Cruz, San Diego, CA) for either MMP-9 or TIMP-1 were diluted at 1:200 and reacted in 4°C. Then biotinylated secondary antimouse antibody and streptavidin horseradish peroxidase (labeled streptavidin-biotinkit, Dako, Glostrup, Denmark) were
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Figure 2. Airway remodeling in lung. A, Peribronchial trichrome staining. B, Peribronchial matrix metalloproteinase 9 (MMP-9) immunostaining. C, Peribronchial tissue inhibitors of metalloproteinase 1 (TIMP-1) immunostaining. Control bronchi from mice challenged with phosphate-buffered saline (PBS) exhibited minimal peribronchial inflammation and fibrosis (a). In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced circumferential inflammation, resulting in peribronchial fibrosis and tissue remodeling (b), which was significantly inhibited by systemic administration of dexamethasone (DEXA) (c). Representative pictures with arrows showing positive staining for trichrome (A), MMP-9 (B), and TIMP-1 (C) in peribronchial lung airway.
added for 1 hour and 30 minutes, respectively. Then the section was reacted with 3,3-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. PAS staining for goblet cell hyperplasia. PAS staining was performed to observe the development of goblet cell hyperplasia. Sections of the middle turbinate were used, and the result was presented as the number of goblet cells expressed per unit length (1 m) in the middle nasal turbinate. H&E staining for submucosal gland hypertrophy. Quantification of submucosal gland hypertrophy in the middle turbinate was performed using an image analysis system. Sections of the middle turbinate were stained with H&E. The
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results were presented as the maximal height of epithelial cells in the submucosal glands. Measurement of TGF- in nasal lavage. The expression of transforming growth factor  (TGF-) was measured in the nasal lavage fluid by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). The sensitivity of TGF- concentration that was measurable through this method was 61 pg/mL. Statistical Analysis Results in the different groups of mice were compared by analysis of variance using the nonparametric Kruskal-Wallis
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Table 1. Parameters of Airway Remodeling in the Lung* Parameter Trichrome stained area, m /m PBS Ovalbumin Ovalbumin and dexamethasone MMP-9 stained area, m2/m PBS Ovalbumin Ovalbumin and dexamethasone TIMP-1 stained area, m2/m PBS Ovalbumin Ovalbumin and dexamethasone
0 mo
1 mo
3 mo
3.40 ⫾ 0.77 4.44 ⫾ 1.54 3.18 ⫾ 0.09
3.38 ⫾ 0.37 2.86 ⫾ 0.51 3.24 ⫾ 0.13
2.99 ⫾ 0.48 18.11 ⫾ 1.84† 2.95 ⫾ 0.29†
5.17 ⫾ 1.71 4.84 ⫾ 0.75 4.03 ⫾ 1.37
3.41 ⫾ 0.85 4.64 ⫾ 1.05 2.78 ⫾ 0.85
3.70 ⫾ 0.57 21.17 ⫾ 9.63† 2.84 ⫾ 1.58†
5.99 ⫾ 1.79 2.77 ⫾ 1.25 4.99 ⫾ 0.40
7.97 ⫾ 1.41 7.26 ⫾ 1.46 5.21 ⫾ 0.35
6.42 ⫾ 0.61 12.97 ⫾ 3.88† 5.30 ⫾ 0.41†
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Abbreviation: MMP-9, matrix metalloproteinase 9; PBS, phosphate-buffered saline; TIMP-1, tissue inhibitors of metalloproteinase 1. * Data are presented as mean ⫾ SD. † P ⬍ .05.
no statistically significant difference was found among the 0-month, 1-month, control, and treatment groups. Immunohistochemical staining for MMP-9 and TIMP-1 showed similar results as those found in the trichrome staining. Expression was significantly increased in the 3-month group compared with the control group (P ⫽ .002), and these findings were significantly inhibited by steroid administration (P ⫽ .02) (Figs 2B and C). The parameters of airway remodeling in the lung are summarized in Table 1.
Figure 3. Eosinophil counts in the nasal mucosa. The mean ⫾ SD eosinophil count in the positive control group was 84.2 ⫾ 15.6, 34.6 ⫾ 10.6, and 35.5 ⫾ 15.7 in the 0-, 1-, and 3-month groups, respectively, and these were significantly different from that of negative control group (P ⫽ .001, P ⫽ .008, and P ⫽ .03, respectively) and the steroid treatment group (P ⫽ .002, P ⫽ .008, and P ⫽ .02, respectively).
test followed by posttesting using the Dunn multiple comparison of means. Statistical analysis was performed using SPSS statistical software for Windows, version 11.5 (SPSS Inc, Chicago, IL), and P ⬍ .05 was considered statistically significant. RESULTS Airway Remodeling in the Lung Subepithelial fibrosis significantly increased in the 3-month group compared with the control group (P ⫽ .03), and this was inhibited by steroid administration (P ⫽ .02) (Fig 2A). Also, subepithelial fibrosis observed in the 3-month group was significantly increased compared with the 0- and 1-month groups, respectively (P ⫽ .03 for both). However,
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Eosinophil Quantification The number of eosinophils in the submucosal area in the whole nasal septum was counted under a light microscope (⫻400 magnification; Fig 3). The mean ⫾ SD eosinophil number in the positive control group was 84.2 ⫾ 15.6, 34.6 ⫾ 10.6, and 35.5 ⫾ 15.7 in the 0-, 1-, and 3-month group, respectively, and it was significantly different from that of the negative control group (P ⫽ .001, P ⫽ .008, and P ⫽ .03, respectively) and the steroid treatment group (P ⫽ .002, P ⫽ .008, and P ⫽ .02, respectively) (Fig 3). Airway Remodeling in the Nose Expression of subepithelial fibrosis. Subepithelial fibrosis significantly increased in the 3-month group compared with the control group (P ⫽ .04), and this was inhibited by steroid administration (P ⫽ .003) (Fig 4). Also, subepithelial fibrosis in the 3-month group showed a significant increase compared with the 0- and 1-month groups, respectively (P ⫽ .001 and P ⫽ .002). However, no significant difference was found among the 0-month, 1-month, control, and treatment groups. Expression of MMP-9 and TIMP-1. The expressions of MMP-9 and TIMP-1 were significantly increased in the 3-month group compared with the control group (P ⫽ .005 and P ⫽ .001), and this was inhibited by steroid administration (P ⫽ .001 and P ⫽ .002) (Fig 5). However, no significant difference was found among the 0-month, 1-month, and control groups. Expression of MMP-9 and TIMP-1 was also significantly increased in the 3-month group compared with
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Figure 4. Subepithelial fibrosis in the nose. A, Subepithelial trichrome staining. Control middle turbinate derived from mice challenged with phosphatebuffered saline (PBS) (a) exhibited minimal subepithelial trichrome staining. In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced subepithelial trichrome staining (arrow) (b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Subepithelial trichrome stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .04, OVA vs PBS) but not 0 or 1 month developed increased subepithelial trichrome staining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced levels of subepithelial trichrome staining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .003, DEXA and OVA vs OVA). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
the 0- and 1-month groups, respectively (MMP-9 and TIMP-1: vs 0 month, P ⫽ .001 and P ⫽ .003, respectively; MMP-9 and TIMP-1: vs 1 month, P ⫽ .002 and P ⫽ .002, respectively). Interestingly, the expression of MMP-9 and TIMP-1 in the 3-month control group was significantly increased compared with the 0- and 1-month control groups, respectively (MMP-9: vs 0- and 1-month control groups, P ⫽ .003 and P ⫽ .02; TIMP-1: vs 0- and 1-month control groups). However, the expression of MMP-9 and TIMP-1 was not significantly different among other groups. Goblet cell hyperplasia. PAS staining performed in the nasal cavity showed that goblet cells were significantly increased in the 3-month group compared with the 3-month control group (P ⫽ .001), and this was significantly inhibited by steroid administration (P ⫽ .002) (Fig 6). However, no significant differences were found among the 0-month, 1-month, control, and treatment groups. Goblet cell was significantly increased in the 3-month group compared with the 0- and 1-month groups, respectively (P ⫽ .001 and P ⫽
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.002). The 3-month control group showed a significant increase in goblet cell when compared with the 0- and 1-month control groups, respectively (P ⫽ .01 and P ⫽ .02). Submucous gland hypertrophy. Submucous gland hypertrophy was significantly increased in the 3-month group compared with the control group (P ⫽ .002), and this was significantly inhibited by steroid administration (P ⫽ .001) (Fig 7). However, no significant differences were found among the 0-month, 1-month, control, and treatment groups. Submucous gland hypertrophy was significantly increased in the 3-month group compared with the 0- and 1-month groups (P ⫽ .001 and P ⫽ .001). However, these findings were not observed in control or treatment groups. Levels of TGF- in the nasal lavage fluid. Mice repetitively challenged with ovalbumin for 3 months did not show significant change of TGF- expression in the nasal lavage fluid compared with control PBS-challenged mice. Mice challenged with ovalbumin and treated with dexamethasone did not exhibit any significant change. Also, no significant
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Figure 5. Expression of matrix metalloproteinase 9 (MMP-9) (A) and tissue inhibitors of metalloproteinase 1 (TIMP-1) (B) in the nose. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited MMP-9 and TIMP-1 immunostaining (yellow). Repetitive ovalbumin (OVA) challenge for 3 months induced an increase in the area of MMP-9 and TIMP-1 immunostaining (arrows, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). C, Subepithelial MMP-9 stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .005, OVA vs PBS) but not 0 or 1 month developed increased MMP-9 immunostaining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the area of subepithelial MMP-9 immunostaining in mice challenged repetitively with OVA, compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .001, DEXA and OVA vs OVA). There was increased MMP-9 immunostaining in the subepithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 or 1 month (P ⫽ .003 and P ⫽ .02, respectively). D, Subepithelial TIMP-1 stained area. Mice repetitively challenged with OVA for 3 months (P ⫽ .001, OVA vs PBS) but not 0 or 1 month developed increased TIMP-1 immunostaining compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the area of subepithelial TIMP-1 immunostaining in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .002, DEXA and OVA vs OVA). There was increased TIMP-1 immunostaining in the subepithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 month (P ⫽ .02). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
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Figure 6. Goblet cell hyperplasia in the nose. A, Periodic acid–Schiff (PAS) staining. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited minimal epithelial PAS staining (red). In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced epithelial PAS staining (arrow, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Goblet cell count. Mice repetitively challenged with OVA for 3 months (P ⫽ .001, OVA vs PBS) but not 0 or 1 month developed increased PAS staining of epithelium compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the PAS staining in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .002, DEXA and OVA vs OVA). There was increased PAS staining in the epithelium of mice challenged repetitively with PBS for 3 months compared with epithelium of mice challenged with PBS for 0 or 1 month (P ⫽ .001 and P ⫽ .02, respectively). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
differences were found in the level of TGF- expression among the 0-, 1-, and 3-month experimental groups. DISCUSSION In this study we demonstrated that repetitive ovalbumin challenge for 3 months in a murine model of allergic rhinitis could induce features of airway remodeling in the nose and the lung. These features include increased subepithelial fibrosis, increased MMP-9 and TIMP-1 expression, goblet cell hyperplasia, and submucous gland hypertrophy. Subepithelial fibrosis is an important feature of the airway remodeling process and can be evaluated through tissue staining with trichrome. Jeffery6 showed that this change in the bronchial mucosa is caused by the deposition of fibrous tissues and extracellular matrix (ECM) under the basement membrane. It has been reported that type I and type III collagens were significantly increased in subepithelial fibro-
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sis of bronchial asthma patients compared with healthy counterparts.7 However, studies of subepithelial fibrosis in the nasal cavity of allergic patients have rarely been performed. It was reported that the amount of type I and type III collagen deposition was increased in the basement membrane of allergic rhinitis patients and thickness of the basement membrane was also increased compared with healthy individuals.8 In this study we have demonstrated that mice repetitively challenged with ovalbumin for 3 months developed increased subepithelial trichrome staining compared with control PBSchallenged mice, strongly suggesting subepithelial fibrosis occurring in the nose in response to long-term allergen challenge. MMP is a zinc- and calcium-dependent substrate-specific endopeptidase, which is synthesized mainly from structural and inflammatory cells. MMP is one of the major proteinases involved in the turnover of ECM and basement membrane
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Figure 7. Submucous gland hypertrophy in the nose. A, Hematoxylin-eosin staining. Control middle turbinate derived from mice challenged with phosphate-buffered saline (PBS) (a) exhibited minimal submucosal gland hypertrophy. In contrast, repetitive ovalbumin (OVA) challenge for 3 months induced submucosal gland hypertrophy (arrow, b), which was significantly inhibited by administration of dexamethasone (DEXA) (c). B, Width of submucosal gland. Mice repetitively challenged with OVA for 3 months (P ⫽ .002, OVA vs PBS) but not 0 or 1 month developed submucosal gland hypertrophy compared with control PBS-challenged mice. Systemic administration of DEXA significantly reduced the submucosal gland hypertrophy in mice challenged repetitively with OVA compared with untreated mice challenged repetitively with OVA for 3 months (P ⫽ .001, DEXA and OVA vs OVA). Open bars indicate no OVA; filled bars, OVA; diagonally striped bars, OVA and DEXA.
proteins in the respiratory epithelium. Therefore, it is considered to be involved in the remodeling process of the tissue after inflammations. Previous studies have reported that MMP-9 and TIMP-1 are abundant in the sputum and bronchial tissues of patients with bronchial asthma, and the expression of MMP-9 significantly correlates with the amount of collagen deposition in the basement membrane.9,11 Furthermore, the concentration of TIMP-1 is increased in the bronchial alveolar fluid of untreated bronchial asthma patients, suggesting that high concentration of TIMP-1 is related to airway remodeling.10 It has been suggested that imbalance between MMP-9 and TIMP-1 causes structural changes in the airway, leading to airway remodeling and obstruction in bronchial asthma and bronchitis.11 The expression of MMP-9 and TIMP-1 in the bronchial tissue in our study is consistent with previous reports. However, previous studies performed in the nasal cavity show inconsistent results. Some reported
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that expression of MMP-9 did not change in allergic rhinitis,12 whereas others reported increased eosinophil cationic proteins and MMP-9 after nasal allergen challenge.13 In this study there was an increase in MMP-9 and TIMP-1 after repetitive allergen challenge. Inflammation caused by repetitive exposure to allergens can lead to epithelial damage, which is subsequently followed by a healing process. We can speculate that a series of these injuries and healing processes can be the mechanism for airway remodeling in the nasal cavity. Normally, goblet cells are not present in terminal bronchioles; however, goblet cell hyperplasia can be observed in bronchial asthma patients. Previous animal experiments have shown findings of goblet cell hyperplasia and mucous hypersecretion in response to repetitive allergen inhalation.9 Contradictory reports exist in the nasal cavity; some report no change in the number of goblet cells,14 whereas some show
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goblet cell hyperplasia and increased number of submucosal glands in patients with allergic rhinitis.15,16 In this study, we have demonstrated an increase in goblet cells and hypertrophy of submucosal glands in response to repetitive allergen stimulation for 3 months. TGF- is produced and secreted from macrophages, fibroblasts, epithelial cells, and eosinophils. It is a common profibrotic cytokine that mediates the synthesis and degradation of ECM and is thought to be the major cytokine involved in the fibrosis process of the lower airway. In vitro experiments have proven that TGF- is involved in the synthesis and secretion of abundant ECM proteins such as collagen type I and III, fibronectin, tenascin, and proteoglycans by stimulating fibroblasts. In asthma patients, the concentration of TGF- in the bronchoalveolar fluid was elevated, and the concentration was higher when exposed to allergen.17 Also, it has been reported that a close correlation exists among expression of TGF-, clinical symptoms, and the degree of subepithelial fibrosis.18 However, the expression of TGF- in allergic rhinitis is not well defined. Some show no change,19 whereas others report an increase of TGF-, suggesting its role in the activation of epithelial cells in response to tissue injury caused by allergic inflammation.20 In this study, the level of TGF- in the nasal lavage fluid in mice repetitively challenged with ovalbumin for 3 months was slightly increased compared with the control group; however, this finding was not statistically significant. Considering the fact that TGF- is a cytokine expressed in cells such as macrophages, fibroblasts, epithelial cells, and eosinophils, the relatively well-maintained integrity of the epithelial cell layer in the nasal cavity can account for this nonsignificant difference. We therefore speculate that although features of airway remodeling in the nose with allergic inflammation resemble the changes in the lung airway, the role of TGF- may not be as significant as in the lung. Further studies to determine the factors that contribute to airway remodeling in the nose are warranted. Interestingly, mice challenged with PBS instead of ovalbumin for 3 months showed a significant increase in MMP-9 and TIMP-1 expression and goblet cell hyperplasia compared with the 0- and 1-month group. This finding was not as remarkable as in the ovalbumin-challenged group, and these findings were not observed in the lung tissue. This result could imply that, though minimal, inflammation and airway remodeling in the nasal mucosa of an already established allergic rhinitis may also occur after nonspecific environmental stimuli, which in this case was PBS. We think that PBS, even though it does not induce direct inflammation, can act as a nonspecific stimulant. The nasal cavity mucosa is constantly exposed to various harmful substances and pathogens in the air and therefore is prone to developing inflammations. As a result of these constant stimuli, although not remarkable, thickening of the basement membrane may occur. This is evidenced by findings of basement membrane thickening found not only in allergic rhinitis patients but also in healthy individuals.21 The absence of airway remodeling in the lung
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can be attributable to the elimination to a certain extent of this nonspecific stimulus by the nasal cavity. However, the possibility that these changes observed in the 3-month group could be the result of a physiologic aging process should be considered. Steroids are commonly used for the treatment of bronchial asthma and allergic rhinitis. They cause reduction in the density of inflammatory cells such as eosinophils, mast cells and lymphocytes, increase of normal ciliated epithelium, reduction of basement membrane thickness, and decrease of tenascin in the basement membrane.22 Steroid administration also decreases subepithelial collagen deposition through regulation of MMP-9 and TIMP-1 expression23 and has also been known to reduce allergen-induced late reactions23,24 in both the lung and nasal cavity. In this study, although airway remodeling occurred in the 3-month and control groups, steroid administration inhibited this process, suggesting that steroids can effectively inhibit airway remodeling caused by allergic reactions in the lung and nasal cavity. In summary, this study demonstrates that long-term, repetitive allergen challenge induces airway remodeling in the nasal cavity and the lung. In addition, long-term nonspecific stimulation of the allergic nasal mucosa may also induce minimal airway remodeling. Steroids can prevent not only the allergic inflammation but also the airway remodeling process in response to long-term allergic challenge in the upper and lower airways. ACKNOWLEDGMENTS This study was partly supported by grant 11 to 2003– 008 from the Samsung Research Fund. REFERENCES 1. Rowe-Jones JM. The link between the nose and lung, perennial rhinitis and asthma—Is it the same disease? Allergy 1997;52(36 suppl):20 –28. 2. Calderon MA, Lozewicz S, Prior A, Jordan S, Trigg CJ, Davies RJ. Lymphocyte infiltration and thickness of the nasal mucous membrane in perennial and seasonal allergic rhinitis. J Allergy Clin Immunol. 1994;93:635– 643. 3. Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A, Olivieri D. Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest. 1997;111: 852– 857. 4. Frieri M. Clinical and basic science research on allergic rhinitis and asthma from Nassau University Medical Center. Allergy Asthma Proc. 2001;22:167–172. 5. Rhee CS, Libet L, Chisholm D, et al. Allergen-independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis. Immunology. 2004;113: 106 –113. 6. Jeffery PK. Pathology of asthma. Br Med Bull. 1992;48:23–39. 7. Chakir J, Laviolette M, Boutet M, Laliberte´ R, Dube´ J, Boulet L-P. Lower airways remodeling in nonasthmatic subjects with allergic rhinitis. Lab Invest. 1996;75:735–744. 8. Sanai A, Nagata H, Konno A. Extensive interstitial collagen deposition on the basement membrane zone in allergic nasal mucosa. Acta Otolaryngol. 1999;119:473– 478.
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9. Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol. 1998;102:783–788. 10. Mautino G, Henriquet C, Jaffuel D, Bousquet J, Capony F. Tissue inhibitor of metalloproteinase-1 levels in bronchoalveolar lavage fluid from asthmatic subjects. Am J Respir Crit Care Med. 1999;160:324 –330. 11. Vignola AM, Riccobono L, Mirabella A, et al. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med. 1998;158:1945–1950. 12. Shaida A, Kenyon G, Devalia J, Davies RJ, MacDonald TT, Pender SL. Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis. J Allergy Clin Immunol. 2001;108:791–796. 13. van Toorenenbergen AW, Gerth van Wijk R, Vermeulen AM. Allergen induced matrix metalloproteinase-9 in nasal lavage fluid. Allergy. 1999;54:293–294. 14. Berger G, Marom Z, Ophir D. Goblet cell density of the inferior turbinates in patients with perennial allergic and nonallergic rhinitis. Am J Rhinol. 1997;11:233–236. 15. Gluck U, Gebbers J. Epithelial changes in seasonal allergic rhinitis throughout the year: evidence of coexistent air pollution and local secretory IgA deficiency? ORL J Otorhinolaryngol Relat Spec. 2000;62:68 –75. 16. Malekzadeh S, Hamburger MD, Whelan PJ, Biedlingmaier JF, Baraniuk JN. Density of middle turbinate subepithelial mucous glands in patients with chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2002;127:190 –195. 17. Redington AE, Madden J, Frew AJ, et al. Transforming growth factor-[beta]1 in asthma. Measurement in bronchoalveolar lavage fluid. Am J Respir Crit Care Med. 1997;156:642– 647. 18. Minshall EM, Leung DY, Martin RJ, et al. Eosinophilassociated TGF-[beta]1 mRNA expression and airways fibrosis
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in bronchial asthma. Am J Respir Cell Mol Biol. 1997;17: 326 –333. Bousquet J, Jacot W, Vignola AM, Bachert C, Van Cauwenberge P. Allergic rhinitis: a disease remodeling the upper airways? J Allergy Clin Immunol. 2004;113:43– 49. Salib RJ, Howarth PH. Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clin Exp Allergy. 2003;33:1629 –1633. Braunstahl GJ, Fokkens WJ, Overbeek SE, KleinJan A, Hoogsteden HC, Prins JB. Mucosal and systemic inflammatory changes in allergic rhinitis and asthma: a comparison between upper and lower airways. Clin Exp Allergy. 2003;33:579 –587. Fish JE, Peters SP. Airway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol. 1999;104: 509 –516. Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol. 1999;104:356 –363. Cockcroft D, Murdock K. Comparative effects of inhaled salbutamol, sodium cromoglycate, and beclomethasone dipropionate on allergen-induced early asthmatic responses, late asthmatic responses, and increased bronchial responsiveness to histamine. J Allergy Clin Immunol. 1987;79:734 –740.
Requests for reprints should be addressed to: Chae-Seo Rhee, MD Department of Otorhinolaryngology-Head and Neck Surgery Seoul National University Hospital 28 Yongon-dong Chongno-gu Seoul 110-744, Korea E-mail: [email protected]
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