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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 50-56, (2003)
© 2003 American Thoracic Society

Original Article

Differential Regulation by Glucocorticoid of Interleukin-13–induced Eosinophilia, Hyperresponsiveness, and Goblet Cell Hyperplasia in Mouse Airways

Atsuko Kibe, Hiromasa Inoue, Satoru Fukuyama, Kentaro Machida, Koichiro Matsumoto, Hiroshi Koto, Tomomi Ikegami, Hisamichi Aizawa and Nobuyuki Hara

Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University; and First Department of Internal Medicine, Kurume University, Fukuoka, Japan

Correspondence and requests for reprints should be addressed to Hiromasa Inoue, M.D., Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: inoue{at}kokyu.med.kyushu-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-13 induces important features of bronchial asthma such as eosinophilic infiltration, airway hyperresponsiveness (AHR), and mucus hypersecretion. Although glucocorticoids suppress airway inflammation and remain the most effective therapy for asthma, the effects of glucocorticoids on the IL-13–dependent features are unknown. We studied the effects of dexamethasone on eotaxin production, eosinophil accumulation, goblet cell hyperplasia, and AHR after IL-13 administration into the airways of mice in vivo. MUC5AC gene expression, a marker of goblet cell hyperplasia, was also analyzed. IL-13 alone dose dependently induced AHR. Treatment with dexamethasone inhibited eotaxin expression and completely abolished eosinophil accumulation, but it did not affect AHR, MUC5AC overexpression, or goblet cell hyperplasia induced by IL-13. The effects of tumor necrosis factor-{alpha} on IL-13–induced AHR were also examined. Tumor necrosis factor-{alpha} did not affect AHR despite marked enhancement of eosinophil infiltration in IL-13–treated mice. These findings suggest that glucocorticoid is not sufficient to suppress IL-13–induced AHR or goblet cell hyperplasia and that eotaxin expression and eosinophilic inflammation do not have a causal relationship to the induction of AHR or goblet cell hyperplasia by IL-13. Control of steroid-resistant features induced by IL-13, including AHR and mucus production, may provide new therapeutic modalities for asthma.

Key Words: airway hyperreactivity • corticosteroid • cytokine • eosinophil • goblet cell metaplasia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bronchial asthma is a disease that is characterized by chronic inflammation, eosinophilic infiltration, reversible airway narrowing, airway hyperresponsiveness (AHR) to nonspecific stimuli, hyperplasia/metaplasia of goblet cells, and subepithelial fibrosis (1, 2). Although the mechanisms underlying these features are complex, CD4+ Th2 lymphocytes and their cytokine products, such as interleukin (IL)-4, IL-5, IL-9, and IL-13, play a crucial role in generating these abnormalities. In patients with asthma, CD4+ T cells producing IL-4, IL-5, and IL-13 have been identified in bronchoalveolar lavage (BAL) and airway biopsy (3). Animal studies have also shown that Th2 cells induced airway eosinophilia, mucus hypersecretion, and AHR (4, 5).

Increased expression of IL-13 has been demonstrated in the airways of patients with asthma (6, 7). Furthermore, local administration of recombinant IL-13 to nonimmunized mice induces eosinophil influx in the airways, goblet cell hyperplasia with mucus hypersecretion, and AHR (8). Chronic overproduction of IL-13 in the airways induces epithelial eotaxin expression, pathologic changes of asthma, and AHR in mice (9). Neutralization of eotaxin reduces airway eosinophilia and AHR in a mouse model of asthma (10). In vivo blockade of IL-13 by a soluble IL-13 receptor {alpha}2-IgGFc fusion protein also reverses allergen-induced AHR (8, 11). These facts suggest that IL-13 plays an important role in the pathogenesis of asthma and that upregulation of epithelial expression of eotaxin may contribute to IL-13–induced eosinophilia and AHR. Eosinophils are considered a critical determinant of AHR (12–15), but their role in causing AHR in asthma is controversial. Several studies have shown that AHR can develop independently from recruitment of eosinophils (4, 16–19). In one study, selective neutralization of IL-13 was shown to ameliorate allergen-induced airway eosinophilia (11), but in another study, it did not appear to affect eosinophils (8). The precise role of eosinophils in IL-13–induced AHR is not clear. IL-13 may induce AHR directly or indirectly via mediators released from resident cells in the airways or from infiltrating inflammatory cells.

Glucocorticoids remain one of the most effective antiinflammatory agents available in treatment of asthma. The inhibitory effect of glucocorticoids on synthesis of inflammatory mediators is considered the central mechanism of their efficacy. Glucocorticoids downregulate eotaxin in human airway mucosa (20) and IL-13–induced eotaxin expression in epithelial cells (21, 22), but their inhibitory effects on the IL-13–dependent features are unknown. We studied the effects of dexamethasone on eosinophil accumulation, goblet cell hyperplasia, and AHR after IL-13 administration to the airways. We also analyzed MUC5AC gene expression, a marker of goblet cell hyperplasia, in a murine model of allergic asthma (23). The numbers of apoptotic cells in lung tissues and in cells obtained by BAL were analyzed using terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) to assess whether dexamethasone may effect apoptosis of eosinophils. The proinflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) also causes AHR in rats and in humans (24, 25), and it stimulates eotaxin expression in airway epithelial and smooth muscle cells in vitro (26–28). Synergistic effects of IL-13 and TNF-{alpha} have been shown for induction of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells (29), eosinophil activation in vitro (30), and eotaxin production in the mouse lung in vivo (31). If IL-13 induces AHR by promoting eotaxin overexpression and subsequent eosinophil recruitment, then TNF-{alpha} or glucocorticoids should affect IL-13–induced AHR via modulation of chemokine expression. Therefore, we studied the effect of TNF-{alpha} on IL-13–induced AHR.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protocol
Male 6-week-old A/J mice received an intratracheal instillation of 50 µl of recombinant murine IL-13 solution (Sigma, St. Louis, MO) or vehicle solution on Days 1, 3, and 5 (11). Dexamethasone (0.5 mg/kg) was administered intraperitoneally 24 hours before and 1 hour before the first instillation of IL-13 and on 4 consecutive days (Day 0 to Day 5, total 6 consecutive days). To examine the interaction between IL-13 and TNF-{alpha}, the highest dose of TNF-{alpha} that did not affect airway responsiveness (subthreshold dose) was used in combination with IL-13.

Measurements of airway responsiveness, BAL, histologic assessment, and total RNA extraction from lung tissues were performed on Day 6, 24 hours after the last instillation.

Measurement of Airway Responsiveness
Animals were anesthetized with a mixture of ketamine and sodium pentobarbital intraperitoneally, and their tracheas were cannulated via tracheostomy. The animals were ventilated mechanically (tidal volume, 0.3 ml; frequency, 120 breaths/minute). A paralytic agent was administered. Airway opening pressure was measured with a differential pressure transducer and was recorded continuously. Stepwise increases in acetylcholine dose (0.6 to 20 mg/ml) were given with an ultrasonic nebulizer (120 breaths). The data were expressed as the provocative concentration 200 (PC200), the concentration at which airway pressure was 200% of its baseline value. PC200 was calculated by log-linear interpolation for individual animals.

BAL, Cell Counting, Enzyme-linked Immunosorbent Assay, and Eosinophil Peroxidase Assay
Mice were given a lethal dose of pentobarbital, and the lungs were gently lavaged three times with saline at 25 cm H2O via the tracheal cannula. Total cell counts and differential counts were performed as previously described (32). Mouse eotaxin, IL-5, and regulated upon activation, normal T cell expressed and secreted in the supernatant of BAL fluid were measured using enzyme-linked immunosorbent assay kits (R&D Systems, Inc., Minneapolis, MN). Eosinophil peroxidase was assayed in the supernatant of BAL fluid (32).

Histologic Assessment
Lungs were fixed in 10% formalin, and tissue sections were stained with Alcian blue/periodic acid-Schiff to determine the presence of mucin glucoconjugates. TUNEL was performed using a commercial kit (Takara Biomedicals, Komatsu, Japan). The number of TUNEL-positive cells in the entire area of the section was counted under the microscope.

Determination of mRNA Expression of MUC5AC and IL-13 Receptor
Total RNA was isolated from lung tissues by the guanidinium thiocyanate-phenol-chloroform method. Reverse transcription-polymerase chain reaction was performed on 1.0 µg of total RNA. An oligo (dT) primer was used for reverse transcription, and cDNA was amplified by polymerase chain reaction using specific primers. The following oligonucleotide primers were used: MUC5AC, 5'-CAGCCGAGAGGAGGGTTTGATCT-3' and 5'-AGTCTCTCTCCGCTCCTCTCAAT-3' (33); IL-13 receptor {alpha}1 (IL-13R{alpha}1), 5'-GAATTTGAGCGTCTCTGTCGAA-3' and 5'-GGTTATGCCAAATGCACTTGAG-3'; IL-13R{alpha}2, 5'-ATGGCTTTTGTGCATATCAGATGCT-3' and 5'-CAGGTGTGCTCCATTTCATTCTAAT-3'; soluble IL-4R and a membrane bound IL-4R, 5'-AGTGAGTGGAGTCCTAGCATC-3' and 5'-GCTGAAGTAACAGAACAGGC-3' (sIL-4R, 241 bp, and mIL-4R, 127 bp product) (34). Amplification was performed as previously reported (34, 35). Primers used for ß-actin (36) internal control were 5'-TCCTGTGGCATCCATGAAACT-3' and 5'-GAAGCACTTGCGGTGCACGAT-3'.

Data Analysis
Values of PC200 were log transformed and are expressed as arithmetic means ± SEM. Differences between groups for PC200 data were analyzed by analysis of variance, and the significance of differences between values was assessed with Bonferroni correction. The Mann-Whitney U-test was used for cell counts and eotaxin levels in BAL fluid. A p value below 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-Ranging Study of TNF-{alpha}
To examine the interaction between IL-13 and TNF-{alpha}, the highest dose of TNF-{alpha} that did not affect airway responsiveness (subthreshold dose) was determined. TNF-{alpha} (0.1, 1.0, and 10 ng; on Days 1, 3, and 5) was administered by instillation, and airway responsiveness to inhaled acetylcholine was measured on Day 6. Intratracheal administration of 10 ng of TNF-{alpha} significantly decreased PC200 values in comparison to control values (Figure 1A) . A subthreshold dose of 1.0 ng of TNF-{alpha} did not affect airway responsiveness or eosinophil counts in BAL fluid (Figures 1A and 1C). Therefore, we used 1.0 ng of TNF-{alpha} to examine its interaction with IL-13.



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  		Figure 1. (A) Airway responsiveness to inhaled acetylcholine after intratracheal instillation of TNF-{alpha}. In the dose-ranging studies, TNF-{alpha} (0.1, 1.0, and 10 ng; on Days 1, 3, and 5) was administered by intratracheal instillation. Airway responsiveness to inhaled acetylcholine was measured 24 hours after the last instillation. Cytokine was dissolved in vehicle (Hank's balanced salt solution with 0.1% bovine serum albumin), and this vehicle was used for control. Under anesthesia with a mixture of ketamine (45 mg/kg) and xylazine (8 mg/kg) intraperitoneally, animals were given either vehicle or cytokine solution intratracheally (n = 7 to 10). {dagger}p < 0.01 compared with vehicle instillation. (B) Airway responsiveness to inhaled acetylcholine after intratracheal instillation of IL-13 alone (open circles) or of IL-13 plus the subthreshold dose of TNF-{alpha} (1.0 ng/body) (closed circles, n = 7 to 10). {dagger}p < 0.01 compared with vehicle instillation. (C) Eosinophil counts in BAL fluids after instillation of TNF-{alpha} or IL-13 or IL-13 plus TNF-{alpha}. The subthreshold dose of TNF-{alpha} synergistically enhances IL-13–induced eosinophilia. (D) Eotaxin concentration in BAL fluids after instillation of IL-13 alone or of IL-13 plus TNF-{alpha} (n = 10 to 15). *p < 0.05 and {dagger}p < 0.01.

 
Influence of TNF-{alpha} on IL-13 Effects on AHR and the Cellular Profile of BAL Fluid
Intratracheal administration of IL-13 alone caused dose-dependent decreases in PC200 values (Figure 1B). Instillation of 0.5 µg of IL-13 increased significantly airway responsiveness compared with control. IL-13 also increased significantly the concentration of eotaxin in BAL fluid and the number of eosinophils (Figures 1C and 1D). Simultaneous instillations of IL-13 and TNF-{alpha} (subthreshold dose, 1.0 ng) were performed on Days 1, 3, and 5 to examine their interactions. Simultaneous instillations of IL-13 and TNF-{alpha} caused marked increases in eosinophil counts and eotaxin levels in BAL fluids; levels were significantly higher than those observed with IL-13 alone (Figures 1C and 1D). The levels of RANTES and IL-5 in BAL fluids were below detectable limits; however, IL-13 plus TNF-{alpha} instillation produced the same effect on the PC200 value as that produced by IL-13 alone (Figure 1B). Instillation of IL-13 plus TNF-{alpha} did not change the numbers of macrophages, neutrophils, and lymphocytes in BAL fluid in comparison to that with IL-13 alone (data not shown). There were no significant differences in baseline values of airway pressure between treatments. We conclude that the subthreshold dose of TNF-{alpha} synergistically enhanced IL-13–induced eosinophilia but not AHR.

Effects of Dexamethasone on Eosinophils and Eotaxin in BAL Fluid and on AHR after IL-13
Instillation of IL-13 increased significantly the number of eosinophils in BAL fluids and the levels of eotaxin and eosinophil peroxidase compared with control (Figure 2) . Treatment with dexamethasone after IL-13 instillation caused significant decreases in IL-13–induced eotaxin levels in BAL fluid, eosinophil accumulation, and elevated eosinophil peroxidase activity (Figure 2). Compared with saline treatment, dexamethasone treatment did not affect PC200 values after IL-13 instillation (Figure 3) . There were few TUNEL-positive signals in lung tissues with or without instillation of IL-13. Dexamethasone treatment did not increase the number of TUNEL-positive cells in lung tissues or in BAL cells after IL-13 instillation (data not shown). Although more intensive treatment with dexamethasone (3 days of pretreatment plus 5 consecutive days of treatment) was also performed, it did not attenuate the IL-13–induced AHR (data not shown). Treatment with dexamethasone did not change the number of macrophages, neutrophils, and lymphocytes in BAL fluid after IL-13 instillation.



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  		Figure 2. Effects of dexamethasone (Dex) treatment on eosinophil counts (A), eotaxin levels in BAL fluid (B), and eosinophil peroxidase (EPO) activity in BAL fluids (C) after IL-13 instillation. IL-13 induces marked eosinophilia and upregulates eotaxin expression. Dexamethasone inhibits significantly IL-13–induced eotaxin production and abolishes eosinophilia (n = 7 to 10). {dagger}p < 0.01.

 


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  		Figure 3. Effect of dexamethasone treatment on airway responsiveness to inhaled acetylcholine (ACh) after intratracheal instillation of IL-13 (n = 10 for all groups). *p < 0.05 compared with vehicle instillation.

 
Effects of Dexamethasone on IL-13–induced MUC5AC Expression and Goblet Cell Hyperplasia
Alcian blue/periodic acid-Schiff staining cells were extremely rare in the airways of naive mice and of animals received vehicle instillations. In mice receiving IL-13 instillations, there were prominent Alcian blue/periodic acid-Schiff staining cells in the trachea, the main bronchi, and in the intrapulmonary bronchi (Figure 4) . Treatment with dexamethasone did not affect increases in Alcian blue/periodic acid-Schiff staining cells after IL-13 instillation. We also analyzed MUC5AC gene expression, a marker of goblet cell hyperplasia (23). There was no difference in MUC5AC expression after vehicle instillation compared with that in naive animals. IL-13 instillation markedly enhanced expression of MUC5AC mRNA in the lung. Dexamethasone treatment did not affect overexpression of MUC5AC induced by IL-13 (Figure 5) .



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  		Figure 4. Light photomicrographs of mouse lung tissue stained with Alcian blue-periodic acid-Schiff to identify goblet cells (original magnification x200). There are marked increases in goblet cells in the epithelium of the trachea and of the intrapulmonary bronchus 24 hours after IL-13 instillation compared with those in vehicle-treated animals. Treatment with dexamethasone (Dex) does not affect the increase in goblet cells observed after IL-13 administration.

 


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  		Figure 5. MUC5AC expression in vehicle-treated, IL-13–treated, and dexamethasone (Dex) plus IL-13–treated mice. Whole lung total RNA was isolated, and MUC5AC levels were amplified by reverse transcription-polymerase chain reaction. Reverse transcription-polymerase chain reaction products for ß-actin are shown for comparison. Amplified DNA was separated by electrophoresis on an agarose gel containing ethidium bromide, illuminated with ultraviolet light, and photographed.

 
To assess whether dexamethasone may modulate expression of IL-13R, we examined mRNA expression of IL-13R{alpha}1, IL-13R{alpha}2, and soluble and membrane IL-4R. IL-13 instillation enhanced expression of IL-13R{alpha}2 mRNA in the lung, but not expression of IL-13R{alpha}1, soluble IL-4R, or membrane bound IL-4R. There were no detectable changes in the expressions of these receptors with or without treatment with dexamethasone (Figure 6) .



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  		Figure 6. Expression of IL-13R{alpha}1, IL-13R{alpha}2, and soluble and membrane IL-4R in the lung after instillation of vehicle or in IL-13-instilled lung with or without dexamethasone (Dex) treatment. Whole-lung total RNA was isolated, and the levels of IL-13R{alpha}1, IL-13R{alpha}2, soluble IL-4R, and membrane bound IL-4R were amplified by reverse transcription-polymerase chain reaction. Reverse transcription-polymerase chain reaction products for ß-actin are shown for comparison. Amplified DNA was separated by electrophoresis on an agarose gel containing ethidium bromide, illuminated with ultraviolet light, and photographed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that TNF-{alpha} enhances eosinophil infiltration in the airways but not AHR in IL-13–treated mice in vivo. We also demonstrated that in IL-13–instilled animals, dexamethasone inhibits eotaxin expression and abolishes eosinophil accumulation but that it does not affect AHR, MUC5AC overexpression, or goblet cell hyperplasia. These findings suggest that glucocorticoid is not sufficient to suppress IL-13–induced AHR and goblet cell hyperplasia despite substantial inhibition of eosinophilic inflammation, at least partly through downregulation of eotaxin expression. Eotaxin expression and eosinophilic inflammation have no causal relationship to the development of AHR after airway administration of IL-13.

The exact mechanism by which IL-13 induces AHR is unknown. L-4R{alpha} and IL-13R{alpha}1 are expressed in airway epithelial cells and smooth muscle cells (37, 38). IL-13 had an enhancing effect on direct stimuli, such as acetylcholine or methacholine, although IL-13 itself did not constrict smooth muscle in vivo or in vitro (our unpublished observation). Therefore, IL-13 may modulate smooth muscle contractility either directly or indirectly through mediators released by surrounding cells other than eosinophils.

Goblet cell hyperplasia and overexpression of MUC5AC after IL-13 administration were unaffected by dexamethasone even though dexamethasone markedly inhibited eosinophil accumulation. This finding is consistent with other work that has shown that airway epithelial mucus production can be induced by Th2 cells in the absence of eosinophils and that signaling through IL-4R{alpha}, a common receptor chain of IL-4R and IL-13R, is required to stimulate mucus production (39).

Glucocorticoid suppresses the expression of CC chemokines in epithelium by both transcriptional and post-transcriptional mechanisms (22). In this study, dexamethasone inhibited eotaxin production and abolished eosinophil accumulation after IL-13 exposure in vivo. The effect of glucocorticoid on MUC5AC expression in human epithelial cells in vitro is controversial. Dexamethasone is reported to attenuate steady-state mRNA levels of MUC5AC (40), whereas it is reported to upregulate MUC5AC expression (41). The mechanisms by which IL-13–induced AHR, MUC5AC overexpression, and goblet cell hyperplasia resistant to glucocorticoid are not clear. The IL-13–induced asthma phenotypes are dependent on the IL-4R{alpha} chain and STAT6 in naive mice (8, 42). Modification of receptor expression by dexamethasone does not seem to be responsible for steroid resistance because IL-13R{alpha}1, IL-13R{alpha}2, and IL-4R expression were not affected by dexamethasone in this study. Although it is possible that apoptotic eosinophils induce AHR after glucocorticoid treatment, dexamethasone did not increase TUNEL-positive cells in the lung after IL-13 instillation, and it attenuated the increased activity of eosinophil peroxidase in mice that received IL-13. These findings indicate that IL-13–induced AHR in dexamethasone-treated animals is not dependent on apoptotic eosinophils or on granule release. Recently, IL-13 was reported to diminish glucocorticoid receptor binding affinity in monocytes (43). This may contribute to the impaired effect of glucocorticoids on IL-13–induced AHR in our experiment. It is suggested that the relative amount of glucocorticoid receptor expressed within a cell determines the magnitude and nature of the response to glucocorticoids (44). The relative ratio of glucocorticoid receptor to other transcription factors within a given cell type determines whether the predominant consequence will be transcription enhancement or repression for certain target genes (45). Furthermore, although IL-13R subunit expressions in the whole lung were not affected by glucocorticoid in this study, it is possible that glucocorticoid differentially regulates the expression of IL-13R subunit in each cell type.

In contrast to our expectation, treatment with dexamethasone did not inhibit AHR induced by exogenous IL-13. AHR is supposed to be linked to the inflammatory processes in the airways of patients with asthma (46). Dexamethasone suppresses allergen-induced airway eosinophilia and AHR in animals (47, 48). It has been reported that IL-13 induces eotaxin expression in epithelial cells in vitro (21, 22) and in the lung in vivo (31) and that glucocorticoid downregulates eotaxin expression induced by IL-13 in vitro (21); however, to the best of our knowledge, there is no report regarding the effect of glucocorticoids on IL-13–induced AHR. Glucocorticoids suppress expression and production of IL-13 by mast cells in vitro (49), and antigen-induced gene expression of IL-13 in peripheral blood mononuclear cells is downregulated by dexamethasone (50). Therefore, we speculate that glucocorticoids inhibit allergen-induced airway eosinophilia and AHR through downregulating proinflammatory cytokines such as IL-13 but not through modulating the actions of overexpressed IL-13.

Subthreshold TNF-{alpha} did not stimulate eosinophil accumulation by itself, whereas it augmented eosinophilic inflammation induced by IL-13, consistent with an earlier report on IL-13–induced eotaxin expression (31). In contrast to this synergistic effect on eosinophilia, TNF-{alpha} did not enhance IL-13–induced AHR. These findings are supported by observations that eotaxin instillation alone does not induce AHR despite the presence of eosinophils in the airways and that additional factors such as IL-5 or platelet activating factor (PAF) are needed to activate eosinophils for the development of AHR (32, 51).

This study has provided an important clue for further research on the treatment of asthma. Although glucocorticoids improve asthma control and AHR, presumably by suppressing airway inflammation (52), the magnitude of reduction in AHR with inhaled steroids varies among patients. Most patients with asthma remain hyperresponsive compared with normal subjects despite a considerable improvement in AHR (53, 54). Some studies have not found any improvement in AHR after use of inhaled steroids (55, 56). It is possible that the some mechanisms underlying AHR in asthma are not susceptible to glucocorticoids. In this study in mice, IL-13 induced eosinophilic inflammation and AHR, but glucocorticoid did not affect AHR despite marked inhibition of eosinophilic inflammation. Additional investigation will be required to clarify the susceptibility of IL-13–dependent component(s) in asthma to glucocorticoid. IL-13 also induces airway remodeling, including goblet cell hyperplasia and subepithelial fibrosis (9). Overexpression of MUC5AC and an increase in goblet cells were resistant to dexamethasone in this study. These findings imply that IL-13 plays a crucial role not only in the development of asthma but also in the persistence of AHR and other pathologic changes.

In conclusion, we provide evidence that neither chemokine expression nor eosinophilic inflammation is related directly to the induction of AHR by IL-13. Glucocorticoid does not suppress IL-13–induced AHR or goblet cell hyperplasia despite its substantial inhibition of eosinophilia. Because AHR in patients with asthma is less susceptible to glucocorticoids, further understanding of the mechanisms of IL-13–induced AHR and mucus overproduction may have potential therapeutic implications for asthma.

Note Added after Submission
After the submission of this manuscript, an article was published reporting drug modulation of asthma phenotypes induced by IL-13 in the hyperimmunoglobulin E BP2 mice (57). The report supports the differential susceptibility to drugs in each parameter.


   Acknowledgments
 
The authors thank Yuki Yoshiura, B.Sc., and Morphology Core, Faculty of Medicine, Kyushu University, for technical assistance.


   FOOTNOTES
 
Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan

Received in original form October 23, 2001; accepted in final form August 22, 2002


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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