Tissue remodeling induced by hypersecreted epidermal growth factor and amphiregulin in the airway after an acute asthma attack

Article Outline

• Abstract
• Methods
  • Study population
• Results
  • Comparison of the levels of EGF and amphiregulin in the sputa of children with asthma collected during an asthma exacerbation and after recovery
  • Comparison of the proliferation-inducing effects of EGF and amphiregulin on NHBE, BSMC, and NHLF
  • Effect of EGF and amphiregulin on MUC5AC mRNA expression in NCI-H292 cells
  • Effect of long-term exposure of NHBE to EGF and amphiregulin on mucous cell metaplasia in the air-liquid interface (ALI) culture
• Discussion
• Acknowledgment
• Methods
  • Sputum collection and processing
  • Culture of NHBE, BSMC, and NHLF
  • Bromodeoxyuridine uptake assay
  • Culture of NHBEs in ALI culture
  • Protocols
  • Western blot analysis
  • Effect of EGF or amphiregulin on mucin gene expression in NCI-H292 cells
  • Real-time quantitative RT-PCR
  • MUC5AC immunofluorescence
  • ELISA
  • Specificity of the ELISA for EGF and amphiregulin in the sputum samples
  • Detection sensitivity of the ELISA for EGF and amphiregulin in the sputum samples
  • Statistical analysis
• Results
  • Specificity of the ELISA for EGF and amphiregulin in the sputum samples
  • Detection sensitivity of the ELISA for EGF and amphiregulin in the sputum samples
  • Effect of EGF and amphiregulin on MUC5AC mRNA expression in NCI-H292 cells
• Fig E1. 
• Fig E2. 
• Table E1. 
• Table E2. 
• Table E3. 
• References
• References
• Copyright

Background

Epidermal growth factor receptor ligands, such as epidermal growth factor (EGF) and amphiregulin, may play key roles in tissue remodeling in asthma. However, the kinetics of EGF and amphiregulin secretion in the airway after an acute asthma attack and the effect of prolonged airway exposure to these ligands on airway remodeling are unknown.

Objective

To measure the EGF and amphiregulin concentrations in sputa obtained from patients with asthma under various conditions, and to examine the effects of EGF and amphiregulin on the proliferation or differentiation of airway structural cells.

Methods

Epidermal growth factor and amphiregulin levels were measured by ELISA in sputum specimens collected from 14 hospitalized children with asthma during an acute asthma attack, 13 stable outpatients with asthma, 8 healthy control children, and 7 children with respiratory tract infections. The effects of EGF and amphiregulin on the proliferation and/or differentiation of normal human bronchial epithelial cells (NHBE), bronchial smooth muscle cells (BSMC), and normal human lung fibroblasts (NHLF) were examined.

Results

The sputum levels of EGF were significantly higher for about a week after an acute asthma attack compared with the levels in stable subjects with asthma and control subjects. In contrast, upregulation of amphiregulin in the sputa of patients with asthma was observed only during the acute attack. EGF caused proliferation of NHBE, BSMC, and NHLF, whereas amphiregulin induced proliferation of only NHBE. Prolonged exposure of NHBE to EGF and amphiregulin induced mucous cell metaplasia in an IL-13–independent manner.

Conclusion

Acute asthma attacks are associated with hypersecretion of EGF and amphiregulin in the airway. Recurrent acute attacks may aggravate airway remodeling.

Key words: Amphiregulin, bronchial asthma, bronchial epithelial cells, epidermal growth factor, tissue remodeling

Abbreviations used: ADAM17/TACE, A membrane disintegrin and metallopeptidase 17/TNF-alpha converting enzyme, ALI, Air-liquid interface, BSMC, Bronchial smooth muscle cells, EGF, Epidermal growth factor, EGFR, Epidermal growth factor receptor, ERK, Extracellular signal-regulated kinase, HB-EGF, Heparin-binding epidermal growth factor–like growth factor, MC, Mast cells, MUC5AC, Mucin 5AC, NHBE, Normal human bronchial epithelial cells, NHLF, Normal human lung fibroblasts

Bronchial asthma is a complex inflammatory disease of the lungs characterized by reversible airway obstruction, chronic airway inflammation, and airway hyperresponsiveness to spasmogenic stimuli.¹ Airway remodeling is defined as structural changes in the airways that may affect their functional properties.² Such structural changes include increased airway smooth muscle mass, mucus gland hypertrophy, deposition of extracellular matrix components, thickening of the reticular basement membrane, and angiogenesis.² Patients with asthma show accelerated loss of lung function over time, and some patients develop progressive fixed airflow obstruction. These features may reflect airway remodeling in severe and chronic asthma.² Airway remodeling is thought to be a consequence of repeated injury and persistent inflammation,² although the remodeling process is thought to begin in the early stage of development of asthma and to occur in parallel with the establishment of persistent inflammation.³

Epithelial cell proliferation contributes to the thickened epithelium and lamina reticularis in severe asthma.⁴ A disease severity–related, corticosteroid-insensitive increase in the expression of epidermal growth factor receptor (EGFR; also called ErbB1) tyrosine kinase in asthmatic bronchial epithelium has been reported.⁵ Bronchial epithelial cells produce several ligands for EGFR, including epidermal growth factor (EGF), TGF-α, heparin-binding EGF-like growth factor (HB-EGF), and amphiregulin.⁶ The effect of exogenous EGF of accelerating airway epithelial repair has been demonstrated in vitro.⁷, ⁸ We previously reported that amphiregulin was secreted by human mast cells (MC) after aggregation of FcεRI.⁹ Upregulation of amphiregulin expression has been observed in the MC of patients with asthma but not of normal control subjects. Amphiregulin induces upregulation of mRNA of mucin 5AC (MUC5AC), which is the dominant mucin gene expressed in goblet cells among a total of 12 mucin genes.¹⁰ Furthermore, upregulation of amphiregulin in the MC was significantly correlated with the extent of goblet cell hyperplasia in the mucosa in patients with bronchial asthma. However, the precise time points at which each of the EGFR ligands is secreted in the asthmatic airway in the course of asthma—that is, whether only during or after an acute attack, or also during the stable phase when the asthma is well controlled—are still unknown. Therefore, we measured the EGF and amphiregulin concentrations in sputum specimens obtained during an acute attack as well as during the stable phase.

Epidermal growth factor has a proliferation-inducing effect on normal human bronchial epithelial cells (NHBE),¹¹ normal human lung fibroblasts (NHLF),¹² and bronchial smooth muscle cells (BSMC).¹³ MUC5AC protein has also been shown to be induced by EGF in mucoepidermoid NCI-H292 cells.¹⁴ Amphiregulin has been reported to be involved in the process of lung branching morphogenesis in mice.¹⁵ However, the effect of amphiregulin on airway remodeling, except for its upregulation of MUC5AC mRNA in NCI-H292 cells, is still unknown. Also, the effects of prolonged exposure of NHBE to high concentrations of EGF and amphiregulin are unknown. Therefore, we examined the effects of EGF and amphiregulin on the proliferation and/or differentiation processes of airway structural cells in vitro.

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Methods 

Study population 

The study was approved by the Ethics Committee of Kanagawa Children's Medical Center and Nihon University Hospital, and all subjects provided written informed consent for participation, in accordance with the Helsinki Declaration of the World Medical Association. Fourteen children with asthma hospitalized for treatment of an acute attack, 13 stable outpatients before and after therapy with inhaled corticosteroids, 8 healthy controls, and 7 outpatients with respiratory tract infections without asthma were enrolled for the study. The demographic characteristics of the patients with asthma and control subjects are shown in Table I. None of the outpatients with asthma had had an exacerbation of the disease within 3 months before entry into the study. One of the hospitalized patients with asthma was being treated with oral corticosteroids. Six of the hospitalized patients with asthma had been receiving inhaled corticosteroid treatment since before hospital admission. The percentage of eosinophils in the sputa obtained from the hospitalized patients with asthma reduced from 18.8% during the acute phase to 3.3% during the recovery phase. After admission for an acute asthma attack, inhaled β-agonists were used in all patients, and intravenous infusion of a steroid was started in 10 of the 14 patients. Oxygen therapy was started in 12 of the 14 patients. The majority of our patients with asthma had comorbid allergic rhinitis. See this article's Methods in the Online Repository at www.jacionline.org for additional information about the experiments and for descriptions of the following methods used in this study.

Table I. Characteristics of the subjects with asthma and controls

F, Female; ICS, inhaled corticosteroid; LABA, long-acting β-agonist; LTRA, leukotriene receptor agonist; M, male; NA, not applicable; ND, not determined.

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Results 

Comparison of the levels of EGF and amphiregulin in the sputa of children with asthma collected during an asthma exacerbation and after recovery 

Initially, we measured the concentrations of EGF, amphiregulin, TGF-α, and HB-EGF in the sputum samples of 6 patients with asthma collected during an exacerbation and after recovery. The concentration of HB-EGF in all the sputum samples was under the detection limit of the ELISA kit. The concentrations of TGF-α in the sputum samples were much lower than those of EGF and amphiregulin. The results are shown in this article's Table E1 in the Online Repository at www.jacionline.org. Therefore, we focused on the analysis of EGF and amphiregulin in this study. Because sputum is loaded with many destructive proteases, we confirmed the specificity of the immunoassays for EGF and amphiregulin (see this article's Table E2 and result section in the Online Repository at www.jacionline.org) and their detection sensitivity when exogenous ligand was added to the sputum (see this article's Table E3 in the Online Repository at www.jacionline.org). The addition of sputum to NCI-H292 cells increased MUC5AC mRNA levels, and the increase was blocked by anti-EGF or anti-amphiregulin blocking antibodies (data not shown), thus the EGF ligands in the sputa have bioactivities. To examine whether the EGF and amphiregulin levels were upregulated in the bronchial mucosa of patients with asthma during an acute asthma attack, we measured the EGF and amphiregulin levels in the sputa of patients with asthma obtained during an acute attack and during the recovery phase after the acute attack (on the day of discharge from the hospital); sputum specimens were also obtained from patients with stable, well controlled asthma, normal healthy controls, and patients with respiratory tract infections. Children with asthma hospitalized for treatment of an acute attack had significantly higher levels of EGF from the day of hospitalization for the acute attack until the recovery phase than stable outpatients with asthma, healthy controls, or children with respiratory tract infections (Fig 1, A; P < .001 or P < .01). In contrast, the upregulation of amphiregulin in the sputa of the hospitalized patients with asthma was transient, lasting only through the duration of the acute attack (Fig 1, B). Cysteinyl leukotrienes have been reported to play an important role in airway remodeling.¹⁶ The atopic status of the patients is an important issue that must be considered in addition to the factors triggering the asthma attacks. To see whether there were any differences in the sputum levels of EGF and amphiregulin between patient groups with atopic and nonatopic asthma, between patient groups showing and not showing elevation of the serum C-reactive protein during an exacerbation as a marker of infection triggering the asthma attack, and between patient groups treated and not treated with leukotriene receptor antagonists, we compared the concentrations of the 2 EGFR ligands during an exacerbation and after recovery. We found no significant differences in the levels of the 2 EGFR ligands between any of the groups (see this article's Fig E1 in the Online Repository at www.jacionline.org). To clarify the kinetics of the EGF and amphiregulin levels in sputum samples from the start of the acute attack to the recovery stage, we measured the levels of EGF and amphiregulin every day after the onset of the acute attack in sputum samples obtained from 8 of the hospitalized children. As a marker of mediators released during the acute attack, the levels of tryptase were also measured. The EGF levels remained high from the day of hospitalization until the day of recovery from the acute attack (Fig 2, A). In contrast, both the amphiregulin and tryptase levels tended to be high during the acute attack and to gradually decrease with recovery from the acute attack (Fig 2, B and C). There was a significant correlation between the amphiregulin and tryptase levels (Fig 2, E; P < .001; r = 0.735) but not between the EGF and amphiregulin or tryptase levels (Fig 2, D and F).

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• Fig 1. 

  Concentrations of EGF (A) and amphiregulin (AREG) (B) in the sputa of patients with asthma after an asthma attack (acute), during the recovery phase (recovery), and during the stable phase (stable); sputa of normal healthy controls; and sputa of patients with respiratory tract infection (^∗∗P < .01; ^∗∗∗∗P < .001). The number of donors is indicated under the figure. Because of the small volume of the sputum samples, the experiments could not be conducted in some cases.

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• Fig 2. 

  Time-course of changes in the sputum concentrations of EGF (A), amphiregulin (AREG) (B), and tryptase (C) in children with asthma hospitalized for treatment of an acute asthma attack during the acute phase and during the recovery phase after treatment (n = 8 donors). Each symbol indicates an individual donor. Correlation between the sputum concentrations of EGF and AREG (D), between those of AREG and tryptase (E), and between those of EGF and tryptase (F). Even if the asterisked point was omitted, there was a significant correlation between the sputum concentrations of AREG and tryptase (E, P < .01, r = 0.416). NS, Not significant.

Comparison of the proliferation-inducing effects of EGF and amphiregulin on NHBE, BSMC, and NHLF 

To compare the proliferation-inducing effect between EGF and amphiregulin on NHBE, BSMC, and NHLF, we conducted the bromodeoxyuridine uptake assay for these cells after short exposure (24 hours) to amphiregulin and EGF (Fig 3, A-C). EGF induced proliferation of NHBE, NHLF, and BSMC. However, amphiregulin had no proliferation-inducing effect on the NHLF. Amphiregulin exerted a proliferation-inducing effect on the BSMC only at a high concentration (100 ng/mL). Amphiregulin, however, in the concentration range of 1 to 100 ng/mL, induced a concentration-dependent increase of NHBE proliferation. At 10 ng/mL, amphiregulin induced a 2-fold increase of the NHBE proliferative activity relative to that observed in the presence of medium alone, although EGF induced a similar degree of increase of the proliferative activity at 1 ng/mL. At 100 ng/mL, both amphiregulin and EGF induced a 3-fold increase in the cellular proliferative activity of NHBE. These results were confirmed by the [³H] thymidine incorporation assay (data not shown). To examine the difference in the cellular signaling after exposure of the NHBE and BSMC to EGF or amphiregulin, we compared the tyrosine phosphorylation level of EGFR after exposure of these cells to EGF or amphiregulin (Fig 3, D). EGF abundantly stimulated EGFR phosphorylation at Tyr992, Tyr1045, and Tyr1068, whereas amphiregulin did not stimulate phosphorylation at Tyr992 in either the NHBE or the BSMC. Amphiregulin induced only marginal phosphorylation of Tyr1045 in the BSMC; in addition, it induced modest phosphorylation of Tyr1068 in both NHBE and BSMC. We confirmed upregulation of c-Fos mRNA levels in parallel with the increase in the proliferative activities of these cells (data not shown). The signaling pathways leading to extracellular signal-regulated kinase (ERK) activation by EGF and amphiregulin in the NHBE and BSMC were also assessed (Fig 3, E). Amphiregulin produced only a marginal increase in the phosphorylation level of ERK in the NHBE and BSMC.

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• Fig 3. 

  EGF and amphiregulin (AREG) induced distinct patterns of cell proliferation. Bromodeoxyuridine uptake in NHBE (A), BSMC (B), and NHLF (C) induced by EGF (filled circles) or AREG (open circles). Data are expressed as means ± SEMs of 3 independent experiments using different donors. ^∗P < .05, ^∗∗P < .01, ^∗∗∗P < .005, and ^∗∗∗∗P < .001 compared with cells not treated with recombinant human EGF or recombinant human AREG. D, EGFR tyrosine phosphorylation in NHBE and BSMC by EGF and AREG. This experiment was repeated 3 times. E, Phosphorylation of ^44/42ERK in NHBE and BSMC by EGF or AREG. Fold induction of phosphorylation of p42 ERK was determined by densitometry and normalized to the respective protein. One representative of 3 individual experiments is shown. Abs, Absorbances.

Effect of EGF and amphiregulin on MUC5AC mRNA expression in NCI-H292 cells 

We confirmed that EGF and amphiregulin significantly upregulated MUC5AC mRNA expression in the NCI-H292 cells in a concentration-dependent and time-dependent manner; however, the degree to which the level was upregulated was not significantly different between EGF and amphiregulin (see this article's Fig E2 in the Online Repository at www.jacionline.org).

Effect of long-term exposure of NHBE to EGF and amphiregulin on mucous cell metaplasia in the air-liquid interface (ALI) culture 

Patients with severe asthma have frequent asthma attacks. To examine the effect of prolonged exposure of the airway to EGF and amphiregulin, we examined mucous cell metaplasia in an ALI culture of NHBE cells treated with EGF or amphiregulin. To examine whether EGF and amphiregulin induced changes in the phenotype of the differentiated NHBE growing in the ALI culture, expression of MUC5AC mRNA and protein was assessed by real-time RT-PCR and ELISA, respectively. In addition, an antibody to MUC5AC was used for the immunofluorescence assay, as described in the Methods section. When NHBE were maintained in medium supplemented with 1 to 100 ng/mL EGF or amphiregulin for 14 days, the expressions of MUC5AC mRNA and protein were upregulated in a concentration-dependent manner, even in the absence of addition of any other molecules such as IL-13 (Fig 4, A and B). There were many mucus-containing goblet cells that stained positively with the MUC5AC-specific antibody in NHBE cultured in the presence of 100 ng/mL EGF or amphiregulin. In contrast, there were no mucus-containing cells in NHBE cultured in the absence of EGF or amphiregulin (Fig 4, C).

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• Fig 4. 

  Long-term exposure of NHBEs to EGF and amphiregulin (AREG) results in mucous cell metaplasia in the ALI culture. After NHBE were cultured in the ALI culture for 2 weeks in the presence of recombinant human EGF or recombinant human AREG, the medium was replenished, and the cells were incubated in the presence or absence of recombinant human EGF or recombinant human AREG for 24 hours (hatched bars) and 48 hours culture (filled or open bars) for analysis of MUC5AC gene expression (A) and for 72 hours for analysis of MUC5 AC protein production (B) and immunocytochemistry (C). Data are expressed as means ± SEMs (n = 3). C, Mucins were measured in the cell lysate and in the supernatants. i, Without second antibody. ii, Non–AREG-treated or EGF-treated cells. iii, 100 ng/mL recombinant human EGF–treated cells. iv, 100 ng/mL recombinant human AREG–treated cells. The data in A, B, and C are representative of similar results obtained from 3 independent experiments using different donors. Green and red staining indicates MUC5AC and nuclei, respectively.

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Discussion 

In this study, we measured, for the first time, the concentrations of EGF and amphiregulin in sputum samples from patients with asthma obtained during an acute attack and also during the recovery phase. We found that the sputum EGF levels in these patients with asthma increased during the acute attack and remained elevated during the recovery phase after the acute attack. In contrast, the sputum concentrations of amphiregulin and tryptase were only transiently elevated during the acute attack (Fig 1, Fig 2). EGF induced proliferation and differentiation of NHBE and also proliferation of BSMC and NHLF, whereas amphiregulin only stimulated cell proliferation of the epithelial cells among the lung structural cells examined (Fig 3). Prolonged exposure of NHBE to EGF or amphiregulin induced changes in the phenotype of the differentiated NHBE grown in ALI culture (Fig 4). These data indicate that EGF and amphiregulin are hypersecreted in the airway of subjects with acute severe asthma both during and after an acute asthma attack. Although EGF and amphiregulin are both EGFR ligands, each of the ligands appears to have a distinct role in airway repair or remodeling.

It is not clear whether the hypersecretion of EGF and amphiregulin after an acute asthma attack is intended for the purpose of airway repair or remodeling. In regard to the role for endogenous ligands released by damaged or adjacent epithelium, mechanical injury of confluent airway epithelial cell monolayers has been reported to induce expression and release of EGF in coordination with repair.¹⁷ It has been thought that the induction of excessive or prolonged release of EGF in an attempt to effect repair would have additional adverse effects on both epithelial and subepithelial cells and structures.¹⁷ Although the sputum concentrations of EGF and amphiregulin were transiently increased after an acute attack in our hospitalized patients with asthma, the patients were appropriately treated and discharged by 7 days after the acute attack. In the absence of appropriate treatment, the attacks might recur, and recurrent acute attacks may be expected to be associated with excessive production and prolonged exposure of the airway to EGF and amphiregulin, and consequently, airway remodeling. Of course, it is assumed that the effects of these EGFR ligands on normal cells can be extrapolated to the corresponding cells in asthmatic airways. Evidence is presented to suggest that the airway epithelium in asthma is fundamentally abnormal and shows increased susceptibility to environmental injury and impaired repair associated with activation of the epithelial-mesenchymal trophic unit.¹⁸ In addition to conversion to an activated phenotype, the barrier function of the epithelium is impaired through defective tight junction formation, thereby facilitating penetration of potentially toxic or damaging environmental insults. Thus, further studies will be required to clarify the effects of EGF and amphiregulin on the epithelium of asthmatic airways.

In a variety of cultured cell model systems, different EGF family ligands that bind to the same receptor can yield divergent biological outcomes. For example, in MCF10A human mammary epithelial cells, amphiregulin was shown to be a more potent stimulant of motility and invasiveness than EGF.¹⁹ Amphiregulin, but not EGF, was shown to stimulate nuclear factor-κB signaling and IL-1 secretion in MCF10A immortalized cells. These findings appear to account for the divergent stimulation of motility and invasiveness by amphiregulin and EGF.²⁰ It has been shown that EGF, but not amphiregulin, can suppress alcohol-induced apoptosis in human placental cytotrophoblast cells.²¹ Some reasons are proposed why amphiregulin at lower concentrations less effectively induced proliferation of NHBE. First, amphiregulin exhibits a lower affinity for the EGF receptor than EGF.²² The mature form of amphiregulin is truncated at the C terminus and lacks the conserved leucine residue known to be essential for high-affinity binding of EGF to EGFR.²² Second, differences in the sites of ligand-induced EGFR tyrosine phosphorylation may underlie the divergent ligand-induced EGFR coupling to signaling effectors and biological responses. EGF abundantly stimulated EGFR phosphorylation at Tyr1045, whereas amphiregulin did not, which is in agreement with the observations of Gilmore et al.²³ Phosphorylation of Tyr1045 creates a canonical binding site for the E3 ubiquitin ligase c-cbl, leading to EGFR ubiquitination and degradation by the 26S proteasome.²⁴, ²⁵ Also in our experiments, whereas EGF abundantly stimulated EGFR phosphorylation at Tyr992 in NHBEs, amphiregulin did not. In contrast, amphiregulin has been reported to stimulate EGFR phosphorylation abundantly, including Tyr992, in the human breast cancer cell line A1 and the epithelial cell line S1.²³ The phosphorylated Tyr992 residue has been reported to bind phospholipase Cγ, leading to increased phospholipase Cγ activity and activation of the mitogen-activated protein kinase pathway. However, why amphiregulin has only a marginal effect on the proliferation of BSMC and NHLF despite ErbB1 being the receptor for both amphiregulin and EGF is not clear. EGF family ligands bind to and activate the ErbB family receptors, which consist of 4 related subtypes—ErbB1/EGFR, ErbB2, ErbB3, and ErbB4²⁶—by inducing the formation of homodimers and heterodimers, resulting in autophosophorylation of specific tyrosine residues. Our preliminary experiments using quantitative real-time PCR of the ErbB family genes showed that ErbB1/EGFR was the most highly expressed gene among the ErbB family receptors in the all airway structural cells examined (NHBE, BSMC, and NHLF; data not shown). Indeed, amphiregulin-induced proliferation and c-Fos mRNA expression in NHBE were completely inhibited by simultaneous treatment with an anti-EGFR neutralizing antibody (data not shown), suggesting the indispensability of ErbB1/EGFR for amphiregulin-mediated responses of the NHBE. We also found only modest expression of ErbB2 mRNA and no expression of ErbB4 mRNA in the NHBE, BSMC, and NHLF. Interestingly, ErbB3 mRNA and protein were preferentially expressed in NHBE but not in the BSMC or NHLF (data not shown). We confirmed the expression of ErbB3 protein in NHBE by Western blot analysis (data not shown). Because ErbB3 requires ErbB1-ErbB3 and/or ErbB2-ErbB3 heterodimer formation to initiate signaling,²⁷ we hypothesized that ErbB1-ErbB3 and/or ErbB2-ErbB3 heterodimers were critically involved in the amphiregulin-mediated responses. To confirm our hypothesis, we knocked down ErbB3 in the NHBE by using the small interfering RNA silencing technique and compared the amphiregulin-induced proliferative activity of ErbB3–knocked down NHBE and control NHBE. However, we did not observe any differences in amphiregulin-mediated responses between the ErbB3–knocked down NHBE and control NHBE. Further studies are clearly needed to elucidate the functional relevance of ErbB3 in amphiregulin-mediated responses. Alternatively, various factors may contribute to ligand specificity, including differences in the timing of the ligand expression, tissue-specific patterns of ligand expression, and differences in posttranslational cleavage and processing. Accessory molecules and coreceptors, such as heparan sulfate proteoglycans, may also contribute to ligand specificity by sequestering high concentrations of these growth factors locally or by controlling their bioavailability, thereby selectively modulating the duration and/or strength of signaling stimulation by members of the EGF family that bind to these molecules.²⁸

In this study, high concentrations of EGF or amphiregulin alone induced mucous cell metaplasia in an ALI culture of human epithelial cells, without any transdifferentiation signals induced by IL-13 or neutrophil elastase. Two ALI culture methods have been reported, culture in the presence of a high concentration of retinoic acid (50 nmol/L)²⁹, ³⁰ and that in the presence of a low concentration of retinoic acid (0.33 nmol/L)³¹; we used the former method. When 0.33 nmol/L retinoic acid was used for the ALI culture, MUC5AC mRNA, whose upregulation has been shown to be correlated with mucous cell metaplasia in the airways,¹⁰ was not induced by EGF or amphiregulin alone under our experimental conditions (data not shown). Mucous cell metaplasia requires neutrophil elastase-induced transdifferentiation signals under the ALI culture condition of a low concentration of retinoic acid.³¹ Retinoic acid receptors mediate transcription of different sets of genes involved in cell differentiation, thus the effect of retinoic acid also depends on the target cells. It is known that vitamin A is essential for maintenance of the mucociliary epithelium in the conducting airways.³² Our data suggest that EGF or amphiregulin alone may be capable of inducing mucous cell metaplasia in a non—IL-13–dependent manner in microenvironments with a high concentration of retinoic acid.

Bronchial epithelial cells produce both EGF and amphiregulin.⁶ Members of the EGF family are generated by a membrane disintegrin and metallopeptidase 17 (ADAM17)/TNF-α converting enzyme (TACE) through cleavage of a membrane-bound ligand rather than by secretion from intracellular stores.³³ We measured the concentrations of ADAM17/TACE in the sputum of 6 patients with asthma during exacerbation and after recovery (Table E1). ADAM17/TACE was detected in the sputa of 3 of the 6 donors with asthma during an exacerbation, suggesting that ADAM17/TACE plays an important role in the upregulation of EGF ligands during an asthmatic exacerbation. The kinetics of sputum EGF and amphiregulin in patients with asthma after an acute asthma attack were different, and there was a significant correlation between the concentrations of tryptase and amphiregulin, even if the highest value point was omitted, but not between the concentrations of tryptase or amphiregulin and EGF, suggesting that some of the amphiregulin in the sputum of patients with acute asthma with elevated amphiregulin levels might be produced by MC. The concentration of amphiregulin in the airways was significantly increased during the acute attack in our patients. In the event of recurrent acute attacks, the airway epithelial cells may be expected to be exposed for longer durations to high concentrations of amphiregulin. Our findings suggested that amphiregulin may play an important role in the pathophysiology of not only acute severe asthma, in which it induces mucus hypersecretion, but also in the aggravation of mucous cell metaplasia and airway remodeling in patients with recurrent acute asthma attacks. The prolonged hypersecretion of EGF after an acute attack may also play a major role in airway remodeling by increasing the airway smooth muscle mass and inducing mucus gland hypertrophy, deposition of extracellular matrix components, and thickening of the reticular basement membrane. Our data lend support to the notion that activated and repairing epithelial-mesenchymal trophic units generate a range of growth factors that are involved in the early-life origin of this disease as well as its progression in the form of mucous metaplasia and airway wall remodeling.¹⁸ Our findings suggest that the airway remodeling process progresses with every acute attack.

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We thank Dr Zenro Ikezawa (Department of Environmental Immuno-Dermatology, Yokohama City University Graduate School of Medicine, Yokohama, Japan) and Dr Mutsuhiko Minami (Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan) for their helpful insights into the subject dealt with in the article.

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Methods 

Sputum collection and processing 

Spontaneously expectorated sputum samples were obtained from each of the hospitalized patients with asthma: 1 sample immediately after admission to the hospital and 1 sample every day thereafter until discharge. Spontaneously expectorated sputum samples were also collected from outpatients who were diagnosed as having upper respiratory tract infections. Induced sputum samples were obtained from outpatients with asthma in the stable phase and healthy children. Sputum induction and processing were performed as previously described.^E1 The subjects were instructed to rinse their mouths thoroughly with water. Then, after being nebulized under close supervision with a 4.5% saline solution using an ultrasonic nebulizer, they were encouraged to cough forcefully at 3-minute intervals. The sputum samples were kept at 4°C for no more than 2 hours before further processing. A portion of the sample was diluted with PBS containing 0.1% dithiothreitol, gently vortexed at room temperature for 20 minutes, and filtered through nylon gauze. After centrifugation at 400g for 5 minutes, the supernatants were stored at –70°C for subsequent ELISA. Total cell counts were performed with a hemocytometer, and slides were prepared with a cytospin and stained with eosin for differential cell counts.

Culture of NHBE, BSMC, and NHLF 

Normal human bronchial epithelial cells, BSMC, and NHLF were purchased from Lonza (Walkersville, Md). When the cells reached 80% confluence in the culture and contained many mitotic figures throughout the flask, they were seeded at a density of 2.5 to 3.5 × 10³ cells/cm² onto a plate. NHBE, BSMC, and NHLF were maintained in bronchial epithelial basal medium (Lonza), smooth muscle cell basal medium (Lonza), and fibroblast basal medium (Lonza), respectively, supplemented with defined growth factors contained in the SingleQuots kit (Lonza) at 37°C in a humidified, 5% CO₂/95% air, water-jacketed incubator. Cells at the second or third passages from multiple donors were used for the subsequent experiments.

Bromodeoxyuridine uptake assay 

Normal human bronchial epithelial cells in a subconfluent state in 96-well plates were starved of insulin and recombinant human EGF (R&D Systems, Minneapolis, Minn) for 24 hours. BSMC and NHLF in a subconfluent state in 96-well plates were also starved by culturing in smooth muscle cell basal medium and fibroblast basal medium alone not containing growth factors, respectively, for 24 hours. The growth-arrested cells were stimulated with recombinant human EGF (1-100 ng/mL; R&D systems) or recombinant human amphiregulin (1-100 ng/mL; R&D Systems). After 24-hour stimulation, cell proliferation was evaluated by the bromodeoxyuridine uptake assay by using a Cell Proliferation ELISA Bromodeoxyuridine Kit (Roche, Basel, Switzerland) in accordance with the manufacturer's instructions.

Culture of NHBEs in ALI culture 

Normal human bronchial epithelial cells were plated onto collagen-coated plastic dishes and grown to confluence in bronchial epithelial basal medium supplemented with defined growth factors contained in the SingleQuots kit to obtain undifferentiated airway epithelial cells. The cells were passaged after enzymatic dissociation with trypsin. Cells from passage 2 were seeded at 5 × 10⁴ cells/insert onto 12-mm-diameter Corning Costar Transwell-Clear inserts with 0.4-μm pores (Corning Costar Corp, Cambridge, Mass) coated with human placental collagen. The ALI culture method has been described previously.^E2, ^E3 The culture medium consisted of 50% Dulbecco modified Eagle medium and 50% bronchial epithelial growth medium supplemented with insulin (5 μg/mL), hydrocortisone (0.5 μg/mL), transferrin (10 μg/mL), epinephrine (0.5 μg/mL), bovine pituitary extract (13 μg/mL), penicillin/streptomycin (100 μg/mL), and retinoic acid (0.05 μmol/L), with or without recombinant EGF (1-100 ng/mL) or amphiregulin (1-100 ng/mL). The medium was changed every 48 hours. The cells were submerged for the first 7 days in culture, and then the apical medium was removed to establish an ALI that was maintained for 2 weeks. The medium was replenished 3 times weekly. The apical surface of the cells was rinsed with PBS (37°C) once a week to remove accumulated mucus and debris. Cells were maintained at 37°C in 5% CO₂ in a humidified incubator protected from light.

Protocols 

After 2 weeks of ALI culture, the medium was replenished, and the cells were incubated in the presence or absence of EGF or amphiregulin. Total RNA was extracted after 24 and 48 hours of culture in the presence of EGF or amphiregulin. For the MUC5AC ELISA, cells were cultured for 72 hours with EGF or amphiregulin and then lysed with 0.05% Tween 20 and 0.5% BSA in PBS. To remove insoluble material, the cell lysates were centrifuged at 15,000g for 5 minutes at 4°C. The cell lysates were then frozen at –20°C for later analysis. For immunocytochemistry, the cells were collected after 72 hours of culture.

Western blot analysis 

Normal human bronchial epithelial cells in a subconfluent state in 6-well plates were starved of insulin and recombinant human EGF for 24 hours. BSMCs in a subconfluent state in 6-well plates were also starved by culturing in smooth muscle cell basal medium alone not containing growth factors for 24 hours. The growth factor–starved cells were stimulated with 10 ng/mL recombinant human EGF or amphiregulin for 5 minutes. The cells were lysed in 200 μL NuPAGE sample buffer (Invitrogen, Carlsbad, Calif) containing 5% β-mercaptoethanol, followed by sonication. Equal amounts of the lysates were used for estimating the total and phosphorylated EGFR levels or total and phosphorylated p44/42 ERK levels on parallel Western blots. The cell lysates were electrophoresed on 5-15% Tris-HCl Ready Gels J (BIO-RAD, Hercules, Calif) and transferred electrophoretically onto nitrocellulose membranes (iBlot Gel Transfer Stacks; Invitrogen). Bound proteins were quantified using ImageJ 1.39u software (http://rsb.info.nih.gov/ij/). All the antibodies used for the Western blot analyses were purchased from Cell Signaling Technology (Beverly, Mass).

Effect of EGF or amphiregulin on mucin gene expression in NCI-H292 cells 

Recombinant human EGF or amphiregulin was added to confluent serum-depleted NCI-H292 cells (ATCC, Manassas, Va). Total RNA was then extracted for quantitative real-time PCR analysis of MUC5AC expression.

Real-time quantitative RT-PCR 

Total RNA was isolated with the RNeasy mini kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. First-strand cDNA was synthesized by using a set of oligo(dT) (12-18mers) primers and SuperScript III (Invitrogen) as the reverse transcriptase. Real-time quantitative RT-PCR analysis was performed with an Applied Biosystems Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, Calif) using TaqMan analysis, as previously described,^E4 or SYBR Green PCR Master Mix (Applied Biosystems). Human gene–specific TaqMan primers and probe sets for GAPDH and MUC5AC were purchased from Applied Biosystems. Each reaction mixture consisted of 10 μL 2x PCR Master Mix (Applied Biosystems), 300 nmol/L forward and 300 nmol/L reverse primers, 175 nmol/L probe, and 100 ng cDNA made up to 20 μL with nuclease-free water. The SYBR Green Primer sets for MUC5AC (sense, 5′- TTCCATGCCCGGATACCTG3′; antisense, 5′- CAGGCTCAGTGTCACGCTCTT-3′) and GAPDH (sense, 5′GAAGGTGAAGGTCGGAGTC-3′; antisense, 5′- GAAGATGGTGATGGGATTTC-3′) were synthesized at Fasmac (Kanagawa, Japan). To determine the exact copy numbers of the target genes, quantified concentrations of the purified PCR products of MUC5AC and GAPDH were serially diluted and used as the standards in each experiment. Aliquots of cDNA equivalent to 50 ng of the total RNA samples were used for each real-time RT-PCR. The levels were normalized to the GAPDH level in each sample.

MUC5AC immunofluorescence 

To visualize the phenotypic changes induced by EGF or amphiregulin in the NHBEs in ALI cultures, anti-MUC5AC antibodies were used to label the goblet cells. Replicate treated cell culture inserts were fixed with 3% paraformaldehyde (Sigma) and permeabilized with 0.2% Triton X (Sigma, St Louis, Mo) for 2 minutes. After blocking with BSA 1% in PBS for 5 minutes, mouse anti-MUC5AC antibody (1:200; Invitrogen) was added to the luminal surface, followed by overnight incubation at 4°C. Cells were washed with PBS, and Alexa 488–labeled antimouse IgG antibody (1:200; Invitrogen) was added. Cells were washed, followed by the addition of propidium iodide solution (1:200; BD Biosciences, San Jose, Calif) for 1 hour at room temperature for nucleus staining. Images were captured with a fluorecence microscope (Olympus IX71; Olympus, Tokyo, Japan) using analysis software (Lumina Vision; Olympus).

ELISA 

Human EGF, amphiregulin, TGF-α, HB-EGF, and ADAM17/TACE were measured with ELISA kits purchased from R&D Systems. The sensitivities of the assays for human EGF, amphiregulin, TGF-α, HB-EGF, and ADAM17/TACE were 3.9, 3.9, 15.6, 31.2, and 625.0 pg/mL, respectively. Tryptase was measured with an ELISA kit purchased from Phadia (Uppsala, Sweden). MUC5AC ELISA assay was performed as previously described.^E5 Briefly, 50 μL of each sample was incubated with bicarbonate-carbonate buffer (50 μL) at 40°C in a 96-well plate (Nunc, Rochester, NY) until dry. The plates were washed 3 times with PBS and blocked with 2% BSA (Sigma) for 1 hour at room temperature. They were again washed 3 times with PBS and then incubated with 50 μL mouse monoclonal anti-MUC5AC antibody (1:100; Labvision, Fremont, Calif), which was diluted with PBS containing 0.05% Tween 20. After 1 hour, the wells were washed 3 times with PBS, and 100 μL horseradish peroxidase–conjugated goat antimouse IgG (1:10,000) was dispensed into each well. After 1 hour, the plates were washed 3 times with PBS. Color reaction was developed with 3,3′,5,5′-tetramethylbenzidine substrate solution (eBioscience, San Diego, Calif) and stopped with 1 mol/L H₂SO₄. The absorbances were read at 450 nm.

Specificity of the ELISA for EGF and amphiregulin in the sputum samples 

Sputum samples from 5 children with asthma were collected. The stored supernatants of the sputa were diluted 5 times with PBS containing 1% BSA. Exogenous EGF (1.25 ng/mL) and amphiregulin (5 ng/mL) were added to some of the supernatants. The sputa with exogenously added EGF and amphiregulin were then incubated for 15 minutes at 37°C. Just before the ELISA, the diluted samples were further diluted 20 times with PBS containing 1% BSA. The final concentrations of exogenous EGF and amphiregulin were 62.5 pg/mL and 250 pg/mL, respectively. The concentrations of endogenous EGF and amphiregulin were measured in each sample by ELISA.

Detection sensitivity of the ELISA for EGF and amphiregulin in the sputum samples 

Sputum samples from 6 children with asthma were collected. The stored supernatants of the sputa were diluted 5 times with PBS containing 1% BSA. Exogenous EGF (0.24 ng/mL) and amphiregulin (1 ng/mL) were added to some of the supernatants. Because proteases in the sputum function effectively at 37°C for 90 minutes,^E6 the sputa with exogenous EGF and amphiregulin were incubated for 90 minutes at 37°C. Just before the ELISA, the diluted samples were further diluted 4 times with PBS containing 1% BSA. The final concentrations of exogenous EGF and amphiregulin were 60 pg/mL and 250 pg/mL, respectively. The concentrations of endogenous EGF and amphiregulin were measured in each sample.

Statistical analysis 

Statistically significant differences among the 5 groups were calculated using the Kruskal-Wallis test. Correlations were analyzed by calculation of Pearson correlation coefficients. Differences in the in vitro data between 2 paired groups were analyzed by the paired Student t test. Differences with a P value <.05 were considered to be statistically significant. Values were expressed as means ± SEM.

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Results 

Specificity of the ELISA for EGF and amphiregulin in the sputum samples 

As shown in Table E2, the endogenous concentration of EGF was 16.4 ± 3.2 pg/mL (mean ± SEM; n = 5). Addition of exogenous EGF (62.5 pg/mL) alone and of both EGF (62.5 pg/mL) and amphiregulin (250 pg/mL) produced an additive effect on the endogenous and exogenous EGF concentrations (80.9 ± 6.8 and 74.3 ± 7.4 pg/mL, respectively). Because the stored supernatants were finally diluted 100 times, the endogenous concentration of amphiregulin in all the samples was under the detection limit (Table E2). Addition of exogenous amphiregulin (250 pg/mL) alone and of both EGF (62.5 pg/mL) and amphiregulin produced an additive effect on the endogenous and exogenous amphiregulin concentrations (217.3 ± 8.4 and 216.9 ± 4.9 pg/mL, respectively).

Detection sensitivity of the ELISA for EGF and amphiregulin in the sputum samples 

As shown in Table E3, the endogenous concentration of EGF was 91.8 ± 17.0 pg/mL (mean ± SEM; n = 6). Addition of both exogenous EGF (60 pg/mL) and amphiregulin (250 pg/mL) produced an additive effect on the endogenous and exogenous EGF concentrations (149.8 ± 15.7 pg/mL). Because the stored supernatants were finally diluted 20 times, the endogenous concentration of amphiregulin was under the detection limit in the sputum samples from 4 of the 6 donors. Addition of both exogenous EGF (60 pg/mL) and amphiregulin (250 pg/mL) produced an additive effect on the endogenous and exogenous amphiregulin concentrations (269.9 ± 16.2 pg/mL).

Effect of EGF and amphiregulin on MUC5AC mRNA expression in NCI-H292 cells 

To examine whether EGF and amphiregulin might have any distinct effects on mucus production from the bronchial epithelial cells, we compared the expression levels of MUC5AC mRNA in NCI-H292 cells treated with amphiregulin and EGF. As shown in Fig E2, A, EGF and amphiregulin significantly upregulated MUC5AC mRNA expression in the NCI-H292 cells in a concentration-dependent manner in the concentration range of 1 to 100 ng/mL; however, the degree to which the level was upregulated was not significantly different between EGF and amphiregulin. We also examined the kinetics of MUC5AC upregulation by EGF and amphiregulin in the NCI-H292 cells and found that EGF induced significant upregulation of MUC5AC at 16 hours, whereas amphiregulin induced significant upregulation of MUC5AC between 8 and 24 hours (Fig E2, B).

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Fig E1. 

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• Comparison of the sputum levels of EGF and amphiregulin (AREG) between patient groups with atopic and nonatopic asthma (A), between patient groups showing and not showing elevation of the serum C-reactive protein (CRP) during an exacerbation as a marker of infection triggering the asthma attack (B), and between patient groups treated and not treated with leukotriene receptor antagonists (LTRAs) (C).

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Fig E2. 

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• Effect of EGF and amphiregulin (AREG) on the MUC5AC expression in NCI-H292 cells. NCI-H292 cells were incubated with 1 to 100 ng/mL recombinant human EGF or AREG for 24 hours (A) or with 10 ng/mL recombinant human EGF or AREG for 0 to 24 hours (B). The results were expressed as fold-change (mean ± SEM) in the MUC5AC mRNA expression levels in the EGF-treated or AREG-treated cells compared with those in non–EGF-treated or AREG-treated cells (n = 4). ^∗P < .05, ^∗∗P < .01, and ^∗∗∗P < .005 compared with cells not treated with recombinant human EGF or AREG.

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Table E1. 

Comparison of the levels of EGF, amphiregulin, TGF-α, HB-EGF, and TACE in the sputa of children with asthma collected during an asthma exacerbation and after recovery

AREG, Amphiregulin; ND, not detected.

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Table E2. 

Specificity of the ELISA for EGF and amphiregulin in the sputum samples

Donor	Endogenous conc	+ EGF	+ AREG	+EGF and AREG
EGF (pg/mL)
A	13.5	64.2	12.4	53.5
B	17.2	68.6	14.1	61.5
C	13.7	77.3	13.0	72.1
D	29.2	105.3	28.4	97.6
E	8.1	88.9	8.9	87.0
Mean ± SEM	16.4 ± 3.2	80.9 ± 6.8	15.3 ± 3.1	74.3 ± 7.4
AREG (pg/mL)
A	ND	ND	213.4	211.4
B	ND	ND	240.6	216.7
C	ND	ND	202.8	212.8
D	ND	ND	239.9	237.3
E	ND	ND	218.7	206.2
Mean ± SEM	ND	ND	217.3 ± 6.8	216.9 ± 4.9

AREG, Amphiregulin; conc, concentrations; ND, not detected.

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Table E3. 

Detection sensitivity of the ELISA for EGF and amphiregulin in the sputum samples

Donor	Endogenous conc	+ EGF and AREG
EGF (pg/mL)
a	122.7	177.1
b	118.0	174.8
c	70.1	125.2
d	125.2	181.3
e	18.3	83.9
f	97.7	156.5
Mean ± SEM	91.8 ± 17.0	149.8 ± 15.7
AREG (pg/mL)
a	6.6	308.5
b	ND	221.6
c	ND	233.4
d	ND	260.7
e	ND	273.9
f	5.2	321.0
Mean ± SEM	NA	269.9 ± 16.2

AREG, Amphiregulin; conc, concentrations; NA, not applicable; ND, not detected.

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