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Published ahead of print on December 30, 2005, doi:10.1164/rccm.200409-1175OC
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American Journal of Respiratory and Critical Care Medicine Vol 173. pp. 729-735, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200409-1175OC

Original Article

A Disintegrin and Metalloproteinase 33 Protein in Patients with Asthma

Relevance to Airflow Limitation

Ji-Yeon Lee*, Sung-Woo Park*, Hee Kyoung Chang, Ho Young Kim, TaiYoun Rhim, June-Hyuk Lee, An-Soo Jang, Eun-Suk Koh and Choon-Sik Park

Genome Research Center for Allergy and Respiratory Diseases, Soonchunhyang University, Bucheon Hospital, Gyeonggi Do, Korea

Correspondence and requests for reprints should be addressed to Choon-Sik Park, M.D., Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University, Bucheon Hospital, 1174, Jung Dong, Wonmi Ku, Bucheon, Gyeonggi Do, 420-021, Korea. E-mail: mdcspark{at}unitel.co.kr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: ADAM33 has been identified as a novel asthma susceptibility gene in genomewide screening and association studies. High-level expression in smooth muscles and fibroblasts suggests that ADAM33 plays a role in airway remodeling in patients with asthma.

Methods: The ADAM33 protein was identified in the bronchoalveolar lavage (BAL) fluids of patients with asthma and normal control subjects using Western blotting antibody against the catalytic domain. ADAM33 expression was analyzed using immunohistochemical staining of mucosal biopsy specimens. The levels of ADAM33 protein in the BAL fluids were measured by dot blotting, and were correlated with the FEV1 values of the patients with asthma.

Results: Western blot analysis revealed the presence of the ADAM33 protein, with a molecular mass of approximately 55 kD in the BAL fluids. ADAM33 was expressed in the smooth muscles and basement membranes of almost all the patients with asthma, but was absent in the normal control subjects. The ADAM33 levels were increased significantly in patients with moderate to severe asthma and in patients with mild asthma, as compared with normal control subjects (p = 0.001 and p = 0.016, respectively). The ADAM33 protein levels correlated inversely with the FEV1% predicted in the patients with asthma (r = –0.486, p = 0.018).

Conclusions: ADAM33 is associated with asthma development, and the levels of ADAM protein are related to asthma severity.

Key Words: ADAM33 • airflow limitation • asthma • basement membrane • smooth muscle

Asthma is a common and heterogeneous respiratory disease that is characterized by intermittent airway obstruction and respiratory symptoms that are related to chronic airway inflammation and remodeling (1). The pathologic features of airway remodeling include goblet cell hyperplasia, subepithelial fibrosis, collagen deposition, mucosal gland hyperplasia, smooth muscle hypertrophy, and changes in the extracellular matrix (2–4). Inflammation and remodeling are the main causes of airway hyperresponsiveness (AHR) and chronic airway obstruction, which are features of the pathophysiology of asthma. The inflammation and remodeling process are linked to aberrant activation of epithelial-mesenchymal-tropic units (5). Many cytokines and inflammatory mediators are involved in the abnormal signaling and proliferation of mesenchymal and epithelial cells (6).

Endogenous proteases are crucial for the activation of cytokines and inflammatory mediators. The ADAM (a disintegrin and metalloproteinase) gene family, which currently comprises 34 members, is a subgroup of the zinc-dependent metalloproteinase superfamily (7, 8). The ADAM proteins have a complex organization, which encompasses a signal sequence, and pro-, catalytic, disintegrin, cysteine-rich, and epidermal growth factor domains, followed by a transmembrane and a cytoplasmic domain with signaling-specific sequences. The presence of the catalytic domain suggests that ADAMs have protease activity, but the biological roles of these proteins have been elucidated in only a few cases. ADAM17 (tumor necrosis factor-{alpha} [TNF-{alpha}]–converting enzyme) releases the active form of TNF-{alpha} from the cell surface (9, 10) by cleaving pro–TNF-{alpha} (11). ADAM33 belongs to the subfamily that also includes ADAM12, ADAM13, and ADAM19, all of which possess proteolytic activities (7, 12). Recently, the catalytic property of the metalloproteinase domain of human ADAM33 expressed in Drosophila cells was demonstrated (13, 14). Furthermore, a genomewide screen revealed ADAM33, which is encoded on chromosome 20p13, as a novel asthma susceptibility gene that plays a role in AHR (15). Case-control association and transmission disequilibrium tests and haplotype analyses have led to the identification of the ADAM33 gene polymorphisms that are associated with asthma susceptibility and airway hyperreactivity (15, 16), whereas other studies have shown the absence of association (17, 18).

Studies on the expression of ADAM33 mRNA in human cells and tissues have revealed exclusive distribution of this protein in cells of mesenchymal origin, such as fibroblasts and smooth muscle cells, which indicates a possible role for ADAM33 in airway remodeling (15, 19, 20).

Considering its genetic association and functional properties, ADAM33 appears to be a prime candidate for asthma development and bronchial hyperreactivity. However, this hypothesis needs to be validated by studies on protein expression in the airways of patients with asthma; no study has yet revealed the relevance of ADAM33 expression to asthma development and physiologic changes. The present study used immunohistochemical staining and immunoblotting to investigate (1) whether ADAM33 expression is higher in the airways of patients with asthma than in normal control subjects and (2) the potential correlation between levels of ADAM33 protein and physiologic changes in patients with asthma.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subject Characterization
Thirty-five subjects with asthma and 10 normal control subjects were enrolled in the study. Detailed methods of examinations are provided in the online supplement. The subjects with asthma were divided into severity categories, according to the previously defined criteria (1). The patients with mild persistent asthma had a prebronchodilator FEV1 of greater than 80% of the predicted value, and were treated with beta-agonist alone on an as-needed basis. Patients with moderate to severe persistent asthma had an FEV1 of 40%–80% of the predicted value, and were administered low to moderate doses of inhaled combined corticosteroids with long-acting beta-agonist (500–1,000 µg fluticasone and 50 µg salmeterol/d) plus as-needed agonists.

Healthy control subjects were recruited from hospital personnel who answered in the negative to a screening questionnaire for respiratory symptoms, and who had FEV1 values of more than 80% of the predicted value, PC20 methacholine of more than 10 mg/ml, and normal findings on simple chest radiograms. Exclusion criteria were evidence of bacterial infections on chest radiographs or hospitalization during the previous 6 wk before this study. The Ethics Committee of Soonchunhyang University Hospital approved the study, and informed, written consent was obtained from each study subject.

Procedures
Bronchoalveolar lavage (BAL) was performed with a flexible fiberoptic bronchoscope (Olympus B2–10; Olympus Optical Co., Tokyo, Japan), as described previously (21). Detail methods of procedures are described in the online supplement.

Cell Culture
MRC-5 cells, the lung fibroblast line no. CCL-171, were obtained from and cultivated as recommended by American Type Culture Collection (Manassas, VA). Cells were grown in Eagle's minimal essential medium with Earle's balanced salt solution and 2 mM L-glutamine containing 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.5 g/L sodium bicarbonate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum at 37°C in a humidified 5% CO2 water-jacketed incubator. Cells were checked for purity and morphologic abnormalities by phase contrast microscopy. When 95% confluent, cells were plated at 4 x 105 cells/well into six-well tissue culture plate (FALCON; BD Labware, Franklin Lakes, NJ) and incubated with fresh serum-free medium. After 24 h, cells were harvested on ice using cell scraper (NUNC Roskilde, Denmark), and then, lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride) was added. Subsequently, pump up was performed by insulin syringe.

RNA Extraction and cDNA Synthesis
Total RNA from cultured MRC-5 fibroblasts was extracted using Trizol reagent (Molecular Research center, Inc., Cincinnati, OH). Detailed methods are described in the online supplement. Total RNA was quantified using spectrophotometry and checked for purity using gel electrophoresis.

First-strand cDNA was prepared using 4 µg of total RNA. Reverse transcriptions were performed using the SuperScript II RNase H– reverse transcriptase (200 U; Invitrogen Co., Carlsbad, CA) and oligo(dT)12–18 primers (500 ng). RNase inhibitor (40 U) was added during reverse transcriptions.

Primer Design and Reverse Transcriptase–Polymerase Chain Reaction
The polymerase chain reaction (PCR) primers were designed using GeneFisher software (bibiserv.techfak.uni-bielefeld.de/genefisher) based on the DNA sequence provided by Genebank (access numbers: AF466287.1) and synthesized by Genotech Co. Ltd., Korea (Table 1). Reverse transcriptase (RT)–PCR was performed on the RNA of MRC-5 fibroblasts using PCR premix. Detailed methods are described in the online supplement. DNA sequencing was done on the PCR-amplified products using an automatic DNA sequencing analyzer.


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  		TABLE 1. DEMOGRAPHIC CHARACTERISTICS OF STUDY SUBJECTS

 
Immunohistochemistry
The rabbit polyclonal anti-human ADAM33 antibody was raised against a keyhole limpet hemocyanin–conjugated synthetic peptide (Peptron, Daejeon, Korea) with amino-terminal cysteines that corresponded to the regions within the catalytic domain (ASP2, amino acids 303–319; NH2-RAFQGATVGLAPVEGMC-COOH) of ADAM33 (13). Detailed methods are described in the online supplement. The antibody was further purified by passing through a non–phosphopeptide (RAFQGATVGLA PVEGMC_-coupled EAH sepharose column (Pharmacia, Uppsala, Sweden).

Immunoblotting for ADAM33
ADAM33 protein expression was analyzed by Western blotting using the affinity-purified rabbit anti-human ADAM33 metalloproteinase domain (ASP2) antibody and the rabbit anti-human cytoplasmic domain (Cyt2) antibody. Detailed methods are described in the online supplement. Quantitation of the ADAM33 protein in BAL fluids was performed using dot blot analysis. The total protein contents of the BAL fluids were measured using the Micro BCA Protein Assay kit (Pierce, Rockford, IL).

The total 6-ug proteins of the BAL fluids were diluted 1:5, 1:25, 1:125, and 1:625 in deionized distilled water and transferred onto a nitrocellulose membrane (Amersham Biosciences, Uppsala, Sweden) using vacuum suction. The immunoreactivity of the samples was visualized using the method described for the Western blots. The optical density (OD) values were measured using automated densitometric analysis with Image Pro Plus (Media Cybernetics, Silver Spring, MD), and the OD values were converted to arbitrary units. One arbitrary unit (AU) was defined as the reciprocal of the dilution factor that showed 50% of the maximum OD of the pooled BAL fluids from the patients with asthma.

Statistical Analyses
The data are reported as the means ± SEM. The SPSS/PC+ program (SPSS, Inc., Chicago, IL) was used for the statistical analyses. Differences between groups were compared using the nonparametric Kruskal-Wallis H test for continuous data, and in cases of significance, the Mann-Whitney U test was applied to compare any two groups. Correlations between the data were assessed using Spearman's rank test. Differences were considered to be statistically significant when the p value was less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence of ADAM33 Protein in MRC-5 Fibroblasts and BAL Fluids from Subjects with Asthma
The subjects' characteristics with respect to age, sex, smoking, atopy, and PC20 methacholine and respiratory function measurements are shown in Table 1. As expected, the value of FEV1% predicted was lower in the moderate–severe asthma group than in the normal control or mild asthma groups (p < 0.05). In the BAL cell profiles, the total cell counts and mean percentages of eosinophils were significantly higher in the mild and moderate–severe asthma groups than in the normal control group (p < 0.05 and p < 0.01, respectively). In Western blot analysis of the BAL fluids using the affinity-purified ASP2 antibody from the subjects with asthma, an intense band of approximately 55 kD appeared (Figure 1B, lanes 3 and 4); this band also appeared weakly in the normal control subjects (Figure 1B, lanes 1 and 2). Conditioned medium of MRC-5 fibroblasts also showed strong immunoreactivity for a band of the same size (Figure 1A, lane 1), when reacted with anti–catalytic domain antibody (ASP2). Preincubation with the immunizing peptide abolished the reactivities of both the conditioned medium of MRC-5 fibroblasts (Figure 1A, lane 3) and the BAL fluids from the subjects with asthma (Figure 1B, lane 5). No band was detected by Cyt2 antibody in Western blot analysis of the conditioned media of MRC-5 fibroblasts (Figure 1A, lane 2) and the BAL fluid (Figure 1B, lanes 7 and 8). The banding patterns from cell lysates differed substantially from those observed using the conditioned media. Four bands were detected by affinity-purified ASP2 antibody (Figure 1A, lane 4) and by Cyt2 antibody (Figure 1A, lane 5) in cell lysates of MRC-5 fibroblasts at 230, 70, 55, and 45 kD. The band at 55 kD is a significant band in BAL fluids from subjects with asthma (Figure 1B, lanes 3 and 4). The subjects with asthma strongly expressed this protein, whereas the normal subjects showed a weak activity (Figure 1B, lanes 1 and 2). The four bands of cell lysates and the one band of BAL fluids using ASP2 antibody were abolished when preincubated with the immunizing peptide (Figure 1A, lane 6, and Figure 1B, lanes 5 and 6).


Figure 1
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  		Figure 1. Western blot findings for ADAM33 in MRC-5 fibroblast and bronchoalveolar lavage fluid (BALF) from the patients with bronchial asthma (BA) and normal control (NC) subjects. (A) Conditioned media (CM) and cell lysates (CL) of MRC-5 fibroblast were reacted with anti–catalytic domain (ASP2) and cytoplasmic domain antibody (Cyt2). Lanes 1–3: conditioned media; lanes 4–6: cell lysates. Lanes 1, 3, 4, and 6: ASP2 antibody reaction; lanes 2 and 5: Cyt2 antibody reaction. (B) BALF from subjects with BA and NC subjects was reacted with anti–catalytic domain (ASP2) and cytoplasmic domain antibody (Cyt2). Lanes 1–6: ASP2 Ab; lanes 7–8: Cyt2 Ab. Lanes 1, 2, 6, and 7: BALF from NC subjects; lanes 3–5 and 8: BALF from BA; lanes 5 and 6: BALF from subjects with BA and NC subjects preincubated with the antibody of immunizing peptide. The immunoreactivity of ADAM-33 was strongly increased at the 55 kD band in the BALF (lanes 3 and 4) from BA and conditioned media of MRC-5 fibroblasts (A, lane 1), but its increase was markedly inhibited by immunizing peptide (A, lane 3, and B, lane 5). (C). BALF from subjects with BA (lanes 1–6) and NC subjects (lanes 7–12) was reacted with anti–catalytic domain (ASP2).

 
Expression of Metalloproteinase Domain and Intracytoplasmic Domain Transcript by MRC-5
ADAM33 gene and binding sites of antibodies for metalloproteinase domain (ASP2, exon J aa: 304–320) and intracytoplasmic domain (Cyt 2, exon U, aa: 778–791) are schematically represented in Figure 2. RT-PCR of MRC-5 fibroblast RNA was performed on the two regions (exon I-L and exon Q-V) using primers (Figure 2A) to confirm the existence of metalloproteinase domain transcript (477 bp) and intracytoplasmic domain transcript (588 bp). PCR products of compatible sizes were well demonstrated in Figures 2C and 2D. DNA sequencing of the PCR products was performed, and revealed that the sequences are identical to the ones published by Tsuyoshi and coworkers (22).


Figure 2
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  		Figure 2. (A) Primer list. (B) Schematic representation of ADAM33 gene and binding sites of antibodies for metalloproteinase domain (ASP2, exon J aa: 304–320) and intracytoplasmic domain (Cyt 2, exon U, aa: 778–791). Reverse transcriptase–polymerase chain reaction (RT-PCR) of MRC-5 cell line RNA was performed on these two regions (exon I–L and exon Q–V) to confirm the existence of MP domain transcript (477 bp) and intracytoplasmic domain (588 bp). Lane 1: I–L = forward primer = I; reverse primer = L. (C, D) PCR products of compatible sizes were well demonstrated. DNA sequencing of the RT-PCR product was performed, and revealed that the sequences are identical to the ones published by Tsuyoshi and coworkers (22). Lane 1: Q–V = forward primer = Q; reverse primer = V. M: 100-bp DNA ladder.

 
Quantitation of ADAM33 Protein Using ASP2 Antibody in BAL Fluids, and the Relationship between ADAM33 Levels and the Physiologic Changes in Asthma
The OD values for the dot blot analysis of serially diluted samples are shown in Figure 3A, and the densitometric analysis of the dots is shown in a scatter plot. The levels of ADAM33 were significantly higher in the patients with moderate–severe and mild asthmas compared with the normal control group (305 ± 112 vs. 5.7 ± 1.1 AU, and 42 ± 9.5 vs. 5.7 ± 1.1 AU; p = 0.001 and p = 0.016, respectively; Figure 3B). Patients with moderate–severe asthma had significantly higher levels of ADAM33 than did those with mild asthma (p < 0.05).


Figure 3
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  		Figure 3. Quantitative analysis of ADAM33 protein in BALF from subjects with BA and NC subjects. (A) BALF from subjects with BA and NC subjects was diluted as 1:5, 1: 25, 1:125, 1:625, and nondiluted concentration. Integrated optical density was measured using automated densitometric analysis, Image Pro Plus, as described in METHODS. (B) The level of ADAM33 identified by dot blotting was quantified using densitometry. The levels of ADAM33 were significantly increased in the subjects with moderate–severe and mild asthma than in the NC group. *p = 0.016, **p = 0.001.

 
Correlation of ADAM33 Levels with Physiological Changes and BAL Cell Profiles
Multiple comparison analysis was performed to determine the association between the ADAM33 levels and physiologic changes and BAL cells in asthma. The ADAM33 levels correlated inversely with the FEV1% predicted values for the subjects with asthma (r = –0.486, p = 0.018; Figure 4), but not with the PC20 methacholine (r = –0.187, p = 0.215). We evaluated the correlation between each cellular component and ADAM33 levels in the BAL fluids, but there was no significant correlation between them (n = 35; total cells: r = 0.282, p = 0.401; neutrophils: r = 0.264, p = 0.433; lymphocytes: r = –0.047, p = 0.863; eosinonophils: r = 0.409, p = 0.212; macrophages: r = 0.218, p = 0.519).


Figure 4
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  		Figure 4. Correlation between ADAM33 levels of BALF and FEV1 (% predicted) in the subjects with asthma. The significant inverse correlation was noted between ADAM33 levels and FEV1 (% predicted; r = –0.456, p = 0.018).

 
Localization of ADAM33 in the Airways of Subjects with Asthma and Normal Control Subjects
ADAM33 was expressed strongly in the smooth muscle layers (85% of the subjects; Figures 5B and 5C) and expressed moderately in the basement membrane layers (85% of the subjects; Figure 5D). The pattern of ADAM33 staining was heterogeneous in the muscle layers and homogeneous in the basement membrane layers. The epithelium also stained positive for ADAM33 protein, although the positivity rate was relatively low (30% of the subjects; Figure 5E). ADAM33 immunoreactivity was not found in any of the normal control subjects (Figure 5A).


Figure 5
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  		Figure 5. Immunohistochemical analysis of ADAM33 protein expression on bronchial mucosal tissue in subjects with asthma. (A) The expression of ADAM33 protein was not noted in the control subjects. (B, D, and E) Immunoreactive ADAM33 was noted in the smooth muscle cells and submucosal gland (B) and basement membrane (D) of all patients, but immunoreactivity was noted in the bronchial ciliated epithelium from 30% of the patients (E). (C) Trichrome staining.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After the identification of ADAM33 as a candidate gene for asthma susceptibility and bronchial hyperreactivity in a genomewide scan of chromosome 20 (15), several case-control studies have confirmed the associations between ADAM33 polymorphisms and asthma development and the physiologic changes seen in subjects with asthma (16, 23–25). However, other studies showed the absence of association (17, 18). In white and African-American patients with childhood asthma, a common haplotype with a frequency of 14.6% in white subjects was associated with asthma (p = 0.006) and two single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium (T1 and T+1) were marginally associated with asthma in the Hispanic cohort (p = 0.04) (17). In adult Mexicans or in Puerto Ricans, none of the transmission disequilibrium test or case-control results showed significant association with the intermediate phenotypes of asthma, including asthma severity, bronchodilator response, or IgE levels (18). The difference in the association results may be due to phenotypic and environmental heterogeneity between cohorts.

The occurrence of ADAM33 in embryonic mesenchymal cells and the presence of several ADAM33 protein isoforms in adult bronchial smooth muscle strongly suggest its importance in smooth muscle development and/or function, such as genetic association with bronchial hyperresponsiveness and airway wall modeling in the early-life origins of asthma (26). In addition, the polymorphisms in ADAM33 predict impaired early-life lung function (27). Furthermore, the SNPs in ADAM33 are associated with accelerated lung function decline in the general population, which suggests one of the risk factors for COPD (28). Thus, ADAM33 may contribute to accelerated airway dysfunction in various airway diseases.

Given that the ADAM proteins have diverse functions that reflect their domain structures, such as ectodomain shedding to the metalloproteinase domain (29) and cell adhesion to the disintegrin domain (30), it may be that the other domains also play important regulatory roles. These functional properties of ADAM family proteins and the genetic effect of ADAM33 on asthma susceptibility strongly suggest that ADAM33 is a candidate molecule responsible for asthma development and bronchial hyperreactivity. However, this should be confirmed by protein expression measurements in the airways of patients with asthma and validated by in vivo and in vitro functional studies.

This study has demonstrated the presence of ADAM33 protein in the BAL fluids of subjects with asthma using Western blot with a rabbit polyclonal affinity-purified antibody for the catalytic domain (ASP2; amino acids 304–320; NH2-RAFQGATVGLAPVEGMC-COOH) and a rabbit polyclonal antibody directed against the synthetic peptide that encompasses a region within the cytoplasmic domain (Cyt2; amino acids 778–791; NH2-[C] DPENSHEPSSHPEK-COOH) of ADAM33, as described by Garlisi and coworkers (13).

The specificity of the antibody was confirmed by the fact that the immunoreactivities of the conditioned media and cell lysates of MRC-5 fibroblasts and BAL fluids disappeared after pretreatment with the synthetic immunizing peptide (Figure 1A, lanes 3 and 6, and Figure 1B, lanes 5 and 6, respectively). To evaluate the clinical relevance of ADAM33, the levels of ADAM33 protein were measured by immunoblotting the BAL fluids using ASP2, and not Cyt2, because no immunoreactivity with Cyt2 was present in BAL fluids (Figure 2B, lanes 7 and 8).

The correlations between the levels of ADAM33 and various physiologic parameters and cellular profiles of the BAL fluids were analyzed. We found a significant correlation between ADAM33 immunoreactivity and FEV1% predicted and symptom severity (Figure 4). These data suggest that ADAM33 plays a role in the development of airflow limitation and disease severity in patients with asthma. To our knowledge, the present study is the first report to describe the increased expression of ADAM33 in the airways of subjects with asthma and the clinical relevance of this protein.

In the present study, ADAM33 was expressed strongly in the smooth muscle layers and basement membranes in more than 80% of the subjects with asthma. In contrast, ADAM33 expression was not detected in the bronchial mucosal layers of the normal control subjects. In previous studies using primary human cell lines and human airways (15, 19), ADAM33 mRNA was expressed in lung fibroblasts and smooth muscle cells, but not in epithelial cells or endothelial cells of human origin. Our findings are consistent with the results of mRNA expression profiling using cell lines and human bronchi, which have shown ADAM33 expression by smooth muscles and fibroblasts in the airways (19). In the present study, ADAM33 protein was expressed on the epithelium, albeit at a frequency of less than 50% that of the subjects with asthma. Umland and coworkers (19) have reported the lack of ADAM33 mRNA expression by primary bronchial epithelial cells and A549 cells. However, they examined only a few cases of asthma. The discrepancy between these studies may be due to the immunostaining of ADAM33 that was deposited on the epithelial surfaces after synthesis in the secretory form by other tissues, such as muscle layers or fibroblasts. Another possibility is that the epithelium may express ADAM33 when activated, as occurs in the asthmatic airway.

This selective expression of the ADAM33 gene in mesenchymal cells strongly suggests that alterations in the activity of this gene may underlie abnormalities in the functions of airway smooth muscle cells and fibroblasts, which are, in turn, linked to AHR and remodeling in asthma. Although remodeling has been considered to be caused by longstanding inflammation, this process actually begins early in the development of asthma and parallels the inflammatory events; it may even be required for the establishment of persistent inflammation (31–33). Bronchial biopsies of children with asthma have shown that collagen deposition and fibroblast proliferation, rather than eosinophilic inflammation, predominate in the lamina reticularis (20). In the present study, the levels of ADAM33 in the BAL fluids correlated with the degree of airway obstruction and the severity of asthma, but not with the PC20 methacholine values. However, immunohistochemical staining revealed that the basement membranes contained ADAM33 protein. Because bronchial myofibroblasts are the main contributors to subepithelial fibrosis (34), our finding of ADAM33 expression in the basement membranes suggests that bronchial myofibroblasts are a major source of ADAM33 protein. Although PC20 methacholine did not correlate with the ADAM33 levels in the BAL fluids, the immunohistochemical staining presented in the present study indicates that ADAM33 is associated with remodeling in asthma.

Interestingly, the size of the protein band detected by ASP2 in our study was apporoximately 55 kD, although the unprocessed full-length ADAM33 and processed ADAM33 proteins have molecular masses of 120 and 100 kD, respectively (13, 19). The conditioned medium of MRC-5 fibroblast also showed a single protein band of similar size to that detected in the BAL fluids, and both immunoreactivities were blocked by pretreatment with the immunizing peptide, which suggests that these are isoforms of ADAM33. To confirm our data, in which ASP2 antibody detected ADAM33 in soluble form, we conducted a Western blot using anti–cytoplasmic domain antibody (Cyt2 antibody), which was developed in the same way as described by Garlisi and colleagues (13). No band was detected by Cyt2 antibody in Western blot analysis of the conditioned media of MRC-5 fibroblasts (Figure 1A, lane 2) and BAL fluids (Figure 1B, lanes 7 and 8).

In contrast, multiple bands were detected by ASP2 antibody and by Cyt2 antibody in the cell lysates of MRC-5 fibroblast (Figure 1A, lanes 4 and 5, respectively). These data suggest that several isoforms of ADAM33 containing metalloprotease domain and cytoplasmic domains were produced in the intracytoplasmic region, and that a soluble form containing metalloprotease domain, but not cytoplasmic domains, was detected in the conditioned medium of MRC-5 fibroblasts and the BAL fluid of subjects with asthma.

Recently, Haitchi and coworkers demonstrated that several ADAM33 protein isoforms (22, 37, 55, and 65 kD) exist in adult bronchial smooth muscle and in human embryonic bronchi and surrounding mesenchyme, strongly suggesting its importance in smooth muscle development and/or function, which could explain its genetic association with bronchial hyperresponsiveness (26). These and our data suggest a possibility of differences in which isoforms are expressed in subjects with asthma compared with normal control subjects.

The potential for multiple alternatively spliced transcripts of ADAM33 is indicated by variants that lack the exons or parts of the exons. Recently, marked differences in the tissue expression profiles of the pro- and protease domains were reported (35). However, the antibody used in our study is raised against a region of the metalloproteinase domain (exon J), for which alternative splicing has not yet been reported (35). Thus, the protein band detected in our Western blots may represent the soluble form of ADAM33 that arises from a 37-bp deletion in exon R (15). In this case, the size of the protein would be approximately 55 kD, which is similar to the size of the protein in our study. The use of an alternative splice site in the EGF domain, which results in a frame-shift in the resulting transcript, is predicted to produce a protein that does not appear to have any means of cellular attachment (ADAM33 S) (35). For the soluble ADAM proteins, it has been suggested that, as the secreted forms have lost the potential to be tightly regulated by the host cells, they may be very active (36). Analysis of primary human airway fibroblasts has shown the presence of several alternatively spliced forms of ADAM33, including one that encodes a putative secreted variant, and many transcripts that lack the metalloproteinase domain (35). However, the roles of the soluble forms and splice variants of ADAM33 in the pathogenesis of asthma remain to be discovered.

In summary, ADAM33 protein is expressed mainly in the smooth muscles and the basement membranes of the airways in patients with asthma, and the amount of ADAM33 produced is associated with the extent of decline in lung function and asthma severity. Polymorphisms within the ADAM33 gene may exert their effects via regulation of the ADAM33 protein. A better understanding of the exact function of ADAM33 in the asthmatic airway may elucidate the process of airway remodeling.


   Acknowledgments
 
The authors thank the editors from textcheck.com, both native speakers of English, for their editing for grammar and typographic errors.


   FOOTNOTES
 
Supported by a grant of the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN14-0003).

* These authors contributed equally to this article. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200409-1175OC on December 30, 2005

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 7, 2005; accepted in final form December 20, 2005


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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