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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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To determine the relationship of epidermal growth factor receptor (EGFR) expression to mucin synthesis in human airways, we examined EGFR and MUC5AC expression at both gene and protein levels using in situ hybridization and immunohistochemical analysis in human bronchi. Bronchial mucosal biopsy specimens were obtained from 12 asthmatic subjects and 11 healthy subjects. In asthmatic airways, EGFR mRNA was expressed in the airway epithelium. EGFR immunoreactivity staining patterns varied among the asthmatic airways: staining was positive mainly in goblet cells, in basal cells, or in both. In contrast, healthy airways showed little expression of EGFR mRNA; EGFR immunoreactivity was observed mainly in goblet cells. In parallel to EGFR expression, MUC5AC mRNA expression was greater in asthmatic airways; mucous glycoconjugates that stained positively with Alcian blue/PAS were also increased in asthmatic airways. Ciliated cells were negative for EGFR and MUC5AC both in asthmatic and in healthy subjects at both mRNA and protein levels. There was a significant positive correlation between EGFR immunoreactivity and the area of MUC5AC-positive staining in both asthmatics and healthy subjects. These findings suggest a sequence of events by which EGFR activation is involved in mucin expression in asthmatic airway epithelium.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The epidermal growth factor receptor (EGFR) is known to play a role in the regulation of cellular growth and differentiation. EGFR tyrosine phosphorylation promotes its association with signaling proteins, leads to membrane-associated Ras activation, and initiates downstream signaling to the nucleus (1, 2). Thus, EGFR activation can regulate gene transcription and subsequent protein synthesis. It has recently been reported that EGFR activation by its ligands leads to mucin MUC5AC synthesis and goblet-cell production in human bronchial epithelial cells in vitro and in rats in vivo; an inhibitor of EGFR tyrosine kinase prevented goblet-cell production in ovalbumin-sensitized rats, implicating EGFR activation in mucin synthesis in asthmatic airways (3). However, it is difficult to confirm that the EGFR activation plays a role in mucin synthesis in human airways because of the present inability to use EGFR tyrosine kinase inhibitors to prevent hypersecretion in humans. We hypothesize that EGFR activation causes goblet cell production in human asthmatics by a mechanism similar to goblet-cell production induced in sensitized animals. In the present study, we examined whether EGFR is upregulated in asthmatic airways and, if so, which cells express EGFR in airway epithelium. To accomplish this, we performed in situ hybridization and immunohistochemical analysis for both EGFR and MUC5AC (as a marker of goblet cell mucin) (3) expression in sections obtained from bronchial biopsies in healthy and in asthmatic airways, and we examined the pattern of immunoreactivity using a semiautomatic morphometric technique.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Subjects
Eleven healthy and 12 asthmatic subjects were recruited, using informed consent for a protocol approved by the Committee for Human
Research at the University of California, San Francisco. The subjects
were characterized by spirometry, airway reactivity to inhaled methacholine, and skin test reactivity as summarized in Table 1. Healthy
subjects had no clinical history of airway obstruction or perennial
rhinitis and had normal pulmonary function test results. They also had
no skin allergies. Asthmatic subjects met clinical diagnostic criteria
for asthma (4) and showed hyperreactivity to inhaled methacholine.
None of the subjects had received inhaled or oral corticosteroids during the 6 wk prior to enrollment in the study. The asthmatic subjects
used 
-agonists intermittently for symptom control. Among the subjects, there were no current or previous smokers, no history of endotracheal intubation within the previous 5 yr, no respiratory tract infection within the previous 6 wk, and no significant cardiac or neurologic disease.
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Tissue Preparation
A bronchoscope was introduced via the mouth and advanced to the right main stem bronchus. Biopsies were obtained from bifurcations of the upper lobe, the middle lobe, and the superior segment of the lower lobe, using spiked, fenestrated biopsy forceps. Biopsy specimens were fixed with 4% paraformaldehyde for 1 h and then placed in 30% sucrose for cryoprotection overnight. The specimens were embedded in either OCT compound or glycolmethacrylate (GMA) resin (Park Scientific, Northampton, UK) and cut into sections 3-µm thick. All sections were stained with Alcian blue/PAS (to visualize goblet cells) and counterstained with hematoxylin (to count the total number of cells). The Alcian blue (1%) was diluted with acetic acid (3%), with a final pH = 2.5.
In Situ Hybridization of EGFR and MUC5AC
In situ hybridization was performed using a human EGFR probe,
which contains a 350-bp cDNA fragment of the human EGFR gene (pTRI-EGF-R-human probe template; Ambion, Austin, TX) and a
human MUC5AC probe, which contains a 298-bp cDNA fragment of
the human MUC5AC gene (generously provided by Dr. Carol Basbaum). A pBluescriptRII SK
vector (Stratagene, La Jolla, CA) was
used for the subcloning of the EGFR fragment. Hybridization was
performed as described previously (5). In brief, frozen sections 4 µm
thick were cut and placed on positively charged glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA). Sections cut in close proximity were used for hybridization with sense and antisense probes.
The specimens were refixed in 4% paraformaldehyde, rehydrated in
0.5 × SSC, and then acetylated in triethanolamine and acetic anhydride. Hybridization was carried out with 2,500 to 3,000 cpm/µl of antisense or sense probe in 50% deionized formamide, 0.3 M NaCl, 20 mM TRIS, 5 mM EDTA, 1 × Denhardt's solution, 20 mM dithiothreitol, 10% dextran sulfate, 0.5 mg/ml yeast tRNA, and 0.5 mg/ml sonicated salmon sperm DNA at 58° C overnight. Posthybridization treatment consisted of washes with 2 × SSC, 1 mM EDTA, 10 mM
-mercaptoethanol at room temperature, incubation with RNase solution (20 µg/ml) for 30 min at room temperature, and further washes
in 0.1 × SSC, 1 mM EDTA, 10 mM -mercaptoethanol at 55° C for 2 h and then in 0.5 × SSC at room temperature for 20 min. Specimens
were dehydrated, air-dried, and covered with Kodak NBT nuclear
track emulsion (Eastman Kodak, Rochester, NY) for autoradiography. After exposure for 7 to 21 d at 4° C, the slides were developed,
fixed, and counterstained with hematoxylin.
Immunohistochemical Analysis of EGFR and MUC5AC
Immunohistochemistry was performed using GMA-embedded sections. Sections were refixed with 4% paraformaldehyde for 5 min. PBS containing 0.05% Tween 20, 2% normal goat serum, and Levamisol (2 mM) was used as diluent for the antibodies. The sections were incubated with mouse monoclonal antibody to EGFR (1:40; Calbiochem-Behring, San Diego, CA) or mouse monoclonal antibody to MUC5AC (clone 45 M1, 1:100; NeoMarkers, Fremont, CA) overnight at room temperature, and then washed three times with PBS to remove excess primary antibody. The sections were then incubated with biotinylated horse antimouse IgG (Vector Laboratories, Burlingame, CA) at 1:200 dilution for 2 h at room temperature. Bound antibody was visualized according to standard protocols for the avidin-biotin-alkaline phosphatase complex method. All immunohistochemical staining included control sections unexposed to primary antibody, with substitution of an unrelated antibody of the same isotype or preincubation of the antibody with a 10-fold excess of immunizing peptide. For immunostaining of EGFR, a rabbit polyclonal antibody to EGFR (1:100; Calbiochem-Behring) was also used to confirm the staining pattern and to perform quenching using EGFR peptide antigen, which corresponds to amino acid residues 1005-1016 of the human EGFR (Calbiochem-Behring). For anti-EGFR antibody, control experiments were carried out by preincubating the antibody with cell lysates prepared from the EGFR-overexpressing A431 cell line.
Morphometric Analysis
Six images of the airway epithelium were captured randomly from the biopsy sections that stained with anti-EGFR Ab or with anti-MUC5AC Ab at magnification ×400. Goblet cell area was assessed by the volume density of MUC5AC immunoreactivity on the epithelial mucosal surface, using a semiautomatic imaging system described elsewhere (6). We measured the positively stained area and the total epithelial area and expressed the data as the percentage of the positively stained area. The analysis was performed with the public domain NIH IMAGE program (developed at the U.S. National Institutes of Health and available by anonymous FTP from . gov or on floppy disk from the National Technical Information Service, Springfield, VA, part no. PB95-500195GEI). EGFR immunoreactivity was analyzed by Stereology Toolbox (version 1.1; Morphometrix, Davis, CA). The number of EGFR-positive cells in the airway epithelium was determined by point-counting, using a cycloid consisting of points and line. The point-counting was performed by an investigator blind to the identity and disease category of the subjects (7).
Statistical Analysis
Statistics were performed using StatView 4.01 (Abacus Concepts, Berkeley, CA). All data are expressed as mean ± SEM. The Mann-Whitney U test was used to determine statistically significant differences between groups; Pearson's linear regression analysis and one-way analysis of variance were used to determine a correlation between variables. A probability of less than 0.05 for the null hypothesis was accepted as indicating a statistically significant difference.
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RESULTS |
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EGFR mRNA
In all asthmatic subjects, in situ hybridization showed expression of EGFR mRNA in airway epithelium (Figure 1, right panel), whereas healthy subjects showed little EGFR mRNA expression (Figure 1, left panel). The EGFR sense probe was uniformly negative (data not shown).
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EGFR Protein
The percent of total epithelial cells that were EGFR-positive was greater in asthmatics than in healthy subjects (p < 0.05) (Figure 2, left columns). In healthy subjects, EGFR immunoreactivity was rare and was almost entirely limited to goblet cells (Figure 3A, arrow). In asthmatic subjects, EGFR immunoreactivity varied. In some subjects, EGFR immunoreactivity was observed only in goblet cells (Figure 3B, arrows) and was also observed in the airway lumen (Figure 3B, arrowheads). In others, EGFR immunoreactivity was localized mainly in basal cells (Figure 3C). Percent EGFR immunoreactivity in basal cells was greater in asthmatics than in healthy subjects; in goblet cells, percent EGFR immunoreactivity was greater in healthy subjects than in asthmatics (Figure 2 and Table 2). However, it has previously been reported that the stained area of goblet cells (Alcian blue/PAS staining) was greater in asthmatics than in healthy subjects in same samples (7). Occasionally, mucous glands were observed in the biopsies. Mucous, but not serous, cells in glands showed EGFR immunoreactivity. Ciliated cells showed no EGFR immunoreactivity either in asthmatic or in healthy subjects. Sections unexposed to primary antibody or with substitution of an unrelated antibody of the same isotype were negative, and EGFR immunoreactivity was diminished by preadsorption of the antibody with excess EGFR protein (data not shown).
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p < 0.01, significantly different from asthmatics), and total
basal cells (right columns; 
p < 0.0002, significantly different from
healthy subjects).
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MUC5AC mRNA
In asthmatic subjects, MUC5AC mRNA was expressed in airway epithelium (Figure 4) in a patchy pattern similar to the distribution of goblet cells. Epithelium from healthy subjects showed only weak expression of MUC5AC mRNA, which was located in the distribution of goblet cells (Figure 4, arrows). The MUC5AC sense probe was uniformly negative. (data not shown).
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Colocalization of MUC5AC and EGFR
The immunoreactivity of MUC5AC and EGFR were colocalized in goblet cells that were stained with Alcian blue/PAS, when the EGFR immunoreactivity was observed in goblet cells (Figure 5).
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Correlation of EGFR Immunoreactivity with MUC5AC
The EGFR immunoreactivity of airway epithelial cells showed a significant positive correlation with the area of MUC5AC-positive staining in airway epithelium among all subjects (n = 23, r = 0.725, p < 0.0001) (Figure 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we found that there was extensive EGFR
mRNA expression in asthmatic airway epithelium. Moreover,
our results showed for the first time immunoreactivity of EGFR
in airway goblet cells: EGFR immunoreactivity was colocalized with MUC5AC protein and Alcian blue/PAS staining in
goblet cells of healthy subjects, and in some goblet cells of
asthmatic subjects. These results are similar to the effects in
ovalbumin-sensitized rats: EGFR was expressed in pregoblet
and goblet cells in sensitized animals (3). This colocalization
of MUC5AC and EGFR protein in goblet cells may help to
explain the mechanism by which mucin synthesis occurs in airway epithelium. The basis of this hypothesis depends on the
following findings. First, EGFR immunoreactivity has been
observed in other mucin-producing cells such as mucous neck
cells and mucous-secreting pyloric gland cells in human gastric mucosa (8), in mucous secreting cells in rabbit endocervix (9), and in submucosal glands in airways (10). Second, EGFR ligands, EGF and TGF
, cause synthesis of mucous glycoconjugates in urothelium (11), in stomach (12) and in airways (3). Third, mucin synthesis in ovalbumin-sensitized rats is inhibited by a selective EGFR tyrosine kinase inhibitor (3). From
these observations, it is suggested that mucin production in
goblet cells may be regulated by the activation of EGFR.
The reason for the variable pattern of the EGFR immunoreactivity in goblet cells is unknown. One reason may be the internalization and endocytosis of activated EGFR. The mechanism of internalization and endocytosis of activated EGFR has been examined previously (13, 14): When cells expressing EGFR are activated with EGF, they undergo a clustering of receptors into clathrin-coated pits and subsequent internalization of the ligand-bound receptors. After internalization into the endosomal compartment, a significant pool of ligands and receptors may escape recycling to the cell surface and may be sorted to the degradation pathway, resulting in a dramatic loss of surface receptors. Thus, the activated phase of EGFR (asthmatic state) might show less EGFR protein depending on the stage of disease. Similar patterns for the regulation of EGFR protein have been reported in the peribronchial submucosa of patients with cystic fibrosis (15) and in naphthalene-induced Clara cell injury in the mouse (16). A second possible explanation is cleavage of EGFR and subsequent secretion into the lumen. In fact, we found EGFR-positive staining in the airway lumen of asthmatic subjects. Thus, "active" disease may result in enzymatic cleavage of surface EGFR and thus loss of staining with an antibody to EGFR. Further studies are required to examine the regulation of EGFR protein under different conditions. Regardless of the explanation of the variability of EGFR expression, the present study has shown that EGFR mRNA is upregulated in asthmatic airway epithelium, including goblet cells, suggesting an important role of EGFR in asthmatic goblet-cell production.
In the present study, we found that the EGFR immunoreactivity in basal cells was observed more frequently in asthmatics than in healthy subjects. A previous report explained a possible sequence for the evolution of goblet-cell production, based on the expression of EGFR in rats (3). Goblet cells are derived from basal cells and nongranulated secretory cells that express EGFR and are stimulated by EGFR ligands to produce mucins. Thus, the EGFR immunoreactivity seen in asthmatic basal cells may be due to a higher rate of cell differentiation from basal cells to goblet cells in the airway epithelium, which results in goblet cell production in asthmatic airways. Concerning the mechanisms involved in goblet cell metaplasia in asthma, recent studies have shown that damage produced by agarose plugs introduced into rat bronchi result in EGFR expression and subsequent goblet cell production (17). Thus, mechanical damage to airway epithelium could play a role in remodeling of airway epithelium to the goblet cell phenotype in asthma. The high immunoreactivity of EGFR in basal cells has also been found in the bronchial epithelium of smokers (18), and we suggest that this sequence is also involved in the development of goblet cells in chronic obstructive pulmonary disease. In the present study, no ciliated cells showed EGFR immunoreactivity in asthmatics or in healthy subjects, confirming a previous report in which EGFR immunoreactivity was detected only in nonciliated cells, including basal cells and Clara cells (19), a pattern similar to our present findings.
In summary, the present study has shown that the expression of EGFR and MUC5AC is upregulated in the epithelium of asthmatic airways at both mRNA and protein levels and are often colocalized in goblet cells. These results suggest the possible role of EGFR activation in mucin synthesis in asthmatic airways.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Jay A. Nadel, M.D., Cardiovascular Research Institute, Box 0130, University of California San Francisco, San Francisco, CA 94143-0130.
(Received in original form January 12, 2000 and in revised form April 18, 2000).
Acknowledgments: The writers gratefully acknowledge valuable assistance from Claudia Ordoñez, who performed research bronchoscopy and biopsy processing, and Trang Dao-Pick, who performed in situ hybridization. They also thank Iris F. Ueki, Pierre-Regis Burgel, and Jae Jeong Shim for their advice and discussions.
Supported by private funds.
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References |
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METHODS
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REFERENCES
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K. Kohri, I.F. Ueki, J-J. Shim, P-R. Burgel, Y-M. Oh, D.C. Tam, T. Dao-Pick, and J.A. Nadel Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor Eur. Respir. J., November 1, 2002; 20(5): 1263 - 1270. [Abstract] [Full Text] [PDF]
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J. C. Bonner The epidermal growth factor receptor at the crossroads of airway remodeling Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L528 - L530. [Full Text] [PDF]
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 M. Perrais, P. Pigny, M.-C. Copin, J.-P. Aubert, and I. Van Seuningen Induction of MUC2 and MUC5AC Mucins by Factors of the Epidermal Growth Factor (EGF) Family Is Mediated by EGF Receptor/Ras/Raf/Extracellular Signal-regulated Kinase Cascade and Sp1* J. Biol. Chem., August 23, 2002; 277(35): 32258 - 32267. [Abstract] [Full Text] [PDF]
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R A O'Donnell and A J Frew Is there more than one inflammatory phenotype in asthma? Thorax, July 1, 2002; 57(7): 566 - 568. [Full Text] [PDF]
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D. J. Tschumperlin, J. D. Shively, M. A. Swartz, E. S. Silverman, K. J. Haley, G. Raab, and J. M. Drazen Mechanotransduction in the Lung: Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression Am J Physiol Lung Cell Mol Physiol, May 1, 2002; 282(5): L904 - L911. [Abstract] [Full Text] [PDF]
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M. J. TOBIN Asthma, Airway Biology, and Nasal Disorders in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 598 - 618. [Full Text] [PDF]
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J. V. FAHY Remodeling of the Airway Epithelium in Asthma Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): S46 - 51. [Abstract] [Full Text] [PDF]
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 P.-R. Burgel, S. C. Lazarus, D. C.-W. Tam, I. F. Ueki, K. Atabai, M. Birch, and J. A. Nadel Human Eosinophils Induce Mucin Production in Airway Epithelial Cells Via Epidermal Growth Factor Receptor Activation J. Immunol., November 15, 2001; 167(10): 5948 - 5954. [Abstract] [Full Text] [PDF]
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