INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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.

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 pBluescript^RII 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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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).

View larger version (68K): [in this window] [in a new window]   Figure 1.   In situ hybridization for EGFR mRNA with antisense [35S]UTP-labeled RNA probe in healthy and asthmatic airway epithelium. EGFR mRNA expression was observed in asthmatic airway epithelium (arrows indicate areas of EGFR mRNA), whereas healthy subjects showed little EGFR mRNA expression. Results are typical of all subjects studied. Original magnification: ×50; insets; original magnification: ×400. Bars = 50 µm.

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).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Distribution of EGFR immunoreactivity in healthy and in asthmatic airway epithelial cells. Percent EGFR immunoreactivity was examined in relation to total epithelial cells (left columns; *p < 0.05, significantly different from healthy subjects), total goblet cells (middle columns; p < 0.01, significantly different from asthmatics), and total basal cells (right columns; p < 0.0002, significantly different from healthy subjects).

View larger version (46K): [in this window] [in a new window]   Figure 3.   Immunohistochemical staining of EGFR in healthy and in asthmatic airway epithelium. In healthy airways, EGFR immunoreactivity was rare and was observed only in goblet cells (A) (arrow). EGFR immunoreactivity in asthmatic airways varied: in some subjects, EGFR immunoreactivity was mainly observed in goblet cells (B); in others, staining was mainly in basal cells (C ) (arrows). Some EGFR immunoreactivity was also recognized in the lumen (arrowheads). Ciliated cells showed no staining for EGFR in either asthmatic or healthy subjects. Original magnification: ×400. Bar = 50µm.

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).

View larger version (70K): [in this window] [in a new window]   Figure 4.   In situ hybridization for MUC5AC mRNA with antisense [35S]UTP-labeled RNA probe and Alcian blue/periodic acid-Schiff (PAS) staining for identification of mucous glycoconjugates in healthy and in asthmatic airway epithelium. MUC5AC mRNA was upregulated markedly in asthmatic airway epithelium. Likewise, staining with Alcian blue/PAS was increased markedly in asthmatic airway epithelium. Healthy subjects showed a low level of expression of MUC5AC mRNA (arrows), and only a few cells stained with Alcian blue/PAS. Original magnification: ×50. Insets; original magnification: ×400. Bar = 50 µm.

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).

View larger version (56K): [in this window] [in a new window]   Figure 5.   Co-localization of immunoreactivity of MUC5AC and EGFR in goblet cells. MUC5AC immunoreactivity was observed in goblet cells (arrows in left column). Similarly, EGFR immunoreactivity was seen in goblet cells (arrows in middle column). Both immunoreactivities overlapped with Alcian blue/PAS-staining that was usually positive in goblet cells (arrows in right column). Original magnification: ×400. Bar = 50µm.

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).

View larger version (16K): [in this window] [in a new window]   Figure 6.   Correlation between EGFR immunoreactivity and MUC5AC production in airway epithelium. EGFR immunoreactivity of airway epithelial cells showed a significant positive correlation with the area of MUC5AC-positive staining in airway epithelium (n = 23, r = 0.725, p < 0.0001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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.