Glucocorticoid Receptors in Bronchial Epithelial Cells in ... - CiteSeerX

Glucocorticoid Receptors in Bronchial Epithelial Cells in Asthma ISABELLE VACHIER, GIUSEPPINA CHIAPPARA, ANTONIO MAURIZIO VIGNOLA, ROSALIA GAGLIARDO, ELENA ALTIERI, BÉATRICE TÉROUANNE, PATRICE VIC, JEAN BOUSQUET, PHILIPPE GODARD, and PASCAL CHANEZ Institut National de la Santé et de la Recherche Médicale U454, Institut Fédératif de Recherche, Clinique des Maladies Respiratoires, Hôpital Arnaud de Villeneuve, Centre Hospitalier Universitaire, Montpellier, France; Istituto di Fisiopatologica Respiratoria, CNR, Palermo, Italy; and Institut National de la Santé et de la Recherche Médicale U439, Montpellier, France

The expression of the glucocorticoid receptor (GR) in untreated or in steroid-dependent asthmatic patients is poorly understood. We therefore studied GR mRNA and protein levels in bronchial biopsies obtained from seven untreated asthmatic patients, seven control volunteers, and seven patients with chronic bronchitis. We also studied in bronchial epithelial cells obtained by brushing from 13 untreated asthmatics, 18 steroid-dependent asthmatics, 11 control volunteers, and 12 patients with chronic bronchitis, GR and heat shock protein 90 kD (hsp90) mRNA as well as the immunoreactivity of GR, intercellular adhesion molecule (ICAM-1), and granulocyte macrophage–colony-stimulating factor (GM-CSF). GR mRNA and protein level was similar in all subject groups in both biopsies and bronchial epithelial cells. Hsp90 mRNA level was also similar in all subject groups. ICAM-1 expression was significantly increased in bronchial epithelial cells from untreated asthmatics, but ICAM-1 was not expressed in those from steroid-dependent asthmatic patients. GM-CSF expression was significantly increased in bronchial epithelial cells from untreated and steroid-dependent asthmatic patients. GR expression within the airways is unaltered by oral long-term steroid treatment in asthma, but the expression of some but not all specific markers for asthma is modified by oral steroid. Vachier I, Chiappara G, Vignola AM, Gagliardo R, Altieri E, Térouanne B, Vic P, Bousquet J, Godard P, Chanez P. Glucocorticoid receptors in bronchial epithelial cells in asthma. AM J RESPIR CRIT CARE MED 1998;158:963–970.

Glucocorticoids are potent drugs that are effective in the treatment of asthma (1). It has been shown that they reduce symptoms and decrease airway obstruction and hyperresponsiveness. Although glucocorticoids are highly effective and widely used in asthma, their in vivo mechanism of action is still matter of debate. In this regard, while most patients respond to glucocorticoids, some patients fail to have a satisfactory response and are termed steroid-resistant (2–4). In addition to steroid-resistant asthma, with clinical definitions that differ between studies, there is another subset of patients with reduced responsiveness who are called steroid-dependent (SD) asthmatics, because they require long-term systemic steroids to control their symptoms (5). Glucocorticoids act through an inactive glucocorticoid receptor (GR; 94 kD). This inactivated GR is bound to a protein complex including to subunits of the 90 kD heat shock protein (hsp90) (6) and a 59 kD immunophilin protein (7). Hsp90 significantly facilitates GR function because its association with the receptor is essential for obtaining the high-affinity steroid (Received in original form October 27, 1997 and in revised form April 3, 1998) Correspondence and requests for reprints should be addressed to Dr. P. Chanez, M.D., Ph.D., Clinique des Maladies Respiratoires, Hôpital Arnaud de Villeneuve, Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. E-mail: [email protected] Am J Respir Crit Care Med Vol 158. pp 963–970, 1998 Internet address: www.atsjournals.org

binding conformation (8). The GR/steroid complex migrates to the nucleus, and forms a homodimer which binds to a consensus cis-acting DNA sequence, i.e., a glucocorticoid responsive element (GRE) in the upstream regulatory region of genes which either inhibit or stimulate transcription of target genes (9, 10). GR may also form complexes with activating transcription factors in the nucleus to inhibit their effects. Proinflammatory transcription factors, such as activator protein-1 (AP-1) (11) and nuclear factor kappa B (NF-kB) can form complexes with GR to modulate transcription of various genes, including GR itself. The presence and localization of GR in the lungs was examined in a single study, which revealed that the amount of messenger RNA (mRNA) was similar in lungs obtained as surgery from three asthmatic and normal subjects. GR analyzed using in situ hybridization and immunohistochemistry was mainly located within the epithelium and bronchial smooth muscle (12). However, this important study did not provide evidence on the localization or amount of GR in bronchial epithelial cells obtained by bronchoscopy. Inhaled glucocorticoids are supposed to act at the bronchial level, indicating the putative importance of the GR machinery in bronchial epithelial cells. Granulocyte macrophage–colony-stimulating factor (GMCSF) is a pluripotent cytokine implicated in the recruitment, activation, and survival of inflammatory cells. GM-CSF was overexpressed by bronchial epithelial cells of atopic asthmat-

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ics and this expression was downregulated using inhaled corticosteroids (13). One of the mechanisms allowing the cell–cell contact is dependent upon the expression of cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1). It has been demonstrated that the percentage of bronchial epithelial cells expressing ICAM-1 was correlated with the severity of asthma and that FEV1 ICAM-1 plays a key role in the dynamic regulation of the mechanisms of cell adhesion and migration responsible for cellular recruitment and activation in tissue inflammation (14). We studied GR regulation in human bronchial epithelial cells from untreated and steroid-dependent asthmatic patients. Control volunteers and patients with chronic bronchitis were used as the control group. The first experiments investigated the presence and localization of GR mRNA and proteins in bronchial biopsies in seven untreated asthmatic patients, seven control volunteers, and seven patients with chronic bronchitis. In order to focus more closely on bronchial epithelial cells, GR and hsp90 mRNA and GR protein levels were then studied in bronchial epithelial cells obtained from 13 untreated asthmatics, 18 steroid-dependent asthmatics, 11 control volunteers, and 12 patients with chronic bronchitis. The ex vivo functionality of the GR was investigated by the evaluation of the immunostaining for GM-CSF and ICAM-1.

METHODS Patients Twenty asthmatic patients ranging in age from 20 to 68 yr (median: 36.5 yr) were selected according to the criteria of the American Thoracic Society (15). All had a reversible airway obstruction assessed either by an increase of 12% in FEV1 after inhalation of 200 mg of salbutamol or a positive carbachol inhalation challenge. All patients had the same clinical evaluation, including pulmonary function tests, allergy skin prick tests, and RAST. Eighteen patients with severe steroid-dependent asthma ranging in age from 29 to 72 yr (median: 59 yr) were included in the study. Asthma was defined as previously described (15). All patients were already followed in our outpatients clinic for at least 1 yr before the beginning of the present study. They are described as steroid-dependent because we failed to wean them from glucocorticoids in the previous 2 yr, and they presented recurrent threatening episodes of acute asthma in the past 2 yr despite optimal treatment and management. Compliance with prednisone therapy was assessed by measuring 8:00 A.M. plasma and saliva cortisol which were always found to be decreased as compared with normal concentrations. They were treated on a regular basis with prednisone (mean dose: 18 6 11 mg) and 1,600 mg of inhaled budesonide, 100 mg of salmeterol, and 10 mg/kg of long-acting theophylline according to recently published guidelines (16). Control of the disease was assessed by measuring peak expiratory flow rate (PEFR) and nocturnal asthma symptoms. The upper 15% variability in PEFR and the occurrence of nocturnal asthma indicated uncontrolled asthma. Eighteen normal volunteers ranging in age from 21 to 73 yr (median: 44 yr) were enrolled in the study. None of them had any previous history of lung or allergic disease. All subjects had a normal lung function test and negative skin allergy testing. None of these subjects tested were smokers or ex-smokers, and all drugs that might affect the interpretation of the study were carefully avoided. In particular, untreated asthmatic patients were excluded if they had been treated with inhaled or systemic steroids for 1 mo, sodium cromoglycate, nedocromyl sodium, antihistamines, and/or theophylline within the previous 2 wk and b2-agonist in the 48 h before the bronchoscopy. Nineteen patients with chronic bronchitis ranging in age from 39 to 72 yr (median: 58 yr) were selected as previously described (17), according to criteria of the American Thoracic Society (15). Chronic bronchitis was defined as cough and sputum on most days of the month for at least 3 mo a year during the previous 2 yr. Smoking was

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carefully checked and a minimum of 25 pack-yr smoked was required (30 to 70 pack-yr). All subjects had given their informed consent for the procedure and this study fulfilled the criteria of the ethics committee of the University of Montpellier.

Fiberoptic Bronchoscopy Fiberoptic bronchoscopy was performed as previously described (18). Briefly, after premedication with 0.5 mg atropine and 5 mg diazepam, local anesthesia with 2% lidocaine was applied to the upper respiratory tract. During bronchoscopy, nasal oxygen was provided 3 L/min), and epinephrine was readily available, and the patient had intravenous infusion to provide venous access. Nebulization with 10 mg of salbutamol was performed systematically in asthmatics and patients with chronic bronchitis after the end of the procedure. All subjects were observed for 6 h in our outpatient clinic, and were given a contact telephone number upon discharge from the hospital. A BTFR Olympus fiberscope (Olympus, Paris, France) was inserted into the trachea and airways were systematically examined. The bronchial biopsies were obtained using alligator forceps (Olympus) on a subsegmental bronchus of the left lower lobe, as previously described (18). Six to eight biopsies were obtained for each subject. Half of them were immediately frozen in liquid nitrogen (21808 C) for subsequent RNA extraction. The other biopsies were fixed in 10% formaldehyde (pH 7.2) and embedded in paraffin blocks. Tissue sections (6 mm) were fixed on microscope slides and deparaffined for current histology and immunohistochemistry analysis. Bronchial brushings were obtained using a protected brush (Olympus) to sample bronchial subsegments. We brushed the airways from the distal part of the subsegment to the proximal part, avoiding loss of visual contact with the brush. We performed 5 to 10 gentle strokes against the bronchial wall avoiding bleeding, and the sample taken was then removed from the airways. The brush was then placed into a sterile conical tube containing sterile medium and the cells were resuspended by a gentle vortexing of the tube; 3 to 6 brushings were performed (19). The procedure was well tolerated by all the subjects without any side effects during or after the procedure. The bronchial epithelial cells were subsequently pelleted by centrifugation at 400 3 g for 10 min. Cell viability was assessed using a trypan blue (4%) exclusion test. Cells were identified with cytocentrifuged cellular strained preparations using anticytokeratin monoclonal antibody (Dako, Versaille, France) (19). We found that up to 95% of the cells obtained were positive using an alkaline phosphatase antialkaline phosphatase (APAAP) technique. The cells were then pelleted and frozen at 2808 C for subsequent RNA extraction.

Isolation of Total RNA Total cellular RNA was obtained by lysing bronchial epithelial cells from brushings and biopsies directly in RNAzol (Bioprobe System, Montreuil, France) following the manufacturer’s protocol, which is derived from the method of Chomczynski and Sacchi (20). Briefly, the extracted RNA in the aqueous phase was obtained after homogenization of cells in the reaction mixture containing RNAzol and chloroform (Prolabo, Paris, France), and after centrifugation of 12,000 3 g for 15 min at 48 C. The RNA solution extract was allowed to precipitate with 1 volume isopropanol at 48 C for 15 min and centrifuged at 12,000 3 g for 15 min at 48 C to form a pellet. The RNA pellet was washed with a 70% ethanol solution, vacuum dried briefly, and then solubilized in 30 ml H2O and stored at 2808 C until subsequent analysis. The quantity of RNA was calculated by OD 260 spectrophotometry. The integrity of the purified RNA was determined by visualization of the 28 S and 18 S ribosomal RNA bands after electrophoresis of 1 to 2 mg of each RNA sample through a 1% agarose–formaldehyde gel.

Reverse Transcriptase and Polymerase Chain Reaction Total RNA samples were subjected to reverse transcription with oligo-dT used as template-primer. First strand and synthesis was carried out for 1 h at 378 C in 25 ml of a reaction mixture using 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT; GIBCO BRL, Glasgow, Scotland) in 13 reverse transcriptase (RT) buffer, 0.5 mM deoxyadenosine triphosphate (dATP), deoxyguanosine

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Vachier, Chiappara, Vignola, et al.: GR in Bronchial Epithelial Cells triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), 10 mM dithiothreitol (DTT), 20 U RNasin ribonuclease inhibitor (Promega France, Lyon, France), and 0.25 mg oligo-dT (Biolabs, Gaithersburg, MD). The reaction mixture was heated to 988 C for 5 min to inactivate RT (21, 22). Polymerase chain reaction (PCR) primers were prepared for both genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 171 bp), GR (201 bp), and hsp90 (303 bp). Much attention was given to their locus on the gene to be used in the RT-PCR, and PCR primer sequence was as follows: GR sense 59CAA AAG AGC AGT GGA AGG ACA39 (location: 1335–1355); GR antisense 59GAG GTT TCT TGT GAG ACT CCT GT39 (location: 1535–1513); hsp90 sense 59GTC TGG GTA TCG GAA AGC AAG 39 (location: 22–43); hsp90 antisense 59 CTG AGG GTT GGG GAT GAT GTC 39 (location: 304–325); GAPDH sense 59 TCG CCA GCC GAG CCA CAT 39 (location: 47–63); GAPDH antisense 59 GGA ACA TGT AAA CCA TGT AGT TG 39 (location: 194–217). Absence of contaminants was checked by RTPCR assay of control samples with no RNA, no RT, or PCR reaction buffer only. PCR assays contained 0.5 U Taq DNA polymerase, 0.5 mM of each oligonucleotide primer, 0.2 M deoxyribonucleoside triphosphate (dNTP), 0.25 or 2.5 ml of the RT reaction mixture, 50 mM KCl, 10 mM TRIS HCl, pH 9, 0.1% Triton X-100, 5 to 15% glycerol, 1.5 mM magnesium chloride in a final volume of 50 ml. An amplification sequence of 28 denaturation cycles at 958 C for 1 min, annealing at 538 C for 1 min and extension at 728 C for 2 min was used. Both sets of RT reaction mixture were analyzed to check that PCR products were obtained during the exponential phase as previously described (23). To evaluate the reproducibility of this experiment, Wi26 cell lines were used with a very low number of cells (0.2 3 106). Variation noted for six different experiments was less than 4% for GR mRNA levels, less than 7% for GAPDH mRNA, and less than 9% for hsp90 mRNA levels. PCR products were analyzed by electrophoresis in TRIS-acetateEDTA (TAE) buffer with ethidium bromide stained (0.5 mM) 2% agarose gel. Band intensities were measured using an ultrasensitive photon counting imaging camera equipped with a computer-assisted image processor (Argus 100; Hamamatsu Photonics, Tokyo, Japan). Each band was selected and the same area was used to measure the numbers of photons emitted. The results are expressed as ratios GR/ GAPDH and hsp90/GAPDH.

Immunohistochemistry Eosinophilic inflammation was studied using two monoclonal antibodies, EG1 and EG2 (Pharmacia Diagnosis AB, Uppsala, Sweden) as previously described (17). Bronchial specimens from different groups of patients were run on the same experimental series, because slight differences may occur when experiments are run at different times. The evaluation was made by two independent persons, unaware of the patients’ data. The numbers of eosinophils were enumerated and ex-

pressed as number of positive cells/mm2 of submucosa. The percentage of epithelial shedding was evaluated as previously described (24). The expression of GR, GM-CSF, and ICAM-1 was studied on biopsy sections or cytospins of bronchial epithelial cells, using the APAAP method (19). A polyclonal rabbit anti-human GR antibody (Affinity Bioreagents, Neshanic Station, NJ), antibody specific for GM-CSF (dilution 1:30; Genzyme, Cambridge, MA), and antibody specific for ICAM-1 (dilution 1:10; K562; Immunotech, Luminy, France) were used as first antibodies in immunostaining. Control slides were treated with unrelated IgG2 antibody of the same IgG isotype. Bronchial specimens from different groups of patients were run with the same experimental series. The evaluation was made by two independent observers, unaware of the patients’ data. Results obtained using the APAAP method for GR expression on bronchial biopsies were expressed as localization and using a semiquantitative evaluation to describe the coloration, e.g., weak (1), mild/moderate (2), and intense (3) staining. Results obtained using the APAAP method for GR, GMCSF, and ICAM-1 expression on bronchial epithelial cells were expressed as percentage of positive cells counted on 500 cells.

Western Blot Analysis Western blot analysis was performed on total cellular extract from four biopsies. They were homogenized with a Kontes all-glass Dounce (Kontes Glass Co., Vineland, NJ) in lysing buffer (10 mM TRIS-HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40). The homogenate was flash-frozen in liquid nitrogen, rapidly thawed, and centrifuged for 15 min at 12,000 3 g. Protein concentration was determined by a BCA protein assay kit (Pierce, Rockford, IL). 50 mg of protein extract were size fractionated on 4 to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. Membrane was blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA), 0.1% Tween 20. Then it was incubated for 1 h at room temperature with 1:100 of the rabbit anti-human GR antibody previously used for immunocytochemistry. After washing, membrane was incubated for 30 min with 1:15,000 goat anti-rabbit peroxidase conjugated, and protein/antibody interactions were visualized by autoradiography.

Statistical Analysis Statistical analyses were done using nonparametric tests. One-way analysis of variance was performed on all data and, when significance was observed, a Mann-Whitney U test was applied to analyze two groups of data with Bonferroni’s corrections. Medians and range are given for each group of subjects.

RESULTS Clinical Features

All subjects’ characteristics are summarized in Table 1. The procedure was well tolerated by all subjects and no asthma ex-

TABLE 1 DEMOGRAPHIC CHARACTERISTICS OF THE SUBJECTS* Controls Biopsies n Age, yr Allergy, % FEV1, % predicted Smoking, packs/yr Brushings n Age, yr Allergy, % FEV1, % predicted Smoking, packs/yr Cells viability, %

Chronic Bronchitis

Asthmatics

7 53 (28–73) 0 108 (105–130)† 0

7 49 (40–67) 0 54 (32–74) 45 (40–85)

7 35 (24–54) 100 82 (61–114) 0

11 40 (28–68) 0 100 (98–112)† 0 42 (10–57)‡

12 57 (43–70) 0 71 (34–100) 48 (35–65) 11 (0–32)

13 40 (20–68) 0 63 (37–100) 0 25 (2–57)

* Results are expressed in medians, with ranges in parentheses. † p , 0.05 significant difference between control subjects and the three other populations. ‡ p , 0.002 significant difference between control subjects and chronic bronchitis or steroid-dependent.

Steroid-dependent

18 58 (29–72) 0 52 (30–105) 0 17 (0–44)

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Figure 1. (A) Expression of GR mRNA extracted from human bronchial biopsies from seven control subjects, seven patients with chronic bronchitis, and seven untreated asthmatic patients. The results are expressed as a ratio of densitometry measurements of bands obtained after RT-PCR for GR and GAPDH genes. (B) Expression of GR mRNA extracted from human bronchial epithelial cells obtained from brushings of 11 control subjects, 12 patients with chronic bronchitis, 13 untreated asthmatics, and 18 steroiddependent asthmatic patients. The results are expressed as a ratio of densitometry measurements of bands obtained after RT-PCR for GR and GAPDH genes.

acerbation occurred at the end. Airway obstruction was assessed by measuring FEV1 and was not significantly different between SD asthmatics, untreated asthmatics, and patients with chronic bronchitis. mRNA Analysis

mRNA extracted from biopsies. There was no significant difference in the amount of transcript for GR between control subjects, untreated asthmatics, and patients with chronic bron-

chitis (Figure 1A). However, there was a trend toward a decreased quantity of mRNA for GR expression in biopsies from untreated asthmatic patients, but this difference was not significant. mRNA extracted from epithelial cells obtained from brushings. There was no significant difference in the GR (Figure 1B) and hsp90 (Figure 2) gene expression between bronchial epithelial cells obtained from untreated and steroid-dependent asthmatic patients and patients with chronic bronchitis. GR gene expression was found to be lower in these three groups of subjects as compared with this expression in bronchial epithelial cells from the control subjects, but this difference was not significant when Bonferroni’s correction was applied. Morphological Analysis of Bronchial Biopsies

Figure 2. Expression of hsp90 mRNA extracted from human bronchial epithelial cells obtained from brushings of 11 control subjects, 12 patients with chronic bronchitis, 13 untreated asthmatic patients, and 18 steroid-dependent asthmatic patients. The results are expressed as a ratio of densitometry measurements of bands obtained after RT-PCR for hsp90 and GAPDH genes.

The thickness of the basement membrane was significantly increased and the percentage of membrane recovered by epithelium was significantly decreased in biopsies obtained from asthmatic subjects (Table 2). The results of the immunohistochemical analysis with EG2 are presented in Table 2. In control subjects, very few cells reacted with EG2, these cells were found deep in the submucosa and were never degranulated. The number of eosinophils stained by EG2 was significantly greater in biopsies from untreated asthmatics and patients with chronic bronchitis. Eosinophils from untreated asthmatic patients were often degranulated with extracellular release of eosinophil cationic protein (ECP) beneath the basement membrane. In untreated asthmatic patients, eosinophils were beneath the basement membrane, whereas in chronic bronchitis patients they were localized more deeply in the submucosa. Eosinophils were never degranulated in patients with chronic bronchitis. Immunostaining

GR immunostaining in biopsies. GR expression did not differ between the three groups of subjects. GR immunostaining was localized either within the epithelium or in the mononuclear

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Vachier, Chiappara, Vignola, et al.: GR in Bronchial Epithelial Cells TABLE 2 MORPHOLOGICAL CHARACTERISTICS OF BRONCHIAL BIOPSIES* Patients Thickness of basement membrane, mm Basement membrane covered by epithelium, % Intraepithelial eosinophils, cells/mm Eosinophils in submucosa, cells/mm2

Controls 6 (5–12) 82.5 (45–100) 0 (0–1) 0 (0–5)

Chronic Bronchitis 6 (5–15) 91 (75–100) 0.25 (0–1) 0 (0–16)

Asthmatics 13 (6-17)† 45 (0–70)† 0.6 (0–4) 10 (0–60)†

* Results are expressed in medians, with ranges in parentheses. † p , 0.05 significant difference between asthmatics and the two other populations.

cells within the submucosa (Figure 3A). Staining was heterogeneous within the biopsies and no correlation was found between GR and the intensity of eosinophilic inflammation, disease severity or control. GR immunostaining in epithelial cells obtained from brushings. The low number of cells recovered by brushing did not permit us to perform immunostaining with cells from all subjects. GR immunostaining was performed only on four control subjects, five patients with chronic bronchitis, five untreated and 12 steroid-dependent asthmatic patients. GR expression did not differ between the four groups of subjects (Figure 3B). GM-CSF and ICAM-1 immunostaining in epithelial cells obtained from brushings. The percentage of bronchial epithelial cells expressing GM-CSF was greater in steroid-dependent as compared with untreated asthmatics, control subjects, and patients with chronic bronchitis (median and range: 39% [1 to 74] versus 23.5% [12 to 42] for untreated, 0 [0–0] p , 0.001 for control subjects, and 12% [5 to 22] p , 0.005 for chronic bronchitis, respectively). ICAM-1 was significantly overexpressed on bronchial epithelial cells of control subjects, patients with chronic bronchitis, and steroid-dependent asthmatics as compared with untreated asthmatic patients (median and range: 41% [16 to 55]

versus 0 [0–0] p , 0.005 for control subjects, 10% [0 to 23] p , 0.005 for chronic bronchitis, 0 [0 to 11] p , 0.0001 for steroiddependent asthmatics, respectively) (Figure 4). Western Blot

Figure 5 shows Western blotting analysis of protein extracts from human bronchial biopsies obtained from four asthmatic patients. The molecular weight markers indicated on the left are BSA (98 kD), glutamic dehydrogenase (64 kD), and alcohol dehydrogenase (50 kD). These data indicate that the human GR protein was found with a molecular weight of 98 kD.

DISCUSSION In the present study, we used advanced technologies to discover that, despite clinical and biological differences between the groups of subjects, there was no difference in the expression of GR in the airways. Moreover, hsp90 expression was not different in all groups. Concerning bronchial biopsies, GR mRNA expression was not different. Moreover, GR protein was localized in the bronchial epithelium and in cells from the submucosa. Western blot analysis was performed and showed that the antibody used was specific for human GR. Moreover,

Figure 3. (A) Expression level of GR protein in human bronchial biopsies from seven control subjects, seven patients with chronic bronchitis, and seven untreated asthmatic patients. Immunohistochemistry was performed using the APAAP method, and the results are expressed using a semiquantitative evaluation to express the staining as weak (1), mild/moderate (2), and intense (3). (B) Expression of the GR protein in human bronchial epithelial cells obtained from bronchial brushings from four control subjects, five patients with chronic bronchitis, five untreated asthmatic patients, and 12 steroid-dependent asthmatic patients. Immunohistochemistry was performed using the APAAP method, and results are expressed as percentage of positive cells.

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Figure 4. GM-CSF (A) and ICAM-1 (B) expression in human bronchial epithelial cells obtained from bronchial brushings from five control volunteers, nine patients with chronic bronchitis, six untreated asthmatic patients, and 13 steroid-dependent asthmatics patients. Immunohistochemistry was performed using the APAAP method, and the results are expressed as percentage of positive cells. Statistical analysis was performed using Mann-Whitney U test.

we found that in steroid-dependent asthmatics and patterns of the expression of GM-CSF and ICAM-1 were distinctively affected by the long-term therapy. ICAM-1 was not present in cells from all steroid-dependent asthmatic patients, whereas GM-CSF was still overexpressed. The presence of GR in all cells reflected the essential homeostatic role of glucocorticoids. These results are in accordance with those previously obtained with lung biopsies from patients undergoing heart transplantation (12). Semiquantitative assessment of the protein within the bronchial wall was not able to detect any difference within the groups of patients, despite marked differences concerning their structure and eosinophilic inflammation. All of these results were not related to the disease status nor to etiologic factors and ongoing disease activity. Glucocorticoids are widely used for asthma treatment because this condition involves chronic inflammation of airways. Their mechanism of action in real clinical situations is still in-

Figure 5. Western blot analyses from four different biopsies were resolved by electrophoresis through 4 to 12% polyacrylamide gels and transferred to nitrocellulose membrane. Rabbit anti-human glucocorticoid receptor antibody was used as specific antibody. After further washings, specific protein/antibody interactions were visualized by autoradiography. The molecular weight markers indicated on the left are bovine serum albumin (98 kD), glutamic dehydrogenase (64 kD), and alcohol dehydrogenase (50 kD).

completely understood. However, there presence in the bronchial wall suggests that this is an important site of steroid action, although their function is uncertain. Quantification of mRNA and proteins in bronchial epithelial cells and bronchial biopsies from asthmatic patients, patients with chronic bronchitis, and control subjects were of considerable interest. Bronchial inflammation may change the GR quantity and turnover, and the two types of inflammatory diseases of the bronchi, namely asthma (mild and severe) and chronic bronchitis, with different cellular patterns have never been compared. Moreover, bronchial epithelial cells are an important site of action of inhaled steroids because steroids may interfere with the cytokine synthesis (13, 25). At this level, bronchial epithelium is at the interface of the environment and submucosa of airways, and it is more than a physical barrier against external aggression as it also plays a role in inflammation (26). Therefore in our study, prednisone treatment fails to inhibit completely bronchial epithelial cell activation in steroid-dependent asthmatics. The sustained production of GM-CSF probably contributes to perpetuate the presence of inflammation in the airways. Peripheral blood mononuclear cells (PBMC) from steroidresistant asthmatic patients have been extensively investigated, and it was found that these cells have a distinct GR binding pattern at cytosolic and nuclear levels (27, 28). Bronchoalveolar lavage (BAL) cells from the same patients have high T helper cell type 2 (Th2)-like cytokines gene expression which could be reversed by a short course of steroids. An interaction between cell activation and steroid unresponsiveness has been postulated. GR gene expression in bronchial epithelial cells obtained from brushings in patients with bronchial diseases such as asthma and chronic bronchitis was identical to the level obtained in control subjects. GR is further downregulated in vitro and ex vivo by corticosteroid treatment (29). In steroiddependent asthmatic patients under control, we did not observe any change in the GR expression. These results suggest

Vachier, Chiappara, Vignola, et al.: GR in Bronchial Epithelial Cells

that the molecular basis for glucocorticoid action was not affected in long-term treated severe asthmatic patients. It is interesting to note that in the present study, the quantity of GR transcripts was not increased in inflammatory diseases of the bronchi. The increased number of inflammatory cells infiltrating the airways did not amplify the number of transcripts detected by RT-PCR, nor was there an increase in immunostaining of GR within the biopsies. Obviously, neither mRNA quantity nor GR immunostaining, as shown in the present study, were directly correlated with the function of GR relative to glucocorticoids within the bronchi of asthmatic patients. In untreated asthmatics, ICAM-1 is overexpressed in bronchial epithelial cells (26) and this expression was specific and related to the severity of the bronchial disease. In the present study, we confirmed our previous findings and investigated steroid-dependent asthmatics. In epithelial cell lines, corticosteroids repressed both constitutive and 12-0-tetradecanoylphorbol-13-acetate–induced expression of ICAM-1 at a transcriptional level (30). We found that prednisone was able to produce the same effect ex vivo since none (except one) patient displayed an ICAM-1 expression on BEC. The present study addresses the question of steroid sensitivity at the airway level showing difference in the regulation of inflammatory partners. The lack of GM-CSF inhibition for steroid-dependent asthmatics might represent one of the reasons for the need for long-term treatment with a potential risk for harmful side effects. Hsp90 is an ubiquitous, abundant, and conserved protein whose rate of synthesis is increased in response to cellular stress (31). Hsp90 is a protein chaperone that associates a range of cellular steroid receptors including GR (32), and its association with GR is essential for obtaining the high-affinity steroid binding conformation (8). It has been described, in studies on the hypothalamic paraventricular nucleus from rats, that chronic exposure to high circulating levels of corticosterone caused significant suppression of hsp90 mRNA concentrations, and it is implicated in the mechanism of GR downregulation (33). In our study, we did not find any difference in hsp90 mRNA expression between the populations studied. The present study suggests that the molecular basis of glucocorticoid action did not differ in the bronchial wall of mild or severe asthmatic patients and in patients with chronic bronchitis. GR expression and localization of GR within the airways did not differ, suggesting that the cellular specificity of glucocorticoids should also be determined, perhaps with respect to the nature of protein–protein interactions between GR and cell-specific transcription factor more than in terms of GR regulation itself. Moreover, the experiments analyzing the functionality of GR showed that the responsiveness was good for ICAM-1 but not present for GM-CSF. There are multiple cytokine pathways which can be responsible for human asthma and these results provide evidence for a GM-CSF pathway leading to the persistence of asthma symptoms despite long-term treatment with corticosteroids. References 1. Barnes, P. 1995. Corticosteroids. In P. O’Byrne and N. Thompson, editors. Manual of Asthma Management. W. B. Saunders, London. 219– 253. 2. Carmichael, J., I. C. Paterson, P. Diaz, G. K. Crompton, A. B. Kay, and I. W. Grant. 1981. Corticosteroid resistance in chronic asthma. Br. Med. J. Clin. Res. 282:1419–1422. 3. Kamada, A. K., D. Y. Leung, and S. J. Szefler. 1992. Steroid resistance in asthma: our current understanding. Pediatr. Pulmonol. 14:180–186. 4. Barnes, P. J. 1995. Inhaled glucocorticoids for asthma. N. Engl. J. Med. 332:868–875. 5. Cypcar, D., and W. W. Busse. 1993. Steroid-resistant asthma. J. Allergy

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