Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Chronic inflammation in asthmatic airways leads to pathophysiologic changes in the structure of the tissues, a process often referred to as airway wall remodeling. Such changes include thickening of the smooth muscle in the airway wall (1, 2), which may influence airway responsiveness (3). Other structural changes that may affect airway function include disruption to the bronchial epithelium (4), subepithelial fibrosis (5), and goblet cell hyperplasia together with increased mucus accumulation in the airways (6, 7). The mechanisms underlying structural remodeling have not been clearly elucidated.

Endothelin (ET)-1 is a potent bronchoconstrictor (8) and a pro-inflammatory agent and growth factor. ET-1 acts as a direct mitogen (9, 10) and also as a comitogen with epidermal growth factor (EGF) to induce airway smooth muscle (ASM) cell proliferation (11, 12). ET-1 is also promitogenic for airway epithelial cells (13) and fibroblasts (14). ET-1 may contribute to subepithelial fibrosis by stimulating collagen synthesis by fibroblasts (15) and can also induce increased mucus secretion from airway submucosal glands (16). In addition, ET-1 may participate in immune responses because it can stimulate the release of a range of cytokines such as interleukin (IL)-6, IL-1, and tumor necrosis factor- from human monocytes (17) and trigger histamine release from guinea pig pulmonary mast cells (18). ET-1 has also been implicated in allergen-induced eosinophil recruitment to the airways in mice (19). In asthmatics, increased levels of ET-1 have been detected in bronchoalveolar lavage fluid (20) and increased ET-1 immunoreactivity has been demonstrated in the airway epithelium of bronchial biopsies (21). ET-1, therefore, has the potential to participate in inflammatory responses and mediate cellular proliferation occurring in the airway wall of asthmatics.

We have recently reported on a model of repeated allergen exposure in sensitized Brown-Norway rats that causes bronchial hyperresponsiveness and induces a number of characteristic features of remodeling that have been described in the airways of chronic asthmatics (22, 23). These features include increased rates of ASM and epithelial cell DNA synthesis, ASM thickening, and increased major basic protein (MBP)⁺ eosinophil and CD2⁺ T-lymphocyte recruitment to the airway walls. There is also evidence of increased collagen and fibrin deposition in the subepithelium and mucus cell hyperplasia and hypertrophy together with increased levels of mucus in the airways. In the present study, we investigated whether ET-1 was expressed in the airways after either a single or repeated allergen exposure. Furthermore, we used a dual ET_A+B receptor antagonist in our repeated allergen exposure model to assess the influence of ET in the pathogenesis of bronchial hyperresponsiveness and ASM and epithelial cell DNA synthesis, which were observed in this model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Sensitization and Challenges

Pathogen-free, male Brown-Norway rats weighing 220 to 250 g (Harlan, Bicester, UK) were actively sensitized on three consecutive days with 1-mg/kg (intraperitoneally) injections of ovalbumin in 0.9% sterile saline containing 100 mg of aluminum hydroxide as adjuvant. Challenges were performed in a 0.8-m³ chamber, with free-breathing animals exposed to either saline or a 1% ovalbumin aerosol mist produced by a DeVilbiss PulmoSonic nebulizer (DeVilbiss Health Care Ltd., Feltham, UK) at 0.6 liter/ min. At all other times, rats were housed in a caging system receiving clean filtered air (Maximizer; Thorens Caging System Inc., Hazleton, PA).

Single Allergen Exposure Protocol

Rats were sensitized with ovalbumin on Days 1, 2, and 3. On Day 21, rats were exposed to either saline or 1% ovalbumin aerosol for 20 min. The rats were then killed and the lung tissue collected for analysis of ET-1 messenger RNA (mRNA) by Northern blotting at times 0, 2, 6, and 24 h postallergen challenge (n = 3 in each group).

Repeated Allergen Exposure Protocol

Sensitization was carried out as for the single allergen procedure on Days 1, 2, and 3 (Figure 1). On Day 4, rats were implanted with subcutaneous, osmotic mini-pumps containing either vehicle or the dual ET_A+B receptor antagonist. On Days 6, 9, 12, 15, 18, and 21, rats were exposed to 1% ovalbumin or saline aerosol for 20 min. Immediately after allergen challenge, rats received an intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU), an S-phase marker nucleotide, with a second dose administered 8 h later. On Day 22, measurement of bronchial responsiveness to acetylcholine was performed 18 to 24 h after the final challenge. Animals were then killed and the lungs collected for measurement of ET-1 mRNA by Northern blot analysis, immunohistochemical detection of cellular DNA synthesis (BrdU), and localization of ET-1 immunoreactivity. Three groups of animals were studied: group A: ovalbumin-sensitized, saline-challenged, vehicle-treated (n = 7); group B: ovalbumin-sensitized, ovalbumin-challenged, vehicle-treated (n = 8); group C: ovalbumin-sensitized, ovalbumin-challenged, dual ET_A+B receptor antagonist-treated (n = 8).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Schematic diagram of repeated allergen exposure protocol. After sensitization, rats were repeatedly exposed to ovalbumin aerosol allergen challenge (AC) every 3 d. BrdU injections were given for measurement of DNA incorporation. The osmotic mini-pump was used to deliver the endothelin antagonist.

Antagonist Treatment

The dual ET_A+B receptor antagonist used in this study was butane-1-sulfonic acid {2-[4-benzo[1,3]dioxoyl-5-yl-2-(3-fluoro-4-methoxy-phenyl)-3-methyl-pyrrolidin-1-yl]-ethyl}-propyl-amide, which has IC₅₀ values (concentrations that produce 50% inhibition) against human ET_A and ET_B receptors of 0.50 and 0.35 nM, respectively. For comparison, reported IC₅₀ values for other dual receptor antagonists are Ro 46-2002, 200 to 500 nM for ET_A and ET_B receptors (24), and bosentan, 4.7 nM for ET_A and 95 nM for ET_B receptor (25).

The dual ET_A+B receptor antagonist or vehicle was administered to rats via a subcutaneous, osmotic mini-pump delivering a dose of 3 mg/kg/d, at a rate of 0.125 mg/kg/h in 2.5 µl of solution. The ET_A+B receptor antagonist was initially dissolved in 1 N NaOH and mixed with polyethyleneglycol 400 for 1 h. Sterile water was used to make up the volume and the pH set at 7.5 using HCl. Osmotic mini-pumps, 2 ml in volume (model no. 2ML4; Charles River, Kent, UK), were filled with either antagonist or vehicle and contained sufficient volume to last for the duration of the study.

We also examined the effect of the antagonist on baseline airway responsiveness. Rats were sensitized to ovalbumin and on Day 4 of the procedure implanted with subcutaneous, osmotic mini-pumps containing either vehicle or the dual ET_A+B receptor antagonist (n = 4 in each group). These rats received saline exposures every three days from Day 6 to Day 21 of the exposure protocol. On Day 22, bronchial responsiveness to acetylcholine was measured and bronchoalveolar lavage performed.

Osmotic Mini-Pump Implantation

Rats were anesthetized using 0.3 ml/kg (intramuscularly) Hypnorm (consisting of 0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone). When adequate anesthesia was achieved, a small patch of fur was removed from the back of the rat and a small skin incision made. The osmotic mini-pump was placed subcutaneously under the skin flap with the delivery nozzle facing forward, and the site of incision was sutured. The implantation procedure was performed employing sterile techniques.

BrdU Dosing

BrdU (Sigma Chemicals, Poole, UK) was dissolved in dimethylsulfoxide (DMSO) and diluted with sterile water, giving a final concentration of DMSO of less than 7%. Rats were injected with 50 mg/kg BrdU in 1 ml (intraperitoneally) immediately after each allergen challenge and received a second dose 8 h later (total of 12 injections).

Measurement of Bronchial Responsiveness to Acetylcholine

Bronchial responsiveness was measured 18 to 24 h after the final allergen challenge as previously described by Elwood and coworkers (26). In brief, after anesthesia a tracheostomy was performed to allow ventilation of the lungs via a small animal respirator. Rats were initially administered intravenously with 1 mg/kg propranolol to inhibit adrenergic responses and 1.5 mg/kg suxamethonium to inhibit spontaneous breathing. Increasing half-log concentrations of acetylcholine were administered by inhalation and lung resistance was simultaneously calculated using a software program (LabVIEW 2; National Instruments, Austin, TX). Challenges were ceased when lung resistance exceeded 200% of initial baseline values. The provocative concentration of acetylcholine required to increase baseline resistance by 200% (PC₂₀₀) was determined by linear interpolation of log concentration-lung resistance curves.

Tissue Collection

Rats were killed using an overdose of sodium pentobarbitone (500 mg/kg, intraperitoneally) and the lungs rapidly removed. The left lung lobe was insufflated with optimal cutting temperature (OCT) Tissue Tek mounting medium (Raymond A. Lamb, London, UK) diluted 1:1 with phosphate-buffered saline, mounted onto a cork block with the main bronchi uppermost, then snap-frozen in melting isopentane, and stored at 25°C. The remaining lung was cut into small pieces, frozen in liquid nitrogen, and stored at 70°C.

Northern Blot Analysis

The pro-ET-1 complementary DNA (cDNA) probe (nucleotides 611 to 917; 307 bp) used for Northern blot analysis was amplified by polymerase chain reaction using designed primers corresponding to the published rat pro-ET-1 (27). The cDNA probes, including rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1.3-kb PstI fragment), were labeled with [³²P]deoxycytidine triphosphate by use of a random primer labeling kit (Amersham, Middlesex, UK). Total cellular RNA was prepared according to the method of Chomczynski and Sacchi (28). Rat lung was homogenized (twice for 10 s) in 10 ml of solution D (4 M guanidium thiocyanate, 2 M mercaptoethanol, 0.5% sodium sarcosyl). RNA was then isolated from the homogenates with a phenol:chloroform:isoamyl alcohol (50:49:1) extraction in the presence of 0.4 M sodium acetate (pH 4.0). RNA (from the upper aqueous phase) was precipitated in an equal volume of isopropanol at 20°C for 1 to 2 h and then centrifuged at 12,000 × g for 15 min at 4°C. The pellet was resuspended in 5 ml of solution D, and a further isopropanol precipitation was performed overnight at 20°C. Residual salt was removed by a further wash in 75% ethanol. Poly A⁺ RNA extraction was performed on 200 µg of total RNA (measured by absorbance at 260 nm) using an mRNA isolation kit (PolyATtract System IV; Promega, Southampton, UK) according to the manufacturer's instructions. Denatured mRNAs were size-fractionated by gel electrophoresis on 1% agarose/ formaldehyde gels containing 20 mM morpholinosulfonic acid, 5 mM sodium acetate, and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.0) before blotting to "Magna" nylon membranes (msi; Westborough ) by capillary action. The filter was incubated at 42°C for at least 4 h in a prehybridization buffer containing 50% formamide, 50 mM Tris-HCl (pH 7.5), 5× Denhardt's solution, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, and 250 µg/ml denatured salmon sperm DNA. The filter was then hybridized with [³²P]-labeled probes at the concentration of 0.5 to 1.0 × 10⁶ cpm/ml at 42°C for 14 to 16 h. After hybridization, the blots were washed to a stringency of 0.2× saline sodium citrate, 0.1% SDS at 60°C before exposure to Kodak X-OMAT film. After suitable exposure times, autoradiographs were analyzed by laser densitometry (Gel Documentation and Analysis System GDS8000; UVP, Cambridge, UK). Specific mRNA levels were expressed as the ratio of pro-ET-1 to GAPDH mRNA.

Immunohistochemistry for BrdU and ASM

As previously described (22, 23), a primary anti-BrdU monoclonal antibody (clone BU-1) solution containing bovine pancreas DNase I (Amersham) was applied to tissue sections at 37°C for 75 min. A secondary biotinylated rat-adsorbed antiserum to mouse immunoglobulin (Ig) G (Vector Laboratories, Peterborough, UK) was then applied for 30 min followed by a 45-min incubation with peroxidase-linked avidin-biotin complex solution (ABC-Elite kit; Vector Laboratories). BrdU-positive cells were visualized using 3,3-diaminobenzidinetetrachloride solution (Sigma Chemicals). Sections were then applied with a primary anti--smooth muscle actin monoclonal antibody (clone 1A4; Sigma Chemicals) for 1 h at room temperature. A secondary biotinylated rat-adsorbed antiserum to mouse IgG (Vector Laboratories) was applied, followed by an avidin-biotin complex reagent conjugated to alkaline phosphatase (Vector Laboratories). The -smooth muscle actin staining was visualized using Sigma FAST (4-chloro-2-methylbenzenediazonium/3-hydroxy-2-naphthoic acid, 2,4-dimethylanilide phosphate [-naphthol AS-MX], and Fast Red TR) in Tris buffer (Sigma Chemicals) to give a red end-product. Nuclei that were not immunoreactive for BrdU were counterstained by application of the fluorescent DNA ligand 4,6-diamidino-2-phenylindole hydrochloride (DAPI) at a concentration of 0.00001% in phosphate-buffered saline (PBS) containing 0.6% NP40 (Sigma Chemicals) for 20 min. Sections were mounted under glass coverslips using 1:1 PBS/glycerol and stored in the dark at 4°C. Negative controls were performed on sections of lung for BrdU and -smooth muscle actin and also for MBP and ET-1 with antibodies of the same immunoglobulin class or in the absence of primary antibody.

Quantification Using Computer-Assisted Image Analysis

The quantification procedure has been previously described (22, 23). Image processing was performed using a Zeiss microscope fitted for both transmitted light and fluorescence imaging. Images were captured using a monochrome camera at maximum sensitivity and analyzed using a Sonata image analysis system (Seescan, Cambridge, UK). Each selected airway was quantified through a ×10 objective lens to measure internal perimeter, internal area, and airway breadth (greatest diameter perpendicular to the longest axis). Images captured through the ×20 objective lens were then used to count BrdU- and DAPI-labeled cells, and measure smooth muscle area and epithelial basement membrane length.

For measurement of BrdU indices, the airway of interest was selected, and the transmitted light image containing BrdU-positive cells captured and converted to a monochrome image. Without moving the section, a red fluorescence image of the alkaline phosphatase-Fast Red-labeled -smooth muscle actin immunoreactivity was captured. A blue fluorescence image for DAPI-positive nuclei was then captured, and all three images were converted to stored monochrome images. The smooth muscle image was used to create a mask that excluded all regions that were not immunoreactive for -actin. This mask was overlaid onto the transmitted light image of the same area and the number of BrdU positively stained nuclei counted. The mask was then overlaid onto the fluorescence image of the DAPI-positive nuclei and the number of nuclei counted. Measurement of epithelial BrdU indices was performed in a similar way by creating an epithelial mask using interactive delineation of the epithelium on DAPI fluorescence images and counting the number of BrdU-positive nuclei within the mask.

BrdU indices in ASM was measured as the number of BrdU immunoreactive nuclei divided by total nuclei (BrdU plus DAPI nuclei) within the -smooth muscle-actin-stained area. Epithelial BrdU index was measured as the number of BrdU-positive cells within the DAPI-defined epithelial mask divided by the basement membrane length. Statistical power calculations have previously been reported (22, 23).

ET-1 Immunohistochemistry

Sections of lung tissue (5 µm thick) were cut and fixed in Bouin's solution (2 parts picric acid to 1 part paraformaldehyde, vol/vol) at 20°C for 10 min. Tissue sections were then washed in PBS for 10 min and placed in methanol containing 0.3% hydrogen peroxide to quench endogenous peroxidase. After a further 10-min wash in PBS, sections were incubated for 1 h with an anti-ET-1 polyclonal antibody raised in rabbit (29) at a dilution of 1:200. Sections were then washed and applied with a goat antirabbit IgG monoclonal antibody at a concentration of 1:100 for 30 min, rinsed for 10 min in PBS, then applied with an avidin-biotin complex conjugated to peroxidase (Vector Laboratories) for 45 min. ET-1 immunoreactivity was visualized using Sigma FAST to give a red end-product. Tissues were stained with a light hematoxylin counterstain and mounted under glass coverslips.

Quantification of the ET-1 immunoreactive area within the bronchial epithelium was performed using computer-assisted image analysis. The epithelium of all conducting airways was visualized under transmitted light with a ×20 objective lens, delineated, and measured. The epithelium was then viewed using red fluorescence imaging to identify ET-1 immunoreactivity labeled with Sigma FAST and the area measured. The percentage of epithelial ET-1 immunoreactive area was calculated for each individual airway within one section of lung tissue. These data were then pooled to give mean values for the large conducting airways (500 to 1,000 µm in diameter) or small airways (200 to 499 µm in diameter) for each individual rat.

Eosinophil MBP Immunocytochemistry and Counting

Staining for eosinophil MBP-positive cells was performed as previously described (23). In brief, a monoclonal antibody against human MBP (clone BMK-13; Monosan, Uden, The Netherlands) was used on lung sections at a concentration of 1:80 for 1 h at room temperature. After labeling with a biotinylated horse antimouse monoclonal antibody, positive cells were visualized by using an avidin-biotin complex reagent conjugated to alkaline phosphatase (Vector Laboratories) and Sigma FAST. Sections were counterstained with hematoxylin (BDH, Lutterworth, UK) and mounted under glass coverslips. Eosinophil influx around the five largest airways in each lung section was assessed as the number of MBP-positive cells in the airway wall (consisting of epithelium, submucosa, smooth muscle, and lamina propria) and expressed per millimeter of basement membrane length.

Analysis of Data

All the counting procedures used in this study were performed with the investigator blinded to treatment groups. BrdU incorporation indices and eosinophil counts were measured as the weighted mean of the indices from the five largest airways in one section of lung tissue. The percentage of ET-1 immunoreactivity was calculated from all the airways in a single lung section divided into either large conducting airways or small airways. All indices were calculated for individual animals. Mean indices were statistically analyzed after logarithmic transformation by one-way analysis of variance followed by t tests with Bonferroni correction used to evaluate significant differences between groups. Values are expressed as means (95% confidence intervals [CI]) or means ± standard error of the mean (SEM), with P values of less than 0.05 considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Northern Blot Analysis for ET-1 mRNA

A single exposure of sensitized rats induced a 14-fold increase in the pro-ET-1:GAPDH mRNA ratio at 2 h after the challenge (P < 0.001), but there was no significant increase at times 0, 6, and 24 h (Figure 2A). In repeatedly exposed sensitized rats, there was a 50% increase in the pro-ET-1:GAPDH mRNA ratio from 1.0 ± 0.18 in the saline-exposed group to 1.58 ± 0.10 in the ovalbumin-exposed rats at 24 h (P < 0.01). Treatment of sensitized and ovalbumin-exposed rats with the dual ET_A+B receptor antagonist did not alter the levels of pro-ET-1 mRNA (Figure 2B).

View larger version (28K): [in this window] [in a new window]   View larger version (37K): [in this window] [in a new window]   Figure 2.   Pro-ET-1 mRNA expression in the lungs after single (A) and repeated (B) allergen exposure of sensitized rats. (Ai) Representative blot of the time course for pro-ET-1 expression after a single saline or allergen exposure of sensitized rats. (Aii) Bar graph showing mean data ± SEM for the pro-ET-1/GAPDH mRNA ratio after a single allergen exposure (solid bars) compared with a single saline exposure (open bars) at times 0, +2, +6, and +24 h postchallenge (n = 3 in each group). ***P < 0.001 compared with saline-exposed control rats; +++ P < 0.001 compared with other time points. (Bi) Representative blot of pro-ET-1 expression in the lungs 24 h after the last of six saline or allergen exposures performed every 3 d on sensitized rats. (Bii) Mean data ± SEM for the pro-ET-1/GAPDH mRNA ratio after vehicle treatment and saline exposure (open bar, n = 7), vehicle treatment and allergen exposure (solid bar, n = 8) or the dual ETA+B receptor antagonist treatment and allergen exposure (hatched bar, n = 8). ** P < 0.01 compared with the saline-exposed rats.

ET-1 Immunoreactivity in Lungs

ET-1 immunoreactivity was hardly detected in the airway epithelium in control rats, but after chronic allergen challenge, there was a marked increased expression in the airway epithelium (Figure 3, panel a). In the large airways, there was a significant increase in ET-1 immunoreactive area from 2.2 ± 0.5% in the saline-exposed group to 14.1 ± 3.20% in the allergen-exposed group (P < 0.001). In the small airways, there was also an increase from 0.5 ± 0.1% in the saline-exposed rats to 1.6 ± 0.4% in the allergen-exposed group (P < 0.05). Treatment of sensitized and allergen-exposed rats with the dual ET_A+B receptor antagonist had no effect on the ET-1 immunoreactive areas in either the large (12.8 ± 2.2%) or small (1.52 ± 0.20%) airways (Figure 3, panel b).

View larger version (86K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Figure 3.   Effect of chronic allergen exposure on ET-1 immunoreactive areas in bronchial epithelium. (a) There was a marked increase in the levels of ET-1 immunoreactivity detected in the airways of sensitized and repeated allergen-exposed rats (C) compared with saline-exposed controls (A). Plates B and D are the same lung tissue sections as seen in A and C, respectively, viewed under red fluorescence. The increased ET-1 immunoreactivity in repeated allergen-exposed rats (arrows) was localized in the bronchial epithelium of the conducting airways and the area quantified from fluorescent images as a percentage of the total epithelial area. Bars indicate 100 µm. (b) Repeated allergen exposure of sensitized rats (solid bars) induced a significant increase in the ET-1 immunoreactive area within the bronchial epithelium in both the large and small airways compared with the saline-exposed control group (open bars). Treatment with the dual ETA+B receptor antagonist (hatched bars) had no significant effect on expression of ET-1 epithelial immunoreactivity compared with vehicle-treated controls. *** P < 0.001, * P < 0.05 compared with vehicle-treated and saline-exposed rats.

Bronchial Responsiveness after Repeated Allergen Exposure

There were no significant differences in the baseline lung resistance values after saline challenge in any of the three experimental groups (data not shown). There was a significant increase in bronchial responsiveness in the sensitized and chronic allergen-exposed rats (PC₂₀₀, 8.32 mM; 7.24 to 9.55; mean, 95% CI) compared with sensitized and chronic saline-exposed rats (PC₂₀₀, 16.2 mM; 9.77 to 26.3; P < 0.05). The dual ET_A+B antagonist had no effect on this increase in bronchial responsiveness (PC₂₀₀, 5.75 mM; 3.55 to 9.33) (Figure 4).

View larger version (14K): [in this window] [in a new window]   Figure 4.   Bronchial responsiveness to acetylcholine (ACh). Rats were given increasing provocative concentrations of ACh until lung resistance reached 200% (PC200) of baseline values. Horizontal bars indicate mean PC200 values with error bars representing the 95% confidence intervals. There was a decrease in PC200 in the vehicle-treated and repeated allergen-exposed rats compared with saline-exposed controls. Treatment of allergen-exposed rats with the dual ETA+B receptor antagonist had no effect on PC200 compared with the vehicle-treated group. * P < 0.05 compared with vehicle-treated and saline-exposed rats.

The dual ET_A+B antagonist did not cause any significant changes in bronchial responsiveness in sensitized and repeated saline-exposed rats (PC₂₀₀, 19.5 mM, 9.06 to 42.0 after antagonist compared with PC₂₀₀, 16.3 mM, 7.82 to 34.0 after control injections). There were also no changes in bronchoalveolar lavage cells (macrophages, eosinophils, neutrophils, and lymphocytes) between the groups. Thus, the number of macrophages was 5.80 ± 0.72 × 10⁶ versus 5.80 ± 0.67 × 10⁶ and eosinophils were 1.40 ± 0.40 × 10⁴ versus 2.33 ± 1.25 × 10⁴ in the dual ET_A+B antagonist- treated compared with the vehicle-treated group, respectively.

ASM and Epithelial Cell DNA Synthesis after Repeated Allergen Exposure

DNA synthesis as measured by BrdU index in ASM cells was 0.9% (0.5 to 1.6) in saline-exposed rats compared with allergen-exposed rats where the BrdU index was increased to 3.2% (2.6 to 3.8; P < 0.001). Treatment with dual ET_A+B antagonist caused a significant attenuation in the ASM BrdU index (1.2%, 0.8 to 2.0; P < 0.001) compared with the repeated allergen-exposed and vehicle-treated rats (Figure 5A). There was a threefold increase in epithelial DNA synthesis in sensitized and repeated allergen-exposed rats with 14.4 (11.7 to 17.7) BrdU-positive cells/mm basement membrane compared with 4.4 (3.2 to 6.1) in the sensitized and repeated saline-exposed group (P < 0.001). Treatment with the dual ET_A+B antagonist reduced epithelial BrdU-positive cells to 6.9 (4.9 to 9.7; P < 0.001) in sensitized and repeated allergen-exposed rats compared with the vehicle-treated group (Figure 5B).

View larger version (14K): [in this window] [in a new window]   Figure 5.   Effect of the dual ETA+B receptor antagonists on ASM cell (A) and epithelial cell (B) BrdU incorporation after repeated allergen challenge. (A) There was a threefold increase in the ASM cell BrdU index after repeated allergen exposure compared with repeated saline exposure of sensitized rats. Treatment with the dual ETA+B antagonist attenuated the ASM cell BrdU index compared with the allergen-exposed and vehicle-treated group. (B) There was an increase in epithelial cell BrdU incorporation per unit length of basement membrane in repeated allergen-exposed rats compared with the repeated saline-exposed group. Treatment with the dual ETA+B antagonist also attenuated epithelial cell BrdU incorporation. *** P < 0.001 compared with vehicle-treated and saline-exposed rats; ### P < 0.001, ## P < 0.01 compared with vehicle-treated and allergen-exposed rats.

Eosinophil Recruitment to the Airways and Parenchyma

Repeated allergen exposure induced a significant recruitment of MBP⁺ eosinophils to the airways compared with saline-exposed controls. Allergen-exposed rats had 24.9 ± 1.8 MBP⁺ cells/mm of basement membrane compared with 3.8 ± 0.6 in saline-exposed rats (P < 0.001). The dual ET_A+B antagonist caused a significant attenuation of MBP⁺ cell recruitment to the airways with 14.9 ± 1.2 (P < 0.05) compared with the allergen-exposed and vehicle-treated control group (Figure 6).

View larger version (24K): [in this window] [in a new window]   Figure 6.   Effect of the dual ETA+B receptor antagonist on MBP+ eosinophil recruitment to the airways. There was a significant increase in MBP+ eosinophils to the airway walls after repeated allergen exposure of sensitized rats compared with saline-exposed controls. Treatment of allergen-exposed rats with the dual ETA+B antagonist significantly attenuated allergen-induced MBP+ eosinophil recruitment to the airways compared with the vehicle-treated rats. ***P < 0.001 compared with vehicle-treated and saline-exposed rats; # P < 0.05 compared with vehicle-treated and allergen-exposed rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, repeated allergen exposure induced an increase in ET-1 mRNA levels in the lungs together with increased levels of ET-1 immunoreactivity that was localized to the bronchial epithelium of the conducting airways. Repeated allergen exposure also induced increased rates of BrdU incorporation in both ASM and epithelial cells, indicative of an increased proliferative response. The potent and specific ET_A+B receptor antagonist significantly attenuated this increased proliferative response and MBP⁺ eosinophil recruitment to the airways. Repeated allergen exposure induced bronchial hyperresponsiveness to acetylcholine, but this was not modified by the ET_A+B antagonist, indicating that it is unlikely that ET-1 is involved in this response. Therefore, the increased expression of ET-1 in the airway epithelium after repeated allergen challenge may contribute to eosinophilic inflammation and to increased rates of DNA synthesis in ASM and epithelial cells.

Previous studies have shown that patients with asthma have increased expression of ET-1 immunoreactivity in the airway epithelium (21, 30). Increased ET-1 levels in the airway epithelium have also been demonstrated in rats exposed to hypoxia (31, 32) and after intratracheal instillation of Sephadex particles (33). We detected an increased expression of ET-1 mRNA 2 h after a single allergen challenge that returned to baseline levels by 6 h postchallenge. With chronic allergen exposure, the levels of ET-1 mRNA remained elevated 24 h after the final challenge. In addition, ET-1 immunoreactivity localized to the bronchial epithelium was also significantly elevated.

ET-1 causes potent contraction of ASM (34) and can induce ASM cell proliferation in vitro (9, 10). In human ASM cells, ET-1 alone is only a weak mitogen, but in the presence of growth factors such as EGF, it can potentiate proliferation of guinea pig and human ASM via the ET_A receptor (11, 12). In this study, upregulation of ET-1 in airway epithelial cells was associated with increased rates of BrdU incorporation in ASM and epithelial cells after repeated exposures to allergen. In addition, inhibition of BrdU incorporation by the dual ET_A+B antagonist indicates that ET-1 may contribute to the increased proliferative response in these cells as a result of chronic allergic inflammation. ET-1 may also contribute to ASM thickening by inducing its proliferation as well as by attenuation of ASM cell apoptosis (37). ET-1 may augment ASM cell proliferation by stimulating the generation of cysteinyl leukotrienes. Thus, in vitro, ET-1 stimulates the release of arachidonic acid and its products from alveolar macrophages (38), mast cells (39), pericardial smooth muscle cells (40), and tracheal epithelial cells (41). Cysteinyl leukotrienes may also potentiate ASM proliferation induced by certain growth factors in vitro (42), and in our in vivo model, a CysLT₁ receptor antagonist significantly attenuated ASM DNA synthesis (23).

ET-1 possesses other pro-inflammatory properties, including actions as a chemoattractant for monocytes (43), neutrophils (44), and pulmonary artery fibroblasts (45). In the present study, the ET_A+B antagonist attenuated eosinophil recruitment to the airways after repeated allergen challenge. These findings are supportive of others. Eosinophil recruitment to the lungs is attenuated after allergen challenge in mice with an ET_A receptor antagonist but not an ET_B receptor antagonist (46). Similarly, after instillation of Sephadex into the lungs, pulmonary eosinophilia is inhibited by the ET_A receptor antagonist bosentan (47). Because ET-1 does not cause eosinophil chemotaxis (48), it is likely that ET-1 induces this effect indirectly. One such mechanism may be through formation of cysteinyl leukotrienes, which are chemotactic for human eosinophils both in vitro (49) and in vivo (50). Eosinophils chemoattracted to the airway wall may contribute to epithelial damage by releasing factors such as MBP, eosinophil peroxidase, and reactive oxygen species, which may cause cellular damage and induce proliferation. Adherence of activated eosinophils to the epithelium may also stimulate further release of ET-1 from epithelial cells (51).

The current study allows us to speculate about the relationship between features of airway wall remodeling and bronchial hyperresponsiveness. In the model of chronic allergen exposure at 24 h after cessation of allergen exposure, we detected an increase in ASM and epithelial cell proliferation but have not found increases in ASM thickness measured at 24 h after the last allergen challenge (22). However, in another study, we found an increase when measured 1 wk after the last allergen exposure (23). It is likely that the increase in ASM thickness takes longer than 24 h to establish itself. Bronchial hyperresponsiveness is already present at 24 h, indicating that this may not be related to increases in ASM thickness, which has been implicated as underlying bronchial hyperresponsiveness in airway wall remodeling (3). Other mechanisms may contribute to the bronchial hyperresponsiveness observed in our model at 24 h, but this does not appear to involve the generation of ET-1, the appearance of eosinophils, or the increased proliferative rates of ASM or epithelial cells. Interestingly, ET receptor antagonists attenuate bronchial hyperresponsiveness induced by single allergen exposure in guinea pigs (52) and sheep (53), which may indicate a differential role of ET-1 in underlying bronchial hyperresponsiveness after acute or chronic exposure to allergen. However, the definitive role of ET-1 in airway wall remodeling and bronchial hyperresponsiveness can only be assessed in our model that expresses increases in thickness of the airway smooth muscle resulting from increased proliferation.

In summary, we have demonstrated that chronic allergen exposure leads to an increased expression of ET-1, particularly in the airway epithelium. ET-1 contributes to the proliferative responses of the ASM and airway epithelium, and in eosinophilic inflammation in the airways after chronic allergic inflammation. However, the role of ET-1 in bronchial hyperresponsiveness is less certain. ET-1 may contribute to certain features of airway wall remodeling in asthma.