INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: airway; apoptosis; corticosteroids; epithelium; Bcl-2; Bcl-x_L; Bax

The airway epithelium is a target of inflammatory and physical stimuli in asthma. Injury to the epithelium is a common finding in pathologic studies of patients with asthma, even when the clinical state is mild (1, 2). Epithelial damage as demonstrated on endobronchial biopsy is seen in approximately half of subjects with mild asthma and in almost all subjects with persistent asthma (3). Shedding of epithelial cells and denudation of the airway mucosa is one of the features of chronic airway remodeling, the hallmark of chronic, persistent asthma. Although environmental factors (4), mediators from inflammatory cells such as eosinophils (5, 6), and signals from other constitutive cells within the airway (7) have been implicated in the genesis of epithelial cell loss, the precise mechanism by which this occurs is unclear.

Corticosteroids elicit apoptosis in inflammatory cells such as eosinophils (8) and T lymphocytes (9). This in part explains the potent anti-inflammatory effects of corticosteroids in asthma (10). However, the effect of corticosteroids upon airway epithelial injury and repair is less clear. Corticosteroids impair keratinocyte repair after injury (11) but also protect mammary gland epithelial cells from apoptotic stimuli (12, 13) and inhibit apoptosis induced by Fas ligation in alveolar epithelial cells (14). These differing effects of corticosteroids make prediction of the effects of these agents on central airway epithelium uncertain.

Daily glucocorticoid therapy is considered essential to asthma control. We hypothesized that corticosteroids could cause cell death of airway epithelium, and the resulting loss of epithelial cells might explain in part the damage to and denudation of the airway mucosa in chronic asthma. As a first step to test this hypothesis, we examined whether corticosteroids could elicit apoptosis in primary airway epithelial cells and an airway epithelial cell line that has characteristics consistent with basal epithelial cell morphology. Our data demonstrate that corticosteroids produce apoptosis of airway epithelial cells in a time-dependent and concentration-dependent manner. This process occurs through disruption of mitochondrial polarity with extrusion of cytochrome c and the subsequent activation of caspase-9, followed by the downstream activation of caspase proteases. Corticosteroid-induced apoptosis of these cells can be prevented by the overexpression of apoptotic regulators such as Bcl-2 and Bcl-x_L. Our data raise the possibility that glucocorticoids may have a deleterious role in the ongoing process of airway damage and the airways remodeling of chronic asthma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials

The CD95-activating monoclonal antibody (mAb), CH11, was purchased from PanVera Corp. (Madison, WI), anti-Apaf-1 (AB16941) was purchased from Chemicon International, Inc., and anti-caspase 9 (Ab-2) was purchased from Oncogene Research Products. Fluorescein isothiocyanate (FITC)-conjugated and Cy³-conjugated goat anti-mouse IgG, were purchased from Molecular Probes (Eugene, OR). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling (TUNEL) TACS II fluorescent assay kits and Annexin V assay kits were purchased from Trevigen, Inc. (Rockville, MD). The anti-human CD95 blocking monoclonal antibody ZB4, the anti-human FasL monoclonal antibody NOK1, the anti-cytochrome c antibodies 6H2.B4 and 7H8.2C12, and the peptides acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin (Ac-IETD- fmk) and acetyl-Ile-Glu-Thr-Asp-aldehyde (Ac-IETD-cho) were purchased from Pharmingen, Inc. (San Diego, CA). An antibody to bromodeoxyuridine (BrdU) was purchased from Dako, Inc. (Carpenteria, CA). Fetal calf serum (FCS) was obtained from Hyclone (Logan, UT) and heat-denatured before use. The anti-actin antibody (A2066), dexamethasone, beclomethasone, budesonide, and triamcinolone, and all other reagents were purchased from Sigma, Inc. (St. Louis, MO), and were of the highest purity available.

Culture of the Airway Epithelial Cell Line

The cell line 1HAEo, a gift of Dieter Gruenert (University of Vermont, Burlington, VT), are human airway epithelial cells transformed with simian vacuolating virus 40 (SV40) (15) that have cell surface markers similar to primary airway basal epithelial cells (16). Cells were grown on collagen-IV (10 µg/ml) coated chamber slides in Dulbecco's modified essential medium containing 10% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin G and incubated at 37° C in 5% CO₂. Cells were used when approximately 90% confluent. Slides were washed twice in fresh culture medium, after which medium was replaced. Cells were kept in 10% FCS during all experiments to prevent confounding of apoptosis results by withdrawal of any needed growth factors. Agents were dissolved in either dimethyl sulfoxide (DMSO), ethanol, or phosphate-buffered saline (PBS). Agents or their appropriate vehicle control were added, and cells were incubated for 1 to 24 h at 37° C. In all experiments, DMSO and ethanol never exceeded 0.1% as a final concentration in the culture media. At the conclusion of experiments, chamber slides were washed once in fresh medium after the incubation period and fixed in 10% neutral buffered formalin for further processing.

Culture of Primary Airway Epithelial Cells

Primary normal human bronchial epithelial (NHBE) cells were purchased from Clonetics, Inc. (Walkersville, MD). These cells are derived from a single donor and are supplied as first passage cells. Cells were placed into defined medium (Clonetics) containing 5 µg/ml insulin, 0.5 µg/ml human epidermal growth factor, 10 mg/ml transferrin, 6.5 µg/ml triiodothyronine, 0.5 mg/ml epinephrine, and 2 ml/L bovine pituitary extract. Cells were subcultured and used between passages 3 and 7 when approximately 60% confluent. Experiments were done as for the 1HAEo cell line, except that cells were kept in defined medium and not 10% FCS.

Assay for DNA Nicking

Apoptosis in fixed monolayers was demonstrated by labeling free 3'-hydroxyl groups of DNA using a Trevigen TUNEL fluorescent assay kit. Slides were counterstained in 1 mM Hoechst 33258 for 45 s and visualized immediately by fluorescent microscopy. Representative images were collected using a Sensys 12-bit cooled charge-coupled device (CCD) camera (Photometrics, Inc., Tucson, AZ) connected to a Nikon fluorescence microscope. Fields were selected at random by one investigator who was different from the investigator performing the experiment. For each slide, the well on the microscope stage was approximately centered under the objective without viewing the field through the eyepiece. Images then were collected in registration, and the microscope stage was moved a short distance in a random direction without observation through the eyepiece, and images again were collected. Obviously inappropriate images were discarded (e.g., no visible cells), and the stage was moved again in a random direction if required for additional images. In this manner, observer bias was minimized. TUNEL-positive nuclei and Hoechst-stained nuclei were counted in each image as the area of the nuclei in pixels after visual thresholding and exclusion of extraneous positive pixels using Spectrum IP software (IP Labs, Vienna, VA) on a Macintosh computer. TUNEL-positive cells were expressed as the percentage of the thresholded area of the TUNEL-stained image divided by the thresholded area of the Hoechst-stained image. The TUNEL counts of two fields in the same well were averaged to produce a single n. Previous experiments (17) demonstrated a high correlation with the manual counted colorimetric method, and demonstrated that changes in cell shape or morphology alone did not alter significantly the ability to detect apoptotic nuclei. Preliminary experiments confirmed that TUNEL-positive cells did not have the morphologic features of necrosis, which may also lead to single-strand DNA nicking (18).

Assay for Expression of Phosphatidylserine Residues on the Outer Cell Membrane by Annexin V Labeling

This assay is used to confirm cell apoptosis as determined by TUNEL. Cells were treated with 3 µm dexamethasone for 1 to 6 h and then processed using a commercial kit (Trevigen, Inc.) according to directions. Slides were counterstained in 1 mM Hoechst 33258 for 45 s and visualized immediately by fluorescent microscopy.

Assay for Mitochondrial Membrane Potential

This method depends upon the perturbation of the electrochemical gradient across mitochondrial membranes, an early intracellular change after the onset of apoptosis (19, 20) which correlates well with subsequent measures of apoptosis, including the TUNEL assay (21). The method uses the fluorescent lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (DePsipher dye) to detect disruption of the membrane potential (22). The assay is completed using a kit from Trevigen, Inc. (Rockville, MD), modified such that the final concentration of DePsipher dye was 10 µg/ml. Cells then were imaged immediately by fluorescent microscopy.

Cell Proliferation Assay

Cells were treated with dexamethasone or vehicle controls for 12 or 24 h. In the final 6 h of incubation, cells were treated with a 1:500 final dilution of BrdU (RPN 201; Amersham Pharmacia, Piscataway, NJ), a thymidine analog. Cell monolayers then were fixed in 10% neutral buffered formalin. Staining for the presence of BrdU was performed using clone B44 (Becton Dickinson, Franklin Labs, NJ) FITC-conjugated. After removal of the fixative, the cells were denatured by the addition of 0.07 N NaCl for 2 min. Slides were washed in PBS before blocking with 3% bovine serum albumin (BSA) plus 0.1% Tween. Using a 1:6 dilution of stock antibody, slides were incubated overnight at 4° C. Monolayers were washed three times, counterstained with 1 mM Hoechst 33258 in water for 45 s, and then imaged by fluorescent microscopy. BrdU-positive nuclei and Hoechst-stained nuclei were counted, and the proliferation rate calculated, using the same imaging techniques as for the TUNEL assay.

Western Blot Assay

To obtain total cellular protein, cells were incubated for 15 min at 4° C in 1% Nonidet P-40 (NP-40), 0.25% sodium deoxycholate (Na-DOC), 150 mM NaCl, 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM Na₃VO₄, and 1 mM NaF. Samples were centrifuged at 14,000 rpm for 10 min at 4° C after which supernatants were frozen at 70° C. Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mini-gel and transferred onto nitrocellulose membranes. Immunodetection was performed using an enhanced chemiluminescence (ECL) protocol. In some experiments, membranes were stripped and reprobed with an antibody for actin to normalize differences in protein loading.

Fluorescent Assay for Cytochrome c Extrusion

This method follows that of Kennedy and colleagues (23). After interventions, cell monolayers were fixed in 4% formalin plus 0.2% saponin in PBS. Monolayers were blocked in 4% normal donkey serum with 0.2% Triton-X 100 in PBS (NDS-TBST) for 1 h. A primary mAb directed against cytosol-specific cytochrome c (6H2.B4, Pharmingen, Inc.), used at 1 µg/ml, plus 0.2% saponin in NDS-TBST, was added for 90 min at room temperature (RT). Monolayers were washed twice and then incubated with 1:2,000 Cy³-labeled goat anti-mouse IgG in NDS-TBST for 2 h at RT in the dark. Monolayers were washed three times, counterstained with 1 mM Hoechst 33258 in water for 45 s, and then imaged by fluorescent microscopy.

Identification of Cytochrome c in Cytosolic and Mitochondrial Cell Fractions

After interventions, adherent cells were collected using trypsin and washed in medium minus FCS. Cells then were resuspended (3 × 10⁶ cells/ml) in fractionation buffer (FB: 20 mM HEPES pH7.4, 10 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, 1.5 mM MgCl₂, 250 mM sucrose, 1 mM PMSF, and 0.15 U/ml aprotinin), without detergents so as to keep all cell fractions intact. Resuspended cells were placed in a 2-ml Dounce (Vineland, NJ) cell homogenizer and disrupted with 10 strokes of Pestle B. Disrupted cells were transferred to 1.5-ml microcentrifuge tubes and centrifuged at 3,000 rpm for 10 min at 4° C. The collected supernatant representing the cytoplasm then was centrifuged again at 11,000 rpm for 15 min at 4° C to collect both the cytoplasmic fraction (supernatant) and the mitochondrial pellet. This pellet was resuspended in 100 µl ice-cold lysis buffer (20 mM Tris-HCL pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 1 mM PMSF, and 0.15 U/ml aprotinin) and incubated at 4° C for 30 min while rotating, followed by centrifugation at 11,000 rpm for 10 min. The resulting supernatant contained the mitochondrial proteins. The cytoplasmic fraction was ultracentrifuged at 50,000 rpm for 60 min at 4° C to complete the isolation of cytoplasmic proteins. Representative samples from mitochondrial and cytosolic proteins were denatured by boiling and resolved by Western blot using the 7H8.2C12 antibody (Pharmingen). In each lane, a fixed proportion of each collected fraction (10%) was run to represent a similar number of cells for both mitochondrial and cytoplasmic pools.

Total RNA Extraction and Purification

Total RNA from samples of cultured cells was isolated using a Qiagen (Valencia, CA) RNeasy Mini Kit with deoxyribonuclease (DNase) treatment according to directions. Concentrations of RNA are calculated from A260 nm/A280 nm ratios.

Northern Blot Analysis

A volume of 18 µg of total RNA was resolved by electrophoresis on 1% agarose-formaldehyde gels and transferred to Hybond Nylon membranes (Amersham-Pharmacia) in 10× saline sodium citrate (SSC). Hybridization probes for Bcl-2, Bcl-x_L, and Bax generated as gel-purified EcoRI restriction fragments from plasmids were labeled with Redivue [-³²P] CTP using Random Prime labeling system (Amersham-Pharmacia). Hybridization was carried out overnight at 42° C using standard procedures. The membranes were washed twice for 15 min at RT with 2× SSC/0.1% SDS, and twice for 15 min at 60° C with 0.2× SSC/0.1% SDS. Membranes were exposed for 3 to 24 h at 70° C to Kodak X-omat AR film.

Transfections

Expression vectors SFFV.neo containing the Bcl-2, Bcl-x_L, Bax, and empty vector were generously provided by Charles Rudin, M.D., Ph.D., Section of Hematology and Oncology, University of Chicago. These were purified using the double-spin cesium chloride method. 1HAEo cells cultured in medium as previously noted were grown to 80% confluence (~ 500,000 cells), and transfected with 3 µg of DNA, 15 µl of lipofectamine (Life Technologies, Inc., Rockville, MD), and 800 µl of OptiMEM (Life Technologies) for 6 h at 37° C. Transfection reagents then were removed and replaced with fresh medium. After 48 h cells were treated with 300 µg/ml geneticin to select neomycin- resistant cells. Subclones were selected based on Bcl-2, Bcl-x_L, or Bax expression as determined by Northern blot using the appropriate gene cut from each plasmid as a probe.

Data Analysis

TUNEL data are expressed as mean ± SEM. Differences were treated by F test followed by Fisher's protected least significant difference test, and were considered significant when p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Corticosteroids Elicit Apoptosis of Airway Epithelial Cells in Culture

Treatment of confluent 1HAEo monolayers with 3 µM dexamethasone elicited apoptosis that was increasing up to 24 h (Figure 1A). A concentration-response was demonstrated with significant apoptosis, by TUNEL-positive rates, at doses < 0.3 µM dexamethasone (Figure 1B). Apoptosis elicited by 3 µM dexamethasone was similar in magnitude to that achieved by ligation of CD95 (Fas) using the CH11 monoclonal antibody (Table 1).

View larger version (16K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   View larger version (56K): [in this window] [in a new window]   Figure 1.   Apoptosis in 1HAEo airway epithelial cells after corticosteroid treatment, as demonstrated by TUNEL stain for single-strand DNA nicking. (A) Time response. Cells were treated with 3 µM dexamethasone for 2 to 24 h before fixation and staining. Apoptosis increased through 24 h. *p = 0.02 and §p < 0.001 versus control. n = 4 to 8 in each group. (B) Concentration response. Cells were treated with 0.03 to 10 µM dexamethasone for 24 h. Apoptosis was significant at concentrations  0.3 µM. *p = 0.01, §p = 0.0005, and p < 0.0001 versus control. n = 8 to 10. (C ) Apoptosis after treatment with 0.1 to 10 µM of beclomethasone, triamcinolone, or budesonide for 24 h. Apoptosis was significant after treatment with  3 µM of each corticosteroid. *p = 0.05, §p = 0.01, and p = 0.002 versus control. n = 4 in each series. (D) Representative images of airway epithelial cells in culture after TUNEL (top set of figures) or Annexin V stain (bottom set of figures) to demonstrate apoptotic cells by single-strand DNA nicking or expression of phosphatidylserine residues in the outer cell membrane, respectively. Staining as described in the METHODS. Cells treated for 24 h with either sham diluent or 3 µM dexamethasone are shown. Dexamethasone elicits a greater number of cells that label for both TUNEL and Annexin V. Corresponding changes in nuclear morphology are seen on Hoechst stain images taken in registration. Bar for all images, 5 µM.

Other corticosteroids with clinical utility in the treatment of airway inflammation also elicited apoptosis in the 1HAEo cell line. In these experiments, cells were treated for 24 h with 0.1 to 10 µM of budesonide, beclomethasone, or triamcinolone. A concentration-response was demonstrated with significant apoptosis, by TUNEL-positive rates, at doses  3 µM of each corticosteroid (Figure 1C). The order of potency in these experiments was dexamethasone  budesonide = beclomethasone  triamcinolone.

Apoptosis was confirmed by the demonstration of phosphatidylserine residues on the outer cell membrane as demonstrated by Annexin V labeling (Figure 1D). Cell death after administration of dexamethasone was additionally confirmed in separate experiments by the inability of cells to exclude trypan blue (data not shown). Cells treated with 0.1% ethanol, the dexamethasone diluent, or 0.1% DMSO, the caspase inhibitor diluent, did not demonstrate changes in apoptotic rates from non-vehicle-treated cells. Cells did not demonstrate necrosis, as the concentration of lactate dehydrogenase did not change appreciably in cells treated with dexamethasone compared with control (data not shown).

Dexamethasone also elicited apoptosis in primary airway epithelial cells (Figure 2). The response at each time point to 3 µM dexamethasone was similar in magnitude to that achieved after Fas ligation using the CH11 ligating monoclonal antibody (Figure 2). Additionally, the dose-response and time-response trends in primary cells and immortalized airway epithelial cell lines were similar (compare Figures 1A and 1B with Figure 2). Apoptosis began within 6 h and increased up to 24 h over a similar dose range of dexamethasone (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Apoptosis in primary human airway epithelial cells after corticosteroid treatment or after ligation of the Fas receptor, as demonstrated by TUNEL stain for single-strand DNA nicking. Cells were treated with 0.3 to 10 µM dexamethasone, or 1 µg/ml of the CH11 Fas-ligating mAb, for 6, 12, or 24 h. Apoptosis increased through 24 h and at concentrations of dexamethasone  3 µM, and was equivalent to that seen with Fas ligation. *p < 0.05, §p < 0.02, p < 0.01, and ¶p < 0.005, versus control. n = 2 to 4 experiments.

Corticosteroids Suppress Epithelial Cell Proliferation

The use of glucocorticoids in epithelial cell culture has been associated with increased survival and proliferation (24, 25). These processes may generate false-positive TUNEL staining. To test whether corticosteroid treatment altered airway epithelial cell proliferation, 1HAEo cells were treated with 0.3 to 10 µM dexamethasone for 12 or 24 h. BrdU was added in the final 6 h. There was a time-dependent and concentration-dependent suppression of BrdU incorporation (Table 2). In additional experiments, inhibition of cell-cycle progression by dexamethasone was confirmed by hypotonic propidium iodide treatment of cultured cells. As measured by flow cytometry there was an increase in the hypodiploid content and an increased ratio of diploid to tetraploid chromosome number (2n:4n) (data not shown). These data are consistent with the appearance of apoptotic nuclei and cell cycle arrest at the G₁/S interface after exposure to dexamethasone.

The use of small concentrations of glucocorticoids in culture medium has been associated with increased epithelial cell survival and proliferation (25, 26). In additional experiments, we examined whether glucocorticoids were required for survival of 1HAEo cells, and whether small concentrations of glucocorticoids in such settings would confound our results. In these experiments, cells were grown in serum-free, defined medium that did not contain glucocorticoids. Apoptosis under these conditions was low and increased substantially when dexamethasone was added in concentrations  3 µM (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Concentration response in serum-free, defined medium. Cells grown to confluence in medium without glucocorticoids were treated with 0.03 to 10 µM dexamethasone for 24 h. There was little cell death in control cells, and significant apoptosis in cells treated with dexamethasone. *p = 0.02 and §p = 0.01 versus control. n = 4.

Dexamethasone Treatment and Fas-associated Apoptosis

Both primary airway epithelial and 1HAEo cells express Fas and Fas ligand (FasL) (27). It is possible that corticosteroid treatment could elicit expression and release of soluble FasL, and thereby initiate apoptosis. To test this hypothesis, 1HAEo cells were pretreated with 10 µg/ml of the Fas-blocking mAb ZB4 and 10 µg/ml of the FasL-absorbing mAb NOK1, concentrations demonstrated previously to block completely apoptosis induced by ligation of the Fas receptor with the CH11 antibody (17). This was followed 30 min later with 3 µM dexamethasone for 24 h. Cells blocked and then treated with dexamethasone demonstrated no significant reduction in TUNEL-positive staining compared with cells treated with dexamethasone alone (Table 3). Similarly, the treatment of the airway epithelial cells with dexamethasone and the CD95-activating CH11 antibody did not demonstrate augmented rates of apoptosis (Table 3). These observations demonstrated that corticosteroid-induced apoptosis did not require binding of CD95 for subsequent activation of the apoptotic cascade.

Involvement of Mitochondria in Corticosteroid-induced Apoptosis of Airway Epithelial Cells

Disruption of mitochondrial polarity is a necessary feature of apoptosis elicited by some stimuli, including Fas ligation (28). We asked whether mitochondrial depolarization occurred in response to corticosteroid treatment. Confluent 1HAEo cells were treated with 3 µM dexamethasone for 1 to 24 h, after which cells were loaded with DePsipher dye. As demonstrated by the continued aggregation of dye (red color) (Figure 4), control cells maintained mitochondrial polarity. Cells treated with dexamethasone demonstrated loss of mitochondrial polarity as early as 1 h and significant disruption within 4 h. This perturbation persisted for 24 h, as demonstrated by the dye in its monomeric form (green color) (Figure 4). Disruption of mitochondrial polarity, as noted by monomeric dye, occurred before significant DNA nicking (Figure 1). These data demonstrate mitochondrial involvement in corticosteroid-induced apoptosis of airway epithelium.

View larger version (38K): [in this window] [in a new window]   Figure 4.   Demonstration of mitochondrial membrane potential after corticosteroid treatment of 1HAEo airway epithelial cells. Cells were treated with control medium or 3 µM dexamethasone for 1, 4, or 24 h. Cells then were loaded with DePsipher dye and mitochondrial membrane potential was assessed by fluorescent microscopy. Preservation of polarity, demonstrated by aggregated (red) dye, was lost with permeability transition as demonstrated by the dye in monomeric form (green) in cells treated with dexamethasone for 4 h. Representative of three experiments.

In mitochondrial-facilitated apoptosis, caspase cascade activation is triggered by the formation of a multimeric Apaf-1/ cytochrome c complex to recruit and activate pro-caspase-9 with subsequent effector caspase activation (29, 30). To determine if this pathway was activated, we examined whether cytochrome c was extruded from the mitochondria after corticosteroid treatment. Confluent 1HAEo cells were treated with 3 µM dexamethasone for 1 to 24 h, after which cytochrome c extrusion was demonstrated by fluorescent microscopy. Significant numbers of cells demonstrated extrusion after 1 h of treatment (Figure 5). In additional experiments, the presence of cytochrome c in cytosolic and mitochondrial cell fractions was examined on Western blot. Substantial release of cytochrome c from the mitochondria into the cytoplasm was demonstrated at each time point after treatment with 3 µM dexamethasone, relative to controls at the same time points (Figure 5).

View larger version (44K): [in this window] [in a new window]   View larger version (61K): [in this window] [in a new window]   Figure 5.   Demonstration of cytochrome c extrusion after corticosteroid treatment of 1HAEo airway epithelial cells. (A) Demonstration by fluorescent staining. Cells were treated with either control medium or 3 µM dexamethasone for 1, 4, or 24 h. Cells were fixed and stained using an anti-cytochrome c antibody. Cell nuclei were visualized using Hoechst 33258 before imaging by fluorescent microscopy. Substantial extrusion of cytochrome c from mitochondria is seen after corticosteroid treatment. Extrusion correlates to diffuse cytoplasmic staining for cytochrome c and expected changes for apoptotic nuclei by Hoechst staining (arrows denote representative apoptotic nuclei). Representative of three experiments. (B) Demonstration by Western blot. Cells were treated with control medium or 3 µM dexamethasone for 1, 4, and 24 h. Cytosolic and mitochondrial fractions were prepared by centrifugation and resolved by SDS-PAGE. The abundance of cytochrome c then was determined using an analogous antibody to that used in A. Significant extrusion of cytochrome c is seen at each time point (increased relative abundance in cytoplasmic fraction compared with mitochondrial fraction) after dexamethasone treatment. Significant extrusion of cytochrome c is seen at 1 and 4 h. Representative of four experiments.

Mitochondrial-facilitated apoptosis can be regulated by several proteins, including Bcl-2, Bcl-x_L, and Bax (31, 32). We generated cells that stably overexpressed each of these regulators, and selected subclones on the basis of increased gene expression as demonstrated by Northern blot. Subcloned cells then were treated with sham diluent or 10 µM of dexamethasone for 24 h, after which RNA was collected. Treatment with this corticosteroid did not alter expression of Bcl-2, Bcl-x_L, or Bax in the empty vector cells (Figure 6A). Further, gene expression for the appropriate Bcl family member in each overexpressing subclone was not altered by treatment with dexamethasone (Figure 6A). Subcloned cells were treated with sham diluent or 10 µM of dexamethasone, budesonide, beclomethasone, or triamcinolone for 24 h, and then fixed before the TUNEL assay. Overexpression of either Bcl-2 or Bcl-x_L blocked corticosteroid-induced apoptosis completely as compared with the empty vector transfection controls (Figure 6B). Overexpression of Bax led to apoptosis that was equal to or higher than that seen in cells transfected only with the empty vector plasmid (Figure 6B), though no differences were significant.

View larger version (25K): [in this window] [in a new window]   View larger version (70K): [in this window] [in a new window]   Figure 6.   Apoptosis after corticosteroid treatment in 1HAEo airway epithelial cells stably overexpressing Bcl-2, Bcl-xL, or Bax by TUNEL staining. (A) Northern blot for overexpression of each gene. Cells were transfected as described in the METHODS and subclones were treated with sham diluent or 10 µM dexamethasone (Dex) for 24 h. RNA then was collected from confluent cells. Increased expression is noted for Bcl-2, Bcl-xL, and Bax in each transfected clone. Treatment with 10 µM dexamethasone does not alter the baseline gene expression of Bcl-2, Bcl-xL, or Bax in the empty vector subclone (EV). Similarly, this treatment does not alter the level of expression of the overexpressed genes in the transfected listed gene. (B) Each transfected cell line was treated with corticosteroid (CS), diluent (no CS), or 10 µM of dexamethasone, budesonide, beclomethasone, or triamcinolone (all represented as 10 µM CS) for 24 h and processed for TUNEL stain. *p = 0.05, §p = 0.03, p = 0.01, and ¶p < 0.002, versus empty vector transfected cells for the response to 10 µM corticosteroid within each group. **p = 0.03, §§p = 0.005, p = 0.002, and ¶¶p = 0.0001, versus sham diluent within each group and set of transfected cells. n = 4 experiments in each series except for empty vector transfected cells where n = 7.

Corticosteroids Elicit Activation of Caspase Proteases

To determine if after corticosteroid administration the observed mitochondrial changes were associated with caspase cascade activation and downstream nuclear fragmentation events, caspase cleavage and inhibition were investigated. One early activator of caspase proteases is caspase-9, required for the initiation of the caspase cascade in association with Apaf-1 and cytochrome c as stimuli are collected by mitochondrial activity (29, 30, 33). Apaf-1 was present in confluent 1HAEo cells in all treatment conditions (data not shown). To examine whether pro-caspase-9 was cleaved and activated after corticosteroid treatment, confluent 1HAEo cells were treated with 3 µM dexamethasone for 0-12 h, after which pro-caspase-9 and cleavage fragments were determined by Western blotting. The appearance of cleavage fragments was seen within 4 h after dexamethasone treatment (Figure 7). Additionally, 1HAEo cells were grown to confluence and treated with 3 µM dexamethasone plus either 100 µM of Ac-IETD-fmk or 100 µM Ac-DEVD-cho tetrapeptides for 24 h. These inhibitors of caspase 9 and 3 respectively, significantly attenuated TUNEL-positive staining induced by dexamethasone (Table 4).

View larger version (36K): [in this window] [in a new window]   Figure 7.   Cleavage of pro-caspase-9 in 1HAEo airway epithelial cells after corticosteroid treatment, as demonstrated by Western blot. Cells were treated with 3 µM dexamethasone for 0-12 h. Protein lysates were separated by SDS-PAGE, transferred onto nitrocellulose, and probed for caspase-9 using an antibody that recognized both the pro-form and active fragments. Cleavage products become apparent within 6 h of treatment. Protein equivalent to 2 × 106 cells was loaded into each lane. Membranes then were stripped and probed for abundance of -actin as control (actin).

Role for Transcription/Translation in Dexamethasone-induced Apoptosis

Glucocorticoid receptor (GR) binding generally leads to the expression of gene products by trans-activation. To determine if gene translation was necessary for dexamethasone-induced apoptosis, confluent 1HAEo cells were treated with both 3 µM dexamethasone and 3, 10, or 30 µg/ml cycloheximide for 24 h, followed by TUNEL analysis. Cells treated with both cycloheximide and dexamethasone demonstrated similar TUNEL-positive staining compared with cells treated with dexamethasone alone (Figure 8). These data demonstrated that transcription with translation was not required for the process of dexamethasone-induced apoptosis in these epithelial cells.

View larger version (29K): [in this window] [in a new window]   Figure 8.   Requirement for translation in corticosteroid-induced apoptosis of 1HAEo airway epithelial cells. Cells were treated with both 3 µM dexamethasone plus 3, 10, or 30 µg/ml cycloheximide (CHX) for 24 h. TUNEL-positive cells were similar in all treatment groups. Control and CHX alone as treatments had similar baseline rates of apoptosis. These data demonstrate that dexamethasone-induced apoptosis is independent of translation. *p= 0.02, p < 0.004, and ¶ p < 0.0002 versus control. n = 4 experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Corticosteroids are the mainstay of controller therapy for asthma. Corticosteroids elicit apoptosis of eosinophils (8) and lymphocytes (9), and suppress production and release of both inflammatory eicosanoids and cytokines (34). However, adequate treatment with corticosteroids may not completely prevent airway inflammation, and some patients may continue to have asthma symptoms and a long-term decline in lung function despite their use (35). Although corticosteroids are recognized to elicit deleterious effects in other organ systems, no report to date has suggested that their use in airway disease may be associated with damage to constitutive cells of the airway. In this study we present the first such report, by demonstrating that dexamethasone elicits apoptosis both in primary airway epithelial cells and in the 1HAEo airway epithelial cell line.

Epithelial damage and other changes, including smooth muscle hypertrophy, basement membrane thickening, and submucosal fibrosis are cardinal features of chronic airway remodeling, the hallmark of long-standing persistent asthma. Impaired healing of denuded epithelium may in part be due to abnormal responses to growth factors during repair. Incomplete repair may lead to the continued production of inflammatory cytokines and excessive allergen passage, which in turn may lead to further, deleterious changes in the airways. Corticosteroid therapy does not necessarily reverse epithelial damage seen in asthma. Although corticosteroid therapy clearly suppresses inflammatory cells and mediator release, subjects with severe asthma receiving regular inhaled corticosteroid therapy still have substantial epithelial destruction at all levels (36). Epithelial cells are seen in bronchoalveolar lavage fluid of asthmatics of varying severity, regardless of corticosteroid use (37). The mechanism by which epithelial cell shedding occurs in chronic asthma is not clear. One possibility is that corticosteroid therapy may contribute to and not prevent this shedding. Our data provide support for this hypothesis, though we caution that in vivo confirmation is required.

Less is known about corticosteroid-induced apoptosis in epithelial cell types but the effect may be substantially different from that seen in hemapoietic cells. Mammary luminal cells treated with corticosteroids are resistant to apoptotic stimuli (12, 13), and removal of the steroid promotes cell death by apoptosis. Type I alveolar epithelial cells can be protected from Fas-induced or interferon-induced apoptosis by concurrent corticosteroid treatment (14). Our present data offer a demonstration that lung airway epithelial cells may respond variably.

The mitochondria can function as a cellular sensor of "stress." These stresses, which can be generated by glucocorticoids, may include redox potentials, oxidative effects resulting from external stimuli, altered cytokine production, and protease activation. The mitochondria also can serve as a "central apoptotic executioner" and allow for the convergence of multiple and diverse induction signals into one final common pathway for the initiation phase of apoptosis (30, 33, 38). As such, data presented here demonstrate a path to apoptosis, including mitochondrial depolarization, cytochrome c extrusion, and caspase 9 activation after dexamethasone treatment. Additionally, as the tetra-peptide DEVD inhibits dexamethasone-induced apoptosis, this demonstrates the downstream activation and requirement of the effector protease, caspase-3. This path to apoptosis is prevented by the overexpression of the antiapoptotic mitochondrial proteins Bcl-2 and Bcl-x_L.

One concern in our study is whether the concentrations of corticosteroids used are representative of the in vivo exposure of airway epithelium to inhaled corticosteroids such as beclomethasone or fluticasone. Inhaled corticosteroids must first dissolve in the periciliary fluid layer atop the airway epithelium before entering cells and binding to the glucocorticoid receptor. The average depth of the periciliary fluid layer in the first 10 generations of central airways is approximately 5 to 45 µm. This makes the total volume in these airways approximately 5 ml (39, 40). Inhalation of 200 µg of corticosteroid, assuming 10 to 30% deposition in the central airways (41), dictates a concentration in the sol layer of approximately 2 to 10 µg/ml, or 6 to 30 µM. This is the functional concentration that the apical face of exposed central airway epithelial cells will experience in their microenvironment. Only two studies to date have attempted to identify the local or "tissue" concentrations of inhaled glucocorticoids. Distribution studies of fluticasone and budesonide in human subjects suggest airway mucosal and tissue concentrations in at least the nanomolar range (42, 43). The concentration range of corticosteroids we used (10⁸ to 10⁶ M) thus approximates that which may be attained in vivo.

A second concern in our study is that airway epithelial cell lines in culture may not represent the same phenotype as in a normal trachea. Although some morphologic features of the cell line used in our study are not the same as the pseudostratified columnar epithelium seen in vivo (44), the monolayers are uniform and have surface markers typical of basal airway epithelial cells (16). Our observations are further confirmed by experiments with primary airway epithelial cells that have a similar morphology in culture. However, neither set of cells in culture duplicate in vivo conditions, and confirmation in such in vivo models will be required. An additional, related concern is that the experimental model presented in this study does not necessarily reflect cellular responses in asthma, and that while the data presented support a potential for glucocorticoid-induced apoptosis in airway epithelium in vivo, these data do not demonstrate that such an effect occurs in asthmatic airways. In addition to the concerns about epithelial cell morphology, there may be differences in the response of airway epithelium to corticosteroids in asthmatic compared with normal subjects. Such differences may further be compounded by the variance in epithelial cells collected from asthmatic subjects as it may relate to disease severity and duration, to location within the airway (e.g., near a carina), and to prior treatment with glucocorticoids or other agents (e.g., -adrenergic agonists) that may influence epithelial cell function. Such differences in the response of airway epithelium to glucocorticoids, particularly potentially deleterious responses, need to be addressed specifically in asthmatic subjects.

Finally, concern may be raised as to the magnitude of the response to corticosteroids in this study. Corticosteroid-induced apoptosis in hemopoietic cells in culture is rapid (< 24 h) and near-complete (> 70% of cells) (45). Although the magnitude of the response in airway epithelial cells is substantially lower (~ 10 to 15% of cells with each of the corticosteroids used in our study), the effect of chronic administration of corticosteroids to cells that have a low proliferation index in vivo (46) may become significant over time. This will need to be addressed in appropriate studies of human airways in vivo.

In summary, we demonstrate that corticosteroids elicit apoptosis in primary airway epithelial cells and in the airway epithelial cell line 1HAEo. Apoptosis was elicited in a concentration range similar to that attainable in vivo, was blocked by caspase-inhibitors, was associated with disruption of mitochondrial polarity and activation of both caspase-9 and caspase-3, and could be blocked by overexpressing the apoptotic regulators Bcl-2 and Bcl-x_L. Our data suggest that the use of corticosteroids may be one factor in the airways remodeling and epithelial damage seen in many patients with chronic, persistent asthma.