{beta}-Adrenergic agonists inhibit corticosteroid-induced apoptosis of airway epithelial cells

Roberta Tse,1 Bertha A. Marroquin,1 Delbert R. Dorscheid,2 and Steven R. White1

1Section of Pulmonary and Critical Care Medicine, Division of Biological Sciences, University of Chicago, Chicago, Illinois 60637; and 2McDonald Research Laboratories and iCAPTURE Centre, University of British Columbia, Vancouver, British Columbia, Canada V6Z 1Y6

Submitted 30 January 2003 ; accepted in final form 22 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Airway epithelial damage is a feature of persistent asthma. Treatment with inhaled and oral corticosteroids may suppress inflammation and gain clinical control despite continued epithelial damage. We have previously demonstrated that corticosteroids elicit apoptosis of airway epithelial cells in culture. {beta}-Adrenergic receptor agonists are commonly used in asthma therapy and can inhibit corticosteroid-induced apoptosis of eosinophils. We tested the hypothesis that {beta}-adrenergic agonists would inhibit corticosteroid-induced airway epithelial cell apoptosis in cultured primary airway epithelial cells and in the cell line 1HAEo-. Albuterol treatment inhibited dexamethasone-induced apoptosis completely but did not inhibit apoptosis induced by Fas receptor activation. The protective effect of albuterol was duplicated by two different analogs of protein kinase A. The protective effect was not associated with increased translocation of the glucocorticoid receptor to the nucleus nor with changes in glucocorticoid receptor-mediated transcriptional activation or repression. We demonstrate that {beta}-adrenergic agonists can inhibit corticosteroid-induced apoptosis but not apoptosis induced by Fas activation. These data suggest that one potential deleterious effect of corticosteroid therapy in asthma can be prevented by concomitant {beta}-adrenergic agonist treatment.

airway epithelium; {beta}-adrenergic receptor agonist; protein kinase A


INJURY TO THE EPITHELIUM is a common finding in pathological studies of patients with asthma, even when the clinical state is mild (4, 31). Epithelial damage as demonstrated on endobronchial biopsy is seen in about half of subjects with mild asthma and in almost all subjects with persistent asthma (54). Although environmental factors (16), mediators from inflammatory cells such as eosinophils (18, 58), and signals from other constitutive cells within the airway (1) have been implicated in the genesis of epithelial cell loss, the precise mechanisms by which this occurs is unclear.

Corticosteroids elicit apoptosis in eosinophils (36, 41) and T lymphocytes (12), mediated through the glucocorticoid receptor (GR) by either the transcriptional activation or repression of genes (25). Binding of ligand causes dimerization and translocation of the receptor to the nucleus (2). The dimerized receptor then binds to glucocorticoid response elements (GRE) in the promoter region of the target genes (50). The exact mechanism by which corticosteroids elicit apoptosis is not known but involves disruption of mitochondrial polarity followed by release of cytochrome c into the cytoplasm and subsequent activation of caspases, which then initiate the sequence of programmed cell death (20, 47). Among the regulators of mitochondrial polarity, and thus apoptosis, is the Bcl family of proteins. Protective regulators such as Bcl-2 and Bcl-xL prevent disruption of mitochondrial polarity and cytochrome c release (20, 40, 52). Proapoptotic regulators such as Bad (Bcl-2/Bcl-xL-associated death promoter) repress Bcl-xL and Bcl-2 function at mitochondrial pores, permitting polarity disruption to occur (59, 60).

{beta}-Adrenergic receptor ({beta}-AR) agonists elicit bronchodilation, suppression of inflammatory mediators, and changes in mucous composition in airways. These responses are mediated by increases in cAMP, which in turn activates protein kinase A (PKA). One potential way in which {beta}-AR agonists may modulate inflammation is by regulating apoptosis. Increases in intracellular cAMP inhibit apoptosis in bone marrow (6), T lymphocytes (24), neutrophils (46), and macrophages (37) and conversely can induce apoptosis in cardiomyocytes (62) and glioma cells (10). Recent studies suggest that {beta}-adrenergic agonists inhibit glucocorticoid-induced apoptosis of eosinophils (30, 39). However, whether {beta}-adrenergic agents antagonize corticosteroid-induced apoptosis of airway epithelial cells is unclear.

We have previously demonstrated that corticosteroids can induce apoptosis of airway epithelial cells (15). Because both {beta}-adrenergic agonists and corticosteroids are used together frequently in asthma therapy, we hypothesized that these agonists could inhibit corticosteroid-induced apoptosis in airway epithelium. We examined whether {beta}2-AR agonists prevent epithelial cell death induced by corticosteroids in primary airway epithelial cells and an airway epithelial cell line. Our data demonstrates that these {beta}2-AR agonists blocked apoptosis. This effect is mediated by PKA but not by changes in transcriptional activation or repression mediated by the GR. These data suggest that {beta}2-AR agonists may modulate epithelial cell survival.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. The Fas/Apo-1/CD95-activating monoclonal antibody CH11 was purchased from PanVera (Madison, WI). A GR antibody (clone P-20) and the FITC-conjugated goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Cy3-conjugated goat anti-rabbit IgG antibody was purchased from Amersham Biosciences (Piscataway, NJ). Terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) TACS II fluorescent assay kits and annexin V assay kits were purchased from Trevigen (Rockville, MD). NE-PER nuclear and cytoplasmic extraction reagents and rhodamine-conjugated goat anti-rabbit IgG were purchased from Pierce (Rockford, IL). Formoterol was a gift of Astra-Zeneca and was dissolved in DMSO for use. Fetal bovine serum was obtained from Hyclone (Logan, UT) and heat-denatured before use. Dulbecco's modified Eagle's medium (DMEM) and phosphate-buffered saline (PBS) were obtained from Mediatech (Herndon, VA). All other reagents were purchased from Sigma (St. Louis, MO).

Plasmids. A full-length rat GR, the LS7 (P493R/A494S) GR mutant (19, 45), XG46TL, and collagenase A-luciferase (ColA-luc) reporter plasmids were generously provided by Michael Garabedian, New York University. These were individually subcloned into the BamHI site of the pCMV-Neo expression vector. We created a control plasmid by cutting the full-length GR using BamHI and religating the vector. The XG46TL reporter plasmid contains two consensus GRE upstream of the thymidine kinase promoter (position -109) fused to a luciferase gene; binding the GRE elicits activation. This was used to examine transcriptional activation of the GR. The ColA-luc reporter plasmid contains the collagenase A promoter fused to a luciferase gene; binding the GRE represses expression of collagenase A (38) and thus luciferase in this assay. This was used to examine transcriptional repression induced by the GR. A pCMV-LacZ plasmid producing {beta}-galactosidase was provided by Blanca Camoretti-Mercado, University of Chicago.

Cell culture. The 1HAEo- cell line, a gift of Dieter Gruenert (University of Vermont, Burlington, VT), are SV40-transformed human airway epithelial cells (13) that have cell surface markers similar to basal cells (14). Cells were grown on collagen IV (10 µg/ml)-coated chamber slides in DMEM 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 ~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.

The use of primary human epithelial cells was approved by the Institutional Review Board at the University of Chicago. Primary human airway epithelial cells were harvested from lungs collected but rejected for use in lung transplantation. Airway sections were incubated in 1% protease in DMEM at 37°C for 2 h, after which epithelial cells were removed from the airway with a soft, plastic spatula. Cells were placed into defined medium (Clonetics, Walkersville, MD) containing 5 µg/ml insulin, 0.5 µg/ml human epidermal growth factor, 10 mg/ml transferrin, 6.5 µg/ml triiodothyrinine, 0.5 mg/ml epinephrine, and 2 ml/l bovine pituitary extract. Cells were subcultured and used between passages 3 and 7 when ~60% confluent. In previous experiments the purity of epithelial cells, as assessed by keratin and vimentin stain, was >99%. Experiments were done as for the 1HAEo- cell line, except that cells were kept in defined medium and not 10% FCS.

Stable transfections. Cells were transfected to overexpress either full-length GR or GR.LS7 by a method we have described previously (15). We selected subclones after six passages on the basis of gene expression on Northern blot assay using the transfected gene as a probe. Transfected cells were maintained in 300 µg/ml geneticin until use.

Immunohistochemistry for GR translocation. After treatment, cells were fixed overnight in 10% neutral buffered formalin. Cells were washed three times in PBS, permeabilized with 0.1% Triton X-100 for 10 min, washed again in PBS, and blocked with 1% BSA and 2% goat serum in PBS for another 10 min. Slides were incubated with an anti-GR antibody in 2% goat serum (1:30) for 1 h at room temperature, washed three times, then incubated with goat anti-rabbit rhodamine in 1% BSA (1:100) for 1 h at room temperature. Cells were counterstained with 1 mM Hoechst 33258 in water for 45 s, then imaged immediately by fluorescence microscopy.

Collection of cytoplasmic and nuclear extracts. Cytoplasmic and nuclear protein were collected using a kit (Pierce) according to directions.

Reporter assays. The XG46TL reporter plasmid was used to assay transcriptional activation, and the ColA-luc reporter plasmid was used to assay transcriptional repression. We transfected cells at 50% confluence with either reporter plus a pCMV-LacZ plasmid using Optimem for 5 h. Cells were washed and 3 h later were incubated for 24 h with 10 µM dexamethasone. Cells were washed twice in PBS and harvested in Reporter lysis buffer (Promega). Luciferase activity was quantified in a reaction mixture containing 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 1 mM ATP, 0.1 mg/ml BSA, and 1 mM dithiothreitol. Luciferase activity was measured in a Bio-Orbit model 1251 luminometer after addition of1mM D-luciferin. Lysates were assayed for {beta}-galactosidase activity as described (9).

Assays for single-strand DNA nicking. We have previously described methods for the TUNEL assay (15, 55). We demonstrated apoptosis in fixed monolayers 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. We collected representative images with a Sensys 12-bit cooled charge-coupled device camera (Photometrics, Tucson, AZ) connected to a Nikon fluorescence microscope. Fields were selected at random by an investigator (not the same investigator performing the experiment). For each slide, we approximately centered the well on the microscope stage 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 (e.g., noncellular debris, or too few cells to count) were discarded, 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 (55) demonstrated a high correlation with manual counting and demonstrated that changes in cell shape or morphology alone do not significantly alter the ability to detect apoptotic nuclei. Preliminary experiments confirmed that TUNEL-positive cells do not have the morphological features of necrosis, which may also lead to single-strand DNA nicking (56).

Annexin V assay. This assay was used to confirm cell apoptosis. Cells were treated with 3 µM dexamethasone for 1–6 h and then processed with a commercial kit (Trevigen) according to directions. Slides were counterstained in 1 mM Hoechst 33258 for 45 s and visualized immediately by fluorescent microscopy.

Western blot. We have previously described this method (15). To obtain total cellular protein, we incubated cells for 15 min at 4°C in 1% Nonidet P-40, 0.25% Na-DOC, 150 mM NaCl, 1 mM EGTA, 1 mM 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 an SDS-PAGE mini-gel and transferred onto nitrocellulose membranes. Immunodetection was performed according to an enhanced chemiluminescence protocol. In some experiments, membranes were stripped and reprobed with an antibody for actin to normalize differences in protein loading.

Data analysis. Data are expressed as means ± SE. Differences were examined by analysis of variance. When significant differences were found, post hoc testing was done by Fisher's protected least significant differences test. Differences were considered significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}-Adrenergic agonists blocked corticosteroid-induced apoptosis in epithelial cells in culture. We have previously demonstrated that corticosteroids elicit programmed cell death in airway epithelial cells (15). To determine if {beta}-AR agonists could block this, 1HAEo- cells grown to 90% confluence were treated with 3 µM dexamethasone alone or concurrently with 0.1–10 µM albuterol for 24 h. Apoptosis was elicited by treatment with dexamethasone as demonstrated by both TUNEL assay and changes in nuclear morphology seen on Hoechst stain (Fig. 1A). TUNEL staining of cells treated with dexamethasone alone was 11.3 ± 1.1 vs. 2.2 ± 0.6% for control (n = 6–8, P < 0.001). Addition of 10 µM albuterol completely inhibited the apoptosis elicited by dexamethasone (4.2% ± 0.6%, n = 10, P < 0.0001). This effect was concentration dependent and was significant at concentrations >0.1 µM (Fig. 1B). Treatment with albuterol alone did not elicit apoptosis. In separate experiments, apoptosis induced by dexamethasone but not dexamethasone plus albuterol was confirmed by the demonstration of phosphatidylserine residues on the outer cell membrane by annexin V labeling (Fig. 1C).



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Fig. 1. A: apoptosis in 1HAEo- airway epithelial cells after dexamethasone (Dex) and albuterol (Alb) treatment as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) stain for single-strand DNA nicking and by nuclear morphology on Hoechst stain. Cells were treated with 3 µM Dex ± 10 µM Alb for 24 h before fixation and staining, after which representative images were collected. Bar, 50 µm for all images. B: apoptosis in 1HAEo- cells after Dex and Alb treatment as measured by TUNEL stain. Cells were treated with 3 µM Dex ± 0.1–10 µM albuterol for 24 h before staining for TUNEL assay. Treatment with Dex elicited apoptosis that was inhibited by concurrent Alb treatment. *P = 0.052, {dagger}P < 0.002, §P < 0.0001 vs. Dex alone; n = 6–10 at each point. C: apoptosis in 1HAEo- cells after Dex and Alb treatment as shown by annexin V stain to demonstrate expression of phosphatidylserine residues in the outer cell membrane. Cells were treated with 3 µM Dex ± 10 µM Alb for 24 h before staining. All slides were stained at the same time with the same reagents. Bar, 50 µm for all images.

 

To examine the role of a {beta}-AR agonist with a significantly longer half-life (34, 44), we treated 1HAEo- cells grown to 90% confluence with 3 µM dexamethasone alone or concurrently with 0.1–10 µM formoterol for 24 h. As in experiments with albuterol, there was a concentration-dependent decrease in apoptosis: treatment with 10 µM formoterol completely inhibited apoptosis elicited by dexamethasone (0.5 ± 0.2% TUNEL-positive cells for 10 µM formoterol plus dexamethasone vs. 4.5 ± 0.5% for dexamethasone alone, n = 4, P < 0.001; Fig. 2).



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Fig. 2. Apoptosis in 1HAEo- cells after Dex and formoterol treatment as measured by TUNEL stain. Cells were treated with 3 µM Dex ± 0.1–10 µM formoterol for 24 h before staining for TUNEL assay. Treatment with Dex elicited apoptosis, which was inhibited by concurrent formoterol treatment. *P = 0.05, {dagger}P <= 0.0003, {ddagger}P < 0.0001 vs. Dex alone; n = 4 at each point.

 

Concurrent treatment with albuterol also blocked dexamethasone-induced apoptosis in primary human epithelial airway cells (Fig. 3A). Primary cells at passage 3 were used in the same manner as described above. Treatment with albuterol in concentrations >0.1 µM completely inhibited cell death (Fig. 3B). TUNEL staining after treatment with dexamethasone alone for 24 h was 9.1 ± 1.5 vs. 2.7 ± 0.7% in cells treated with 3 µM dexamethasone plus 10 µM albuterol (n = 7, P < 0.0001).



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Fig. 3. A: apoptosis in primary airway epithelial cells after Dex and Alb treatment as demonstrated by TUNEL stain for single-strand DNA nicking and by nuclear morphology on Hoechst stain. Cells were treated with 3 µM Dex ± 10 µM Alb for 24 h before staining, after which representative images were collected. Bar, 50 µm for all images. B: apoptosis in primary airway epithelial cells after Dex and Alb treatment as measured by TUNEL stain. Cells were treated with 3 µM Dex ± 0.1–10 µM Alb for 24 h before staining for TUNEL assay. Treatment with Dex elicited apoptosis, which was inhibited by concurrent Alb treatment. *P <= 0.001, {dagger}P < 0.0001 vs. Dex alone; n = 7 at each point.

 

The protective effect of albuterol on dexamethasone-induced apoptosis was lost if albuterol was added >4 h after addition of dexamethasone. In these experiments, 1HAEo- cells grown to 90% confluence were treated with 3 µM dexamethasone. When 10 µM albuterol was added 2 or 4 h later, apoptosis was significantly reduced, but when albuterol was added 6 h later, there was no significant protective effect: 24 h later, there were 4.7 ± 0.8% TUNEL-positive cells compared with 4.6 ± 0.7% TUNEL-positive cells in cells treated with dexamethasone alone and 0.4 ± 0.1% in cells treated with neither agent (n = 6–8 at each data point, P < 0.0001 for control vs. either dexamethasone alone or dexamethasone plus albuterol added 6 h later; Fig. 4).



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Fig. 4. Apoptosis in 1HAEo- cells: effect of late addition of Alb on Dex-induced apoptosis. Cells were treated with 3 µM Dex, and 10 µM Alb was added immediately or 2, 4, or 6 h later. After 24 h, cells were fixed and stained for TUNEL assay. The protective effect of albuterol was lost if added >4 h after addition of Dex. *P = 0.005, {dagger}P = 0.0001 vs. control; n = 6–8 at each point.

 

Propranolol, a competitive {beta}-AR antagonist, was used to demonstrate that the inhibition of apoptosis caused by albuterol is due to its binding to the {beta}-AR. 1HAEo- cells were pretreated with 10–30 µM of propranolol 15 min before addition of 3 µM dexamethasone and 10 µM albuterol and then incubated for 24 h. Apoptosis as measured by TUNEL assay was 7.1 ± 0.3% for cells treated with 30 µM propranolol plus dexamethasone and albuterol vs. 1.4 ± 0.4% for dexamethasone and albuterol only (n = 4, P < 0.0001; Fig. 5).



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Fig. 5. Apoptosis in 1HAEo- cells after pretreatment with 10 or 30 µM propranolol (Prop) for 15 min, followed by treatment with 3 µM Dex and 10 µM Alb for 24 h. Cells then were stained for TUNEL assay. Addition of Prop completely blocked the protective effect of Alb on corticosteroid-induced apoptosis. *P < 0.001, {dagger}P < 0.0001 vs. Dex plus Alb; n = 4 at each point.

 

In some cell types such as cardiomyocytes, the {beta}-AR may mediate both pro- and antiapoptotic effects: coupling of the stimulatory G protein (Gs) to the {beta}-AR is proapoptotic, whereas coupling of the inhibitory G protein (Gi) to the {beta}-AR is antiapoptotic (57). To examine whether {beta}-adrenergic agonists exhibit dual coupling to both Gs and Gi in airway epithelial cells that could mediate both survival and apoptosis, we pretreated 1HAEo- cells with 0.03–1 µg/ml pertussis toxin, an inhibitor of Gi (61), for 15 min followed by 3 µM dexamethasone ± 10 µM albuterol for 24 h. Apoptosis as measured by TUNEL assay was 1.5 ± 0.2% for cells treated with 1 µg/ml pertussis toxin plus dexamethasone and albuterol vs. 1.0 ± 0.2% for dexamethasone and albuterol only [n = 4, P = not significant (NS); Fig. 6]. These data suggest that there is no significant Gi signal being transduced after albuterol treatment.



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Fig. 6. Apoptosis in 1HAEo- cells after pretreatment with 0.03–1 µg/ml pertussis toxin for 15 min, followed by treatment with 3 µM Dex and 10 µM Alb for 24 h. Cells then were stained for TUNEL assay. Pertussis toxin at any concentration did not inhibit the protective effect of Alb, suggesting that inhibitory G protein signaling is not involved (n = 4 at each point).

 

{beta}-Adrenergic agonists did not prevent Fas-induced apoptosis. The death receptor Fas (CD95) and its ligand are expressed in primary central airway epithelial cells and cell lines in culture (21), and they initiate cell death via a rapid, ordered activation of caspases (47). We asked whether {beta}-AR agonists also blocked Fas-mediated apoptosis. To answer this, we treated 1HAEo- cells with 1 µg/ml of the Fas-activating monoclonal antibody CH11 alone or in combination with 10 µM albuterol for 24 h. Apoptosis as measured by TUNEL assay after Fas ligation was 8.5 ± 2.9 vs. 0.1 ± 0.0% for untreated cells (n = 4, P < 0.03; Fig. 7A). Concurrent treatment with CH11 and 10 µM albuterol did not decrease the percentage of TUNEL-positive cells (10.9 ± 2.6%, n = 4, P < 0.01 vs. control and P = NS vs. Fas ligation alone) compared with CH11 treatment alone. In additional experiments, 1HAEo- cells were treated with 1 µg/ml of CH11 alone or in combination with 10 µM formoterol for 24 h. Treatment with formoterol did not alter apoptosis induced by Fas ligation (Fig. 7B). These data suggest that {beta}-adrenergic agonists did not protect epithelial cells from Fas-induced apoptosis.



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Fig. 7. A: apoptosis in 1HAEo- cells after treatment with 1 µg/ml of the CD95-activating CH11 monoclonal antibody ± 10 µM albuterol for 24 h. Cells then were stained for TUNEL assay. Treatment of Alb did not reverse apoptosis induced by Fas ligation. *P < 0.03, {dagger}P < 0.01 vs. control; n = 4 at each point. B: apoptosis in 1HAEo- cells after treatment with 1 µg/ml of the CD95-activating CH11 monoclonal antibody ± 10 µM formoterol for 24 h. Cells then were stained for TUNEL assay. Treatment of Alb did not reverse apoptosis induced by Fas ligation. *P < 0.03 vs. control; n = 4 at each point.

 

{beta}-AR agonists do not alter translocation of the GR to the nucleus. {beta}-AR stimulation increases GR translocation in vascular smooth muscle cells and in fibroblasts (17). It is possible that the ability of {beta}-AR agonists to mediate GR translocation up or down in different cell types might explain differences in the effect of these agonists on final function elicited by corticosteroids. To test this hypothesis, we examined whether {beta}-AR agonists downregulate GR translocation to the nucleus as a mechanism for its protective effect. After treatment with 3 µM dexamethasone alone or with 10 µM albuterol for 15–60 min, cells were fixed and examined for GR localization by immunohistochemistry. GR localization was not different in cells treated with both dexamethasone and albuterol compared with dexamethasone treatment alone (Fig. 8A). Because immunofluorescence might not reveal fine changes in translocation, Western blots were generated to examine GR abundance in nuclear extracts after treatment with 3 µM dexamethasone ± 10 µM albuterol. Abundance of the GR in the nuclear extracts was negligible in untreated cells but increased substantially within 30 min of treatment with either dexamethasone alone or dexamethasone and albuterol (Fig. 8B). These data suggest that the protective effect of {beta}-AR agonists clearly was not mediated by any downregulation of GR translocation to the nucleus.



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Fig. 8. A: representative images of the translocation of the glucocorticoid receptor (GR). Cells were treated with 3 µM Dex ± 10 µM Alb for 30 min, followed with fixation and staining. All slides were stained at the same time with the same reagents, and images were taken with the same exposure times. Clusters of GR become localized to and near the nucleus after treatment with Dex. Concurrent treatment with Alb did not change translocation appreciably. Bar, 10 µm for all images. B: abundance of the GR in nuclear extracts after treatment with Dex and Alb. Cell fractions were collected after treatment for 30 min–6 h with 3 µM Dex ± 10 µM Alb. Nuclear extracts were separated by SDS-PAGE and then immunoblotted for abundance of GR. Membranes were reprobed for abundance of actin to control for lane loading. Addition of albuterol did not change abundance of the GR in nuclear fractions compared with Dex alone.

 

{beta}-AR agonists do not alter transcriptional activity of the GR. It is possible that {beta}-AR agonists may downregulate GR binding to response elements and thus downregulate either transcriptional activation or repression, independently of effects on translocation. To test this, we used reporter assays for both functions. We used the 1HAEo-.GR+ cell line, which stably expressed ectopic GR, to ensure an adequate signal. After transfection with either reporter plasmid, cells were treated with 3 µM dexamethasone ± 10 µM albuterol for 24 h. Repression reporter activity was 18 ± 2% of control after addition of dexamethasone and 31 ± 3% after addition of both agents (n = 3). Activation reporter activity was increased by 12.2 ± 1.3-fold over control after addition of dexamethasone and 12.1 ± 0.6-fold over control after addition of both agents (n = 2). These data demonstrate that the protective effect of albuterol was not mediated by alterations in GR transcriptional ability.

We then examined whether preventing transcriptional activation completely would block the protective effect of albuterol. For these experiments, we used the 1HAEo-.GR.LS7 cell line, which stably overexpressed a GR with deficient trans-activation activity and normal trans-repression activity (45). We first examined GR transcriptional activity in response to 10 µM dexamethasone: in three experiments, repression reporter activity, measured using the XAP1TL plasmid, was 46 ± 1% of no treatment, and activation reporter activity, measured using the XG46TL plasmid, was 1.04 ± 0.07-fold over control. This confirmed the activity of the GR.LS7 mutation expressed in the 1HAEo- cell line. In separate experiments, this cell line was treated with 3 µM dexamethasone ± 0.1–10 µM albuterol for 24 h. Apoptosis elicited by dexamethasone was progressively decreased by albuterol treatment: 5.9 ± 1.3% for 1HAEo-.GR.LS7 cells treated with 3 µM dexamethasone alone vs. 0.5 ± 0.2% for control and 0.5 ± 0.1% for cells treated with dexamethasone and 10 µM albuterol (n = 4 in each group, P < 0.0001; Fig. 9). These data further demonstrate that the protective effect of albuterol was not the result of blocking GR-mediated transcriptional activation.



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Fig. 9. Apoptosis in the 1HAEo-.GR.LS7 cell line after treatment with 3 µM Dex ± 10 µM Alb for 24 h. Cells then were stained for TUNEL assay. Treatment with Alb inhibited apoptosis induced by Dex in trans-activation-deficient cells. *P < 0.0001 vs. Dex alone; n = 4 at each point.

 

Effect of albuterol may be mediated by PKA. To examine whether the effect of {beta}-AR agonists was the result of activation of PKA, we did additional experiments using the agents forskolin and dibutyryl cAMP, both of which activate PKA directly (48, 49). In these experiments, cells were pretreated with either agent for 15 min, followed by dexamethasone for up to 24 h. Treatment with either PKA activator blocked apoptosis induced by dexamethasone (Fig. 10, A and B). Addition of 10 µM forskolin in cells treated with 3 µM dexamethasone decreased the proportion of apoptotic cells to 0.5 ± 0.1 from 4.4 ± 0.8% (n = 4–8, P < 0.001). Addition of 10 µM dibutyryl cAMP in cells treated with 3 µM dexamethasone decreased the proportion of apoptotic cells to 1.0 ± 0.2 from 4.4 ± 0.8% (n = 4–8, P < 0.001).



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Fig. 10. A: apoptosis in 1HAEo- cells after pretreatment with 0.1–10 µM forskolin for 15 min, followed by treatment with 3 µM Dex for 24 h. Cells then were stained for TUNEL assay. *P <= 0.005, {dagger}P < 0.0001 vs. Dex alone; n = 4 at each point. B: apoptosis in 1HAEo- cells after pretreatment with 0.1–10 µM dibutyryl cAMP for 15 min, followed by treatment with 3 µM Dex for 24 h. Cells then were stained for TUNEL assay. *P <= 0.005, {dagger}P < 0.001, {ddagger}P < 0.0001 vs. Dex alone; n = 4 at each point. Treatment with either PKA agonist inhibited apoptosis induced by Dex. C: apoptosis in 1HAEo- cells after pretreatment with 0.3–3 µM of the PKA inhibitor H-89 for 15 min, followed by 3 µM Dex ± 10 µM Alb for 24 h. Cells then were stained for TUNEL assay. *P < 0.001 vs. Dex alone, {dagger}P < 0.0003 vs. Dex plus Alb, {ddagger}P < 0.0001 vs. Dex plus Alb; n = 6–8 at each point. Treatment with H-89 reversed the inhibitory effect of albuterol on Dex-induced apoptosis.

 

In additional experiments, we examined the effect of a PKA inhibitor, H-89 (22). Cells were pretreated with 0.3–3 µM H-89 for 15 min, followed by subsequent addition of 3 µM dexamethasone and 10 µM albuterol for 24 h. Apoptosis as measured by TUNEL assay was 4.4 ± 0.5% for cells treated with 3 µM H-89, dexamethasone, and albuterol vs. 2.1 ± 1.0% for cells treated with dexamethasone and albuterol alone (n = 6–8, P < 0.0001; Fig. 10C). These data, combined with the response to forskolin and dibutyryl cAMP, suggest that the effect of albuterol was mediated via PKA.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Corticosteroids are a main controller therapy for asthma. In addition to suppression of inflammatory mediators and cytokines (27, 33), corticosteroids elicit apoptosis of inflammatory cells such as eosinophils (36) and T lymphocytes (12). Although corticosteroids suppress inflammation of the airways, subjects with severe asthma receiving regular inhaled glucocorticoid therapy may have significant evidence of epithelial damage and shedding (8, 35). Corticosteroids also elicit apoptosis in airway epithelium (15). It is not clear whether epithelial cell apoptosis is required for mucosal repair to proceed in asthma or whether such cell death is deleterious, contributing to chronic damage and airway remodeling seen in some asthma patients.

Binding the {beta}-AR elicits multiple responses in airways, including bronchodilation, changes in ciliary beat frequency, mucous composition, suppression of inflammatory mediators (7, 11), and changes in submucosal blood vessel number (42) and blood flow (43). Balanced against this are effects that are potentially problematic in asthma, such as the reversal of corticosteroid-induced apoptosis in eosinophils (30, 39). Because corticosteroids and {beta}-AR agonists are commonly used in combination for the treatment of asthma, it is possible that {beta}-AR agonists may elicit other responses in the airway epithelium apart from its effects on ciliary beat frequency and mucous rheology. We examined one potential response: the ability to block corticosteroid-induced apoptosis. Our data demonstrate clearly that treatment with albuterol was able to inhibit corticosteroid-induced epithelial cell death. This was true in both primary cells and the 1HAEo- cell line and was both concentration dependent and inhibited by a receptor blocker. {beta}-Adrenergic agonists elicit many of their actions via activation of PKA (32), and PKA mediated the inhibition of apoptosis in our experiments. Two different PKA activators, forskolin and dibutyryl cAMP, blocked dexamethasone-induced apoptosis, and an inhibitor of PKA, H-89, blocked the protective effect of albuterol. The effect required concurrent or near-concurrent treatment with albuterol, because treatment >4 h after treatment with corticosteroid was not protective. This suggests that the effect of {beta}-adrenergic agonists occurs early in the signal transduction mediated by the GR.

After binding ligand, the GR translocates to the nucleus, and this translocation can be upregulated by {beta}-adrenergic agonists in both vascular smooth muscle cells and fibroblasts (17). We were not able to detect increased GR translocation following the addition of albuterol, nor were we able to demonstrate differences in transcriptional activation or repression mediated by the GR. This suggests important cell-type differences in the modulatory role of the {beta}-AR on the GR in airway cells.

A potential concern of our study is that the magnitude of apoptosis elicited by either Fas ligation or corticosteroid treatment is relatively small. This raises a question of whether corticosteroid-induced apoptosis has any significance in asthmatic airways. The proportion of apoptotic epithelial cells in our experiments is ~5–15%. Although this proportion is relatively small compared with apoptosis elicited by the same agents in hemapoietic cells, ongoing epithelial damage over time may lead to a significant degree of airway mucosal damage. One recent study demonstrated the presence of apoptotic epithelial cells in endobronchial biopsies collected from asthmatic subjects, as measured by localization of activated caspase-3, both in subjects treated with inhaled corticosteroids and in subjects not so treated (5). Another recent study (51) has also demonstrated apoptosis in asthmatic airway epithelium collected by endobronchial biopsy, though epithelial cell apoptosis was not seen in a third study (53). Whether apoptosis was contributory to mucosal damage or an early process in mucosal repair cannot be ascertained either in single-point biopsy studies or in studies of cultured cells. To the extent that apoptosis represents ongoing damage and not a necessary, early event in airway repair, understanding how {beta}-AR agonists might prevent or lessen such damage would contribute to a better understanding of how these agents may benefit asthma patients.

A second potential concern relates to the timing of the protective effect of albuterol, an agent with a relatively short half-life of biological effect (~1 h) when inhaled (23), though other effects may be more prolonged, reflecting the innate time required for a given effect to be manifested (29). Formoterol, a {beta}-AR agonist with substantially longer half-life (34, 44), elicited a similar result. Corticosteroid-induced apoptosis of eosinophils requires >=6 h (36) and in airway epithelium requires >=12 h (15), and the half-life of corticosteroid interaction with the GR is 4–10 h (28). Our data suggest that the protective effect of albuterol occurs via signaling an early event that blocks subsequent signaling by the GR.

Another limitation in our study is that cultured primary epithelium and epithelial cell lines in culture may not represent the same phenotype seen in normal trachea. However, the cell lines grew as uniform monolayers and have surface markers typical of basal airway epithelial cells (15). Although our experiments demonstrate a protective effect of {beta}-AR agonists on corticosteroid-induced cell death, further confirmation in in vivo models is needed.

Finally, the concentrations of corticosteroid used in this study represent concentrations at the high end of what might be achieved in the clinical setting (15). A similar analysis of the concentration of albuterol that may be achieved at the apical surface of the central airway epithelium suggests, with 1) a total volume of periciliary fluid in the first 10 generations of airways ~5 ml (3, 26), 2) inhalation of 84 µg of albuterol, and 3) 10% deposition of the delivered dose into the central airways, a final concentration at the apical surface of ~1.5 µg/ml, or 6 µM. Therefore, concentrations of albuterol that could be achieved in a clinical setting could elicit a protective effect similar to that demonstrated in the present study.

In summary, we demonstrated that a {beta}-AR agonist can inhibit corticosteroid-induced apoptosis in primary airway epithelial cells and in the airway epithelial cell line 1HAEo-. The effect of albuterol was concentration dependent, was blocked by propranolol, and was mediated by PKA. Furthermore, this protective effect was not accompanied by changes in GR transcriptional activity. These data suggest that {beta}-AR agonists can ameliorate one potentially deleterious effect of glucocorticoids on airway epithelium.


    ACKNOWLEDGMENTS
 
We thank Dr. Michael Garabedian, New York University, for the GR, GR.LS7, and reporter plasmids used in this study; Dr. Blanca Camoretti-Mercado, University of Chicago, for the LacZ plasmid; Dr. Nicholas Dulin, Dr. Nidhi Undevia, and Dr. Michael Moore, University of Chicago, for advice; Oscar Estrada for technical assistance; and Dr. Julie Hoag and Astra-Zeneca for the formoterol.

This work was presented in part at the 2002 International Conference of the American Thoracic Society, Atlanta, Georgia, May 16, 2002, and as part of an undergraduate biology honors thesis presentation by R. Tse at the University of Chicago, April 11, 2002.

DISCLOSURES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-63300, the Blowitz-Ridgeway Foundation, the American Lung Association of Metropolitan Chicago, and Canadian Institute of Health Research Grant 43898.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. White, Univ. of Chicago, Section of Pulmonary and Critical Care Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637 (E-mail: swhite{at}medicine.bsd.uchicago.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adamson IY, Hedgecock C, and Bowden DH. Epithelial cell-fibroblast interactions in lung injury and repair. Am J Pathol 137: 385–392, 1990.[Abstract]
  2. Adcock IM. Glucocorticoid-regulated transcription factors. Pulm Pharmacol Ther 14: 211–219, 2001.[ISI][Medline]
  3. Anderson SD, Daviskas E, and Smith CM. Exercise-induced asthma: a difference in opinion regarding the stimulus. Allergy Proc 10: 215–226, 1989.[ISI][Medline]
  4. Beasley R, Roche WR, Robets JA, and Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 139: 806–817, 1989.[ISI][Medline]
  5. Benayoun L, Letuve S, Druilhe A, Boczkowski J, Dombret MC, Mechighel P, Megret J, Leseche G, Aubier M, and Pretolani M. Regulation of peroxisome proliferator-activated receptor {gamma} expression in human asthmatic airways. Relationship with proliferation, apoptosis and airway remodeling. Am J Respir Crit Care Med 164: 1487–1494, 2001.[Abstract/Free Full Text]
  6. Berridge MV, Tan AS, and Hilton CJ. Cyclic adenosine monophosphate promotes cell survival and retards apoptosis in a factor-dependent bone marrow-derived cell line. Exp Hematol 21: 269–276, 1998.
  7. Borger P, Hoeskstra Y, Esselink MT, Postma DS, Zaagsma J, Vellenga E, and Kauffman HF. {beta}-Adrenoceptor-mediated inhibition of IFN-{gamma}, IL-3 and GM-CSF mRNA accumulation in activated human T lymphocytes is solely mediated by the {beta}2-adrenoceptor subtype. Am J Respir Cell Mol Biol 19: 400–407, 1998.[Abstract/Free Full Text]
  8. Bousquet J, Jeffery PK, Busse WW, Johnson M, and Vignola AM. Asthma: from bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 161: 1720–1745, 2000.[Free Full Text]
  9. Camoretti-Mercado B, Forsythe SM, LeBeau MM, Espinosa R III, Vieira JE, Halayko AJ, Willadsen S, Kurtz B, Ober C, Evans GA, Thweatt R, Shapiro S, Niu Q, Qin Y, Padrid PA, and Solway J. Expression and cytogenetic localization of the human SM22 gene (TAGLN). Genomics 49: 452–457, 1998.[ISI][Medline]
  10. Chen TC, Hinton DR, Zidovetzki R, and Hofman FM. Upregulation of the cAMP/PKA pathway inhibits proliferation, induces differentiation, and leads to apoptosis in malignant gliomas. Lab Invest 78: 165–174, 1998.[ISI][Medline]
  11. Chong LK, Cooper E, Vardey CJ, and Peachell PT. Salmeterol inhibition of mediator release from human lung mast cells by {beta}-adrenoceptor-dependent and independent mechanisms. Br J Pharmacol 123: 1009–1015, 1998.[Abstract]
  12. Compton MM and Cidlowski JA. Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology 118: 38–45, 1986.[Abstract]
  13. Cozens AL, Yezzi MJ, Kunzelman K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, and Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 38–47, 1994.[Abstract]
  14. Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and White SR. Characterization of cell surface lectin-binding patterns of human airway epithelium. Histochem J 31: 145–151, 1999.[ISI][Medline]
  15. Dorscheid DR, Wojcik KR, Sun S, Marroquin B, and White SR. Apoptosis of airway epithelial cells induced by corticosteroids. Am J Respir Crit Care Med 164: 1939–1947, 2001.[Abstract/Free Full Text]
  16. Dye JA, Adler KB, Richards JH, and Dreher KL. Epithelial injury induced by exposure to residual oil fly-ash particles: role of reactive oxygen species? Am J Respir Cell Mol Biol 17: 625–633, 1997.[Abstract/Free Full Text]
  17. Eickelberg O, Roth M, Lörx R, Bruce V, Rüdiger J, Johnson M, and Block LH. Ligand-independent activation of the glucocorticoid receptor by {beta}2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells. J Biol Chem 274: 1005–1010, 1999.[Abstract/Free Full Text]
  18. Frigas E, Loegering DA, and Gleich GJ. Cytotoxic effects of the guinea pig eosinophil major basic protein on tracheal epithelium. Lab Invest 42: 35–43, 1980.[ISI][Medline]
  19. Godowski PJ, Sakai DD, and Yamamoto KR. Signal transduction and transcriptional regulation by the glucocorticoid receptor. UCLA Symp Mol Cell Biol 95: 197–210, 1989.
  20. Green DR. Apoptotic pathways: paper wraps stone blunts scissors. Cell 102: 1–4, 2000.[ISI][Medline]
  21. Hamann KJ, Dorscheid DR, Ko FD, Conforti AE, Sperling AI, Rabe KF, and White SR. Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium. Am J Respir Cell Mol Biol 19: 537–542, 1998.[Abstract/Free Full Text]
  22. Harada H, Becknell B, Wilm M, Mann M, Huang LJ, Taylor SS, Scott JD, and Korsmeyer SJ. Phosphorylation and activation of BAD by mitochondria-anchored protein kinase A. Mol Cell 2: 413–422, 1999.
  23. Harris JB, Ahrens RC, Milavetz G, Annis L, Ries R, and Hendricker C. Comparison of the intensity and duration of effects of inhaled bitolterol and albuterol on airway caliber and airway responsiveness to histamine. J Allergy Clin Immunol 85: 1043–1049, 1990.[ISI][Medline]
  24. Hoshi S, Furutani-Seiki M, Seto M, Tada T, and Asano Y. Prevention of TCR-mediated apoptosis by the elevation of cAMP. Int Immunol 6: 1081–1089, 1994.[Abstract]
  25. Jaffuel D, Demoly P, Gougat C, Balaguer P, Mautino G, Godard P, Bousquet J, and Mathieu M. Transcriptional potencies of inhaled glucocorticoids. Am J Respir Crit Care Med 162: 57–63, 2000.[Abstract/Free Full Text]
  26. Jayaraman S, Song Y, Vetrivel L, Shankar L, and Verkman AS. Noninvasive in vivo fluorescence measurement of airway-surface liquid depth, salt concentration, and pH. J Clin Invest 107: 317–324, 2001.[Abstract/Free Full Text]
  27. John M, Lim S, Seybold J, Jose P, Robichaud A, O'Connor B, Barnes PJ, and Chung KF. Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma. Am J Respir Crit Care Med 157: 256–262, 1998.[ISI][Medline]
  28. Johnson M. Development of fluticasone propionate and comparison with other inhaled corticosteroids. J Allergy Clin Immunol 101: S434–S439, 1998.[ISI][Medline]
  29. Johnson M. The beta-adrenoceptor. Am J Respir Crit Care Med 158: S146–S153, 1998.[Abstract/Free Full Text]
  30. Kankaanranta H, Lindsay MA, Giembycz MA, Zhang X, Moilanen E, and Barnes PJ. Delayed eosinophil apoptosis in asthma. J Allergy Clin Immunol 106: 77–83, 2000.[ISI][Medline]
  31. Laitinen LA, Heino M, Laitinen A, Kava T, and Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 131: 599–606, 1985.[ISI][Medline]
  32. Liggett SB. Molecular and genetic basis of beta2-adrenergic receptor function. J Allergy Clin Immunol 104: S42–S46, 1999.[ISI][Medline]
  33. Lim S, Jatakanon A, John M, Gilbey T, O'Connor BJ, Chung KF, and Barnes PJ. Effect of inhaled budesonide on lung function and airway inflammation. Assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med 159: 22–30, 1999.[Abstract/Free Full Text]
  34. Lofdahl CG and Svedmyr N. Formoterol fumarate, a new beta 2-adrenoceptor agonist. Acute studies of selectivity and duration of effect after inhaled and oral administration. Allergy 44: 264–271, 1989.[ISI][Medline]
  35. Lozwicz S, Wells C, Gomez E, Ferguson H, Richman P, Devalia J, and Davies RJ. Morphological integrity of the bronchial epithelium in mild asthma. Thorax 45: 12–15, 1990.[Abstract]
  36. Meagher LC, Cousin JM, Seckl JR, and Haslett C. Opposing effects of glucocorticoids on the rate of apoptosis in neutrophilic and eosinophilic granulocytes. J Immunol 156: 4422–4428, 1996.[Abstract]
  37. Messmer UK, Lapetina EG, and Brune B. Nitric oxide induced apoptosis in RAW 265.7 macrophages is antagonized by protein kinase C and protein kinase A activating compounds. Mol Pharmacol 47: 757–765, 1995.[Abstract]
  38. Meyer T, Starr DB, and Carlstedt-Duke J. The rat glucocorticoid receptor mutant K461A differentiates between two different mechanisms of transrepression. J Biol Chem 272: 21090–21095, 1997.[Abstract/Free Full Text]
  39. Nielson CP and Hadjokas NE. Beta-adrenoceptor agonists block corticosteroid inhibition in eosinophils. Am J Respir Crit Care Med 157: 184–191, 1998.[ISI][Medline]
  40. O'Gorman DM and Cotter TG. Molecular signals in anti-apoptotic survival pathways. Leukemia 15: 21–34, 2001.[ISI][Medline]
  41. Ohta K and Yamashita N. Apoptosis of eosinophils and lymphocytes in allergic inflammation. J Allergy Clin Immunol 104: 14–21, 1999.[ISI][Medline]
  42. Orsida BE, Ward C, Li X, Bish R, Wilson JW, Thien F, and Walters EH. Effect of a long-acting {beta}2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 164: 117–121, 2001.[Abstract/Free Full Text]
  43. Proud D, Reynolds CJ, Lichtenstein LM, Kagey-Sobotka A, and Togias A. Intranasal salmeterol inhibits allergen-induced vascular permeability but not mast cell activation or cellular infiltration. Clin Exp Allergy 28: 868–875, 1998.[ISI][Medline]
  44. Rabe KF, Jorres R, Nowak D, Behr N, and Magnussen H. Comparison of the effects of salmeterol and formoterol on airway tone and responsiveness over 24 hours in bronchial asthma. Am Rev Respir Dis 147: 1436–1441, 1993.[ISI][Medline]
  45. Rogatzky I, Hittelman AB, Pearce D, and Garabedian MJ. Distinct glucocorticoid receptor transcriptional regulatory surfaces mediate the cytotoxic and cytostatic effects of the glucocorticoid. Mol Cell Biol 19: 5036–5049, 1999.[Abstract/Free Full Text]
  46. Rossi AG, Cousin JM, Dransfield I, Lawson MF, Chilvers ER, and Haslett C. Agents that elevate cAMP inhibit human neutrophil apoptosis. Biochem Biophys Res Commun 217: 892–899, 1995.[ISI][Medline]
  47. Scaffidi C, Fulda S, Srinivasan C, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, and Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17: 1675–1687, 1998.[Abstract/Free Full Text]
  48. Schwede F, Maronde E, Genieser H, and Jastorff B. Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol Ther 87: 199–226, 2000.[ISI][Medline]
  49. Seamon KB and Daly JW. Forskolin: its biological and chemical properties. Adv Cyclic Nucleotides Protein Phosphorylation Res 20: 1–150, 1986.[ISI][Medline]
  50. Taylor DR and Hancox RJ. Interactions between corticosteroids and {beta}-agonists. Thorax 55: 595–602, 2000.[Free Full Text]
  51. Trautmann A, Schmid-Grendelmeier P, Krüger K, Crameri R, Akdis M, Akkaya A, Bröcker EB, Blaser K, and Akdis CA. T cells and eosinophils cooperate in the induction of bronchial epithelial cell apoptosis in asthma. J Allergy Clin Immunol 109: 329–337, 2002.[ISI][Medline]
  52. Tsujimoto Y and Shimizu S. Bcl-2 family: life or death switch. FEBS Lett 466: 6–10, 2000.[ISI][Medline]
  53. Vignola AM, Chanez P, Campbell AM, Fouques F, Lebel B, Enander I, and Bousquet J. Airway inflammation in mild intermittent and in persistent asthma. Am J Respir Crit Care Med 157: 403–409, 1998.[ISI][Medline]
  54. Vignola AM, Chiappara G, Siena L, Bruno A, Gagliardo R, Merendino AM, Polla BS, Arrigo AP, Bonsignore G, Bousquet J, and Chanez P. Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma. J Allergy Clin Immunol 108: 738–746, 2001.[ISI][Medline]
  55. White SR, Williams P, Wojcik KR, Sun S, Hiemstra PS, Rabe KF, and Dorscheid DR. Initiation of apoptosis by actin cytoskeletal derangement with cytochalasin D and jasplakinolide in human airway epithelial cells. Am J Respir Cell Mol Biol 24: 282–294, 2001.[Abstract/Free Full Text]
  56. Wyllie AH, Kerr JFR, and Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 68: 251–305, 1980.[Medline]
  57. Xiao RP. Cell logic for dual coupling of a single class of receptors to Gs and Gi proteins. Circ Res 87: 635–637, 2000.[Free Full Text]
  58. Yukawa T, Read RC, Kroegel C, Rutman A, Chung KF, Wilson R, Cole PJ, and Barnes PJ. The effects of activated eosinophils and neutrophils on guinea pig airway epithelium in vitro. Am J Respir Cell Mol Biol 2: 341–353, 1990.[ISI][Medline]
  59. Zha J, Harada H, Osipov K, Jockel J, Waksman G, and Korsmeyer SJ. BH3 domain of Bad is required for heterodimerization with Bcl-xL and pro-apoptotic activity. J Biol Chem 272: 24101–24104, 1997.[Abstract/Free Full Text]
  60. Zha J, Harada H, Yang E, Jockel J, and Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factors results in binding to 14–3-3 not Bcl-xL. Cell 87: 619–628, 1996.[ISI][Medline]
  61. Zheng M, Zhang SJ, Zhu W-Z, Ziman B, Kobilka BK, and Xiao R-P. {beta}2-Adrenergic receptor-induced p38 MAPK activation is mediated by protein kinase A rather than by Gi or Gs in adult mouse cardiomyocytes. J Biol Chem 275: 40635–40640, 2000.[Abstract/Free Full Text]
  62. Zhu W-Z, Zheng M, Koch WJ, Lefkowitz RJ, Kobilka BK, and Xiao R-P. Dual modulation of cell survival and cell death by {beta}2-adrenergic signaling in adult mouse cardiac myocytes. Proc Natl Acad Sci USA 98: 1607–1612, 2000.[Abstract/Free Full Text]




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