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
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ABSTRACT |
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airway epithelium; -adrenergic receptor agonist; protein kinase A
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).
-Adrenergic receptor (
-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
-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
-adrenergic agonists inhibit glucocorticoid-induced apoptosis of
eosinophils (30,
39). However, whether
-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
-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
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
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
2-AR agonists may modulate epithelial cell
survival.
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MATERIALS AND METHODS |
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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 -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 -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 16 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.
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RESULTS |
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To examine the role of a -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 0.110 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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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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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 = 68 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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Propranolol, a competitive -AR antagonist, was used to demonstrate
that the inhibition of apoptosis caused by albuterol is due to its binding to
the
-AR. 1HAEo- cells were pretreated with 1030 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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In some cell types such as cardiomyocytes, the -AR may mediate both
pro- and antiapoptotic effects: coupling of the stimulatory G protein
(Gs) to the
-AR is proapoptotic, whereas coupling of the
inhibitory G protein (Gi) to the
-AR is antiapoptotic
(57). To examine whether
-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 0.031 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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-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
-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
-adrenergic agonists did not protect epithelial cells from
Fas-induced apoptosis.
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-AR agonists do not alter translocation of the GR to the
nucleus.
-AR stimulation increases GR translocation in vascular
smooth muscle cells and in fibroblasts
(17). It is possible that the
ability of
-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
-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 1560 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
-AR agonists clearly was not mediated by any downregulation of GR
translocation to the nucleus.
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-AR agonists do not alter transcriptional activity of the
GR. It is possible that
-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 ± 0.110 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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Effect of albuterol may be mediated by PKA. To examine whether the
effect of -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 = 48, 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 = 48, P < 0.001).
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In additional experiments, we examined the effect of a PKA inhibitor, H-89 (22). Cells were pretreated with 0.33 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 = 68, 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.
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DISCUSSION |
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Binding the -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
-AR agonists are commonly used in combination for the treatment of
asthma, it is possible that
-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.
-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
-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 -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
-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 515%. 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
-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
-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 410 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 -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 -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
-AR agonists can
ameliorate one potentially deleterious effect of glucocorticoids on airway
epithelium.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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