1 Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115; 2 GSF National Research Center for Environment and Health, Institute for Inhalation Biology, D-85764 Oberschleissheim, Germany; and 3 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19102
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ABSTRACT |
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We have previously reported that interleukin
(IL)-1 decreases responsiveness of cultured human airway smooth
muscle (HASM) cells to
-agonists. The purpose of this study was to
determine whether glucocorticoids inhibit this IL-1
effect. Dexamethasone (Dex;
10
6 M) had no effect on
concentration-related decreases in cell stiffness in response to
isoproterenol (Iso) in control cells as measured by magnetic twisting
cytometry but prevented the decreased responsiveness to Iso observed in
IL-1
(20 ng/ml)-treated cells. In addition, Dex had no effect on
Iso-stimulated cAMP formation in control cells but prevented the
IL-1
-induced reduction in Iso-stimulated cAMP formation. Similar
effects on cell stiffness and cAMP responses were seen after
pretreatment with the glucocorticoid fluticasone proprionate (FP). Dex
and FP also prevented IL-1
-induced hyporesponsiveness to
PGE2 stimulation. In contrast,
neither IL-1
nor glucocorticoids had any effect on cell stiffness
responses to dibutyryl cAMP. We have previously reported that the
IL-1
effect on
-adrenergic responsiveness is mediated through
cyclooxygenase-2 expression and prostanoid formation. Consistent with
these observations, IL-1
-induced cyclooxygenase-2 expression was
virtually abolished by FP at concentrations of
10
10 M and greater, with a
resultant decrease in PGE2
formation. However, Dex did not inhibit IL-1
-induced nuclear
translocation of nuclear factor-
B or activator protein-1 in HASM
cells. In summary, our results indicate that, in HASM cells,
glucocorticoids alone do not alter responses to
-agonists but do
inhibit IL-1
-induced
-adrenergic hyporesponsiveness.
Glucocorticoids mediate this effect by inhibiting prostanoid formation
but without altering nuclear factor-
B or activator protein-1 translocation.
interleukin-1;
2-adrenergic receptor; magnetic
twisting cytometry; adenosine 3',5'-cyclic monophosphate; cyclooxygenase-2; prostaglandin
E2; nuclear factor-
B
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INTRODUCTION |
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DECREASED -ADRENERGIC
RESPONSIVENESS is a characteristic feature of asthma. The
bronchodilator response to
-agonists is reduced in airways of
asthmatic subjects both in vivo (5) and in vitro (4) and in animal
models of asthma (13). The mechanistic basis for this
hyporesponsiveness has not been established, but cytokines released in
the asthmatic airway may contribute to this phenomenon. For example,
interleukin (IL)-1
, a cytokine elevated in bronchoalveolar lavage
fluid of patients with symptomatic asthma (41), decreases
-adrenergic responses in a number of cell types (20, 45). Shore et
al. (39) have recently demonstrated that IL-1
also
decreases the responses of cultured human airway smooth muscle (HASM)
cells to
-agonists.
Glucocorticoids are the mainstay of treatment for asthma. The
mechanistic basis for the efficacy of glucocorticoids is not completely
established but includes their ability to decrease expression of
proinflammatory cytokines and to inhibit the influx and activation of
inflammatory cells. Our hypothesis is that an additional important way
in which glucocorticoids may be effective in asthma is through direct
effects on smooth muscle, which result in inhibition of IL-1-induced
hyporesponsiveness to
-agonists. In support of this hypothesis,
glucocorticoids have been shown to inhibit responses to IL-1
in
other cell systems (9, 10).
To evaluate this hypothesis, we measured the responses of cultured HASM
cells treated with glucocorticoids and/or IL-1 to the
-agonist
isoproterenol (Iso). Responses to Iso were assessed with magnetic
twisting cytometry (MTC), which measures changes in cytoskeletal
stiffness of adherent cells (43, 44). Responses to Iso in these same
groups were also assessed by measuring cAMP formation because cAMP is
the second messenger involved in the airway smooth muscle relaxation
response. We report here that the glucocorticoids dexamethasone (Dex)
and fluticasone propionate (FP) ablated the ability of IL-1
to
decrease HASM cellular responses to Iso using either cell stiffness
responses or cAMP formation as outcome indicators.
Laporte et al. (22) have previously reported that the effect of IL-1
on
-adrenergic responsiveness of HASM cells is mediated through
increased prostanoid formation. We have provided evidence for this
mechanism by showing that IL-1
leads to cyclooxygenase (COX)-2
expression and subsequent PGE2
release in HASM cells, that prolonged exposure of the cells to elevated
levels of PGE2 mimicked the
effects of IL-1
, and that inhibitors of COX-2 blocked IL-1
-induced
-adrenergic hyporesponsiveness as measured by both MTC and cAMP formation. Because glucocorticoids have been shown to
inhibit cytokine-induced COX-2 expression in a number of cell systems
(7, 34), we postulated that glucocorticoid inhibition of prostanoid
formation might be the mechanism by which glucocorticoids inhibit
IL-1
effects in HASM cells. To examine this hypothesis, we measured
the concentration-related effects of glucocorticoids on COX-2 protein
expression and PGE2 formation in
HASM cells treated with IL-1
. We report here that IL-1
-induced
COX-2 expression and PGE2
formation are inhibited by both Dex and FP in a concentration-dependent manner and that this inhibition occurs at concentrations of
glucocorticoid consistent with those that prevent IL-1
-induced
-adrenergic hyporesponsiveness.
Glucocorticoids have been shown to inhibit nuclear factor (NF)-B and
activator protein (AP)-1 DNA binding in a number of cell types,
including those in the human lung (1), and the ability of
glucocorticoids to interact with these transcription factors has been
shown to be a mechanism by which the activated glucocorticoid-receptor
complex has effects independent of binding to glucocorticoid response
elements (27, 36). Because the promoter region of the
COX-2 gene has no
identifiable glucocorticoid response elements (46) but does have
consensus sequences for NF-
B and AP-1 (3, 46), we speculated that
the effects of glucocorticoids on COX-2 expression might occur through
inhibition of IL-1
-induced NF-
B or AP-1 translocation. Our data
suggest that this is not the case in HASM cells.
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MATERIALS AND METHODS |
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Cell culture. Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania (Philadelphia) Committee on Studies Involving Human Beings. Tracheal smooth muscle cells were harvested from the tracheae as previously described (32). Briefly, a segment of trachea just proximal to the carina was dissected under sterile conditions, with ~1 g of wet tissue obtained from each donor. Once the trachealis muscle was isolated, the tissue was minced, centrifuged, and then incubated for 90 min in a shaking bath at 37°C in 10 ml of buffer containing 0.2 mM CaCl2, 640 U of collagenase, 10 mg of soybean trypsin inhibitor, and 10 U/ml of elastase. The cell suspension was filtered through 125-µm Nytex mesh, and the filtrate was washed with an equal volume of cold Ham's F-12 medium supplemented with 10% FCS. The cells were then plated in plastic flasks at 104 cells/cm2 in Ham's F-12 medium with 10% FCS that was also supplemented with 102 U/ml of penicillin, 0.1 mg/ml of streptomycin, 12 mM NaOH, 2.5 µg/ml of amphotericin B, 1.7 mM CaCl2, 2 mM L-glutamine, and 25 mM HEPES. Medium was replaced every 3-4 days, and the cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days.
Under these culture conditions, HASM cells grow, with the population doubling every 1.5 days, and reach confluence in ~9-10 days. The cells grow with the hill-and-valley appearance characteristic of smooth muscle cells in culture and are elongated and spindle shaped with a central nucleus. HASM cells cultured in this manner respond to bradykinin, histamine, substance P, leukotriene D4, platelet-activating factor, thrombin, and, more variably, cholinergic agonists, with increases in intracellular Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation (11, 15, 31, 32). The cells also respond to Iso, PGE2, and forskolin with increases in cAMP (14-16, 32). These observations indicate that the cells in culture have the requisite receptor and/or second messenger systems necessary to support both contractile and relaxant responses. The expression of smooth muscle-specific contractile proteins decreases when smooth muscle cells are placed in culture (14, 15, 30, 32) but increases again once the cells reach confluence. At confluence, the cells stain prominently for smooth muscle-specific actin and myosin. Expression of smooth muscle-specific contractile proteins also increases in response to serum-free medium (30, 33). For these reasons, confluent serum-deprived HASM cells were used in the studies described below.
We have previously reported that HASM cells respond to Iso and PGE2 with a concentration-related decrease in cell stiffness even in the absence of added contractile agonists (39). These results suggest that the cells have some degree of active prestress. Dilator agonists such as Iso also cause a decrease in cell stiffness in HASM cells in which contractile agonists are first added to increase cell stiffness (17). For these reasons, Iso and PGE2 were used to study changes in cell stiffness in the studies described in Experimental protocol.
Specifically, confluent cells were serum deprived and supplemented with 5.7 µg/ml of insulin and 5 µg/ml of transferrin 24-36 h before use. On the day of the experiment, cells were passaged and plated in a serum-free hormone-supplemented medium 1) at 20,000 cells/well on collagen I (500 ng/cm2)-coated bacteriological plastic dishes (96-well Removawells, Immulon 2, Dynatech Laboratories, Chantilly, VA) for use in the MTC experiments or 2) at 100,000 cells/well in 24-well plates for cAMP and PGE2 measurements. Cells in passages 4-7 from nine different donors were used in the studies described in Experimental protocol.
Experimental protocol. We examined the
effects of Dex on cell stiffness, cAMP formation, and
PGE2 release in IL-1-treated HASM cells. For each set of experiments, four flasks of confluent HASM
cells from the same passage of the same cell line were serum deprived
and hormone supplemented as described in Cell
culture. Approximately 5 h later, two flasks were
treated with Dex (10
6 M)
and the other two flasks served as controls. The vehicle in which Dex
was dissolved was added to the control flasks. Three hours after Dex
treatment, one flask in each group was treated with IL-1
(20 ng/ml).
On the morning of the experimental day (20 h after Dex or control
treatment), the cells were passaged and plated at 20,000 cells/well on
collagen-coated Removawells for cell stiffness measurements or at
100,000 cells/well in 24-well plates for measurement of cAMP or
PGE2. In both cases, Dex and IL-1
were readded to the wells after the cells were plated. Cell stiffness measurements were made 2-10 h after plating, alternating between wells of the four groups. cAMP and
PGE2 assays were performed 4.5 h
after plating in the manner described below.
For cell stiffness measurements with MTC, cumulative
concentration-response curves to Iso,
PGE2, or dibutyryl cAMP (DBcAMP) were obtained as follows. First, two to four measurements of cell stiffness were made under baseline conditions. Beginning 1 min after
addition of the desired agonist, two to four measurements of cell
stiffness were again obtained. This procedure was repeated with
increasing concentrations of the agonist over the desired range
(108 to
10
5 M Iso,
10
9 to
10
6 M
PGE2, and
10
4 to 3 × 10
3 M DBcAMP). Only one
agonist was studied per well.
For cAMP measurements, four to eight wells of HASM cells were plated
for each group. Cells were allowed to readhere for 4 h at 37°C, at
which time the medium was replaced with 0.5 ml of PBS
containing 0.1 mM IBMX (to prevent degradation of cAMP by phosphodiesterases) and 300 µM ascorbic acid (to prevent oxidation of
Iso). Thirty minutes later, the cells were either treated with Iso
(107 to
10
5 M) or left untreated to
measure basal cAMP formation. Cells in all wells were incubated for an
additional 10 min and then placed on ice, with ice-cold ethanol (1 ml)
added to lyse the cells. The lysate was centrifuged at 2,000 g for 15 min at 4°C, with the
resulting supernatant removed, evaporated to dryness, and stored at
20°C until assayed. cAMP was assayed with a Rainen cAMP
125I radioimmunoassay kit (NEN,
Boston, MA).
For PGE2 measurements, four to
eight wells of HASM cells were plated for each group. Four hours after
being plated, the cells were washed with fresh serum-free,
hormone-supplemented medium. Fifteen minutes later, the supernatants
were harvested and stored at 20°C until subsequent analysis
with a PGE2 enzyme immunoassay kit
(Cayman Chemical, Ann Arbor, MI).
These experiments were repeated to measure the effects of the
glucocorticoid FP at five different concentrations
(1011 to
10
7 M).
For experiments in which we measured COX-2 protein expression, six
flasks of HASM cells from the same passage of the same donor cells were
grown to confluence and serum deprived. Eight hours later, four of the
flasks were treated with FP, each at a different concentration
(1012 to
10
9 M), and two of the
flasks were left untreated. Then 2 h later, these four flasks were each
treated with IL-1
(20 ng/ml), one flask that did not receive FP was
treated with IL-1
, and the other remained untreated (control).
Approximately 24 h later, cells from these six flasks were harvested
for measurement of COX-2 protein as described in
Western blots for COX-2. This same protocol was used to measure COX-2 protein expression after treatment with Dex. In that set of experiments, five flasks of HASM cells were
used, and three of the flasks were treated with Dex, each at a
different concentration
(10
10 to
10
8 M).
For experiments in which we prepared nuclear extracts and performed
electrophoretic mobility shift assays, four flasks of HASM cells from
the same passage of the same donor cells were grown to confluence and
serum deprived. Nineteen hours later, two of the flasks were treated
with Dex (106 M) and two of
the flasks were left untreated. Then 3 h after Dex treatment, one flask
in each group was treated with IL-1
(20 ng/ml). Approximately 2 h
later, cells from these four flasks were harvested for preparation of
nuclear extracts as described in Nuclear protein
extracts and electrophoretic mobility shift assays for
NF-
B and AP-1.
MTC. MTC was used to measure
cytoskeletal mechanics of adherent cells as previously described (17,
22, 39, 43, 44). In our studies, ferromagnetic beads (4.5 µm in
diameter) were coated with a synthetic Arg-Gly-Asp (RGD)-containing
peptide (50 µg · ml1 · mg
bead
1; PepTite 2000, Telios) by incubation in carbonate buffer (pH 9.0) overnight at
4°C. The beads were then washed twice in serum-free medium
containing 1% BSA. Approximately 5 × 104 beads were added to wells
containing 2 × 104 HASM
cells and allowed to incubate at 37°C for 20 min to permit binding
of beads to cells through integrin receptors that recognize the RGD
sequence. Nonadherent beads were then removed by washing the cells with
serum-free medium.
The wells containing the cells were placed within the magnetometer,
where the beads were then magnetized with a brief 1,000-gauss pulse so
that their magnetic moments were aligned in one direction parallel to
the surface on which the cells were plated. Subsequent application of a
much smaller external magnetic field orthogonal to the first field
applied a magnetic torque (or twisting force) causing the bead to
rotate as a compass needle would. Bead rotation was opposed, however,
by reaction forces that developed within the cytoskeleton to which the
beads were bound through the integrin receptors. MTC measured the
resulting angular rotation (strain) of the magnetic bead in relation to
the applied twisting stress, and the ratio of applied stress to strain
was defined as the cell stiffness. Bead rotation increased with the
strength of the applied twisting field and was inversely proportional
to the resistance of the cell to shape distortion (17, 44). Shear
stress (), which was directly proportional to the strength of the
magnetic twisting field, was defined as torque/bead volume and is
expressed in dynes per square centimeter. Because the angle between the twisting field and the magnetic moment vector of the bead decreased as
the beads rotated toward the twisting field,
decreased with angular
strain;
was calibrated by placing beads in a known viscosity standard and quantifying the relationship between twisting field and
applied stress.
The measurement protocol in this study was that 20 s after pulse
magnetization, the twisting field was applied for 1 min. The angular
strain () caused by the twisting field was defined as the rotation
of beads during this 1-min twist and was determined by measuring the
magnetic field of the beads 20 and 80 s after pulse application
(M20 and
M80, respectively). Therefore,
= cos
1(M80/M20).
Apparent stiffness was defined as the ratio between
and
, both
measured at the end of stress application or 80 s after the initial
pulse magnetization. With the definitions of
and
, apparent
stiffness = (
0cos
)/
. In
this study, the strength of the twisting field was set so that
initially a
0 of 80 dyn/cm2 was applied to the cells.
The magnetic field obtained after pulsing but without the second
imposed twisting field was used to control for the normal decay of the
magnetic field induced by random movement of the beads. This decay was
usually very small when compared with bead rotation due to twisting and
was >5% of the original magnetic field over the 1.5-min period
during which measurements were made.
Western blots for COX-2. Multiple
flasks of HASM cells from the same passage of the same donor cells were
grown to confluence, serum deprived, and, 4 h later, treated with Dex
(1010 to
10
8 M), FP
(10
12 to
10
9 M), or vehicle. Twenty
hours later, the medium was removed, and the cells were washed with
PBS. HASM cells were harvested by a brief exposure to 0.25% trypsin
and 1 mM EDTA and then washed with Ham's F-12 medium. The cells were
incubated with extraction buffer [10 mM
Tris · HCl buffer with 50 mM NaCl, 50 mM NaF, 10 mM
D-serine, 1 mM EDTA, 1 mM EGTA,
1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.2 mM
phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 1 µg/ml of
pepstatin, and 10
2 U/ml of
aprotinin] and then were passed through a 25
-gauge needle. Cell lysates were clarified by centrifugation of the
supernatants at 4,000 g for 10 min to
remove cellular debris. Protein concentrations were determined with a
Bio-Rad (Richmond, CA) dye reagent. Supernatants of the cell lysates
were mixed with equal volumes of loading buffer [0.062 M
Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 5%
2-mercaptoethanol, and 0.01% (wt/vol) bromphenol
blue] and then boiled for 5 min. Equal amounts of solubilized
proteins (100 µg/lane) were separated by SDS-polyacrylamide gel
electrophoresis (125 V for 2 h) on 12% acrylamide Tris-glycine gel
(Novex, San Diego, CA) under nonreducing conditions and transferred
electrophorically (16 V for 1 h) to a nitrocellulose membrane
in transfer buffer (Pierce, Rockford, IL). Recombinant COX-2 (Oxford
Biomedical Research, Oxford, MI) was also loaded on the gel as a
positive control.
After transfer to nitrocellulose, the blot was incubated in blocking solution (10 mM Tris containing 150 mM NaCl, 0.1% Tween 20, and 4% BSA) and then primed with rabbit polyclonal antibody raised to COX-2 protein (dilution of 1:1,000; Oxford Biomedical Research) for 2 h. The blot was then incubated with a goat anti-rabbit IgG linked to peroxidase (dilution 1:20,000; Calbiochem, La Jolla, CA) for 1 h and then visualized by light emission on film with enhanced chemiluminescent substrate (Pierce). The band visualized at 70 kDa was quantified with a laser densitometer. Band density values are expressed in arbitrary optical density units.
Nuclear protein extracts and electrophoretic mobility
shift assays for NF-B and AP-1. Nuclear
extraction was carried out with standard methods (21, 26). Briefly,
confluent HASM cells were harvested by scraping and centrifuging (3,000 rpm for 5 min) at 4°C in PBS containing a protease inhibitor
cocktail (1 µg/ml of aprotinin, 1 µg/ml of leupeptin, 10 µg/ml of
soy bean trypsin inhibitor, and 1 µg/ml of pepstatin). The
supernatant was removed, and the pellet was washed twice with 1 ml of
ice-cold buffer A [10 mM HEPES,
pH 7.9, 1.5 mM MgCl2, 10 mM KCl,
0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride
(PMSF)] and centrifuged as described above. The supernatant was
removed again, and the nuclei were isolated by treating this pellet
with 60 µl of buffer A that also
contained 0.1% Nonidet P-40 for 5 min on ice, followed by
centrifugation at 14,000 rpm for 10 min at 4°C. The crude nuclear pellet was resuspended in 10 µl of buffer
B [20 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 0.42 M NaCl, 0.2 mM EDTA,
pH 8.0, 25% glycerol (vol/vol), 0.5 mM DTT, and 0.5 mM
PMSF] for 15 min on ice and clarified by centrifugation at 14,000 rpm for 10 min at 4°C. This supernatant containing the
nuclear protein extract was subsequently diluted with 10 µl of
modified buffer D [20 mM HEPES,
pH 7.9, 50 mM KCl, 20% glycerol (vol/vol), 0.5 mM DTT, and 0.5 mM
PMSF]. Protein concentrations were determined by the
bicinchoninic acid system. Nuclear extracts were stored at
70°C.
A double-stranded oligonucleotide probe containing the NF-B or AP-1
consensus sequence (Gel Shift Assay System, Promega, Madison, WI) was
end labeled with
[
-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; NEN) with T4 kinase. A total of 4 µg of nuclear
extract was incubated with binding buffer [50 mM
Tris · HCl, 5 mM
MgCl2, 250 mM NaCl, 2.5 mM EDTA,
2.5 mM DTT, 20% glycerol, 0.25 µg poly(dI-dC)] for 10 min at
room temperature and then with radiolabeled probe for another 20 min at
room temperature. The reaction volume was held constant at 10 µl.
Where indicated, unlabeled competitive oligonucleotide (NF-
B or
AP-1) or an irrelevant oligonucleotide (SP1) was added 10 min before
addition of the radiolabeled probe. DNA-protein complexes were resolved
by electrophoresis on a 6% nondenaturing polyacrylamide gel (Novex,
San Diego, CA) at 65 V. The gels were then dried and exposed to X-ray
film by audioradiography, which was quantified by densitometry.
Reagents. Tissue culture reagents and
drugs used in this study were obtained from Sigma (St. Louis, MO), with
the exception of amphotericin B and trypsin-EDTA solution, which were
purchased from GIBCO BRL (Life Technologies, Grand Island, NY);
IL-1, which was acquired from Genzyme (Cambridge, MA);
PGE2, which was bought from BIOMOL
(Philadelphia, PA); Dex, which was purchashed from Calbiochem (La
Jolla, CA); and FP, which was a gift from GlaxoWellcome (Research
Triangle, NC). FP was dissolved at
10
2 M in dimethylacetamide
with 1% Tween 80. PGE2 was
dissolved at 10
2 M in DMSO
(with a final concentration of DMSO of <0.1% in the wells). Dex was
dissolved at 10
1 M in DMSO
(again, with final concentration of DMSO of <0.1% in the wells).
DBcAMP was dissolved at 10
1
M in distilled water. The concentrated FP,
PGE2, and DBcAMP were each frozen
in aliquots and diluted appropriately in medium on the day of use. Iso
was dissolved at 10
1 M in
distilled water each experimental day, and because Iso is rapidly
oxidized, dilutions of Iso in medium were made immediately before the
cells were treated.
Statistics. The effect of the
combination of glucocorticoids (Dex or FP) and IL-1, glucocorticoid
alone, or IL-1
alone on baseline cell stiffness measurements,
baseline cAMP formation, and PGE2
release was examined by ANOVA, with experimental day and drug treatment
as main effects. The effect of the combination of glucocorticoids (Dex
or FP) and IL-1
, glucocorticoid alone, or IL-1
alone on changes
in stiffness induced by agonists was examined by repeated-measures
ANOVA, again with experimental day and drug treatment as main effects.
The effects of drug treatment on cAMP formation induced by agonists was
assessed by ANOVA, with experimental day, drug treatment, and agonist
dose as main effects. The effects of IL-1
treatment on NF-
B and
AP-1 translocation compared with those in control cells, was assessed
by paired t-tests for optical
densitometry measurements. The Bonferroni rule was used to correct for
multiple comparisons. A P value < 0.05 was considered significant.
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RESULTS |
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Figure 1 shows cell stiffness changes
induced by Iso in control HASM cells and in cells pretreated with
IL-1 (20 ng/ml for 20 h) alone, Dex
(10
6 M for 23 h) alone, and
a combination of IL-1
and Dex. Baseline cell stiffness did not
differ significantly among these groups. Baseline stiffness averaged
129 ± 8.8 dyn/cm2 in control
cells (n = 23), 134 ± 6.3 dyn/cm2 in IL-1
-treated cells
(n = 23), 131 ± 6.8 dyn/cm2 in Dex-treated cells
(n = 23), and 128 ± 5.7 dyn/cm2 in cells
(n = 24) treated with the combination
of IL-1
and Dex. In addition, neither IL-1
, Dex, nor their
combination affected the rate of magnetic field decay when no twisting
field was applied. In control cells, Iso caused a dose-related decrease
in cell stiffness. This decrease in cell stiffness was reversible with
removal of Iso (data not shown). However, as previously reported (39), in cells pretreated with IL-1
, cell stiffness decreased lessin response to all concentrations of Iso examined than in control cells
(P < 0.001 by repeated-measures
ANOVA). Pretreatment with Dex alone did not alter Iso-induced cell
stiffness changes in control cells. In contrast, Dex restored
-adrenergic responsiveness to cells treated with IL-1
.
Iso-induced decreases in cell stiffness were not significantly
different in cells treated with IL-1
and Dex compared with cells
treated with Dex alone but were significantly different from the
response obtained in cells treated with IL-1
alone
(P < 0.001 by repeated-measures
ANOVA).
|
To determine whether the ability of Dex to restore -adrenergic
responsiveness to IL-1
was specific to Dex or represented a general
effect of glucocorticoids, we repeated these experiments with FP and
examined the concentration dependence of the response to FP
(10
11 to
10
7 M; Fig.
2). The results with FP were similar to the
results with Dex. FP at higher concentrations
(10
9 to
10
7 M) restored
-adrenergic responsiveness to cells treated with IL-1
; in cells
pretreated with the combination of FP
(10
9 to
10
7 M) and IL-1
, Iso
caused a dose-related decrease in cell stiffness that was not
significantly different from the cell stiffness response induced by Iso
in cells treated with FP alone (Fig. 2,
C-E)
but was significantly different from the response obtained in
IL-1
-treated cells (P < 0.005 for
10
8 and
10
7 M FP;
P < 0.05 for
10
9 M FP, both by
repeated-measures ANOVA). FP
(10
10 M) had an
intermediate effect on the reduced
-adrenergic responsiveness induced by IL-1
. Specifically, in cells pretreated with FP
(10
10 M) and IL-1
, Iso
caused a dose-related decrease in cell stiffness that was not
significantly different from the cell stiffness response in the
FP-treated cells (Fig. 2B) or the
IL-1
-treated cells. However, FP
(10
11 M) did not inhibit
the IL-1
effect. Pretreatment with FP
(10
11 M) and IL-1
caused
a significant reduction in the cell stiffness response to Iso compared
with the cell stiffness response in cells treated with FP alone
(P < 0.05 by repeated-measures
ANOVA; Fig. 2A). In these cells, the
response to Iso was not significantly different from the response
obtained in cells treated with IL-1
alone. As with Dex, FP at any
concentration had no effect on baseline cell stiffness or on cell
stiffness responses to Iso.
|
To determine whether the ability of glucocorticoids to inhibit
IL-1-induced
-adrenergic hyporesponsiveness was specific to
-agonists or was also observed with other agonists that have the
capacity to cause smooth muscle relaxation, we examined the effect of
the combination of Dex and IL-1
on cell stiffness responses to
PGE2 and DBcAMP, a cell-permeant
analog of cAMP. As previously observed (39), in this set of
experiments, PGE2 caused a
dose-related decrease in cell stiffness, and this response to
PGE2 was virtually abolished in
cells pretreated with IL-1
(P < 0.001 by repeated-measures ANOVA). Dex
(10
6 M) alone did not alter
the dose-related decrease in cell stiffness induced by
PGE2 in control cells but restored
responsiveness to PGE2 in cells
pretreated with IL-1
(Fig.
3). Cell stiffness responses to DBcAMP were not significantly different among cells treated with Dex
(10
6 M), IL-1
, or their
combination (Fig. 4). Similar results were obtained with FP (10
7 M;
data not shown).
|
|
IL-1 has been previously shown (36) to cause a slight elevation in
basal cAMP levels in HASM cells but to substantially reduce Iso- and
PGE2-induced increases in cAMP
formation. To determine whether glucocorticoids altered the effect of
IL-1
on basal cAMP levels and on cAMP formation in response to Iso,
HASM cells were pretreated with the combination of Dex
(10
6 M for 23 h) and
IL-1
(20 ng/ml for 20 h), Dex alone, or IL-1
alone or were
untreated (control). ANOVA indicated a significant effect of drug
treatment on baseline cAMP (P < 0.003; Fig. 5). Follow-up analysis
indicated that the treatment effect lay in the cells treated with
IL-1
in which cAMP levels were significantly increased above those
in cells of the other three groups (P < 0.05 in each case). In contrast, there were no significant
differences in baseline cAMP among the other three groups. Similar
results were obtained with
10
7 and
10
8 M FP (data not shown).
|
We also measured cAMP formation in response to Iso
(107 to
10
5 M) in these same four
groups of HASM cells (Fig. 5). ANOVA indicated a significant effect of
Iso dose (P < 0.001) as well as a
significant effect of drug treatment
(P < 0.001) on cAMP formation.
Follow-up analysis indicated that the drug treatment effect lay in the
cells treated with IL-1
alone in which Iso-induced cAMP formation
was significantly less than in all three other groups
(P < 0.002 in each case), whereas
the cAMP responses in the three other groups were not significantly
different from each other. Thus Dex alone did not alter Iso-induced
cAMP formation compared with that in control cells but did prevent the
decrease in cAMP formation observed in cells treated with IL-1
.
Similar results were obtained with PGE2 as the relaxant agonist and
when cells were pretreated with FP
(10
8 and
10
7 M; data not shown).
Because this IL-1 effect on
-adrenergic responsiveness is
mediated through COX-2 expression and prostanoid formation (22), we
wanted to determine whether glucocorticoids inhibited, in a concentration-dependent manner, the increase in COX-2 protein expression and resultant PGE2
formation that is observed in HASM cells treated with IL-1
. To
measure COX-2 expression, HASM cells were treated with the combination
of FP at increasing concentrations (10
12 to
10
9 M for 24 h) and IL-1
(20 ng/ml for 22 h) or IL-1
alone or were untreated (control). After
the proteins were isolated from these cells, COX-2 expression was
detected by Western immunoblot analysis. Figure
6 shows a representative Western blot from
a single experimental day. No COX-2 expression was observed in control
cells, but IL-1
induced prominent COX-2 expression. FP caused a
dose-dependent inhibition of IL-1
-induced COX-2 expression (Fig.
6A). When quantified by laser
densitometry for 4 experimental days, this inhibition by FP of COX-2
expression was significant at the two higher concentrations (10
10 to
10
9 M; Fig.
6B). To measure
PGE2 release, HASM cells were
treated with the combination of FP
(10
10 or
10
8 M for 24 h) and IL-1
(20 ng/ml for 22 h), FP alone, or IL-1
alone or were untreated
(control). IL-1
caused a marked increase in basal
PGE2 release compared with that in
control cells. FP did not alter basal
PGE2 release in control cells but
at both concentrations of FP inhibited the marked increase in
PGE2 formation induced by IL-1
(Fig. 7). Dex at higher
concentrations (10
10 to
10
8 M) also caused a
dose-dependent inhibition of IL-1
-induced COX-2 formation and
PGE2 release that was not observed
at lower concentrations (10
12 to
10
11 M).
|
|
Because glucocorticoids have been shown to inhibit NF-B and AP-1 DNA
binding in a number of cell types, including those in the human lung
(1), and because the promoter region of the COX-2 gene has consensus sequences for
both NF-
B and AP-1 (3, 46), we wanted to determine whether
glucocorticoids inhibit IL-1
-induced NF-
B and AP-1 nuclear
translocation in HASM cells. In this set of experiments, HASM cells
were treated with the combination of Dex
(10
6 M for 5 h) and IL-1
(20 ng/ml for 2 h), Dex alone, or IL-1
alone or were untreated
(control). With nuclear protein extracts obtained from these four
groups, electrophoretic mobility shift assays for NF-
B and AP-1 were
performed. Figure 8 shows a representative gel shift assay for AP-1. IL-1
caused an increase in nuclear translocation of AP-1 compared with that in control cells. Dex alone
did not affect AP-1 translocation in control cells nor did it inhibit
this IL-1
-induced AP-1 translocation (Fig.
8A). When quantified by laser
densitrometry for 5 experimental days, IL-1
caused a significant
increase in the nuclear translocation of NF-
B (Fig.
8B) and AP-1 (Fig.
8C), and this IL-1
-induced
translocation of both NF-
B and AP-1 was not inhibited by Dex.
|
![]() |
DISCUSSION |
---|
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---|
Our results indicate that the glucocorticoids Dex and FP ablate
IL-1-induced hyporesponsiveness of HASM cells to the
-agonist Iso
whether relaxation in cell stiffness (Figs. 1 and 2) or cAMP formation
in response to Iso was measured (Fig. 5). Similar results were obtained
with PGE2 as the dilating agonist
(Fig. 3). In contrast, neither glucocorticoid, IL-1
, nor their
combination had any effect on the ability of DBcAMP to decrease cell
stiffness (Fig. 4). Glucocorticoids also inhibited IL-1
-induced
COX-2 expression (Fig. 6) and PGE2
formation (Fig. 7) in a concentration-dependent manner at the same
concentrations at which IL-1
-induced
-adrenergic hyporesponsiveness is inhibited. Glucocorticoids have no effect on
IL-1
-induced NF-
B or AP-1 DNA binding (Fig. 8).
Glucocorticoids are the mainstay of treatment for asthma. The precise
mechanistic basis for the efficacy of glucocorticoids has not been
established but may include decreasing expression of a number of
proinflammatory cytokines and inhibition of the influx and activation
of inflammatory cells. Shore et al. (39) have previously shown that in
HASM cells IL-1 decreases cell stiffness and cAMP responses to Iso.
We now demonstrate that glucocorticoids inhibit this IL-1
effect in
response to Iso. These results indicate that in addition to their
anti-inflammatory effects, glucocorticoids may also have direct effects
on airway smooth muscle cells that are important in reversing
inflammatory bronchoconstriction.
Responses to Iso were assessed by measuring cAMP and measuring cytoskeletal stiffness with MTC. MTC as performed here measures the resistance to shape distortion of the cytoskeleton, including actin and myosin. Theoretical modeling studies of such networks indicate that increasing the interconnectedness of the members, as would occur during actomyosin interactions, increases the stiffness of the network (40). Indeed, using MTC, Hubmayr et al. (17) have previously shown that contractile agonists, known to increase intracellular calcium or Ins(1,4,5)P3 formation in HASM cells, increase cytoskeletal stiffness and that the rank order of stiffness responses to these agonists reflects their ability to increase Ca2+. In addition, agonists known to increase cAMP or cAMP formation decrease cell stiffness (17). Similar results have been obtained with vascular smooth muscle cells (23). Transfection of NIH/3T3 fibroblasts with a tonically active myosin light chain kinase resulted not only in increased myosin phosphorylation but also in increased cell stiffness as measured by MTC, whereas cells transfected with an empty plasmid, where there was no increase in myosin phosphorylation, had no increase in cell stiffness (8). However, although stiffness measurements by MTC most likely reflect the effects of contractile and dilator agonists on actomyosin interactions, stiffness is likely to be a surrogate marker rather than a direct measure of the contraction and shortening of smooth muscle.
Iso and PGE2 mediate relaxation in
smooth muscle cells through a second messenger system involving cAMP.
Specifically, Iso acts on
2-adrenergic receptors that
couple to the stimulatory protein
Gs, the
-subunit of which
activates adenylyl cyclase to produce cAMP.
PGE2 also causes increased cAMP
formation, presumably through activation of
PGE2 receptors (EP2 or EP4), which
also couple to Gs (28, 38).
Increased cAMP activates protein kinase (PK) A, and potentially PKG as
well, which results in relaxation of airway smooth muscle cells through
effects on K+ channels,
Na+-K+-ATPase,
Ca2+ sequestration,
Ca2+ sensitivity of myosin, and
Ins(1,4,5)P3
formation. We demonstrated that the reduction in cell stiffness
responses to Iso, observed when cells are treated with IL-1
, is
ablated by glucocorticoids. Measuring cell stiffness responses to
DBcAMP, a cell-permeant analog of cAMP that directly activates PKA,
helps identify where IL-1
and glucocorticoids alter
-adrenergic
responses. Neither IL-1
, glucocorticoids, nor their combination
affect cell stiffness responses to DBcAMP, indicating that IL-1
and
glucocorticoids do not influence either the ability of cAMP to activate
PKA or PKG or the targets of PKA and PKG activation that ultimately
decrease cell stiffness. Furthermore, IL-1
attenuates Iso-induced
cAMP formation, and glucocorticoids restore the ability of cells
treated with IL-1
to augment cAMP formation in response to Iso.
Taken together, these results suggest that the glucocorticoid effects on cell stiffness responses to Iso and
PGE2 in IL-1
-treated cells are
likely to involve cAMP formation rather than cAMP action.
Glucocorticoids not only inhibit IL-1-induced hyporesponsiveness to
Iso but also inhibit IL-1
-induced hyporesponsiveness to
PGE2 (Fig. 3). Although the
mechanism through which IL-1
can attenuate the
2-adrenergic
response involves COX-2 expression (22), presumably
through PKA phosphorylation of
-adrenergic receptors, we do not know
the mechanism of IL-1
effects on receptors for
PGE2. Bastepe and Ashby
(6) demonstrated that a mutant form of the
PGE2 receptor EP4 displayed
sustained activation of adenylyl cyclase on exposure to
PGE2, whereas the wild-type
receptor showed decreased cAMP production over 20 min (6). Because the mutant receptor lacked 36 of the 38 serine and threonine residues in
the long cytoplasmic carboxy terminus present in the wild type, they
speculated that phosphorylation of the receptors may be involved in the
hyporesponsiveness that they observed. Perhaps IL-1
-induced phosphorylation of EP2 and EP4 receptors is also responsible for hyporesponsiveness to prostanoid stimulation. Thus glucocorticoids may
inhibit IL-1
-induced hyporesponsiveness to
PGE2 by the same mechanism with
which they inhibit IL-1
-induced
-adrenergic hyporesponsiveness.
Shore et al. (39) have previously reported that there is no evidence of
the involvement of Gi or changes
in Gs expression in the
IL-1-induced decreases in HASM cell responses to Iso. In addition,
the effect of IL-1
occurs without altering the expression or
activity of adenylyl cyclase because forskolin-stimulated cAMP formation is not different in IL-1
-treated cells from that in control cells. Instead, our results indicate that IL-1
-induced
-adrenergic hyporesponsiveness is mediated through increased prostanoid formation (22). Specifically, we show that IL-1
results
in COX-2 protein expression and
PGE2 formation in HASM cells, that
PGE2 mimics the effects of IL-1
on the responses to Iso, and that inhibitors of COX-2 block
IL-1
-induced
-adrenergic hyporesponsiveness. The elevation in
PGE2 elevates basal cAMP, as we
have demonstrated, which presumably activates PKA and ultimately results in phosphorylation and desensitization of the
-adrenergic receptor. A report by Pang et al. (35) confirms these observations.
Recent studies (7, 34, 35) have demonstrated that Dex at high
concentrations (106 to
10
5 M) inhibits
IL-1
-induced COX-2 expression and
PGE2 synthesis in HASM cells. Our
work extends these observations by showing that Dex and FP, both at
concentrations as low as
10
10 M, inhibit these
IL-1
effects. Given the demonstrated role of COX-2 expression in
IL-1
-induced
-adrenergic hyporesponsiveness, the observation that
both Dex and FP inhibited IL-1
-induced COX-2 expression and
PGE2 formation is sufficient to
explain the effects of glucocorticoids on IL-1
-induced
-adrenergic hyporesponsiveness. The observation that the
concentrations of FP that inhibited COX-2 expression and
PGE2 formation were very similar
to the concentrations of FP that inhibited IL-1
effects on cell
stiffness responses further supports the hypothesis that
glucocorticoids inhibit IL-1
effects on
2-adrenergic
responses in HASM cells through their effects on COX-2 expression.
Glucocorticoids have been shown to inhibit NF-B and AP-1 DNA binding
in a number of cell types, including those in the human lung (1),
although in a more recent report, Newton et al. (29) saw no effect of
Dex on NF-
B binding in a transfected epithelial cell line. The
ability of glucocorticoids to interact with the nuclear transcription
factors NF-
B and AP-1 has been shown to be a mechanism by which the
activated glucocorticoid-receptor complex has effects independent of
binding to glucocorticoid response elements (27, 36). Because the
promoter region of the COX-2 gene has
no identifiable glucocorticoid response elements (46) but does have
consensus sequences for NF-
B and AP-1 (3, 46), we speculated that
the effects of glucocorticoids on COX-2 expression might occur through
inhibition of IL-1
-induced NF-
B or AP-1. However, this is not the
case. We now report that in HASM cells IL-1
causes nuclear
translocation of NF-
B (Fig. 8B).
Tumor necrosis factor-
has similar effects (2). IL-1
also causes
AP-1 translocation (Fig. 8C).
However, Dex at maximally effective concentrations with respect to
COX-2 expression does not inhibit these IL-1
-induced responses.
These findings suggest that the effects of glucocorticoids in HASM
cells do not involve interference with NF-
B or AP-1 binding.
Although glucocorticoids do not alter NF-B or AP-1 translocation in
HASM cells, they have been shown to have other transcriptional effects
that could account for their inhibition of IL-1
-induced COX-2
expression. For example, Dex has been shown to induce the human
adenovirus E4 protein binding protein (E4BP4), which negatively regulates COX-2 expression in ID13 mouse fibroblasts (42). The role of
E4BP4 has not been investigated in HASM cells. Because electrophoretic
mobility shift assays measure translocation only, it is also possible
that gene expression by Dex may be regulated not in the binding of
NF-
B or AP-1 DNA but rather through transcriptional modulation after
its binding (18). In addition, glucocorticoids have been shown in
airway epithelial cells to induce expression of intracellular
IL-1-receptor antagonist type I (24), a specific IL-1-receptor
antagonist that binds to IL-1 receptors without inducing agonist
activity, thereby blocking the biological effects of IL-1. Finally,
HASM cells have been demonstrated to produce numerous cytokines in
response to IL-1
and tumor necrosis factor-
(12, 19). The
contribution of these cytokines to IL-1
-induced
-adrenergic
hyporesponsiveness is not known, but glucocorticoids have been shown to
have effects on the transcription of a number of cytokines (19, 37).
Whether glucocorticoids have effects on responses to any of these other
cytokines in HASM cells remains to be established.
Because glucocorticoids have been shown to increase
2-adrenergic receptor
expression in a number of cell systems including human lung (25),
conceivably increased
2-adrenergic-receptor expression could account for the ability of glucocorticoids to inhibit
IL-1
-induced
-adrenergic hyporesponsiveness in HASM cells
independent of their effects on COX-2 expression. However, we believe
this to be an unlikely explanation. First, effects on
-adrenergic
receptor expression require concentrations of glucocorticoids higher
than those at which we have observed effects. Second, Shore et al. (39)
have previously shown that this IL-1
-induced
-adrenergic
hyporesponsiveness occurs without a decrease in total
-adrenergic
receptor number. Nevertheless, the method we used to perform the
binding experiments does not distinguish between cell surface and
internalized receptors, so that it is possible that there were fewer
receptors on the cell surface. Third, glucocorticoids alone do not
enhance cell stiffness or cAMP responses to Iso compared with those in
control cells. Thus even if glucocorticoids increase the number of
2-adrenergic receptors, the
excess receptors do not result in additional functional effects.
In summary, our results indicate that glucocorticoids inhibit the
decreased response to -agonists that is observed in HASM cells after
IL-1
treatment. Although glucocorticoids have a number of actions in
the asthmatic airway, this direct action of glucocorticoids on airway
smooth muscle cells suggests another important mechanism by which
glucocorticoids have beneficial effects in asthma. We also provide
evidence that glucocorticoids inhibit IL-1
-induced COX-2 expression
and PGE2 formation at the same
concentrations at which they inhibit IL-1
-induced
-adrenergic
hyporesponsiveness but that this effect is not through inhibition of
IL-1
-induced activation of NF-
B or AP-1. Given our previous
observation that IL-1
-induced COX-2 expression is required for
IL-1
effects on
-adrenergic responses, the effects of
glucocorticoids on COX-2 expression are sufficient to explain their
effects on
-adrenergic responsiveness.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dale Youngkin and Joseph Abraham for technical assistance, Dr. Ben Fabry for assistance with the magnetic twisting cytometer, G. Chad Blain and Dr. Shinsakua Ueda for assistance with electrophoretic mobility shift assays, and Dr. Jeffrey Drazen for critical review of this manuscript.
![]() |
FOOTNOTES |
---|
This study was supported by National Heart, Lung, and Blood Institute Grants HL-56383, HL-33009, and R01-HL-55301; National Aeronautics and Space Administration Grant NRA-94-OLMSA-02; the American Lung Association (Career Investigator Award to R. Panettieri); GlaxoWellcome (Research Triangle, NC); and the Foundation for Fellows in Asthma Research.
J. Laporte was the recipient of a Canadian Lung Association and Medical Research Council of Canada fellowship and an American Lung Association fellowship.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. A. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Received 9 December 1998; accepted in final form 15 June 1999.
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