Glucocorticoids ablate IL-1beta -induced beta -adrenergic hyporesponsiveness in human airway smooth muscle cells

Paul E. Moore1, Johanne D. Laporte1, Sonia Gonzalez1, Winfried Moller2, Joachim Heyder2, Reynold A. Panettieri Jr.3, and Stephanie A. Shore1

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


    ABSTRACT
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We have previously reported that interleukin (IL)-1beta decreases responsiveness of cultured human airway smooth muscle (HASM) cells to beta -agonists. The purpose of this study was to determine whether glucocorticoids inhibit this IL-1beta 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-1beta (20 ng/ml)-treated cells. In addition, Dex had no effect on Iso-stimulated cAMP formation in control cells but prevented the IL-1beta -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-1beta -induced hyporesponsiveness to PGE2 stimulation. In contrast, neither IL-1beta nor glucocorticoids had any effect on cell stiffness responses to dibutyryl cAMP. We have previously reported that the IL-1beta effect on beta -adrenergic responsiveness is mediated through cyclooxygenase-2 expression and prostanoid formation. Consistent with these observations, IL-1beta -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-1beta -induced nuclear translocation of nuclear factor-kappa B or activator protein-1 in HASM cells. In summary, our results indicate that, in HASM cells, glucocorticoids alone do not alter responses to beta -agonists but do inhibit IL-1beta -induced beta -adrenergic hyporesponsiveness. Glucocorticoids mediate this effect by inhibiting prostanoid formation but without altering nuclear factor-kappa B or activator protein-1 translocation.

interleukin-1beta ; beta 2-adrenergic receptor; magnetic twisting cytometry; adenosine 3',5'-cyclic monophosphate; cyclooxygenase-2; prostaglandin E2; nuclear factor-kappa B


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DECREASED beta -ADRENERGIC RESPONSIVENESS is a characteristic feature of asthma. The bronchodilator response to beta -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)-1beta , a cytokine elevated in bronchoalveolar lavage fluid of patients with symptomatic asthma (41), decreases beta -adrenergic responses in a number of cell types (20, 45). Shore et al. (39) have recently demonstrated that IL-1beta also decreases the responses of cultured human airway smooth muscle (HASM) cells to beta -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-1beta -induced hyporesponsiveness to beta -agonists. In support of this hypothesis, glucocorticoids have been shown to inhibit responses to IL-1beta in other cell systems (9, 10).

To evaluate this hypothesis, we measured the responses of cultured HASM cells treated with glucocorticoids and/or IL-1beta to the beta -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-1beta 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-1beta on beta -adrenergic responsiveness of HASM cells is mediated through increased prostanoid formation. We have provided evidence for this mechanism by showing that IL-1beta 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-1beta , and that inhibitors of COX-2 blocked IL-1beta -induced beta -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-1beta 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-1beta . We report here that IL-1beta -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-1beta -induced beta -adrenergic hyporesponsiveness.

Glucocorticoids have been shown to inhibit nuclear factor (NF)-kappa 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-kappa B and AP-1 (3, 46), we speculated that the effects of glucocorticoids on COX-2 expression might occur through inhibition of IL-1beta -induced NF-kappa B or AP-1 translocation. Our data suggest that this is not the case in HASM cells.


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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-1beta -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-1beta (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-1beta 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 (10-8 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 (10-7 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 (10-11 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 (10-12 to 10-9 M), and two of the flasks were left untreated. Then 2 h later, these four flasks were each treated with IL-1beta (20 ng/ml), one flask that did not receive FP was treated with IL-1beta , 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 (10-6 M) and two of the flasks were left untreated. Then 3 h after Dex treatment, one flask in each group was treated with IL-1beta (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-kappa 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 · ml-1 · 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 (sigma ), 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, sigma  decreased with angular strain; sigma  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 (phi ) 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, phi  = cos-1(M80/M20). Apparent stiffness was defined as the ratio between sigma  and phi , both measured at the end of stress application or 80 s after the initial pulse magnetization. With the definitions of sigma  and phi , apparent stiffness = (sigma 0cos phi )/phi . In this study, the strength of the twisting field was set so that initially a sigma 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 (10-10 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<FR><NU>5</NU><DE>8</DE></FR>-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-kappa 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-kappa B or AP-1 consensus sequence (Gel Shift Assay System, Promega, Madison, WI) was end labeled with [gamma -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-kappa 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-1beta , 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-1beta , glucocorticoid alone, or IL-1beta 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-1beta , glucocorticoid alone, or IL-1beta 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-1beta treatment on NF-kappa 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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Figure 1 shows cell stiffness changes induced by Iso in control HASM cells and in cells pretreated with IL-1beta (20 ng/ml for 20 h) alone, Dex (10-6 M for 23 h) alone, and a combination of IL-1beta 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-1beta -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-1beta and Dex. In addition, neither IL-1beta , 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-1beta , 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 beta -adrenergic responsiveness to cells treated with IL-1beta . Iso-induced decreases in cell stiffness were not significantly different in cells treated with IL-1beta and Dex compared with cells treated with Dex alone but were significantly different from the response obtained in cells treated with IL-1beta alone (P < 0.001 by repeated-measures ANOVA).


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Fig. 1.   Effects of combination of dexamethasone (Dex; 10-6 M for 23 h) and interleukin (IL)-1beta (20 ng/ml for 20 h), Dex alone, and IL-1beta alone on changes in cell stiffness induced by increasing concentrations of isoproterenol (Iso). Results are expressed as percent baseline stiffness before addition of Iso and are means ± SE from 8 control wells, 8 wells treated with IL-1beta , 8 wells treated with Dex, and 9 wells treated with Dex + IL-1beta on 4 experimental days. ** P < 0.005 compared with wells treated with Dex + IL-1beta .

To determine whether the ability of Dex to restore beta -adrenergic responsiveness to IL-1beta 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 beta -adrenergic responsiveness to cells treated with IL-1beta ; in cells pretreated with the combination of FP (10-9 to 10-7 M) and IL-1beta , 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-1beta -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 beta -adrenergic responsiveness induced by IL-1beta . Specifically, in cells pretreated with FP (10-10 M) and IL-1beta , 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-1beta -treated cells. However, FP (10-11 M) did not inhibit the IL-1beta effect. Pretreatment with FP (10-11 M) and IL-1beta 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-1beta alone. As with Dex, FP at any concentration had no effect on baseline cell stiffness or on cell stiffness responses to Iso.


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Fig. 2.   Effect of combination of fluticasone propionate (FP; for 23 h) and IL-1beta (20 ng/ml for 20 h) compared with effect of FP alone on changes in cell stiffness induced by increasing concentrations of Iso. A: 9 wells treated with FP + IL-1beta and 8 wells treated with FP alone on 3 experimental days. B: 9 wells treated with FP + IL-1beta and 9 wells treated with FP alone on 4 experimental days. C: 10 wells treated with FP + IL-1beta and 10 wells treated with FP alone on 6 experimental days. D: 16 wells treated with FP + IL-1beta and 21 wells treated with FP alone on 12 experimental days. E: 8 wells treated with FP + IL-1beta and 9 wells treated with FP alone on 4 experimental days. base, Baseline. Results are means ± SE. * P < 0.05 compared with FP alone.

To determine whether the ability of glucocorticoids to inhibit IL-1beta -induced beta -adrenergic hyporesponsiveness was specific to beta -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-1beta 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-1beta (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-1beta (Fig. 3). Cell stiffness responses to DBcAMP were not significantly different among cells treated with Dex (10-6 M), IL-1beta , or their combination (Fig. 4). Similar results were obtained with FP (10-7 M; data not shown).


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Fig. 3.   Effect of combination of Dex (10-6 M for 23 h) and IL-1beta (20 ng/ml for 20 h), Dex alone, and IL-1beta alone on changes in cell stiffness induced by increasing concentrations of PGE2. Results are expressed as percent baseline stiffness before addition of PGE2 and are means ± SE from 9 control wells, 9 wells treated with IL-1beta , 9 wells treated with Dex, and 8 wells treated with Dex + IL-1beta on 5 experimental days. * P < 0.05 compared with wells treated with Dex + IL-1beta . ** P < 0.001 compared with wells treated with Dex + IL-1beta . # P < 0.05 compared with wells treated with Dex.



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Fig. 4.   Effect of combination of Dex (10-6 M for 23 h) and IL-1beta (20 ng/ml for 20h), Dex alone, and IL-1beta alone on changes in cell stiffness induced by increasing concentrations of dibutyryl cAMP (DBcAMP). Results are expressed as percent baseline stiffness before addition of DBcAMP and are means ± SE from 6 control wells, 6 wells treated with IL-1beta , 6 wells treated with Dex, and 6 wells treated with Dex + IL-1beta on 3 experimental days. No significant differences were observed among groups.

IL-1beta 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-1beta 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-1beta (20 ng/ml for 20 h), Dex alone, or IL-1beta 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-1beta 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).


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Fig. 5.   Effect of combination of Dex (10-6 M for 23 h) and IL-1beta (20 ng/ml for 20 h), Dex alone, and IL-1beta alone on basal cAMP levels and Iso (10-7 to 10-5 M)-induced changes in cAMP formation. Each well of human airway smooth muscle (HASM) cells was treated with a single concentration of Iso for 10 min or was left untreated. Values are means ± SE from 11-13 wells on 6 experimental days.

We also measured cAMP formation in response to Iso (10-7 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-1beta 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-1beta . 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-1beta effect on beta -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-1beta . 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-1beta (20 ng/ml for 22 h) or IL-1beta 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-1beta induced prominent COX-2 expression. FP caused a dose-dependent inhibition of IL-1beta -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-1beta (20 ng/ml for 22 h), FP alone, or IL-1beta alone or were untreated (control). IL-1beta 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-1beta (Fig. 7). Dex at higher concentrations (10-10 to 10-8 M) also caused a dose-dependent inhibition of IL-1beta -induced COX-2 formation and PGE2 release that was not observed at lower concentrations (10-12 to 10-11 M).



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Fig. 6.   Measurement of cyclooxygenase-2 (COX-2) protein expression in HASM cells treated with IL-1beta (20 ng/ml for 22 h) and FP at indicated concentrations (for 24 h) or IL-1beta alone and in untreated cells (control). A: representative Western blot. ST, COX-2 standard. +, Presence; -, absence. B: laser densitometry quantification. Results are expressed as percent inhibition of COX-2 expression in cells treated with both FP and IL-1beta compared with cells from the same passage of the same donor lines studied on the same day but treated with IL-1beta alone. Values are means ± SE from 4 different experimental days. ** P < 0.001 compared with COX-2 expression of cells treated with IL-1beta alone.



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Fig. 7.   Effect of combination of FP at 2 concentrations (for 24 h) and IL-1beta (20 ng/ml for 22 h), FP alone, and IL-1beta alone on PGE2 release in HASM cells. Values are means ± SE from 2 HASM cell wells in each case on 4 experimental days for 10-10 M FP and 3 experimental days for 10-8 M FP. ** P < 0.001 compared with IL-1beta -treated cells.

Because glucocorticoids have been shown to inhibit NF-kappa 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-kappa B and AP-1 (3, 46), we wanted to determine whether glucocorticoids inhibit IL-1beta -induced NF-kappa 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-1beta (20 ng/ml for 2 h), Dex alone, or IL-1beta alone or were untreated (control). With nuclear protein extracts obtained from these four groups, electrophoretic mobility shift assays for NF-kappa B and AP-1 were performed. Figure 8 shows a representative gel shift assay for AP-1. IL-1beta 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-1beta -induced AP-1 translocation (Fig. 8A). When quantified by laser densitrometry for 5 experimental days, IL-1beta caused a significant increase in the nuclear translocation of NF-kappa B (Fig. 8B) and AP-1 (Fig. 8C), and this IL-1beta -induced translocation of both NF-kappa B and AP-1 was not inhibited by Dex.



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Fig. 8.   Effect of combination of Dex and IL-1beta , Dex alone, and IL-1beta alone on nuclear translocation of nuclear factor (NF)-kappa B and activator protein (AP)-1 in HASM cells. A: representative electrophoretic mobility shift assay for AP-1. Lane 1, negative control (no nuclear extract); lane 2, positive control (HeLa nuclear extract alone); lane 3, competitive inhibitor (HeLa nuclear extract with AP-1 oligonucleotide); lane 4, noncompetitive inhibitor (HeLa nuclear extract with SP-1 oligonucleotide); lane 5, control HASM cells; lane 6, HASM cells treated with IL-1beta alone; lane 7, HASM cells treated with Dex alone; lane 8, HASM cells treated with Dex + IL-1beta . B: laser densitometry quantification. Results are expressed as relative optical density (OD) units for NF-kappa B binding. Values are means ± SE from 5 experimental days. ** P < 0.005 compared with control cells by paired t-test. C: laser densitometry quantification. Results are expressed as relative OD units for AP-1 binding. Values are means ± SE from 5 experimental days. * P < 0.05 compared with control cells by paired t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results indicate that the glucocorticoids Dex and FP ablate IL-1beta -induced hyporesponsiveness of HASM cells to the beta -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-1beta , nor their combination had any effect on the ability of DBcAMP to decrease cell stiffness (Fig. 4). Glucocorticoids also inhibited IL-1beta -induced COX-2 expression (Fig. 6) and PGE2 formation (Fig. 7) in a concentration-dependent manner at the same concentrations at which IL-1beta -induced beta -adrenergic hyporesponsiveness is inhibited. Glucocorticoids have no effect on IL-1beta -induced NF-kappa 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-1beta decreases cell stiffness and cAMP responses to Iso. We now demonstrate that glucocorticoids inhibit this IL-1beta 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 beta 2-adrenergic receptors that couple to the stimulatory protein Gs, the alpha -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-1beta , is ablated by glucocorticoids. Measuring cell stiffness responses to DBcAMP, a cell-permeant analog of cAMP that directly activates PKA, helps identify where IL-1beta and glucocorticoids alter beta -adrenergic responses. Neither IL-1beta , glucocorticoids, nor their combination affect cell stiffness responses to DBcAMP, indicating that IL-1beta 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-1beta attenuates Iso-induced cAMP formation, and glucocorticoids restore the ability of cells treated with IL-1beta 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-1beta -treated cells are likely to involve cAMP formation rather than cAMP action.

Glucocorticoids not only inhibit IL-1beta -induced hyporesponsiveness to Iso but also inhibit IL-1beta -induced hyporesponsiveness to PGE2 (Fig. 3). Although the mechanism through which IL-1beta can attenuate the beta 2-adrenergic response involves COX-2 expression (22), presumably through PKA phosphorylation of beta -adrenergic receptors, we do not know the mechanism of IL-1beta 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-1beta -induced phosphorylation of EP2 and EP4 receptors is also responsible for hyporesponsiveness to prostanoid stimulation. Thus glucocorticoids may inhibit IL-1beta -induced hyporesponsiveness to PGE2 by the same mechanism with which they inhibit IL-1beta -induced beta -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-1beta -induced decreases in HASM cell responses to Iso. In addition, the effect of IL-1beta occurs without altering the expression or activity of adenylyl cyclase because forskolin-stimulated cAMP formation is not different in IL-1beta -treated cells from that in control cells. Instead, our results indicate that IL-1beta -induced beta -adrenergic hyporesponsiveness is mediated through increased prostanoid formation (22). Specifically, we show that IL-1beta results in COX-2 protein expression and PGE2 formation in HASM cells, that PGE2 mimics the effects of IL-1beta on the responses to Iso, and that inhibitors of COX-2 block IL-1beta -induced beta -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 beta -adrenergic receptor. A report by Pang et al. (35) confirms these observations.

Recent studies (7, 34, 35) have demonstrated that Dex at high concentrations (10-6 to 10-5 M) inhibits IL-1beta -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-1beta effects. Given the demonstrated role of COX-2 expression in IL-1beta -induced beta -adrenergic hyporesponsiveness, the observation that both Dex and FP inhibited IL-1beta -induced COX-2 expression and PGE2 formation is sufficient to explain the effects of glucocorticoids on IL-1beta -induced beta -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-1beta effects on cell stiffness responses further supports the hypothesis that glucocorticoids inhibit IL-1beta effects on beta 2-adrenergic responses in HASM cells through their effects on COX-2 expression.

Glucocorticoids have been shown to inhibit NF-kappa 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-kappa B binding in a transfected epithelial cell line. The ability of glucocorticoids to interact with the nuclear transcription factors NF-kappa 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-kappa B and AP-1 (3, 46), we speculated that the effects of glucocorticoids on COX-2 expression might occur through inhibition of IL-1beta -induced NF-kappa B or AP-1. However, this is not the case. We now report that in HASM cells IL-1beta causes nuclear translocation of NF-kappa B (Fig. 8B). Tumor necrosis factor-alpha has similar effects (2). IL-1beta also causes AP-1 translocation (Fig. 8C). However, Dex at maximally effective concentrations with respect to COX-2 expression does not inhibit these IL-1beta -induced responses. These findings suggest that the effects of glucocorticoids in HASM cells do not involve interference with NF-kappa B or AP-1 binding.

Although glucocorticoids do not alter NF-kappa 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-1beta -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-kappa 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-1beta and tumor necrosis factor-alpha (12, 19). The contribution of these cytokines to IL-1beta -induced beta -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 beta 2-adrenergic receptor expression in a number of cell systems including human lung (25), conceivably increased beta 2-adrenergic-receptor expression could account for the ability of glucocorticoids to inhibit IL-1beta -induced beta -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 beta -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-1beta -induced beta -adrenergic hyporesponsiveness occurs without a decrease in total beta -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 beta 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 beta -agonists that is observed in HASM cells after IL-1beta 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-1beta -induced COX-2 expression and PGE2 formation at the same concentrations at which they inhibit IL-1beta -induced beta -adrenergic hyporesponsiveness but that this effect is not through inhibition of IL-1beta -induced activation of NF-kappa B or AP-1. Given our previous observation that IL-1beta -induced COX-2 expression is required for IL-1beta effects on beta -adrenergic responses, the effects of glucocorticoids on COX-2 expression are sufficient to explain their effects on beta -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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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 277(5):L932-L942
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