Prostanoids mediate IL-1beta -induced beta -adrenergic hyporesponsiveness in human airway smooth muscle cells

Johanne D. Laporte1, Paul E. Moore1, Reynold A. Panettieri2, Winfried Moeller3, Joachim Heyder3, and Stephanie A. Shore1

1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19002-2209; and 3 GSF National Research Center for Environment and Health Institute of Inhalation Biology, Oberschleissheim, Germany

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We have previously reported that pretreatment of cultured human airway smooth muscle (HASM) cells with interleukin-1beta (IL-1beta ) results in decreased beta -adrenergic responsiveness. The purpose of this study was to determine whether prostanoids released as a result of cyclooxygenase-2 (COX-2) induction by IL-1beta contribute to this effect of the cytokine. Confluent serum-deprived HASM cells were studied in passages 4-7. IL-1beta (20 ng/ml for 22 h) reduced the ability of the beta -agonist isoproterenol (Iso) to decrease stiffness of HASM cells as measured by magnetic twisting cytometry. The effect of IL-1beta on Iso-induced changes in cell stiffness was abolished by nonselective [indomethacin (Indo), 10-6 M] and selective (NS-398, 10-5 M) COX-2 inhibitors. Indo and NS-398 also inhibited both the increased basal cAMP and the decreases in Iso-stimulated cAMP production induced by IL-1beta . IL-1beta (20 ng/ml for 22 h) caused an increase in both basal (15-fold) and arachidonic acid (AA)-stimulated (10-fold) PGE2 release. Indo blocked basal and AA-stimulated PGE2 release in both control and IL-1beta -treated cells. NS-398 also markedly reduced basal and AA-stimulated PGE2 release in IL-1beta -treated cells but had no significant effect on AA-stimulated PGE2 release in control cells. Western blot analysis confirmed the induction of COX-2 by IL-1beta . Exogenously administered PGE2 (10-7 M, 22 h) caused a significant reduction in the ability of Iso to decrease cell stiffness, mimicking the effects of IL-1beta . Cycloheximide (10 µg/ml for 24 h), an inhibitor of protein synthesis, also abolished the effects of IL-1beta on Iso-induced cell stiffness changes and cAMP formation. In summary, our results indicate that IL-1beta significantly increases prostanoid release by HASM cells as a result of increased COX-2 expression. The prostanoids appear to contribute to beta -adrenergic hyporesponsiveness, perhaps by heterologous desensitization of the beta 2 receptor.

prostaglandin E2; cytoskeletal mechanics; indomethacin; NS-398; adenosine 3',5'-cyclic monophosphate; interleukin-1beta

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

beta -adrenergic hyporesponsiveness is a characteristic feature of asthma. Decreased bronchodilator responses to beta -agonists have been observed in asthmatic subjects and also in animal models of asthma (2, 3, 14, 33). It is possible that cytokines are involved in mediating this phenomenon. Increased levels of interleukin-1beta (IL-1beta ) have been observed in bronchoalveolar fluid of patients with symptomatic asthma (32, 50), and IL-1beta has been shown to decrease beta -adrenergic responsiveness in a number of cells and tissues (11, 17, 25, 53), including cultured human airway smooth muscle (HASM) cells (45).

The cyclooxygenase (COX) enzyme converts arachidonic acid (AA) to PGH2 (19), which is then metabolized to various PGs, such as PGE2, PGD2, and PGI2, and thromboxane A2 (TxA2). It is now recognized that two COX enzymes exist. COX-1 was initially cloned from ovine seminal vesicles (13). The second isoenzyme, COX-2, was initially identified as a member of a group of genes in which expression is elevated in chicken fibroblasts after transformation by Rous sarcoma virus (46). The mouse homologue of COX-2 gene was later identified as one of several inducible proteins from 3T3 fibroblasts (26). The major difference between these isoenzymes appears to be their regulation. COX-1 is expressed constitutively in most cells and is probably responsible for the production of prostaglandins under physiological conditions (47). In contrast, COX-2 is induced in cells by proinflammatory stimuli such as mitogens (23), cytokines (13, 5, 47), and bacteria lipopolysaccharide (27, 34). One of the cytokines that induces COX-2 is IL-1beta . IL-1beta has been shown to induce the expression of COX-2 in a variety of cell types, including airway smooth muscle cells (15, 16, 31, 35, 47, 48). PGE2 is the primary prostanoid produced by airway smooth muscle, and IL-1beta increases its synthesis (39, 49).

In some systems, prostanoids generated as a result of COX-2 induction appear to mediate the effects of IL-1beta . For example, IL-1beta activates endothelial cells of the brain vasculature to induce COX-2 expression, which is possibly responsible for the elevated level of prostaglandins important during fever (10). The enhancement of the production of PGE2 by human dermal fibroblasts and sinovial fibroblast cells by IL-1beta may play an important role in the process of the inflammatory response and the persistence of the inflammation in the chronic inflammatory diseases (9).

We have previously reported that IL-1beta decreases the ability of cultured HASM cells to respond to beta -agonists (45). Responses to isoproterenol (Iso) were assessed by measuring cAMP formation and by measuring changes in cytoskeletal stiffness by magnetic twisting cytometry (22, 45, 51, 52). Using this technique, we have previously shown that HASM cells decrease their stiffness in response to any of a panel of bronchodilator agonists known to cause relaxation of airway smooth muscle, whereas stiffness increases in response to contractile agonists known to increase cytosolic calcium concentrations in these cells (22, 45). To determine whether COX products are involved in this effect of IL-1beta , we first examined the effect of nonselective [indomethacin (Indo)] and selective (NS-398) COX-2 inhibitors on IL-1beta -induced changes in HASM cell responses to the beta -agonist Iso. To confirm previous reports of COX-2 induction by IL-1beta in our system and to verify the efficacy of the inhibitors used, we also examined the effect of IL-1beta on PGE2 release from HASM cells and measured IL-1beta -induced COX-2 expression by Western blot analysis. Because our results indicated that COX products are implicated in IL-1beta -induced changes in the response to beta -agonists, we examined the effect of prolonged pretreatment with PGE2 on HASM cell stiffness in response to Iso. Because our results also indicated that induction of COX-2 by IL-1beta appears to be required for the cytokine to exert its effect on beta -adrenergic responsiveness, we examined the effect of the protein synthesis inhibitor cycloheximide on IL-1beta -induced changes in HASM cell stiffness and cAMP formation responses to Iso.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. Human tracheas were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Tracheal smooth muscle cells were harvested from trachea as previously described (38). Briefly, a segment of trachea just proximal to the carina was dissected under sterile conditions, and the trachealis muscle was isolated. Approximately 1 g of wet tissue was obtained from each donor. The tissue was minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 units of collagenase, 10 mg of soybean trypsin inhibitor, and 10 U/ml elastase. Tissue was incubated with enzymes for 90 min in a shaking water bath at 37°C. The cell suspension was then filtered through 127-µm Nytex mesh, and the filtrate was washed with an equal volume of cold Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS). Cells were plated in plastic flasks at 1.0 × 104 cells/cm2 in Ham's F-12 medium supplemented with 10% FBS, penicillin (103 U/ml), streptomycin (1 mg/ml), amphotericin B (2 mg/ml), NaOH (12 mM), CaCl2 (1.7 µM), L-glutamine (2 mM), and HEPES (25 mM). Medium was replaced every 3-4 days. Cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days. Confluent cells were serum deprived and supplemented with 5.7 µg/ml insulin and 5 µg/ml tranferrin 24 h before use. Cells from 13 different donors studied in passages 4-7 were used in the studies.

Experimental protocol. For experiments in which we examined the effect of the COX inhibitor Indo on IL-1beta -induced changes in cell stiffness responses to Iso or dibutyryl-cAMP (DBcAMP), four flasks of HASM cells from the same passage of the same donor cells were serum deprived and hormone supplemented. Approximately 10 h later, two flasks were treated with Indo (10-6 M). Two hours later, IL-1beta was added to one of the flasks treated with Indo and also to an untreated flask. Eighteen hours later, cells were harvested by brief exposure to trypsin and EDTA, resuspended in serum-free medium with or without IL-1beta and Indo, and plated at 20,000 cells/well on collagen I (500 ng/cm2)-coated bacteriological plastic dishes (6.4-mm, 96-well Removawells, Immunlon II). Two to six hours later, measurements of cell stiffness were made using magnetic twisting cytometry. Cumulative concentration-response curves to Iso or DBcAMP were performed as follows: first, three to five measurements of cell stiffness were made under baseline conditions. After these measurements, 2 µl of a solution containing the agonist Iso or DBcAMP were added to the well that contained 200 µl of medium. After a 1-min incubation with agent, two to four measurements of cell stiffness were obtained again. This procedure was repeated with increasing concentration of the agent. The concentration ranges used were as follows: Iso, 10-8 to 10-5 M; DBcAMP, 10-4 to 3 × 10-3 M. Only one agonist was studied per well. The effects of the selective COX-2 inhibitor NS-398 (10-5 M) and the protein synthesis inhibitor cycloheximide (10 µg/ml) were examined in an identical fashion. Details of the methodology for magnetic twisting cytometry are found in Magnetic twisting cytometry.

We also examined the effect of exogenous PGE2 on cell stiffness responses to Iso. For these experiments, two flasks of confluent serum-deprived, hormone-supplemented HASM cells were used. One flask was treated with PGE2 (10-7 M), and one served as a control. Twenty hours later, the cells were harvested and used to obtain cell stiffness dose responses to Iso as described above. PGE2 was washed from the cells ~20 min before the initiation of cell stiffness measurements. The dose of PGE2 (10-7 M) was chosen for the following reason. In some experiments, we harvested supernatant of the IL-1beta -treated cells just before harvesting of the cells for plating in Removawells. In those supernatants, which we believed were representative of what the cells were continuously exposed to during their IL-1beta treatment period, the PGE2 concentration was 10-7 M or higher, depending on the donor line used.

For experiments in which we examined the effects of Indo, NS-398, and cycloheximide on IL-1beta -induced changes in cAMP formation, cells were treated identically as in the cell stiffness experiments, except that, once harvested, the cells were plated at 105/well in 24-well plates. cAMP generation in response to Iso was measured 4 h after plating, at which time the medium was replaced with 0.5 ml of phosphate-buffered saline (PBS) containing 0.1 mM IBMX (to prevent degradation of cAMP by phosphodiesterases), ascorbic acid (300 µM; to prevent oxidation of Iso), Indo (10-6 M), NS-398 (10-5 M), and cycloheximide (10 µg/ml). Thirty minutes later, Iso (10-7 to 10-5 M) was added to the cells. Only one concentration of agonist was used per well. Some cell wells were used to measure basal cAMP release and did not receive Iso. Cells were incubated for an additional 10 min and then were removed from the incubator and placed on ice. Ice-cold ethanol (1 ml) was added to lyse the cells. The lysate was centrifuged at 2,000 g for 15 min at 4°C, and the supernatant was removed, evaporated to dryness, and stored at -70°C. On the day of the assay, the cell lysate was resuspended in 200-500 µl of assay buffer. Some samples were diluted to ensure that they fell within the limits of the standard curve. cAMP was assayed using a Rainen cAMP 125I radioimmunoassay kit (New England Nuclear).

We examined the effect of IL-1beta on PGE2 release from HASM cells in the presence or absence of Indo or NS-398. For these experiments, the cells were treated with IL-1beta and COX inhibitors and harvested as described in Experimental protocol. The cells were plated at 105 cells/well in 24-well plates. COX inhibitors and IL-1beta were readded to the cells, and the cells were incubated for 4 h. The medium was then replaced with 0.5 ml of fresh medium. Indo and NS-398 were readded to each treated well. The effect of IL-1beta on COX activity was examined by adding AA (10-5 M) to some of the cell wells at this time. Fifteen minutes later, the supernatants were harvested and stored at -20°C until subsequent assay with a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). The antibody to PGE2 had <1% cross-reactivity to 6-keto-PGF1alpha and <0.01% to TxB2 and other prostaglandins according to the manufacturer's specifications.

In other experiments, we assessed the effect IL-1beta on the expression of COX-2 by Western blotting. For these experiments, two flasks of HASM cells from the same passage of the same donor cells were grown to confluence and serum deprived and 10 h later were treated with IL-1beta (20 ng/ml) or control medium. Approximately 24 h later, cell medium was removed, and cells were washed with PBS. HASM cells were harvested by brief exposure to 0.25% trypsin and 1 mM EDTA and then were washed with Ham's F-12 medium. 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 leupeptin, 1 µg/ml pepstatin, and 10-2 U/ml aprotinin] and then were passed through a 25.625-gauge needle. Cell lysates were clarified by centrifugation at 4,000 g for 10 min to remove debris. Protein concentration of the supernatant was determined with Bio-Rad dye reagent (Bio-Rad, Richmond, CA).

Western blotting. Supernatant of cell lysates from control and IL-1beta -treated cells were mixed with equal volumes of loading buffer [0.062 M Tris · HCl (pH 6.8), 10% glycerol, 2% SDS, 5% beta -mercaptoethanol, and 0.01% (wt/vol) bromphenol blue] and then were boiled for 5 min. Solubilized proteins (100 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis (125 V, 2 h) on 12% Tris-glycine gel (Novex, San Diego, CA) under nonreducing conditions and transferred electrophorically (16 V, 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 saline buffer containing 150 mM NaCl, 0.1% Tween 20, and 4% bovine serum albumin) 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 using a laser densitometer. Band density values were expressed in arbitrary optical density units.

Magnetic twisting cytometry. Details of the magnetic twisting cytometry technique have been previously described (51, 52). The principle of magnetic twisting cytometry is as follows: ferromagnetic beads are first coated with a prescribed ligand [Arg-Gly-Asp (RGD)-containing peptides in this case] and then bound to the surface of the cells through the corresponding receptor system (integrins in this case). Individual wells containing adherent cells bound to ligand-coated ferromagnetic beads are placed into the magnetic twisting chamber, are maintained in defined medium, and are held at 37°C using a circulating water bath that is built into the system. The cell well is oriented horizontally, rotated around the vertical axis (10 Hz), and shielded with four external superalloy cylinders to minimize the disturbances from external magnetic field and hence the signal-to-noise ratio. The attached beads are magnetized with a brief 1,000-G pulse so that their magnetic moments are aligned in one direction, parallel to the surface on which the cells are plated. The magnetic field vector generated by the beads in the horizontal direction is measured by the in-line magnetometer. The sensitivity of the magnetometer is 0.1 nT (1,000 G = 0.1 mT). Subsequently, a much smaller magnetic field is applied in the vertical direction to the first applied torque (or twisting stress) using coils 1.9 cm in diameter. This twisting stress causes the beads to rotate as would a compass needle, but bead rotation is opposed by reaction forces developed within the cytoskeleton to which the beads are bound through the integrin molecules. Magnetic twisting cytometry measures the applied twisting stress and the resulting angular rotation of the magnetic bead and expresses the ratio as cell stiffness. Bead rotation increases with the strength of the applied twisting field and is inversely proportional to the cell's resistance to shape distortion.

Ferromagnetic beads (4.5 µm in diameter, Fe3O4) were coated with synthetic RGD-containing peptide (Peptite 2000, Telios) by incubation overnight at 4°C with 50 mg · ml peptide-1 · mg beads-1 in carbonate buffer (pH 9.0). Beads were then washed two times in serum-free medium containing 1% BSA. Approximately 5 × 104 beads were added to wells containing 20,000 HASM cells and were allowed to incubate 20 min at 37°C 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, and the wells containing the cells were placed within the magnetometer and maintained at 37°C.

Magnetic twisting cytometry was used to characterize HASM cell mechanics on the basis of three variables: angular strain, applied shear stress (sigma ), and apparent stiffness (51, 52). The sigma , which is directly proportional to the strength of the magnetic twisting field, was defined as torque per bead volume and is expressed as dynes per square centimeter. In this study, we set the strength of the twisting field so that, initially, a sigma  of 80 dyn/cm2 was applied to the cells. Because the angle between the twisting field and the bead's magnetic moment vector decreases as the beads rotate toward the twisting field, sigma  decreases with angular strain (and time). The sigma  was calibrated by placing beads in known viscosity standard, and thus a relationship between the twisting field and applied stress was obtained (52). Average angular strain was defined as the fractional rotation of beads during a 1-min twist and was expressed as the angle (phi ) between the bead's original magnetic moment and the magnetic moment 1 min after the twisting field was applied. Therefore, phi  = cos-1 (M80/M20) where M80 is the magnetic field measured at the end of the 1-min twist (i.e., 80 s after the original pulse) and M20 is the magnetic field 20 s after the original pulse (i.e., just before the application of the twisting field). Apparent stiffness is defined as the ratio between sigma  and shear strain, both measured 60 s after the twisting field is applied. Thus stiffness = sigma ocosphi /phi , where sigma o is the magnitude of the stress at the instant when it is applied. Stiffness is analogous to the shear modulus of the cell and is also expressed in dynes per square centimeter. 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 is usually very small when compared with the bead's rotation due to twisting and is usually <3% of the original magnetic field over the 2.5-min period during which measurements are made.

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-ETDA solution which were purchased from GIBCO (Grand Island, NY), IL-1beta which was obtained from Genzyme (Cambridge, MA), and PGE2 (10-2 M in DMSO) which was obtained from BioMol (Philadelphia, PA). DBcAMP was dissolved at 10-1 M in distilled water, frozen in aliquots, and diluted appropriately in medium on the day of use. Iso (10-1 M in distilled water) was made fresh each day. Because Iso is rapidly oxidized, dilution of Iso in medium was made immediately before addition to the cells. Indo and cycloheximide were dissolved in ethanol and diluted in medium on the day of use. NS-398 was dissolved in DMSO at 10-2 M and diluted in medium.

Statistics. The effect of the inhibitors (cycloheximide, Indo, and NS-398) on IL-1beta -induced changes in cell stiffness responses to Iso was examined by repeated-measures ANOVA, using treatment (control, IL-1beta , inhibitor, and IL-1beta  + inhibitor) and experimental day as main effects. Follow-up t-tests were used to determine where the treatment effect lay. Repeated-measures ANOVA was also used to determine the effect of pretreatment with PGE2 on Iso-induced changes in cell stiffness. Changes in cAMP formation induced by Iso were expressed as percent of basal cAMP and log transformed, and ANOVA was performed using experimental day and drug treatment (control, IL-1beta , inhibitor, and IL-1beta  + inhibitor) as main effects. Changes in basal and AA-stimulated PGE2 release induced by IL-1beta were examined by repeated-measures ANOVA, using treatment and experimental day as main effects. A P value <0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

We have previously reported that IL-1beta decreases the ability of cultured HASM cells to respond to beta -agonists. To determine whether COX products are involved in this effect of IL-1beta , we first examined the effect of nonselective (Indo) and selective (NS-398) COX-2 inhibitors on IL-1beta -induced changes in HASM cell responses to Iso. Results are shown in Fig. 1. Neither Indo, IL-1beta , nor their combination had any effect on baseline cell stiffness. Baseline stiffness averaged 108.03 ± 7.85 dyn/cm2 in control, 122.59 ± 12.01 dyn/cm2 in IL-1beta -treated cells, 116.02 ± 9.2 dyn/cm2 in Indo-treated cells, and 125.84 ± 7.21 dyn/cm2 in IL-1beta  + Indo-treated cells. Acute treatment with Indo (5-30 min) also did not change the baseline cell stiffness. In control cells, Iso caused a dose-related decrease in cell stiffness (Fig. 1). Repeated-measures ANOVA indicated a significant effect of drug treatment (P < 0.001) on Iso-induced changes in cell stiffness. Follow-up analysis indicated that the treatment effect lay in the response to IL-1beta , which reduced the capacity of Iso to decrease cell stiffness, as previously described. Compared with control treatment, Indo caused a more pronounced decrease in cell stiffness at 10-8 and 10-5 M Iso. Importantly, Indo abolished the effect of IL-1beta on Iso-induced changes in cell stiffness.


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Fig. 1.   Effect of indomethacin (Indo; 1 µM for 24 h) on interleukin-1beta (IL-1beta ; 20 ng/ml for 22 h)-induced changes in cell stiffness responses to isoproterenol (Iso). Results are expressed as %baseline stiffness values before addition of Iso and are means ± SE of data from 6 experimental days from 11 control wells, 10 IL-1beta -treated wells, 11 Indo-treated wells, and 11 Indo + IL-1beta -treated wells obtained from 4 different donor cells. * P < 0.05 compared with control, # P < 0.05 compared with IL-1beta , and @ P < 0.05 compared with control.

We have previously reported that, whereas IL-1beta significantly inhibits the ability of HASM cells to decrease their stiffness in response to Iso, it has no effect on cell stiffness responses to DBcAMP (45), suggesting that the effect of IL-1beta lies upstream of the effects of activation of protein kinase A (PKA) by cAMP. To ensure that the observed effects of Indo (Fig. 1) were not related to any downstream effects of the drug, we also examined the effects of Indo and IL-1beta on cell stiffness responses to DBcAMP. DBcAMP induced a concentration-related decrease in cell stiffness in all four groups of cells, and an effect of neither IL-1beta nor Indo was observed (Fig. 2).


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Fig. 2.   Effect of Indo (1 µM for 24 h) or IL-1beta (20 ng/ml for 22 h) on changes in cell stiffness induced by increasing concentrations of dibutyryl-cAMP (DBcAMP). Results are expressed as %baseline stiffness values before addition of DBcAMP and are means ± SE of data from 3 experimental days from 5 control wells, 5 IL-1beta -treated wells, 6 Indo-treated wells, and 5 Indo + IL-1beta -treated wells obtained from 3 different donor cells.

To determine whether it was COX-2-induced prostanoid formation that was important in IL-1beta -induced changes in beta -adrenergic responses of HASM cells, we examined the effect of the more specific COX-2 inhibitor NS-398 (10-5 M, 24 h) on IL-1beta -induced changes in cell stiffness responses to Iso (Fig. 3). Neither NS-398, IL-1beta , nor their combination had any effect on baseline cell stiffness. Baseline stiffness averaged 87.6 ± 2.9 dyn/cm2 in control cells, 85.5 ± 1.1 dyn/cm2 in NS-398-treated cells, 85.4 ± 5.9 dyn/cm2 in IL-1beta -treated cells, and 97.4 ± 5.3 dyn/cm2 in NS-398 + IL-1beta -treated cells [not significant (NS)]. Repeated-measures ANOVA indicated a significant effect of drug treatment on cell stiffness responses to Iso (P < 0.001; Fig. 3). Follow-up analysis indicated that, as previously observed, the treatment effect lay in the response to IL-1beta (20 ng/ml), which reduced the capacity of Iso to decrease cell stiffness (P < 0.001). Compared with control cells, NS-398 alone had no effect on cell stiffness responses to any concentration of Iso. However, as with Indo treatment, NS-398 abolished the effects of IL-1beta on cell stiffness responses to Iso.


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Fig. 3.   Effect of NS-398 (10 µM for 24 h) on IL-1beta (20 ng/ml for 22 h)-induced changes in cell stiffness responses to Iso. Results are expressed as %baseline stiffness values before addition of Iso and are means ± SE of data from 4 experimental days from 8 control wells, 7 IL-1beta -treated wells, 8 NS-398-treated wells, and 8 NS-398 + IL-1beta -treated wells obtained from 3 different donor cells. * P < 0.001 compared with control and dagger  P < 0.001 compared with IL-1beta .

We have previously reported that IL-1beta causes a slight (~30%) increase in basal cAMP formation but substantially decreases the ability of Iso to stimulate cAMP in HASM cells in culture (45). To determine whether prostanoids might be involved in these effects and, if so, whether the prostanoids were derived from COX-2, we examined the effect of Indo and NS-398 on changes in basal and Iso-stimulated cAMP formation induced by IL-1beta . Figure 4 shows the effect of Indo and NS-398 on basal cAMP formation in control and IL-1beta (2 ng/ml for 22 h)-treated cells. In both experiments, ANOVA indicated a significant treatment effect (P < 0.05 for NS-398 experiment and P < 0.001 for Indo experiment). IL-1beta increased basal cAMP formation (P < 0.05). Indo reduced basal cAMP in both control (P < 0.05) and IL-1beta -treated cells (P < 0.001) such that there was no effect of IL-1beta on basal cAMP in Indo-treated cells. NS-398 had no significant effect on basal cAMP in control cells but reduced basal cAMP formation in IL-1beta -treated cells (P < 0.02).


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Fig. 4.   Effect of IL-1beta (2 ng/ml for 22 h) on changes in basal cAMP levels in control human airway smooth muscle (HASM) cells and HASM cells pretreated with 1 µM Indo (A) or 10 µM NS-398 (B). Results are means ± SE of data from 10 control, 11 IL-1beta -treated, 11 Indo-treated, and 11 Indo + IL-1beta -treated wells (A) and 9 control, 10 IL-1beta -treated, 9 NS-398-treated, and 9 NS-398+ IL-1beta -treated wells. In each case, data represent experiments from 6 different experimental days on cells from 4 different donors.

The effects of Indo and NS-398 on IL-1beta -induced changes in Iso-stimulated cAMP formation are shown in Fig. 5. As shown, Iso (10-7 and 10-6 M) caused approximately twofold and threefold increases in cAMP formation over baseline in control cells, respectively. In both experiments, ANOVA indicated a significant drug treatment effect (P < 0.001 for Indo experiment and P < 0.03 for NS-398 experiment). Follow-up analysis indicated that, in both cases, the treatment effect lay in the IL-1beta -treated cells, in which cAMP formation was reduced relative to the controls (P < 0.01). Neither Indo nor NS-398 alone had any effect on Iso-stimulated cAMP formation. However, Indo blocked the effect of IL-1beta such that Iso (10-6 and 10-7 M)-stimulated cAMP formation in cells treated with IL-1beta  + Indo was not different from that in cells treated with Indo alone. NS-398 also blocked the effect of IL-1beta such that Iso (10-6 M)-stimulated cAMP formation was significantly different in cells treated with IL-1beta  + NS-398 compared with cells treated with IL-1beta , and Iso (10-7 M)-stimulated cAMP formation was not different in cells treated with IL-1beta  + NS-398 compared with cells treated with NS-398 alone.


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Fig. 5.   Effect of IL-1beta (2 ng/ml for 22 h) on changes in cAMP formation induced by Iso (10-7 and 10-6 M) in control HASM cells and HASM cells pretreated with 1 µM Indo (A) or 10 µM NS-398 (B). Results are expressed as %changes above basal levels measured in wells from the same flask of cells. In each case, results are means ± SE of data from 6 experimental days from 7-10 wells and represent cells from 3 different donors. * P < 0.05 and ** P < 0.01 compared with control.

To verify the efficacy of the inhibitors used and to confirm, in our system, previous reports of increased PGE2 release by IL-1beta , we also examined the effect of IL-1beta (20 ng/ml for 22 h) on basal and AA (10 µM)-stimulated release of PGE2 by HASM cells in the presence or absence of the COX inhibitors Indo and NS-398 (Fig. 6). Compared with control cells, IL-1beta caused a significant increase in both basal PGE2 release (15-fold increase) and in AA-stimulated PGE2 release (10-fold increase; P < 0.0001 in each case). Indo markedly reduced basal and AA-stimulated PGE2 release in both control and IL-1beta -treated cells (P < 0.0001 in each case). NS-398 caused a marked and significant reduction in basal and AA-stimulated PGE2 release in IL-1beta -treated cells (P < 0.0001 in each case), but NS-398 had no significant effect on AA-stimulated PGE2 release in control cells. Western blot analysis confirmed the induction of COX-2 by IL-1beta (20 ng/ml for 24 h; Fig. 7). In four different IL-1beta -stimulated HASM cell extracts, the COX-2 antibody revealed one band corresponding to molecular mass of 70 kDa, as described by others (15, 39, 50). In contrast, in control cells taken from the same passages of the same donor cells, no band corresponding to molecular mass of 70 kDa was observed.


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Fig. 6.   Release of PGE2 by control HASM cells (A) and HASM cells treated with IL-1beta (20 ng/ml for 22 h; B). Some cells were also pretreated for 24 h with 1 µM Indo or 10 µM NS-398. Basal release and arachidonic acid (AA; 10 µM)-stimulated PGE2 release over a 15-min period were assessed. Results are means ± SE of data from 8 HASM cell wells in each case and were obtained on 4 experimental days in cells from 2 different donors. ** P < 0.0002 compared with control cells, dagger dagger P < 0.0002 compared with AA-stimulated cells, ## P < 0.0001 compared with IL-1beta -treated cells, and ▵▵ P < 0.001 compared with IL-1beta -treated and AA-stimulated cells.


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Fig. 7.   Western blot showing cyclooxygenase-2 (COX-2) expression in control HASM cells (C) and HASM cells from the same passages of the same donor cells treated with IL-1beta (20 ng/ml) for 24 h. Paired control and IL-1beta cells are grouped from left to right. Cells from 4 different experimental days are represented. Lane on the far right is a COX-2 standard.

Because our results suggested that prostanoid formation was involved in IL-1beta -induced changes in beta -adrenergic responses of HASM cells, we examined the effect of exogenous PGE2 (10-7 M for 22 h) on changes in cell stiffness responses to Iso. PGE2 was washed from the cells before the initiation of cell stiffness measurements. Baseline stiffness averaged 126.52 ± 16.4 dyn/cm2 in control and 125.6 ± 7.9 dyn/cm2 in PGE2-treated cells (NS). PGE2 caused a significant reduction in the cell stiffness response to Iso (P < 0.01 by repeated-measures ANOVA; Fig. 8). Follow-up t-tests indicated that the effect of PGE2 was observed at all concentrations of Iso examined.


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Fig. 8.   Effect of PGE2 (100 nM for 22 h) on changes in cell stiffness induced by increasing concentrations of Iso. Results are expressed as %baseline stiffness of each well before addition of Iso and are means ± SE of data from 3 experimental days from 8 control wells and 11 PGE2-treated wells obtained from 2 different donor cells. * P < 0.005 compared with control.

Because our results suggested that IL-1beta -induced COX-2 synthesis was important in IL-1beta -induced changes in beta -adrenergic responses of HASM cells, we examined the effect of the protein synthesis inhibitor (cycloheximide: 10 µg/ml for 24 h) on IL-1beta -induced changes in HASM cell stiffness responses to Iso (Fig. 9). In contrast to experiments described above in which cell treatment did not alter baseline cell stiffness, baseline stiffness was significantly decreased by treatment with cycloheximide (P < 0.02 by repeated-measures ANOVA). Baseline stiffness averaged 123.4 ± 11.6, 132.31 ± 8.4, 90.6 ± 5.9, and 97.5 ± 4.7 dyn/cm2 in control cells, cells treated with IL-1beta alone, cells treated with cycloheximide alone, and cells treated with cycloheximide + IL-1beta , respectively. A possible explanation is that cycloheximide might influence the formation of molecules important for cell adhesion to the extracellular matrix and thereby influence basal cell stiffness (22). As previously observed, IL-1beta (20 ng/ml) reduced the capacity of Iso to decrease cell stiffness (P < 0.001). Cycloheximide blocked this effect of IL-1beta such that there was no significant difference in cell stiffness responses to Iso between control and IL-1beta -treated cells that had been pretreated with the protein synthesis inhibitor.


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Fig. 9.   Effect of cycloheximide (10 µg/ml for 24 h) on IL-1beta (20 ng/ml for 22 h)-induced changes in cell stiffness responses to Iso. Results are expressed in dyn/cm2 and are means ± SE of data from 6 experimental days from 8 control wells, 11 IL-1beta -treated wells, 6 cycloheximide-treated wells, and 13 cycloheximide + IL-1beta -treated wells from 3 different donors. ** P < 0.001 compared with control.

The effects of cycloheximide on IL-1beta -induced changes in Iso (10-6 M)-stimulated cAMP formation are shown in Fig. 10. ANOVA indicated a significant drug treatment effect (P < 0.0001). Follow-up analysis indicated that the treatment effect lay in the IL-1beta -treated cells, in which cAMP formation was reduced relative to the controls (P < 0.003). Cycloheximide alone had no effect on Iso-stimulated cAMP formation. However, cycloheximide blocked the effect of IL-1beta such that Iso-stimulated cAMP formation in cells treated with IL-1beta  + cycloheximide was not different from that in cells treated with cycloheximide alone.


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Fig. 10.   Effect of IL-1beta (20 ng/ml for 22 h) on changes in cAMP formation induced by Iso (10-6 M) in control HASM cells and HASM cells pretreated with cycloheximide (Cyclo; 10 µg/ml). Results are expressed as %changes above basal levels measured in wells from the same flask of cells. In each case, results are means ± SE of data studied on 6 experimental days from 9 control wells, 9 IL-1beta -treated wells, 9 cycloheximide-treated wells, and 9 cycloheximide + IL-1beta -treated wells from 3 different donors. * P < 0.05 compared with control.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Our results indicate that Indo and NS-398 inhibited the effects of IL-1beta on cell stiffness and cAMP responses to beta -agonists (Figs. 1 and 3-5), whereas pretreatment of HASM cells with PGE2 mimicked the effects of IL-1beta (Fig. 8). IL-1beta significantly increased PGE2 release, and both nonselective (Indo) and selective (NS-398) COX-2 inhibitors (Fig. 6) prevented this augmented release, suggesting that it was the result of the increased COX-2 expression induced by IL-1beta (Fig. 7). A protein synthesis inhibitor prevented the IL-1beta -induced changes in beta -adrenergic responses of HASM cells (Figs. 9 and 10). Taken together, the results support the hypothesis that prostanoids released as a result of increased COX-2 expression contribute to the beta -adrenergic hyporesponsiveness induced in HASM cells by IL-1beta .

We have previously reported that IL-1beta decreased the ability of HASM cells to both reduce their stiffness and to increase cAMP formation in response to Iso (45). We have also shown that IL-1beta decreases cell stiffness and cAMP generation responses to PGE2 (45). In contrast, HASM cell responses to the cell-permeant cAMP analog DBcAMP and to forskolin, which directly activates adenylyl cyclase, were not altered. The latter results suggest that neither changes in PKA activation and responses nor changes in adenylyl cyclase expression contributed to the effects of IL-1beta . Pretreatment with IL-1beta had no significant effect on beta 2-adrenoceptor number, nor was there any significant effect of IL-1beta on Gs expression. Furthermore, the effect of IL-1beta was not altered by pretreatment of cells with pertussis toxin, indicating that the effect of IL-1beta was not mediated by changes in Gi. We suggested that the beta -adrenergic hyporesponsiveness induced by IL-1beta was mediated by uncoupling of beta -receptors from Gs-induced activation of adenylyl cyclase. In this report, we have shown that COX inhibitors block the ability of IL-1beta to reduce cell stiffness responses of HASM cells to Iso, suggesting that prostanoids contribute to the decreased responsiveness to beta -agonists that is induced by IL-1beta . A unifying explanation for these observations is outlined schematically in Fig. 11. IL-1beta induces COX-2 expression and markedly increases the production of PGE2 by HASM cells. The release of PGE2 is sufficient to increase cAMP formation by the cells activating PKA. PKA causes phosphorylation of the beta -receptor, inhibiting its ability to bind and activate Gs and resulting in decreased ability of Iso to induce responses.


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Fig. 11.   Potential mechanism whereby IL-1beta leads to beta -adrenergic hyporesponsiveness. PKA, protein kinase A.

In support of this hypothesis, our results indicate that IL-1beta causes a marked increase in PGE2 formation in HASM cells. Basal and AA-stimulated PGE2 release increased ~15- and 10-fold, respectively, in response to IL-1beta . The observation that the COX-2 inhibitor NS-398 blocked the increase in basal and AA-stimulated PGE2 release (Fig. 6) in conjunction with the observation that IL-1beta induced expression of COX-2 (Fig. 7) supports the hypothesis that the increase in PGE2 release results from increased COX-2 activation. NS-398 also blocked the basal release of PGE2 but had no effect on AA-stimulated release in control cells. These results may suggest that NS-398 is a weak inhibitor of COX-1 inhibitor, at least at very low concentrations of the substrate. Alternatively, it is possible that NS-398 interferes with the assay of low concentrations of PGE2. Our results are consistent with those of Pang and Knox (39), who demonstrated that 1 and 10 ng/ml IL-1beta increased PGE2 release by ~10-fold and induced COX-2 expression in HASM cells after 24 h of treatment. Vigano et al. (49) have shown that IL-1beta (1-50 U/ml) induced COX-2 expression and increased PGE2 formation in HASM cells sevenfold after 6 h of treatment. Other investigators have reported that COX-2 expression is also induced by phorbol esters and by tumor necrosis factor-alpha in a human bronchial smooth muscle cell line and in rat tracheobronchial smooth muscle cells (1, 39, 47).

The observation that cycloheximide prevented IL-1beta -induced changes in both cell stiffness and cAMP responses to beta -agonists indicates that this effect of IL-1beta is dependent on de novo protein synthesis. These experiments do not allow us to pinpoint the precise protein whose synthesis is important to the effect of IL-1beta . However, we believe that inhibition of COX-2 synthesis is the basis for the efficacy of cycloheximide. We have also observed that anti-inflammatory steroid treatment, such as dexamethasone, which inhibits IL-1beta -induced COX-2 expression and PGE2 release (5, 35, 49), also reverses the effect of IL-1beta on beta -adrenergic responses of HASM cells (37).

In support of the hypothesis outlined in Fig. 11, our results reported both here (Fig. 4) and previously (45) indicate that IL-1beta causes a small but significant increase in basal cAMP formation in HASM cells. In airway smooth muscle cells, PGE2, like Iso, results in cAMP formation (18, 45), and it is likely that the small increase in basal cAMP induced by IL-1beta (Fig. 4) is the result of increased PGE2 formation by these cells; both Indo and NS-398 prevented IL-1beta from augmenting basal cAMP (Fig. 4). IL-1beta has also been shown to induce cAMP formation in other types (25, 36, 44). In addition, IL-1beta -induced cAMP formation was also associated with a marked increase in prostacyclin production in human vascular smooth muscle cells and 3T3 fibroblasts (4, 8).

PGE2 has the capacity to induce beta -adrenergic hyporesponsiveness in HASM cells. We observed reduced cell stiffness responses to beta -agonists after prolonged exposure to PGE2 (Fig. 6). This occurred without any effect of PGE2 on baseline cell stiffness, suggesting that the PGE2 had been effectively washed from the cells at the time of Iso application. Similarly, Hall et al. (18) have reported that the same concentration of PGE2 (10 µM) produced a 44% attenuation of subsequent cAMP responses to Iso (10-8 to 10-5 M). PGE2 causes cAMP formation, and it has been shown that activation of PKA by cAMP can phosphorylate the beta 2-adrenoceptor and induce desensitization of the receptor (6). The beta 2-adrenoceptor possesses sites for PKA phosphorylation in the COOH-terminal portion of its third intracellular loop and in its cytoplasmic tail. These regions are important in the receptor's interaction with Gs and appear to be involved in the heterologous densitization of the receptor by agonists that induce cAMP formation (12, 21, 29, 28, 41). In contrast, phosphorylation of the receptor by beta -adrenergic receptor kinase, which permits binding to beta -arrestin, requires receptor occupation with ligand and appears to be important in homologous desensitization (30, 43).

HASM cells produce other prostanoids such as PGI2, TxA2, and PGF2alpha in response to IL-1beta . Although the amounts released are smaller than for PGE2 (39), PGI2alpha , like PGE2 and Iso, can act on cell surface receptors that couple to Gs protein and may also contribute to the increased basal cAMP induced by IL-1beta . Another potential mechanism whereby prostanoids might uncouple beta -receptors from Gs-induced activation of adenylyl cyclase is phosphorylation of the beta -receptor or Gs by protein kinase C (7, 24, 40, 42). Both TxA2 and PGF2 act on cell surface receptors that couple to Gq and activate phospholipase C. Phospholipase C can cause diacylglycerol formation and activation of protein kinase C (20).

Cytoskeletal stiffness, as defined here, is a measure of the ability of cells to resist distortions of shape in response to shear stresses applied through magnetic beads linked to the cytoskeleton via integrin receptors. Actin and myosin form part of the cytoskeleton, and cross-bridge formation appears to increase cytoskeletal stiffness, since application of a variety of contractile agonists to HASM cells results in increased stiffness, whereas bronchodilating agonists reduce stiffness (22, 45). Changes in cell adhesion to the extracellular matrix can also influence cytoskeletal stiffness (51), and we have previously reported that HASM cells plated on high-density collagen are more spread out and develop more pronounced decreases in cell stiffness in response to Iso than cells plated on low-density collagen matrix (22). Although it is theoretically possible that COX inhibitors might influence cell adhesion and thereby influence cell stiffness responses to Iso, we believe that such an explanation is very unlikely. First, changes in cell adhesion influence basal cell stiffness (22, 51), but neither IL-1beta , COX inhibitors, nor their combination altered baseline cell stiffness in these experiments. Second, such changes would have been expected to alter cell stiffness responses to any dilating agonist, but responses to DBcAMP were unaffected either by IL-1beta or by Indo. Finally, the observation that COX inhibitors also reversed the effects of IL-1beta on Iso-induced cAMP formation suggests that the effect of the prostanoids lies upstream of the mechanical responses to the PKA activation.

In summary, our results indicate that IL-1beta significantly increases PGE2 release by HASM cells as a result of increased COX-2 expression. The increased PGE2 formation appears to contribute to beta -adrenergic hyporesponsiveness perhaps by heterologous desensitization of the beta -receptor. beta -Adrenergic hyporesponsiveness is a characteristic feature of human asthma. Although we do not know that IL-1beta contributes to this phenomenon in asthma, the observations that IL-1beta is produced in greater amounts in airways of asthmatic than of normal subjects and that IL-1beta can decrease responses of HASM cells to beta -agonists suggest that it may be important. Understanding the role of prostanoids in the mechanism of action of cytokines in leading to beta -adrenergic receptor dysfunction may provide new avenues for improvements in the safety and efficacy of these agents.

    ACKNOWLEDGEMENTS

We thank Dale Youngkin, Joseph Abraham, and Hadi Danaee for technical assistance.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-55301 and HL-33009. J. Laporte was supported by a fellowship from the Medical Research Council of Canada and the Canadian Lung Association.

Address for reprint requests: J. Laporte, Physiol. Prgm., Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.

Received 3 November 1997; accepted in final form 19 May 1998.

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Am J Physiol Lung Cell Mol Physiol 275(3):L491-L501
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