Johanne D. Laporte

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

We have previously reported that pretreatment of cultured human airway smooth muscle (HASM) cells with interleukin-1β (IL-1β) results in decreased β-adrenergic responsiveness. The purpose of this study was to determine whether prostanoids released as a result of cyclooxygenase-2 (COX-2) induction by IL-1β contribute to this effect of the cytokine. Confluent serum-deprived HASM cells were studied in passages 4-7. IL-1β (20 ng/ml for 22 h) reduced the ability of the β-agonist isoproterenol (Iso) to decrease stiffness of HASM cells as measured by magnetic twisting cytometry. The effect of IL-1β 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-1β. IL-1β (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-1β-treated cells. NS-398 also markedly reduced basal and AA-stimulated PGE2release in IL-1β-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-1β. 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-1β. Cycloheximide (10 μg/ml for 24 h), an inhibitor of protein synthesis, also abolished the effects of IL-1β on Iso-induced cell stiffness changes and cAMP formation. In summary, our results indicate that IL-1β significantly increases prostanoid release by HASM cells as a result of increased COX-2 expression. The prostanoids appear to contribute to β-adrenergic hyporesponsiveness, perhaps by heterologous desensitization of the β2 receptor.

Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1β

We have previously reported that interleukin (IL)-1beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1beta (20 ng/ml) alone. Pretreating cells with the mitogen-activated protein kinase kinase inhibitor PD-98059 or U-126 (2 h before IL-1beta treatment) decreased ERK phosphorylation. IL-1beta (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal PGE(2) release 28-fold (P < 0.001). PD-98059 (100 microM) and U-126 (10 microM) each decreased COX-2 expression when administered before IL-1beta treatment. In control cells, PD-98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 microM)-stimulated PGE(2) release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 microM)-stimulated PGE(2) release, consistent with a role for ERK in the activation of phospholipase A(2) by BK. In IL-1beta-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK- and AA-stimulated PGE(2) release, respectively (P < 0.01), whereas administration of PD-98059 20 h after IL-1beta resulted in only 38 and 43% decreases in basal and BK-stimulated PGE(2) release, respectively (P < 0.02) and had no effect on AA-stimulated PGE(2) release. IL-1beta attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1beta induces PGE(2) synthesis and beta-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase A(2).We have previously reported that interleukin (IL)-1β causes β-adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1β (20 ng/ml) alone. Pretreating cells with the mitogen-activated protein kinase kinase inhibitor PD-98059 or U-126 (2 h before IL-1β treatment) decreased ERK phosphorylation. IL-1β (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal PGE2 release 28-fold ( P < 0.001). PD-98059 (100 μM) and U-126 (10 μM) each decreased COX-2 expression when administered before IL-1β treatment. In control cells, PD-98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 μM)-stimulated PGE2 release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 μM)-stimulated PGE2 release, consistent with a role for ERK in the activation of phospholipase A2 by BK. In IL-1β-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK- and AA-stimulated PGE2 release, respectively ( P < 0.01), whereas administration of PD-98059 20 h after IL-1β resulted in only 38 and 43% decreases in basal and BK-stimulated PGE2release, respectively ( P < 0.02) and had no effect on AA-stimulated PGE2 release. IL-1β attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1β induces PGE2 synthesis and β-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase A2.

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

We have previously reported that interleukin (IL)-1β decreases responsiveness of cultured human airway smooth muscle (HASM) cells to β-agonists. The purpose of this study was to determine whether glucocorticoids inhibit this IL-1β 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-1β (20 ng/ml)-treated cells. In addition, Dex had no effect on Iso-stimulated cAMP formation in control cells but prevented the IL-1β-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-1β-induced hyporesponsiveness to PGE2 stimulation. In contrast, neither IL-1β nor glucocorticoids had any effect on cell stiffness responses to dibutyryl cAMP. We have previously reported that the IL-1β effect on β-adrenergic responsiveness is mediated through cyclooxygenase-2 expression and prostanoid formation. Consistent with these observations, IL-1β-induced cyclooxygenase-2 expression was virtually abolished by FP at concentrations of 10-10 M and greater, with a resultant decrease in PGE2formation. However, Dex did not inhibit IL-1β-induced nuclear translocation of nuclear factor-κB or activator protein-1 in HASM cells. In summary, our results indicate that, in HASM cells, glucocorticoids alone do not alter responses to β-agonists but do inhibit IL-1β-induced β-adrenergic hyporesponsiveness. Glucocorticoids mediate this effect by inhibiting prostanoid formation but without altering nuclear factor-κB or activator protein-1 translocation.