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. Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz. J Appl Physiol 89: 1619-1632, 2000.-We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (GЈ) and loss modulus (GЉ) from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, GЈ increased weakly with frequency, whereas GЉ was independent of the frequency. The cell was predominantly elastic, with the ratio of GЉ to GЈ (defined as ) being ϳ0.35 at all frequencies. GЈ and GЉ increased together after contractile activation and decreased together after deactivation, whereas remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. GЈ and GЉ decreased with disruption of the actin fibers by cytochalasin D, but increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton. cytoskeleton; storage modulus; viscoelasticity; contraction; structural damping; magnetic twisting cytometry PHYSICAL FORCES ACTING ON cells and the resulting cell deformations affect critical features of cell function, including proliferation, differentiation, wound healing, protein and DNA synthesis, apoptosis, cell shape, and cell motility (3,5,6,22). When deformed, a cell stores mechanical energy, and this stored energy permits subsequent recovery of shape. The cell also dissipates mechanical energy through mechanical friction. The structural origin for cell elasticity is widely believed to originate in the network of actin fibers within the cytoskeleton (50). However, the origin for energy dissipation is less clear but is often thought to arise from viscous mechanisms associated with shear of the cytoplasmic fluids (2,26,45,51).A common method to separate elastic from dissipative behavior for any material is to measure responses to oscillatory loads (21). Although the oscillatory mechanics of reconstituted gels of the cytoskeletal filaments have been well studied (24,32,36), there have been relatively few investigations of the oscillatory mechanics of the intact living cell. In studies of the oscillatory mechanics, the cell was probed with the use of a variety of techniques: through the cell surface by atomic force microscopy (46), from the interior by oscillating intracellular granuoles using laser tracking (60), along the cell longitudinal axis using glass manipulators (47), or in a cell pellet using oscillating disk rheometry (12,23). However, no study has yet reported the oscillatory mechanics of the cell by accessing the cytoskeleton through direct attachments to focal adhesions. Focal adhesions are the mechanical connections linking the cytoskeleton of t...

Decreased beta-adrenergic responsiveness is a characteristic feature of asthma. In order to determine whether cytokines released in the asthmatic airway contribute to this phenomenon, we measured changes in stiffness of cultured human airway smooth muscle (HASM) cells induced by isoproterenol (ISO) in control HASM cells and HASM cells pretreated with IL-1 beta (20 ng/ml for approximately 42 h). Stiffness was measured by magnetic twisting cytometry. HASM cells were obtained from normal tracheal tissue obtained at lung transplant, and studied in passages 4-7. In control cells, ISO caused a dose-related decrease in cell stiffness. IL-1 beta had no effect on baseline cell stiffness. However, IL-1 beta caused a rightward shift in the concentration-response curve to ISO and decreased the maximal effectiveness of this agonist. Decreased responses to ISO were also obtained with 2 ng/ml IL-1 beta, or when cells were pretreated with IL-1 beta (20 ng/ml) for 22 h. This effect of IL-1 beta was not altered by pretreatment of the cells with pertussis toxin (100 ng/ml throughout the IL-1 beta exposure period). IL-1 beta also significantly attenuated the ability of prostaglandin E2 (PGE2) to decrease cell stiffness. In contrast, IL-1 beta had no effect on cell stiffness responses to dibutryl cAMP, a cell permeant analog of cAMP suggesting that the cytokine does not influence either the ability of cAMP to activate kinases, or the targets of these kinases which ultimately mediate cell relaxation. IL-1 beta (20 ng/ml for 40 h) caused a small (30%) but significant (P < 0.02) increase in basal cAMP, but also resulted in a 2-3-fold decrease in the changes in cAMP formation induced by either ISO or PGE2. In contrast, IL-1 beta had no effect on cAMP formation in response to forskolin, suggesting that IL-1 beta does not mediate its effects via changes in the expression or activity of adenylyl cyclase. Pretreatment with IL-1 beta had no significant effect on beta 2 adrenoceptor number assessed by [125I]-CYP binding in these cells, nor was there any significant effect of IL-1 beta on Gsa expression assessed by Western blot. In summary, our results indicate that IL-1 beta causes a concentration- and time-dependent decrease in responses of HASM cells to ISO and are consistent with the hypothesis that the effects of IL-1 beta are mediated by uncoupling of beta-receptors from Gs-induced activation of adenylyl cyclase.

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.

We examined the influence of two common polymorphic forms of the beta(2)-adrenergic receptor (beta(2)AR): the Gly16 and Glu27 alleles, on acute and long-term beta(2)AR desensitization in human airway smooth muscle (HASM) cells. In cells from 15 individuals, considered without respect to genotype, pretreatment with Isoproterenol (ISO) at 10(-7) M for 1 h or 24 h caused approximately 25% and 64% decreases in the ability of subsequent ISO (10(-6) M) stimulation to reduce HASM cell stiffness as measured by magnetic twisting cytometry. Similar results were obtained with ISO-induced cyclic adenosine monophosphate (cAMP) as the outcome indicator. Data were then stratified post hoc by genotype. Cells containing at least one Glu27 allele (equivalent to presence of the Gly16Glu27 haplotype) showed significantly greater acute desensitization than did cells with no Glu27 allele, whether ISO-induced cell stiffness (34% versus 19%, p < 0.03) or cAMP formation (58% versus 11%, p < 0.02) was measured. Likewise, cells with any Glu27 allele showed greater long-term desensitization of cell stiffness and cAMP formation responses than did cells without the Glu27 allele. The distribution of genotypes limited direct conclusions about the influence of the Gly16 allele. However, presence of the Gly16Gln27 haplotype was associated with less acute and long-term desensitization of ISO-induced cAMP formation than was seen in cells without the Gly16Gln27 haplotype (14% versus 47%, p < 0.09 for short-term desensitization; 32% versus 84%, p < 0.01 for long-term desensitization), suggesting that the influence of Glu27 is not through its association with Gly16. The Glu27 allele was in strong linkage disequilibrium with the Arg19 allele, a polymorphic form of the beta(2)AR upstream peptide of the 5'-leader cistron of the beta(2)AR, and this polymorphism in the beta(2)AR 5'-flanking region may explain the effects of the Glu27 allele. Cells with any Arg19 allele showed significantly greater acute and long-term desensitization of ISO-induced cAMP formation than did cells without the Arg19 allele (54% versus 2%, p < 0.01 for short-term desensitization; 73% versus 35%, p < 0.05 for long-term desensitization). Similar results were obtained for ISO-induced changes in cell stiffness. Thus, the presence of the Glu27 allele is associated with increased acute and long-term desensitization in HASM.

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)-1 beta causes beta-adrenergic hyporesponsiveness in cultured human airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2 expression. The purpose of this study was to determine whether p38 mitogen-activated protein (MAP) kinase is involved in these events. IL-1 beta (2 ng/ml for 15 min) increased p38 phosphorylation fourfold. The p38 inhibitor SB-203580 (3 microM) decreased IL-1 beta-induced COX-2 by 70 +/- 7% (P < 0.01). SB-203580 had no effect on PGE(2) release in control cells but caused a significant (70-80%) reduction in PGE(2) release in IL-1 beta-treated cells. IL-1 beta increased the binding of nuclear proteins to the oligonucleotides encoding the consensus sequences for activator protein (AP)-1 and nuclear factor (NF)-kappa B, but SB-203580 did not affect this binding, suggesting that the mechanism of action of p38 was not through AP-1 or NF-kappa B activation. The NF-kappa B inhibitor MG-132 did not alter IL-1 beta-induced COX-2 expression, indicating that NF-kappa B activation is not required for IL-1 beta-induced COX-2 expression in HASM cells. IL-1 beta attenuated isoproterenol-induced decreases in HASM stiffness as measured by magnetic twisting cytometry, and SB-203580 abolished this effect. These results are consistent with the hypothesis that p38 is involved in the signal transduction pathway through which IL-1 beta induces COX-2 expression, PGE(2) release, and beta-adrenergic hyporesponsiveness.

In human cultured airway smooth muscle cells, interleukin (IL)-1 beta increases cyclooxygenase (COX)-2 expression and PGE(2) release, ultimately resulting in decreased beta-adrenergic responsiveness. In this study, we aimed to determine whether tumor necrosis factor-alpha (TNF-alpha) synergizes with IL-1 beta in the induction of these events. TNF-alpha alone, at concentrations up to 10 ng/ml, had no effect on COX-2 protein expression; at concentrations as low as 0.1 ng/ml, it significantly enhanced the ability of IL-1 beta (0.2 ng/ml) to induce COX-2 and to increase PGE(2) release. IL-1 beta and TNF-alpha in combination also significantly enhanced COX-2 promoter activity, indicating that synergism between the cytokines is mediated at the level of gene transcription. Although IL-1 beta and TNF-alpha each increased nuclear factor-kappa B activation and induced extracellular regulated kinase and p38 phosphorylation, combined administration of the cytokines did not enhance either nuclear factor-kappa B or mitogen-activated protein kinase activation. Combined administration of IL-1 beta (0.2 ng/ml) and TNF-alpha (0.1 or 1.0 ng/ml) reduced the ability of isoproterenol to decrease human airway smooth muscle cell stiffness, as measured by magnetic twisting cytometry, even though individually these cytokines, at these concentrations, had no effect on isoproterenol responses. Treatment with the selective COX-2 inhibitor NS-398 abolished the synergistic effects of TNF-alpha and IL-1 beta on beta-adrenergic responsiveness. Our results indicate that low concentrations of IL-1 beta and TNF-alpha synergize to promote beta-adrenergic hyporesponsiveness and that effects on COX-2 expression and PGE(2) are responsible for these events. The data suggest that the simultaneous release in the airway, of even very small amounts of cytokines, can have important functional consequences.