Philip R. Cooper

Steroids completely reverse albuterol-induced β2-adrenergic receptor tolerance in human small airways

BACKGROUND Evidence suggests that chronic stimulation of beta(2)-adrenergic receptors (beta(2)-ARs) induces receptor tolerance that limits the efficacy of beta-agonists in the treatment of asthma. The precise mechanisms that induce beta(2)-AR tolerance remain unclear. OBJECTIVE We sought to determine whether steroids modulate albuterol-induced beta(2)-AR tolerance in human small airways. METHODS beta(2)-AR responsiveness to isoproterenol was characterized in human precision-cut lung slices (PCLSs) precontracted to carbachol after pretreatment with albuterol. RESULTS Incubation of PCLSs with albuterol for 3, 6, or 12 hours attenuated subsequent isoproterenol-induced relaxation in a dose- and time-dependent manner. A 40% decrease (P < .0001) in maximum relaxation and a 45% decrease (P = .0011) in airway sensitivity from control values occurred after the maximum time and concentration of albuterol incubation. Desensitization was not evident when airways were relaxed to forskolin. Dexamethasone pretreatment of PCLSs (1 hour) prevented albuterol-induced beta(2)-AR desensitization by increasing the maximum drug effect (P = .0023) and decreasing the log half-maximum effective concentration values (P < .0001) from that of albuterol alone. Albuterol (12-hour incubation) decreased the beta(2)-AR cell-surface number (P = .013), which was not significantly reversed by 1 hour of preincubation with dexamethasone. CONCLUSION These data suggest that beta(2)-AR desensitization occurs with prolonged treatment of human small airways with albuterol through mechanisms upstream of protein kinase A and that steroids prevent or reverse this desensitization. Clarifying the precise molecular mechanisms by which beta(2)-AR tolerance occurs might offer new therapeutic approaches to improve the efficacy of bronchodilators in asthma and chronic obstructive pulmonary disease.

TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation

Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyperreactivity. Toll-like receptor 3 (TLR3) recognizes viral double-stranded (ds) RNA such as polyinosinic-polycytidylic acid [poly(I:C)] and stimulates innate immune responses. The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and beta-adrenergic receptor agonists in human lung slices. Human airway smooth muscle (ASM) was incubated for 24 h in poly(I:C) +/- TNFalpha and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250-microm thickness) from healthy human lungs containing a small airway were incubated in 0, 10, or 100 microg/ml poly(I:C) for 24 h. Intravital microscopy of lung slices was used to quantify contractile and relaxation responsiveness to carbachol and isoproterenol, respectively. Supernatants of ASM and PCLS were analyzed for cytokine secretion using a 25-multiplex bead assay. In human ASM, poly(I:C) (0.5 microg/ml) increased macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 and 100 microg/ml) had little effect on the log EC(50) or maximum drug effect (E(max)) for contraction and relaxation in response to carbachol and isoproterenol, respectively. The responsiveness of the same human PCLS to poly(I:C) incubation was confirmed by the robust increase in chemokines and cytokines. In separate experiments, incubation of PCLS with IL-13 or TNFalpha (100 ng/ml) increased airway sensitivity to carbachol. Poly(I:C) promotes inflammatory mediator release that was not associated with enhanced bronchoconstriction or attenuated bronchodilation in normal healthy human lung slices. Transduction at the TLR3 initiated by dsRNA stimulates downstream innate immune responses.

Expression and activation of the oxytocin receptor in airway smooth muscle cells: Regulation by TNFα and IL-13

BackgroundDuring pregnancy asthma may remain stable, improve or worsen. The factors underlying the deleterious effect of pregnancy on asthma remain unknown. Oxytocin is a neurohypophyseal protein that regulates a number of central and peripheral responses such as uterine contractions and milk ejection. Additional evidence suggests that oxytocin regulates inflammatory processes in other tissues given the ubiquitous expression of the oxytocin receptor. The purpose of this study was to define the role of oxytocin in modulating human airway smooth muscle (HASMCs) function in the presence and absence of IL-13 and TNFα, cytokines known to be important in asthma.MethodExpression of oxytocin receptor in cultured HASMCs was performed by real time PCR and flow cytomery assays. Responses to oxytocin was assessed by fluorimetry to detect calcium signals while isolated tracheal rings and precision cut lung slices (PCLS) were used to measure contractile responses. Finally, ELISA was used to compare oxytocin levels in the bronchoalveloar lavage (BAL) samples from healthy subjects and those with asthma.ResultsPCR analysis demonstrates that OXTR is expressed in HASMCs under basal conditions and that both interleukin (IL)-13 and tumor necrosis factor (TNFα) stimulate a time-dependent increase in OXTR expression at 6 and 18 hr. Additionally, oxytocin increases cytosolic calcium levels in fura-2-loaded HASMCs that were enhanced in cells treated for 24 hr with IL-13. Interestingly, TNFα had little effect on oxytocin-induced calcium response despite increasing receptor expression. Using isolated murine tracheal rings and PCLS, oxytocin also promoted force generation and airway narrowing. Further, oxytocin levels are detectable in bronchoalveolar lavage (BAL) fluid derived from healthy subjects as well as from those with asthma.ConclusionTaken together, we show that cytokines modulate the expression of functional oxytocin receptors in HASMCs suggesting a potential role for inflammation-induced changes in oxytocin receptor signaling in the regulation of airway hyper-responsiveness in asthma.

Efflux of monoclonal antibodies from rat brain by neonatal Fc receptor, FcRn.

Monoclonal antibody (mAb) engineering that optimizes binding to receptors present on brain vascular endothelial cells has enabled them to cross through the blood-brain barrier (BBB) and access the brain parenchyma to treat neurological diseases. However, once in the brain the extent to which receptor-mediated reverse transcytosis clears mAb from the brain is unknown. The aim of this study was to determine the contribution of the neonatal Fc-receptor (FcRn) in rat brain efflux employing two different in vivo drug delivery models. Two mAb variants with substantially different affinities to FcRn, and no known neuronal targets, (IgG1 N434A and H435A) were administered to rats via intranasal-to-central nervous system (CNS) and intra-cranial dosing techniques. Levels of full-length IgG were quantified in serum and brain hemispheres by a sensitive enzyme-linked immunosorbent assay (ELISA). Following intra-nasal delivery, low cerebral hemisphere levels of variants were obtained at 20min, with a trend towards faster clearance of the high FcRn binder (N434A); however, the relatively higher serum levels confounded analysis of brain FcRn contribution to efflux. Using stereotaxic coordinates, we optimized the timing and dosing regimen for injection of mAb into the cortex. Levels of N434A, but not H435A, decreased in the cerebral hemispheres following bilateral injection into the rat cortex and higher levels of N434A were detected in serum compared to H435A after 24h. Immunohistochemical staining of human IgG1 in sections of cortex was consistent with these results, illustrating relatively less intense immunostaining in N434A than H435A dosed animals. Using two in vivo methods with direct cranial administration, we conclude that FcRn plays an important role in efflux of IgG from the rat brain.

Formoterol and salmeterol induce a similar degree of β2‐adrenoceptor tolerance in human small airways but via different mechanisms

Steroids prevent and reverse salbutamol‐induced β2‐adrenoceptor tolerance in human small airways. This study examines the effects of the long‐acting β2 agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization.

20-HETE Mediates Ozone-Induced, Neutrophil-Independent Airway Hyper-Responsiveness in Mice

Background Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways. Methodology/Principal Findings Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 µm thickness) containing an intrapulmonary airway (∼0.01 mm2 lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC50 and Emax values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment. Conclusions/Significance These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.

An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma

In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.

β-Agonist-associated Reduction in RGS5 Expression Promotes Airway Smooth Muscle Hyper-responsiveness

Although short-acting and long-acting inhaled β2-adrenergic receptor agonists (SABA and LABA, respectively) relieve asthma symptoms, use of either agent alone without concomitant anti-inflammatory drugs (corticosteroids) may increase the risk of disease exacerbation in some patients. We found previously that pretreatment of human precision-cut lung slices (PCLS) with SABA impaired subsequent β2-agonist-induced bronchodilation, which occurred independently of changes in receptor quantities. Here we provide evidence that prolonged exposure of cultured human airway smooth muscle (HuASM) cells to β2-agonists directly augments procontractile signaling pathways elicited by several compounds including thrombin, bradykinin, and histamine. Such treatment did not increase surface receptor amounts or expression of G proteins and downstream effectors (phospholipase Cβ and myosin light chain). In contrast, β-agonists decreased expression of regulator of G protein signaling 5 (RGS5), which is an inhibitor of G-protein-coupled receptor (GPCR) activity. RGS5 knockdown in HuASM increased agonist-evoked intracellular calcium flux and myosin light chain (MLC) phosphorylation, which are prerequisites for contraction. PCLS from Rgs5−/− mice contracted more to carbachol than those from WT mice, indicating that RGS5 negatively regulates bronchial smooth muscle contraction. Repetitive β2-agonist use may not only lead to reduced bronchoprotection but also to sensitization of excitation-contraction signaling pathways as a result of reduced RGS5 expression.

Involvement of IL-13 in tobacco smoke-induced changes in the structure and function of rat intrapulmonary airways.

Chronic obstructive pulmonary disease (COPD) involves disease of small airways with an increase in airway smooth muscle sensitivity to spasmogens and with structural changes described as airway remodeling. We investigated the effect of tobacco smoke (TS) exposure on the structure and function of small airways in rats and the role of IL-13 in this response. Precision-cut lung slices (230-280 microm) were prepared from male Sprague-Dawley rats after acute (3 d) or chronic (8 or 16 wk) daily exposure to TS or air. Carbachol (CCh) and 5-hydroxytryptamine (5HT) concentration responses were performed on airways (50-400 microm diameter). The effect of IL-13 in vitro on small airway sensitivity to CCh and 5HT was also determined. Acute exposure to TS did not affect the sensitivity of the intrapulmonary airways to either spasmogen. After 8 weeks of TS exposure, airway hyperresponsiveness (AHR) to CCh was evident (log EC(50) CCh: air = 0.22 microM; TS = -0.12 microM; P = 0.019); AHR to 5HT was also observed after the 16-week exposure to TS (air = -0.85 microM; TS = -1.06 microM; P = 0.038). Chronic TS exposure increased airway wall SMA content, which correlated with increased expression of IL-13 and transforming growth factor (TGF)-beta(1) in the lung tissues. In vitro incubation with IL-13, but not TGF-beta(1), induced changes in small airway sensitivity to CCh and 5HT. Chronic TS exposure induces increased responsiveness in intrapulmonary airways of rats that may be mediated in part by an increase in IL-13.

C-027 inhibits IgE-mediated passive sensitization bronchoconstriction and acts as a histamine and serotonin antagonist in human airways.

Atopic asthma is poorly controlled by current therapies. Newer therapies and novel antihistamines are, therefore, required to treat patients whose atopic asthma is not controlled. For the first time, C-027 is shown to antagonize histamine, IgE-mediated and serotonin-induced contraction in human airways and vessels. Human precision-cut lung slices (PCLS, 250 μm thick), containing an airway or blood vessel, were pretreated with either C-027 (2 hours) or with vehicle alone and were contracted with histamine or serotonin. Known antihistamine was used as a comparator in antihistamine studies. Also, human airways were contracted via IgE passive sensitization in the presence or absence of C-027 or fexofenadine. Affinity of C-027 toward human G-protein coupled receptors was also determined, as well as the drugs biodistribution in murine model. C-027 was shown to have the highest affinity toward human histamine and serotonin receptors. Subsequently, C-027 was shown to antagonize histamine- and serotonin-induced airway and vascular smooth muscle contraction, respectively, and histamine-released bronchocontraction mediated by IgE passive sensitization in human small airways. C-027 also inhibited histamine-mediated single-cell calcium ion release. Low levels of C-027 were found in murine brain tissue. Collectively, these data suggest that C-027 markedly inhibits IgE-induced bronchoconstriction and antagonizes histamine and serotonin-contraction with little biodistribution in the brain. The compound may offer a future therapy for allergen-induced airway hyperresponsiveness in patients with asthma.