Judith S. Grunstein

Mechanism regulating proasthmatic effects of prolonged homologous β2-adrenergic receptor desensitization in airway smooth muscle

Use of long-acting beta(2)-adrenergic receptor (beta2AR) agonists to treat asthma incurs an increased risk of asthma morbidity with impaired bronchodilation and heightened bronchoconstriction, reflecting the adverse effects of prolonged homologous beta2AR desensitization on airway smooth muscle (ASM) function. Since phosphodiesterase 4 (PDE4) regulates ASM relaxation and contractility, we examined whether the changes in ASM function induced by prolonged homologous beta2AR desensitization are attributed to altered expression and action of PDE4. Cultured human ASM cells and isolated rabbit ASM tissues exposed for 24 h to the long-acting beta2AR agonist salmeterol exhibited impaired acute beta2AR-mediated cAMP accumulation and relaxation, respectively, together with ASM constrictor hyperresponsiveness. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity due to enhanced expression of the PDE4D5 isoform and were prevented by pretreating the ASM preparations with the PDE4 inhibitor rolipram or with inhibitors of either PKA or ERK1/2 signaling. Extended studies using gene silencing and pharmacological approaches demonstrated that: 1) the mechanism underlying upregulated PDE4D5 expression following prolonged beta2AR agonist exposure involves PKA-dependent activation of G(i) protein signaling via its betagamma-subunits, which elicits downstream activation of ERK1/2 and its induction of PDE4D5 transcription; and 2) the induction of PDE4 activity and consequent changes in ASM responsiveness are prevented by pretreating the beta2AR agonist-exposed ASM preparations with inhibitors of G(i)-betagamma signaling. Collectively, these findings identify that the proasthmatic changes in ASM function resulting from prolonged homologous beta2AR desensitization are attributed to upregulated PDE4 expression induced by G(i)-betagamma-mediated cross-talk between the PKA and ERK1/2 signaling pathways.

Prolonged heterologous β2-adrenoceptor desensitization promotes proasthmatic airway smooth muscle function via PKA/ERK1/2-mediated phosphodiesterase-4 induction

Beta2-adrenergic receptor (beta2AR) agonists acutely relieve bronchoconstriction via cAMP-mediated relaxation of airway smooth muscle (ASM). Airway constrictor responsiveness may be significantly heightened, however, following protracted exposure to these agents, presumably reflecting the effects of beta2AR desensitization in ASM accompanying prolonged cAMP signaling. Because cAMP phosphodiesterase (PDE) activity can significantly modulate ASM contractility, we investigated the mechanism regulating PDE expression and its potential role in mediating changes in agonist-induced constrictor and relaxation responsiveness in ASM following its heterologous beta2AR desensitization by prolonged exposure to cAMP-elevating agents. Isolated rabbit ASM tissues and cultured human ASM cells treated for 24 h with the receptor- or nonreceptor-coupled cAMP-stimulating agent, prostaglandin E(2) (PGE(2)) or forskolin, respectively, exhibited constrictor hyperresponsiveness to acetylcholine and impaired beta2AR-mediated relaxation and cAMP accumulation. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity, reflective of increased transcription of the PDE4D5 isoform, and were prevented by pretreatment of the ASM with a PDE4 inhibitor. Extended studies using gene silencing and pharmacological approaches to inhibit specific intracellular signaling molecules demonstrated that the mechanism underlying PGE(2)-induced transcriptional upregulation of PDE4D5 involves PKA-dependent activation of G(i) protein signaling via the betagamma-subunits, the latter eliciting downstream activation of ERK1/2 and its consequent induction of PDE4D5 transcription. Collectively, these findings identify that beta2AR desensitization in ASM following prolonged exposure to cAMP-elevating agents is associated with proasthmatic-like changes in ASM responsiveness that are mediated by upregulated PDE4 expression induced by activated cross talk between the PKA and ERK1/2 signaling pathways.

Th2 cytokine-induced upregulation of 11β-hydroxysteroid dehydrogenase-1 facilitates glucocorticoid suppression of proasthmatic airway smooth muscle function

The anti-inflammatory actions of endogenous glucocorticoids (GCs) are regulated by the activities of the GC-activating and -inactivating enzymes, 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-1 and 11beta-HSD2, respectively, that catalyze the interconversion of the inert GC, cortisone, and its bioactive derivative, cortisol. Proinflammatory cytokines regulate 11beta-HSD1 expression in various cell types and thereby modulate the bioavailability of cortisol to the glucocorticoid receptor (GR). Since endogenous GCs reportedly attenuate the airway asthmatic response to allergen exposure, we investigated whether airway smooth muscle (ASM) exhibits cytokine-induced changes in 11beta-HSD1 expression that enable the ASM to regulate its own bioavailability of GC and, accordingly, the protective effect of GR signaling on airway function under proasthmatic conditions. Human ASM cells exposed to the primary proasthmatic T helper type 2 (Th2) cytokine, IL-13, exhibited upregulated expression of 11beta-HSD1, an effect that was attributed to activation of the transcription factor, AP-1, coupled to MAPK signaling via the ERK1/2 and JNK pathways. The induction of 11beta-HSD1 expression and its oxoreductase activity by IL-13 (also IL-4) served to amplify the conversion of cortisone to cortisol by the cytokine-exposed ASM and, hence, heighten GR-mediated transcriptional activation. Extended studies demonstrated that this amplified 11beta-HSD1-dependent GC activation enabled physiologically relevant concentrations of cortisone to exert enhanced protection of ASM tissues from the proasthmatic effects of IL-13 on ASM constrictor and relaxation responsiveness. Collectively, these novel findings identify a Th2 cytokine-driven homeostatic feedback mechanism in ASM that enhances its responsiveness to endogenous GCs by upregulating 11beta-HSD1 activity, thereby curtailing the adverse effects of the proasthmatic cytokine on airway function.

Mechanism of glucocorticoid protection of airway smooth muscle from proasthmatic effects of long-acting β2-adrenoceptor agonist exposure

BACKGROUND Chronic use of long-acting beta2-adrenergic receptor agonists (LABAs), resulting in beta2-adrenergic receptor desensitization, has been associated with increased asthma morbidity. When LABAs are used in combination with inhaled glucocorticoids, however, asthma control is improved, raising the following question: Do glucocorticoids inhibit the proasthmatic mechanism that mediates altered contractility in LABA-exposed airway smooth muscle (ASM)? OBJECTIVE This study aimed to identify the potential protective role and mechanism of action of glucocorticoids in mitigating the effects of prolonged LABA exposure on ASM constrictor and relaxation responsiveness. METHODS Cultured human ASM cells and isolated rabbit ASM tissues were examined for induced changes in agonist-mediated cyclic adenosine monophosphate accumulation, constrictor and relaxation responsiveness, and expression of specific glucocorticoid-regulated molecules after 24-hour exposure to the LABA salmeterol in the absence and presence of dexamethasone. RESULTS Salmeterol-exposed ASM exhibited impaired cyclic adenosine monophosphate and relaxation responses to isoproterenol and increased acetylcholine-induced contractility. These proasthmatic effects of prolonged LABA exposure were attributed to upregulated phosphodiesterase 4 (PDE4) activity and were ablated by pretreatment with dexamethasone. Further studies demonstrated that (1) dexamethasone suppressed activation of the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2), which upregulate PDE4 expression in salmeterol-exposed ASM; and (2) the inhibitory actions of dexamethasone on salmeterol-induced ERK1/2 activation and resultant PDE4-mediated changes in ASM responsiveness were prevented by gene silencing or pharmacologic inhibition of dexamethasone-induced expression of mitogen-activated protein kinase phosphatase 1, an endogenous deactivator of ERK1/2 signaling. CONCLUSION Glucocorticoids prevent the adverse proasthmatic effects of prolonged LABA exposure on airway responsiveness as a result of glucocorticoid-induced upregulation of mitogen-activated protein kinase phosphatase 1, which inhibits proasthmatic ERK1/2 signaling in the LABA-exposed ASM.

Superantigen Presentation by Airway Smooth Muscle to CD4+ T Lymphocytes Elicits Reciprocal Proasthmatic Changes in Airway Function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4+ T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4+ T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4+ T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.

G Protein βγ-Subunit Signaling Mediates Airway Hyperresponsiveness and Inflammation in Allergic Asthma

Since the Gβγ subunit of Gi protein has been importantly implicated in regulating immune and inflammatory responses, this study investigated the potential role and mechanism of action of Gβγ signaling in regulating the induction of airway hyperresponsiveness (AHR) in a rabbit model of allergic asthma. Relative to non-sensitized animals, OVA-sensitized rabbits challenged with inhaled OVA exhibited AHR, lung inflammation, elevated BAL levels of IL-13, and increased airway phosphodiesterase-4 (PDE4) activity. These proasthmatic responses were suppressed by pretreatment with an inhaled membrane-permeable anti-Gβγ blocking peptide, similar to the suppressive effect of glucocorticoid pretreatment. Extended mechanistic studies demonstrated that: 1) corresponding proasthmatic changes in contractility exhibited in isolated airway smooth muscle (ASM) sensitized with serum from OVA-sensitized+challenged rabbits or IL-13 were also Gβγ-dependent and mediated by MAPK-upregulated PDE4 activity; and 2) the latter was attributed to Gβγ-induced direct stimulation of the non-receptor tyrosine kinase, c-Src, resulting in downstream activation of ERK1/2 and its consequent transcriptional upregulation of PDE4. Collectively, these data are the first to identify that a mechanism involving Gβγ-induced direct activation of c-Src, leading to ERK1/2-mediated upregulation of PDE4 activity, plays a decisive role in regulating the induction of AHR and inflammation in a rabbit model of allergic airway disease.

IL-13-induced changes in endogenous glucocorticoid metabolism in the lung regulate the proasthmatic response

Endogenous glucocorticoid (GC) activation is regulated by the intracellular GC-activating and -inactivating enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)1 and 11β-HSD2, respectively, that catalyze interconversion of inert cortisone and its bioactive metabolite cortisol. Because endogenous GCs are critically implicated in suppressing the asthmatic state, this study examined the roles of the 11β-HSD enzymes in regulating GC activation and bronchoprotection during proasthmatic stimulation. Airway hyperresponsiveness to methacholine and inflammation were assessed in rabbits following inhalation of the proasthmatic/proinflammatory cytokine IL-13 with and without pretreatment with the 11β-HSD inhibitor carbenoxolone (CBX). Additionally, IL-13-induced changes in 11β-HSD isozyme expression and GC metabolism were examined in epithelium-intact and -denuded tracheal segments and peripheral lung tissues. Finally, the effects of pretreatment with CBX or 11β-HSD2-targeted siRNAs were investigated with respect to cortisol prevention of IL-13-induced airway constrictor hyperresponsiveness and eotaxin-3 production by airway epithelial cells. IL-13-exposed rabbits exhibited airway hyperresponsiveness, inflammation, and elevated bronchoalveolar lung fluid levels of eotaxin-3. These responses were inhibited by pretreatment with CBX, suggesting a permissive proasthmatic role for 11β-HSD2. Supporting this concept, extended studies demonstrated that 1) IL-13-treated tracheal epithelium and peripheral lung tissues exhibit upregulated 11β-HSD2 activity, 2) the latter impairs cortisone-induced cortisol accumulation and the ability of administered cortisol to prevent both IL-13-induced heightened airway contractility and eotaxin-3 release from epithelial cells, and 3) these proasthmatic responses are prevented by cortisol administration in the presence of 11β-HSD2 inhibition. Collectively, these data demonstrate that the proasthmatic effects of IL-13 are enabled by impaired endogenous GC activation in the lung that is attributed to upregulation of 11β-HSD2 in the pulmonary epithelium.

1660 MATURATIONAL RESPIRATORY RESPONSE TO ENDOGENOUS OPIATES IN NEWBORN RABBITS

Significant changes in respiratory control occur in the human neonate during the early post-natal period. These changes may be related to the action of endorphins, which have been isolated from amniotic fluid and cord blood, and are known to affect respiration. To determine the maturational effects of these agents on respiration, we measured ventilation (VE), tidal volume (VT) and respiratory frequency (f) in 18 rabbit pups (age range: 1 to 19 days) before and after systemic administration of D-Ala2,D-Leu5 enkephalin (ENK). Tracheostomy and carotid artery catheterization were performed under light ether anesthesia. After anesthetic recovery the pups were placed in a body plethysmograph. Saline infusion had no effect. Twenty minutes after infusion of 0.5 ug/g ENK, however, VE was significantly reduced in all pups, ranging from 25 to 80% below control. The VE changes were secondary to decreases in f, although VT increased (+15 to +65%) due to prolongation of inspiratory duration. The degree of VE depression varied inversely with age, being most marked in pups < 4 days old. Indeed, in 4 of 7 pups < 4 days old, ENK produced periodic breathing with apnea. All effects of ENK were abruptly reversed with infusion of naloxone (0.8μg/g), an opiate antagonist. These data provide evidence that: a) opiate-like peptides depress ventilation but their effect diminishes with age; b) early in post-natal life these agents may cause periodic breathing with apnea.

1747 OPIOID EFFECTS ON LUNG MATURATION IN FETAL RABBITS

Based on the observations that the incidence of respiratory distress syndrome is less in infants of opiate-addicted mothers, and that amniotic fluid and fetal cord blood endorphins are increased during late gestation, we tested the hypothesis that endogenous opioids modulate fetal lung development. Pregnant rabbits received daily intramuscular injections, from days 18 to 28 of gestation, of either morphine sulfate (MS) (1.0 mg/kg), naloxone (NLX) (0.4 mg), or saline (C). Their fetuses, delivered prematurely on day 28 (term ∼ 31 days), were assessed for differences in their lung static pressure-volume (P-V) characteristics and morphology. The mean (± SE) deflation lung volume at transthoracic pressure (PT) = 5 cm H2O (V5), expressed as a percentage of the lung volume at PT = 40 (V40), amounted to 70.9 (± 4.3), 52.9 (± 5.8), and 33.9 (± 7.3)% in the MS, C, and NLX fetuses, respectively. Thus, relative to the C group, the MS fetuses had P-V curves displaced significantly (p < .05) upward and to the left, indicating increased alveolar stability; whereas the curves of the NLX fetuses were displaced significantly (p < .05) downward and to the right, demonstrating decreased alveolar stability. Moreover, relative to the C lungs, the MS lungs also appeared more mature histologically, having thinner alveolar septa and a greater airspace-to-tissue ratio. In contrast, the NLX lungs were less developed. These data provide evidence that opiate agonists accelerate intrauterine lung development, whereas opiate antagonists delay lung maturation. These findings suggest that lung development may be significantly modulated by endogenous opioids.

1829 VENTILATORY EFFECTS OF PERIPHERAL CHEMO-RECEPTOR STIMULATION IN MATURING RABBITS

While it is well known that significant changes in ventilatory control occur during postnatal development, the maturational influence of peripheral chemoreceptor stimulation (PCS) during normoxia (N) and hypoxia (H) have not been systematically evaluated. Accordingly, in the present study, we separately assessed the ventilatory responses to PCS with sodium cyanide (NaCN), both during steady-state inhalation of 21% (N) and 10% (H) O2. Following tracheostomy and carotid artery catheterization under light ether anesthesia, pulmonary ventilation (VE) was continually monitored in newborn rabbits (age range: 1-30 days) placed in a body plethysmograph. During N, while saline had no effect, infusions of NaCN (dose range: 0.01 to 0.4 mg/kg) established reproducible dose-dependent increases in VE. The maximal percent increases in VE (VEmax) with NaCN systematically increased with age; however, “sensitivity” to NaCN, represented by the dose of NaCN producing 50% of VEmax, was not age related. In marked contrast to N, during steady-state H, lower doses of NaCN (i.e., less than 0.1 mg/kg) produced less VE stimulation, while higher doses produced VE depression resulting in some cases in acute apnea. Bilateral carotid body denervation abolished the VE responses to NaCN. These findings in maturing rabbits indicate that: (1) during normoxia, PCS with NaCN produces ventilatory stimulation; however, (2) during steady-state hypoxia PCS produces ventilatory depression which may lead to apnea.