Scott M. O'Grady

THE DANGER SIGNAL, EXTRACELLULAR ATP, IS A SENSOR FOR AN AIRBORNE ALLERGEN AND TRIGGERS IL-33 RELEASE AND INNATE TH2-TYPE RESPONSES

The molecular mechanisms underlying the initiation of innate and adaptive proallergic Th2-type responses in the airways are not well understood. IL-33 is a new member of the IL-1 family of molecules that is implicated in Th2-type responses. Airway exposure of naive mice to a common environmental aeroallergen, the fungus Alternaria alternata, induces rapid release of IL-33 into the airway lumen, followed by innate Th2-type responses. Biologically active IL-33 is constitutively stored in the nuclei of human airway epithelial cells. Exposing these epithelial cells to A. alternata releases IL-33 extracellularly in vitro. Allergen exposure also induces acute extracellular accumulation of a danger signal, ATP; autocrine ATP sustains increases in intracellular Ca2+ concentration and releases IL-33 through activation of P2 purinergic receptors. Pharmacological inhibitors of purinergic receptors or deficiency in the P2Y2 gene abrogate IL-33 release and Th2-type responses in the Alternaria-induced airway inflammation model in naive mice, emphasizing the essential roles for ATP and the P2Y2 receptor. Thus, ATP and purinergic signaling in the respiratory epithelium are critical sensors for airway exposure to airborne allergens, and they may provide novel opportunities to dampen the hypersensitivity response in Th2-type airway diseases such as asthma.

Proteases Induce Production of Thymic Stromal Lymphopoietin by Airway Epithelial Cells through Protease-Activated Receptor-2

Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells and triggers dendritic cell-mediated Th2-type inflammation. Although TSLP is up-regulated in epithelium of patients with asthma, the factors that control TSLP production have not been studied extensively. Because mouse models suggest roles for protease(s) in Th2-type immune responses, we hypothesized that proteases from airborne allergens may induce TSLP production in a human airway epithelial cell line, BEAS-2B. TSLP mRNA and protein were induced when BEAS-2B cells were exposed to prototypic proteases, namely, trypsin and papain. TSLP induction by trypsin required intact protease activity and also a protease-sensing G protein-coupled receptor, protease-activated receptor (PAR)-2; TSLP induction by papain was partially dependent on PAR-2. In humans, exposure to ubiquitous airborne fungi, such as Alternaria, is implicated in the development and exacerbation of asthma. When BEAS-2B cells or normal human bronchial epithelial cells were exposed to Alternaria extract, TSLP was potently induced. The TSLP-inducing activity of Alternaria was partially blocked by treating the extract with a cysteine protease inhibitor, E-64, or by infecting BEAS-2B cells with small interfering RNA for PAR-2. Protease-induced TSLP production by BEAS-2B cells was enhanced synergistically by IL-4 and abolished by IFN-γ. These findings demonstrate that TSLP expression is induced in airway epithelial cells by exposure to allergen-derived proteases and that PAR-2 is involved in the process. By promoting TSLP production in the airways, proteases associated with airborne allergens may facilitate the development and/or exacerbation of Th2-type airway inflammation, particularly in allergic individuals.

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current ( I sc; 60%) was inhibited by amiloride (IC50 0.96 μM) or benzamil (IC50 0.5 μM). Basolateral addition of terbutaline (2 μM) produced a rapid decrease in I sc, followed by a slow recovery back to its initial amplitude. When Cl- was replaced with methanesulfonic acid, the basal I sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of -28 mV. Increasing Cl- concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl- conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that β-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl- permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

Adrenergic regulation of ion transport across adult alveolar epithelial cells: effects on Cl- channel activation and transport function in cultures with an apical air interface.

Abstract. The effect of β-adrenergic receptor stimulation on Cl− channel activation was investigated in alveolar epithelial cells grown in monolayer culture and in freshly isolated cells. Monolayers cultured under apical air interface conditions exhibited enhanced amiloride-sensitive Na+ transport compared to apical liquid interface monolayers. Amiloride or benzamil inhibited most (66%) of the basal short circuit current (Isc) with half-maximal inhibitory concentration (IC50) values of 0.62 μm and 0.09 μm respectively. Basolateral addition of terbutaline (2 μm) produced a rapid decrease in Isc followed by a slow recovery that exceeded the basal Isc. When Cl− was replaced with methanesulfonate in either intact monolayers or basolateral membrane permeabilized monolayers, the response to terbutaline (2 μm) was completely inhibited. No effect of terbutaline on amiloride-sensitive Na+ current was detected. β-Adrenergic agonists and 8-chlorothiophenyl cyclic adenosine monophosphate (8-ctp cAMP) directly stimulated a Cl− channel in freshly isolated alveolar epithelial cells. The current was blocked by glibenclamide (100 μm) and had a reversal potential of −22 mV. No increase in amiloride-sensitve current was detected in response to terbutaline or 8-cpt cAMP stimulation. These data support the conclusion that β-adrenergic agonists produce acute activation of apical Cl− channels and that monolayers maintained under apical air interface conditions exhibit increased Na+ absorption.

Regulation of human eosinophil NADPH oxidase activity: A central role for PKCδ

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin‐5 (IL‐5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL‐5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+‐sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL‐5 activation of NADPH oxidase. Additionally, PKCδ inhibitors block PMA, IL‐5 and LTB4 mediated superoxide formation. Interestingly, the PKCδ‐selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL‐5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3‐kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G‐protein‐coupled LTB4 receptor and the IL‐5 cytokine receptor converge on a common signaling pathway involving PI 3‐kinase and PKCδ to regulate NADPH oxidase activity in human eosinophils.

Cystic fibrosis transmembrane conductance regulator is involved in airway epithelial wound repair.

The role of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial wound repair was investigated using normal human bronchial epithelial (NHBE) cells and a human airway epithelial cell line (Calu-3) of serous gland origin. Measurements of wound repair were performed using continuous impedance sensing to determine the time course for wound closure. Control experiments showed that the increase in impedance corresponding to cell migration into the wound was blocked by treatment with the actin polymerization inhibitor, cytochalasin D. Time lapse imaging revealed that NHBE and Calu-3 cell wound closure was dependent on cell migration, and that movement occurred as a collective sheet of cells. Selective inhibition of CFTR activity with CFTR(inh)-172 or short hairpin RNA silencing of CFTR expression produced a significant delay in wound repair. The CF cell line UNCCF1T also exhibited significantly slower migration than comparable normal airway epithelial cells. Inhibition of CFTR-dependent anion transport by treatment with CFTR(inh)-172 slowed wound closure to the same extent as silencing CFTR protein expression, indicating that ion transport by CFTR plays a critical role in migration. Moreover, morphologic analysis of migrating cells revealed that CFTR inhibition or silencing significantly reduced lamellipodia protrusion. These findings support the conclusion that CFTR participates in airway epithelial wound repair by a mechanism involving anion transport that is coupled to the regulation of lamellipodia protrusion at the leading edge of the cell.

Cyclic AMP-dependent Cl secretion is regulated by multiple phosphodiesterase subtypes in human colonic epithelial cells

The role of phosphodiesterase (PDE) isoforms in regulation of transepithelial Cl secretion was investigated using cultured monolayers of T84 cells grown on membrane filters. Identification of the major PDE isoforms present in these cells was determined using ion exchange chromatography in combination with biochemical assays for cGMP and cAMP hydrolysis. The most abundant PDE isoform in these cells was PDE4 accounting for 70-80% of the total cAMP hydrolysis within the cytosolic and membrane fractions from these cells. The PDE3 isoform was also identified in both cytosolic and membrane fractions accounting for 20% of the total cAMP hydrolysis in the cytosolic fraction and 15-30% of the total cAMP hydrolysis observed in the membrane fraction. A large portion of the total cGMP hydrolysis detected in cytosolic and membrane fractions of T84 cells was mediated by PDE5 (50-75%). Treatment of confluent monolayers of T84 cells with various PDE inhibitors produced significant increases in short-circuit current (Isc). The PDE3-selective inhibitors terqinsin, milrinone and cilostamide produced increases in Isc with EC50 values of 0.6 nM, 8.0 nM and 0.5 microM respectively. These values were in close agreement with the IC50 values for cAMP hydrolysis. The effects of the PDE1-(8-MM-IBMX) and PDE4-(RP-73401) selective inhibitors on Isc were significantly less potent than PDE3 inhibitors with EC50 values of >7 microM and >50 microM respectively. However, the effects of 8-MM-IBMX and terqinsin on Cl secretion were additive, suggesting that inhibition of PDE1 also increases Cl secretion. The effect of PDE inhibitors on Isc were significantly blocked by apical treatment with glibenclamide (an inhibitor of the CFTR Cl channel) and by basolateral bumetanide, an inhibitor of Na-K-2Cl cotransport activity. These results indicate that inhibition of PDE activity in T84 cells stimulates transepithelial Cl secretion and that PDE1 and PDE3 are involved in regulating the rate of secretion.

Events in the immortalizing process of primary human mammary epithelial cells by the catalytic subunit of human telomerase.

The in vitro immortalization of primary human mammary epithelial (HME) cells solely by the exogenous introduction of the catalytic subunit of human telomerase (hTERT) has been achieved. Early passage hTERT-transfected HME (T-HME) cells continuously decreased the length and density of telomeres even in the presence of telomerase activity, with a significant number of cells staining positive for senescence-associated beta-galactosidase (SA-beta-gal). Subsequently, with the increase in cell passages, the copy number of the exogenously transfected hTERT gene and the percentage of SA-beta-gal positive cells were found to decrease. Eventually, a single copy of the exogenous hTERT gene was observed in the relatively later passage T-HME cells in which telomere length was elongated and stabilized without obvious activation of endogenous hTERT and c-Myc expression. In T-HME cells, the expression of two p53 regulated genes p21(WAF) and HDM2 increased (as in primary senescent HME cells), and was found to be further elevated as the function of p53 was activated by treatment with DNA-damaging agents. p16(INK4a) was shown to be significantly higher in the primary senescent HME and the early passage T-HME cells when compared with the primary presenescent HME cells, with a dramatic repression of p16(INK4a) observed in the later passage T-HME cells. In addition, the expression of E2F1 and its transcription factor activity were found to be significantly higher in the later passage T-HME cells when compared with the earlier passage T-HME cells. Together, our results indicate that in vitro immortalization in HME cells may require the activation of the function of telomerase and other genetic alterations such as the spontaneous loss of p16(INK4a) expression.

Regulation of eosinophil membrane depolarization during NADPH oxidase activation

Protein kinase C (PKC) activation in human eosinophils increases NADPH oxidase activity, which is associated with plasma membrane depolarization. In this study, membrane potential measurements of eosinophils stimulated with phorbol ester (phorbol 12-myristate 13-acetate; PMA) were made using a cell-permeable oxonol membrane potential indicator, diBAC4(3). Within 10 minutes after PMA stimulation, eosinophils depolarized from– 32.9±5.7 mV to +17.3±1.8 mV. The time courses of depolarization and proton channel activation were virtually identical. Blocking the proton conductance with 250 μM ZnCl2 (+43.0±4.2 mV) or increasing the proton channel activation threshold by reducing the extracellular pH to 6.5 (+44.4±1.4 mV) increased depolarization compared with PMA alone. Additionally, the protein kinase C (PKC) δ-selective blocker, rottlerin, inhibited PMA-stimulated depolarization, indicating that PKCδ was involved in regulating depolarization associated with eosinophil NADPH oxidase activity. Thus, the membrane depolarization that is associated with NADPH oxidase activation in eosinophils is sufficient to produce marked proton channel activation under physiological conditions.

Stable knockdown of CFTR establishes a role for the channel in P2Y receptor-stimulated anion secretion.

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+‐dependent and PKC‐dependent Cl− channels were activated following treatment with uridine triphosphate (UTP) (5 µM). Current‐voltage relationships comparing calcium‐dependent and PKC‐dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl− and I− for the PKC‐dependent conductance (PI/PCl = 0.76), but not for Ca2+‐dependent conductance (PI/PCl = 1.02). The I−/Cl− permeability ratio for the PKC‐dependent conductance was identical to that measured for 8‐cpt cAMP. Furthermore, PKC stimulation using phorbol 12‐myristate 13‐acetate (PMA) activated an apical membrane Cl− conductance that was blocked by the CFTR selective inhibitor, CFTRinh‐172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC‐activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl− secretion, and provided a more sustained secretory response compared to conditions where only Ca2+‐activated Cl− channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium. J.Cell.Physiol.