Oxidative stress serves as a key checkpoint for IL-33 release by airway epithelium.

BACKGROUND: Interleukin (IL)-33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL-33 release is controlled. The transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a key role in antioxidant response regulation. OBJECTIVE: The goal of this project was to investigate the role of cellular oxidative stress in controlling IL-33 release in airway epithelium. METHODS: Complementary approaches were used that included human bronchial epithelial cells and mouse models of airway type-2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) was used to evaluate the role of Nrf2-induced antioxidant molecules. RESULTS: Human bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N-acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL-33 release. Administration of CDDO-Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO-Me treatment reduced allergen-induced ATP secretion and IL-33 release by airway epithelial cells in vitro and protected mice from IL-33 release and asthma-like pathological changes in the lungs. CONCLUSIONS: The balance between oxidative stress and antioxidant responses plays a key role in controlling IL-33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Basolateral Cl- transport is stimulated by terbutaline in adult rat alveolar epithelial cells.

Stimulation of adult rat alveolar epithelial cells with terbutaline was previously shown to activate Cl- channels in the apical membrane. In this study, we show that terbutaline stimulates net transepithelial (apical-to-basolateral) Cl- absorption from 0.19 +/- 0.13 to 1.43 +/- 0.31 mmol x cm-2 x hr-1. Terbutaline also increases net Cl- efflux across the basolateral membrane under conditions where an outward [K+] gradient exists and the membrane voltage is clamped at zero mV. When the [K+] gradient is eliminated, the effect of terbutaline on net Cl- efflux is inhibited to the extent that no significant Cl- efflux can be detected across the basolateral membrane. RT-PCR experiments detected mRNA for three KCl cotransport isoforms (KCC1, KCC3 and KCC4) in monolayer cultures of alveolar epithelial cells. Western blot analysis using antibodies to the four cloned isoforms of KCl cotransporters revealed the presence of KCC1 and KCC4 isoforms in monolayer cultures of these cells. These results provide evidence suggesting a role for KCl cotransport in terbutaline-stimulated transepithelial Cl- absorption.

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.