Airway epithelial anion secretion and barrier function following exposure to fungal aeroallergens: role of oxidative stress

Abstract

Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca²⁺-activated Cl^- channels. Stimulation of anion secretion was dependent on Ca²⁺ uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca²⁺] evoked by AlternariaAlternaria also decreased transepithelial resistance, and a portion of this effect was dependent on the increase in ROS. However, the Alternaria-induced increase in unidirectional dextran (molecular mass = 4,000 Da) flux across the epithelium could not be accounted for by increased oxidative stress. These results support the conclusion that oxidative stress induced by Alternaria was responsible for regulating Ca²⁺-dependent anion secretion and tight junction electrical resistance that would be expected to affect mucociliary clearance.

Keywords: ATP release; chloride secretion; intracellular calcium; tight junctions.

Fig. 1.

Alternaria exposure stimulates transepithelial anion secretion. A: kinetics of the short-circuit current (I_sc) response following apical exposure to 50 and 100 μg/ml Alternaria extract. B: concentration-response relationship for Alternaria (Alt)-evoked increases in steady-state I_sc (n = 4; *significantly different from the basal short-circuit current). C: kinetics of apical DIDS (250 μM) posttreatment and pretreatment on the Alternaria-evoked (50 μg/ml) I_sc response (n = 6). D: concentration-response relationship showing the inhibitory effects of apical DIDS on the Alternaria-induced (50 μg/ml) increase in I_sc (n = 5). The IC₅₀ = 1.23 × 10⁻⁴ M. E: kinetics of apical CaCC_inh (40 μM) and DIDS posttreatment on the Alternaria-evoked I_sc response (n = 6). F: summary bar graph showing the effects of CFTR_inh-172 (20 μM), DIDS (250 or 100 μM), and CaCC_inh (40 μM) on steady-state I_sc increase evoked by Alternaria (50 μg/ml). *Significant difference between the basal and Alternaria-stimulated I_sc. †Significant differences between the Alternaria-stimulated and inhibitor (CFTR_inh-172, CaCC_inh-Ano1, or DIDS)-treated I_sc values.

Fig. 2.

Alternaria-induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]). A: images of 16HBE14o⁻ cell monolayers labeled with fura-2 AM before and after 700 s of stimulation with 50 μg/ml Alternaria extract and after pretreatment with 1 mM EGTA in the extracellular solution. Scale bar = 20 μm. B: kinetics of the Alternaria effect on [Ca²⁺]_i in the absence and presence of 1 mM EGTA in the extracellular solution (n = 25 cells for each condition). C: effects of BAPTA-AM pretreatment (50 and 100 μM) on Alternaria-evoked increases in [Ca²⁺]_i (n = 25 cells for each condition). D: BAPTA pretreatment significantly inhibits the effect of Alternaria on [Ca²⁺]_i (n = 25 cells) and I_sc (n = 8 monolayers). *Significantly different from the basal I_sc. †Significant differences between basal and Alternaria-treated conditions. E: pretreatment with 250 μM DIDS blocks the Alternaria-evoked increase in [Ca²⁺]_i but does not inhibit ionomycin-induced increases in [Ca²⁺]_i (n = 25 cells for each condition). F: summary of the effects of DIDS on Alternaria and ionomycin-induced increases in [Ca²⁺]_i (n = 25 for each condition).

Fig. 3.

Alternaria induces oxidative stress in 16HBE14o⁻ cells. A: basal fluorescence of cells loaded with CellROX orange. B: Alternaria-evoked (50 μg/ml) increase in fluorescence after 15 min of exposure to the extract. C: pretreatment with 5 mM GSH blocks the increase in fluorescence produced by Alternaria exposure. D: summary of the changes in relative fluorescence intensity obtained from images of basal, Alternaria-stimulated, and 5 mM GSH + Alternaria-stimulated conditions (n = 25 cells for each condition). *Significantly different from the basal condition. Scale bar = 20 μm.

Fig. 4.

ROS scavengers and DPI inhibit the effects of Alternaria on ATP release and [Ca²⁺]_i. A: kinetics of ATP release evoked by Alternaria (100 μg/ml). B: effects of DPI (100 μM) and GSH (5 mM) pretreatment (5 min before Alternaria addition) on ATP release evoked by 50 μg/ml Alternaria. *Significantly different from the Alternaria-treated condition. C: GSH inhibits Alternaria-evoked increase in [Ca²⁺]_i (n = 25 cells for each condition). D: DPI inhibits Alternaria-evoked increase in [Ca²⁺]_i (n = 25 cells for each condition). E: summary of the effects of GSH and DPI on [Ca²⁺]_i (n = 25 cells for each condition). *Significantly different from the Alternaria-treated condition. F: concentration-response effects of N-acetyl cysteine (NAC) on [Ca²⁺]_i (n = 25 cells for each condition).

Fig. 5.

Alternaria and H₂O₂ exposure reduces transepithelial resistance (TER) and increases macromolecule permeability of 16HBE14o⁻ cell monolayers. A: time-dependent decreases in TER following exposure to apical Alternaria (100 μg/ml) or H₂O₂ (0.5 mM) compared with untreated controls (n = 6 for each condition). B: pretreatment with 5 mM GSH completely blocks the inhibitory effect of H₂O₂ on TER and partially inhibits the effects of Alternaria on TER (n = 6). C: effects of serine protease exposure (trypsin, 7 μg/ml) or Alternaria (100 μl/ml) on TER before and after pretreatment with the serine protease inhibitor PMSF (0.5 mM) or the cysteine protease inhibitor E64 (10 μM) (n = 3 for each condition). D: effects of Alternaria and H₂O₂ on apical-to-basolateral unidirectional FITC-Dextran (molecular mass  = 4,000 Da) fluxes at time 0, 1 h after treatment, and 4 h after treatment. *Significantly different from time 0 (n = 6 for each condition).

Fig. 6.

Effects of Alternaria and H₂O₂ on tight junction complexes in monolayers of 16HBE14o⁻ cells. A: immunocytochemistry showing tight junction expression of E-cadherin, β-catenin, and ZO1 under untreated control conditions. B: exposure to Alternaria for 4 h produced no measurable change in the expression pattern for E-cadherin, β-catenin, or ZO1. C: treatment with 500 μM H₂O₂ for 4 h diminishes the intensity of β-catenin and ZO1 labeling along the lateral membranes. Scale bar = 20 μm.

Fig. 7.

A model summarizing the key findings of this study. See discussion for details.