O’Grady, Kelly E.

Aeroallergens produced by Alternaria alternata have been shown to induce life threatening exacerbations of airway inflammation in sensitized patients with severe asthma. Inflammation produced by Alternaria is associated with ATP release and purinergic receptor activation. In the present study the effects of Alternaria on signaling pathways and ion transport mechanisms associated with mucociliary clearance were investigated in an immortalized human bronchial epithelial cell line (16HBE14o ‐ cells). Cells were grown on Snapwell TM membrane filters until a stable transepithelial resistance (>500 Ω) was established. Cell monolayers were then voltage clamped at zero mV and apically stimulated with 50 μM Alternaria extract to induce a time‐dependent increase in short circuit current (Isc) consistent with anion secretion. Treatment with the CFTR channel blocker, CFTR inh ‐172 significantly reduced the Isc by ~25%. Furthermore, addition of the disulfonic stilbene compound DIDS (250 μM) abolished the Isc response. Measurements of the effect of Alternaria on intracellular Ca 2+ concentration ([Ca 2+ ] i ) showed that the extract caused a persistent increase in [Ca 2+ ] i that was blocked by chelating extracellular Ca 2+ with EGTA. Pretreatment with 250 μM DIDS also abrogated the effects of Alternaria on [Ca 2+ ] i but did not affect the increase in [Ca 2+ ] i produced by 4 μM ionomycin. Similarly, pretreatment of monolayers with glutathione also blocked the Alternaria induced increase in [Ca 2+ ] i . These results indicate that Alternaria aeroallergens are capable of stimulating CFTR and Ca 2+ ‐dependent anion secretion. Moreover, the increase in [Ca 2+ ] i appears to involve a DIDS‐sensitive uptake mechanism that is blocked when oxidant scavenger molecules are present in the extracellular solution. Grant Funding Source : Supported by NIH: HL110539

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 Alternaria. Alternaria 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.