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A7558 - Ozone Stimulates Basolateral Release of ATP from Normal and Asthmatic Primary Human Bronchial Epithelial Cells
Author Block: S. L. Tilley1, Z. A. German2, C. M. Jania2, S. D. Budney2, C. Van Heusden1, E. Lazarowski1; 1Univ of N Carolina At Chapel Hill, Chapel Hill, NC, United States, 2Medicine, Univ of N Carolina At Chapel Hill, Chapel Hill, NC, United States.
Rationale: Mast cells are important intermediaries of asthma exacerbations. Since mast cells largely reside beneath the epithelium, epithelial derived mediators must be secreted basolaterally to stimulate mast cell activation. Epithelia can release substantial amounts of ATP following stimulation; however, it is generally believed that ATP is secreted apically rather than basolaterally. The objective of this study was to test the hypothesis that ozone can stimulate both apical and basolateral secretion of ATP, and activate mast cells on the basolateral side. Methods: Ozone-induced nucleotide release was measured using primary human bronchial epithelial cells (HBECs) from normal donors, primary HBECs from allergic asthmatics, and from the lungs in vivo from naïve and HDM-allergic mice. Passage 1 HBECs were cultured for 28-35 days at ALI, and then exposed to ozone 0.4 PPM or air for 3 hours; mice were exposed to ozone 2 PPM or air for 3 hours. Following ozone exposures, ATP, ADP, AMP, and adenosine levels were measured in the basolateral media, apical wash, and BAL fluid by ethenoderivatization/HPLC. HBEC-mast cell co-culture experiments were also performed to determine if activation of mast cells in the basolateral media could result from apical exposure of HBECs to ozone. Results: Ozone exposure resulted in a rise in nucleotide levels in the apical wash and basolateral media from HBECs, and in the BAL fluid from mice. Significant amounts of ATP, ADP, AMP, and adenosine were found in the basolateral media, and nucleotide levels from asthmatic HBECs were similar to levels from normal HBECs (Total nucleotides: 2400 + 200 nM vs. 2400+/- 800 nM). In contrast, approximately 10-fold greater total nucleotide levels were found in the BAL from HDM-allergic mice compared to naïve mice (5000 + 1000 nM vs. 500 + 50 nM). Finally, histamine levels in the basolateral media increased following ozone exposure in HBEC-mast cell co-cultures. Conclusions: These findings support a model in which ATP release from airway epithelia following ozone exposure activates sub-epithelial mast cells, and contributes to the pathogenesis of ozone-induced asthma exacerbation.