Role of Ca2+ in responses of airway epithelia to Pseudomonas aeruginosa, flagellin, ATP, and thapsigargin.

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration (Ca) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-kappaB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca(2+) pump, releases endoplasmic reticulum Ca(2+), and triggers Ca(2+) entry through plasma membrane channels) both increased Ca but hardly stimulated NF-kappaB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-kappaB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer Ca) or Ca(2+)-free solution reduced increases in Ca due to ATP or thapsigargin and also reduced NF-kappaB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)beta/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-kappaB-binding site was critical for activation by all agonists and for activation by Ca. Thus increased Ca was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased Ca was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in Ca synergized with flagellin or P. aeruginosa, and buffering or reducing Ca reduced these responses. Thus Ca plays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 microM and [flagellin] >10(-8) g/ml and during steady increases rather than oscillations in Ca.