Role of Ca²⁺in responses of airway epithelia toPseudomonas aeruginosa, flagellin, ATP, and thapsigargin

Abstract

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca(2+) concentration ([Ca](i)) 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](i) 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](i)) or Ca(2+)-free solution reduced increases in [Ca](i) 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](i). Thus increased [Ca](i) was not required for P. aeruginosa- or flagellin-activated NF-kappaB and IL-8 expression and secretion, and increased [Ca](i) was only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca](i) synergized with flagellin or P. aeruginosa, and buffering or reducing [Ca](i) reduced these responses. Thus [Ca](i) 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](i).

Redox-independent Activation of NF-κB by Pseudomonas aeruginosa Pyocyanin in a Cystic Fibrosis Airway Epithelial Cell Line

“…Because increases in Ca i potently synergize with flagellin in stimulating NF-B in airway epithelial cells (19), this Ca i -stimulating effect of PYO could be particularly important when the bacteria also produce and release flagellin into the airway surface liquid. We therefore tested whether PYO or H 2 O 2 triggered changes in Ca i in CF15 cells.…”

“…Treatments with agonists were made by diluting stock solutions into Ringer's solution at the concentrations stated in the text. Fluorescence ratio imaging measurements of cytosolic Ca i were performed using methods that have been reported previously (19,31). Briefly, a Nikon Diaphot inverted microscope was used with a 40ϫ Neofluar objective (1.4 NA).…”

“…research showed 80 -300 M PYO triggered increases in Ca i in human bronchial epithelial cells and A549 cells (20) and that increases in Ca i synergized markedly with flagellin in activating NF-B and IL8 secretion (19). We therefore tested whether the synergism noted between flagellin and PYO in activating NF-B involved similar changes in Ca i in CF15 cells.…”

“…PYO activation of IL8 production may occur through effects on cellular signaling leading to activation of the transcription factors AP-1, NF-IL6/C-EBP, and/or NF-B, which control IL8 production (19). It is widely assumed that the effects of PYO on signaling are mediated at least in part through its ability to redox cycle with cellular NADPH and/or GSH leading to the production of ROS (9,13,20,21) and oxidation of the cytosol and/or mitochondria (21,22).…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Purinergic receptors that are activated by ATP include P2X receptor channels and P2Y G-protein coupled receptors that mediate the activation of phospholipase C, intracellular Ca 2+ mobilization and activation of PKC (139). Several P2X and P2Y isoforms are expressed in airway epithelial cells and are involved in regulating a number of epithelial properties including epithelial anion transport (139,147), ciliary beat frequency (139), mucin secretion (148-151), production of inflammatory mediators such as CCL20, IL-8, and MMP-9 (87,(152)(153)(154), as well as epithelial cell migration and wound repair (155)(156)(157). Since P2 receptor stimulation induces Ca 2+ mobilization, it is not surprising that extracellular ATP is capable of stimulating DUOX-mediated H 2 O 2 production by airway epithelial cells.…”