Rapid Induction of Signal Transduction in Respiratory Epithelial Cells in Response to Pseudomonas aeruginosa Ligands ♦ 898

Abstract

Studies of bronchoalveolar lavage from infants with cystic fibrosis (CF) have demonstrated increased amounts of proinflammatory cytokines and polymorphonuclear leukocytes even in the absence of detectable infection. Decreased clearance of secretions in the respiratory tree may lead to exposure of epithelial cells to environmental organisms that would be efficiently cleared in a normal host. We hypothesized that even transient exposure to Pseudomonas aeruginosa (PA) ligands, which recognize specific glycolipid receptors on respiratory epithelial cells, can lead to activation of host signal transduction pathways. Fluxes in intracellular calcium concentration ([Ca2+]i) are common components of proinflammatory signalling cascades and can be readily monitored by fluorescence imaging. We studied the kinetics of changes in [Ca2+]i of epithelial cells in response to wild-type PA as well as to several mutant strains, purified PA ligands, and antibody to the pilin receptor. Single cell imaging was performed on primary human nasal polyp, bovine tracheal epithelial cells, and SV-40 immortalized respiratory cell lines (1HAEo- and 16HBE). The cells were loaded with fura-2/AM, a calcium-sensitive, ratiometric fluorescent dye. Stimulation of the cells with wild-type PA (strain PAO1) triggered a 100-fold increase in [Ca2+]i that was maintained for several minutes. UV-killed organisms did not elicit a change in [Ca2+]i. Stimulation with a non-piliated strain (PAO-NP) or a non-piliated non-flagellated strain (PAO-NP fliA) led to a greatly attenuated calcium response by the epithelial cells. Purified PAO1 flagellin induced a more rapid increase in [Ca2+]i that returned to baseline in less than one minute. Treatment with purified pilin protein or antibody to asialoGM1 (the pilin receptor) led to a typical [Ca2+]i oscillatory response in the epithelial cells. All of the above responses were attenuated but not eliminated by chelation of extracellular calcium with EGTA, indicating that intracellular stores are likely to be responsible for these signals. These results demonstrate activation of signal transduction pathways in respiratory cells in response to only brief exposure to P. aeruginosa ligands.