Flagellin‐triggered NF‐kappaB in airway epithelia: Role for calcium?

We tested the role of cytosolic [Ca], Cai, in controlling innate immune responses of airway epithelia to P. aeruginosa (PA) by measuring Cai (fura‐2 and imaging microscopy), IL8 secretion (by ELISA), IL8 promoter activity (luciferase) and NF‐kB activation (using NF‐kB‐activated luciferase) in two airway cell lines, JME (nasal cell, cystic fibrosis) and Calu‐3 (serous‐like gland cell, non‐CF). ATP (purinergic agonist) and thapsigargin (TG, SERCA pump blocker) both increased epithelial Cai, and these were reduced or abolished by BAPTA/AM (Ca‐chelator) or Ca‐free bathing solution. In contrast, FLG or PA increased Cai only infrequently (< 10% of cells). IL8 promoter activity and secretion were increased slightly during treatments of JME or Calu‐3 by ATP and TG and to a much greater extent by FLG or PA, but not by PAfliC (do not express FLG structural gene). Responses to FLG or PA were augmented synergistically by ATP and, especially, TG, and these were inhibited by BAPTA or Ca‐free solution. Mutation analysis of the IL8 promoter showed that the NF‐kB binding site was required to obtain activation in response to FLG or PA, alone or in combination with ATP and TG, while AP‐1 and NF‐IL6 sites exerted modulatory roles. We conclude: Activation of IL8 expression and secretion in airway epithelia by PA requires FLG expression in the bacteria and NF‐kB activation in the epithelia. Although increases in Cai are not required to activate NF‐kB and IL8 expression and secretion in response to PA or FLG, Cai plays an important modulatory role in FLG‐triggered activation of NF‐kB and subsequent IL8 production. Ca entry into cells across the plasma membrane is required to obtain maximal increases in Cai and innate immune responses. Support: UCB, CF Foundation and CF Research, Inc.