Ps. a. filtrate induced tolerance to a second Ps. a. challenge and cross-tolerance with IL-1β but not TNFα. The apical surface of well-differentiated, day-21 hTBE cell cultures was challenged with Ps. a. filtrate or TSB as schematically illustrated in (A) and IL-8 production was measured in the basolateral medium at 24 or 48 hours. The results of a representative experiment are given as means ± SD in panel (B) and are from duplicate assays of three replicate wells. Similar results were obtained in 3 separate experiments with cell cultures from 3 different donors. C) The apical surface of well-differentiated, day-21 hTBE cell cultures (triplicate wells) was challenged with Ps. a. filtrate or TSB. Following washing and basolateral media change, the cells were re-challenged as indicated. IL-8 production during the second 24 hour period was normalized by dividing the Ps. a.-pretreated IL-8 value by the corresponding value in TSB-pretreated cultures (eg. Ps. a. → Ps. a./TSB → Ps. a.). The results are the average ± SEM of three independent experiments using cells from different donors. P values are based on ANOVA and Tukeys test, n.s. = not significant.