Tracheal aspirate IL-8 concentration and airway epithelial cell IL-8 expression are each increased in premature infants undergoing mechanical ventilation. We sought to determine the cytokines responsible for IL-8 expression in this context. Tracheal aspirates were collected from 18 mechanically ventilated premature infants. IL-8 protein abundance was high in tracheal aspirates from ventilated premature infants (mean, 5806 +/- 4923 pg/mL). IL-1 alpha (mean, 20 +/- 6 pg/mL), IL-1 beta (mean 67 +/- 46 pg/mL), and tumor necrosis factor (TNF)-alpha (mean, 8 +/- 2 pg/mL) were also found. Incubation of tracheal aspirates with 16HBE14o- human bronchial epithelial cells increased IL-8 protein in both cell lysates and supernatants, as well as transcription from the IL-8 promoter. Aspirates also induced nuclear factor (NF)-kappa B activation. Mutation of the IL-8 promoter NF-kappa B site abolished aspirate-induced IL-8 transcription. Endotoxin concentrations in the tracheal aspirates were negligible and incapable of inducing IL-8 promoter activity. Finally, incubation of tracheal aspirates with a neutralizing antibody against IL-1 beta reduced epithelial cell IL-8 production, whereas neutralizing antibodies against IL-1 alpha and TNF-alpha had no effect. We conclude that airway fluid from mechanically ventilated premature infants contains soluble factors capable of inducing airway epithelial cell IL-8 expression via a NF-kappa B-dependent pathway, and that IL-1 beta plays a specific role in this process.