FIG. 1.

Time course of 9HTEo− cytokine IL-6 and IL-8 gene expression and secretion in response to different stimuli. Cultured human tracheal epithelial cells (9HTEo−) were coincubated with tissue culture medium alone (⧫), 20 ng of IL-1β per ml (■), 1 μg of LOS per ml (▵), and 5 × 10⁷ CFU of NTHi strains AAr176p⁺ (×), 11 (□), and AA238 (●) per ml. An NTHi cell density of 5 × 10⁷ CFU/ml corresponds to a 100::1 ratio of bacteria to epithelial cells. Based on the calculations of Gu et al. (18), 5 × 10⁷ CFU of NTHi per ml contains approximately 1 μg of LOS per ml. Total RNA and cell culture fluid were isolated from epithelial cells after 0, 1, 2, 4, 8, and 16 h of coincubation with the stimulus. Levels of IL-6 (A) and IL-8 (B) gene expression were subsequently tested by multiprobe RNase protection assays. Representative IL-6 and IL-8 time courses are shown as fold increase over the unstimulated control. Secreted cytokines IL-6 (C) and IL-8 (D) were assayed by ELISA using commercially available kits. The results are shown as picograms of IL-6 and IL-8 per milliliter of culture fluid.