Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae

Abstract

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.

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.

FIG. 2

Dose response of 9HTEo− cytokine gene expression to increasing amounts of NTHi AAr176p⁺ LOS. Epithelial cells were coincubated with 0.01, 0.1, 1.0, 10, and 100 μg of purified LOS per ml from strain AAr176p⁺ for 4 h. Cytokine gene expression was analyzed as for Fig. 1. Cytokine IL-6, IL-1α, IL-1β, TNF-α, IL-8, and MCP-1 expression is shown as fold increase over the unstimulated control. The data shown are from one representative experiment of four performed.

FIG. 3

Dose response of 9HTEo− cytokine gene expression to increasing amounts of NTHi AAr176p⁺ whole cells. Epithelial cells were coincubated with 5 × 10⁵, 5 × 10⁶, 5 × 10⁷, and 5 × 10⁸ CFU of strain AAr176p⁺ per ml. Cytokine IL-6, IL-1α, IL-1β, TNF-α, IL-8, and MCP-1 expression is shown as fold increase over the unstimulated control. The data shown are from one representative experiment of four performed.

FIG. 4

Quantitation of cell bound LOS on NTHi strain AAr176p⁺ after SDS-PAGE and silver staining. Lanes 1 and 7, molecular weight standards; lane 2, 3 × 10⁸ CFU of strain AAr176p⁺; lanes 3 through 6, 3, 1.5, 0.75, and 0.375 μg, respectively, of purified LOS from strain AAr176p⁺.

FIG. 5

Inhibition of NTHi LOS stimulation of 9HTEo− IL-8 secretion. Monolayers of cultured human tracheal epithelial cells (3 × 10⁶ cells/well in six-well tissue culture plates; serum-free culture medium) were coincubated with tissue culture medium alone, IL-1β (20 ng/ml), LOS (0.21 μg/ml), and NTHi strain AAr176p⁺ cells (5 × 10⁷ CFU/ml) with or without polymyxin B (0.05, 0.5, 5.0, and 50.0 μg/ml). Culture fluid was harvested after 16 h of coincubation with various stimuli, and secreted IL-8 was measured using commercially available ELISAs. The results are shown as nanograms of IL-8 per milliliter of culture fluid. Error bars each represent the standard error of the mean of three different wells each analyzed in triplicate by ELISA. Unstimulated control wells showed no IL-8 secretion.

FIG. 6

Screening of H. influenzae clinical isolates with 9HTEo− cells for epithelial IL-8 secretion. Monolayers of cultured human tracheal epithelial cells (3 × 10⁶ cells/well in six-well tissue culture plates; serum-free culture media) were coincubated with tissue culture medium alone, IL-1β (20 ng/ml), LOS (0.21 μg/ml), and H. influenzae clinical isolates (5 × 10⁷ CFU/ml). Culture fluid was harvested after 16 h of coincubation with various stimuli, and secreted IL-8 was measured using commercially available ELISAs. The results are shown as nanograms of IL-8 per milliliter of culture fluid. Error bars each represent the standard error of the mean of three different wells each analyzed in triplicate by ELISA. IL-8 concentrations were 0.317 ± 0.006 ng/ml in unstimulated control wells and 14.25 ± 4.87 ng/ml in IL-1β-stimulated control wells. NTHi strains used in this study were derived from wild-type strains AAr176p⁺ (Pili), 11 (Hia), 12 (HMW1/2), N187 (Hap), and Rd. See Table 1 for strain details.