Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells

Abstract

Helicobacter pylori infection is associated with altered gastric epithelial cell turnover. To evaluate the role of oxidative stress in cell death, gastric epithelial cells were exposed to various strains of H. pylori, inflammatory cytokines, and hydrogen peroxide in the absence or presence of antioxidant agents. Increased intracellular reactive oxygen species (ROS) were detected using a redox-sensitive fluorescent dye, a cytochrome c reduction assay, and measurements of glutathione. Apoptosis was evaluated by detecting DNA fragmentation and caspase activation. Infection with H. pylori or exposure of epithelial cells to hydrogen peroxide resulted in apoptosis and a dose-dependent increase in ROS generation that was enhanced by pretreatment with inflammatory cytokines. Basal levels of ROS were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from uninfected individuals. H. pylori strains bearing the cag pathogenicity island (PAI) induced higher levels of intracellular oxygen metabolites than isogenic cag PAI-deficient mutants. H. pylori infection and hydrogen peroxide exposure resulted in similar patterns of caspase 3 and 8 activation. Antioxidants inhibited both ROS generation and DNA fragmentation by H. pylori. These results indicate that bacterial factors and the host inflammatory response confer oxidative stress to the gastric epithelium during H. pylori infection that may lead to apoptosis.

FIG. 1.

Induction of ROS in gastric epithelial cells after exposure to H. pylori or oxygen metabolites. (A) DCFH₂-DA-treated Kato III cells were exposed to H₂O₂ at concentrations from 0 to 1,000 μM or infected with H. pylori (Hp) at a ratio of bacteria to epithelial cells of 300:1. Intracellular DCF fluorescence was measured by flow cytometry at intervals up to 20 min after stimulation. ROS accumulated in proportion to the concentration of H₂O₂ added to the cells, and bacteria stimulated the production of ROS in epithelial cells in a time-dependent manner. A representative experiment is shown. (B) Superoxide anion was measured by the cytochrome c reduction assay method at 0, 0.5, and 1 h after various concentrations of H. pylori strain 26695 (equivalent to ratios of bacteria to epithelial cells of 0:1, 300:1, 600:1, and 1,000:1) were added to wells containing media alone (dotted line) or media with Kato III gastric epithelial cells (continuous line). A dose-dependent increase in superoxide anion generation was measured with increasing concentrations of bacteria both with and without epithelial cells. Data represent superoxide anion production expressed as means ± SEM (n = 8 to 12). *, P < 0.05 compared to bacteria alone; #, P < 0.05 compared to Kato III cells alone. (C) Kato III and NCI-N87 gastric epithelial cells were exposed to H. pylori (ratio of bacteria to epithelial cells of 300:1), harvested at 6 or 24 h poststimulation, and assayed for levels of intracellular GSH using a colorimetric assay. Oxidative stress generated by H. pylori results in a sustained decrease in GSH levels. The data are means ± SEM, expressed as percentages of control levels. *, P < 0.05 compared to control (n = 5 to 7).

FIG. 2.

Detection of ROS in freshly isolated human gastric epithelial cells. Gastric epithelial cells were isolated from gastric biopsy specimens, stained with DCFH₂-DA, and analyzed via flow cytometry. The y axes of panels A and B reflect the frequency of events on a linear scale, while the x axes indicate increasing levels of ROS as estimated by the DCF fluorescence on a logarithmic scale. (A) A representative experiment in which ROS levels were measured in cells from a subject infected with H. pylori and from an uninfected subject. (B) In another set of experiments, cells isolated from an uninfected subject were assayed for ROS before (resting) or after (stimulated) exposure to 400 mM H₂O₂. Treatment with H₂O₂ markedly increased intracellular ROS. (C) Mean levels of fluorescence ± SEM measured by fluorimeter in DCFH₂-DA-treated cells isolated from three uninfected and three infected subjects. *, P < 0.05 compared to results for uninfected subjects. Hp, H. pylori.

FIG. 3.

Effect of proinflammatory cytokines on ROS generation. Kato III cells were treated overnight with media alone (no cytokine) or media containing 10 ng/ml TNF-α, 100 U/ml IFN-γ, or 10 ng/ml IL-1β before stimulation with media alone (control), 400 μM H₂O₂, or H. pylori at a ratio of bacteria to epithelial cells of 300:1. Peak increases in ROS levels (measured as increases in DCF fluorescence) are depicted as means ± SEM (n = 3 to 6). All three cytokines increased basal levels of fluorescence and ROS responses to H. pylori, while IFN-γ and IL-1β also increased ROS generation after H₂O₂ stimulation. *, P < 0.05 compared to cells without cytokine treatment.

FIG. 4.

Induction of ROS by cag PAI-bearing strains in gastric epithelial cells. (A) Kato III cells were infected with cag PAI-positive strains 26695 and 84-183 (solid bars) or their isogenic cag PAI-negative mutants, 8-1 and 2-1, respectively (open bars), at comparable concentrations. Peak increases in DCF fluorescence occurring within 40 min of infection are expressed as percentages of levels in uninfected control cells and depicted as means ± SEM (n = 5 to 7). *, P < 0.05 for PAI⁺ strains compared to their PAI⁻ counterparts and to control levels. (B) Kato III cells were infected with the cag PAI-positive strain 26695 or its isogenic mutant, 8-1, at comparable concentrations. The amount of superoxide anion released was measured by the cytochrome c assay. Values at 30 and 60 min after infection are depicted as means ± SEM (n = 6, three replicates each). **, P < 0.0001; *, P < 0.05 (compared to control levels for both the PAI⁺ strain and its PAI⁻ counterpart); #, P < 0.01 for cells infected with the PAI⁺ strain compared to the PAI⁻ strain at both time points.

FIG. 5.

Inhibition of ROS induction by NAC. (A) DCFH₂-DA-loaded Kato III cells were treated with 10 mM NAC or media alone 1 h before stimulation with media, 400 μM H₂O₂, or H. pylori at a ratio of bacteria to epithelial cells of 300:1. Data are depicted as mean levels of maximal DCF fluorescence within 20 min of stimulation in NAC-treated cells expressed as percentages of fluorescence in cells without NAC (means ± SEM; n = 4 to 6 experiments). *, P < 0.05 compared to cells without NAC pretreatment. (B) Identical experiments performed with epithelial cells isolated from subjects without H. pylori infection (n = 3). *, P < 0.05 compared to cells without NAC pretreatment.

FIG. 6.

Effects of antioxidants on H. pylori-induced ROS production in Kato III cells. DCFH₂-DA-loaded Kato III cells were treated with various antioxidants or media alone 1 h before stimulation with H. pylori strain 26695 at a ratio of bacteria to epithelial cells of 300:1. Antioxidants tested in these experiments were 10 mM GSH, 50 mM DMTU, 5 mM DESF, and DTPA. Data are depicted as mean levels of DCF fluorescence 30 min after stimulation with H. pylori in untreated cells (H. pylori) or in antioxidant-treated cells (H. pylori + drug), as percentages of values for uninfected, untreated control cells, ± SEM (n = 3 or 4 experiments). The effects of drugs alone are also shown. *, P < 0.05; **, P < 0.01 (compared to H. pylori alone using a paired Student t test).

FIG. 7.

Effects of the xanthine oxidase inhibitor allopurinol on H. pylori-induced superoxide anion in Kato III cells. Cells were incubated with various concentrations of allopurinol (10 to 100 μM) for 1 h before stimulation with H. pylori strain 26695 at a ratio of bacteria to epithelial cells of 300:1. Superoxide anion was assessed by cytochrome c assay 30 min after stimulation. Each experiment was performed in triplicate. Values are means ± SEM of three separate experiments. *, P < 0.05 compared to control cells without allopurinol using a paired Student t test.

FIG. 8.

Induction of apoptosis in gastric epithelial cells and its inhibition by antioxidants. (A) NCI-N87 cells were exposed to H₂O₂ at concentrations from 0 to 400 μM with cell viability (expressed as % of control) measured by trypan blue exclusion and apoptosis (shown as apoptotic index), determined by detection of endonucleosomes via ELISA. Cells exposed to 800 U/ml IFN-γ for 6 h followed by 100 μg/ml anti-Fas antibody were used as a positive control. Dose-dependent changes were observed with decreases in cell viability and increases in apoptosis significantly different from those of control cells at doses of 100 to 400 μM H₂O₂ (P < 0.05). Data shown as means ± SEM (n = 3 to 5). (B) Kato III cells were treated with H. pylori (300 bacteria per epithelial cell), 400 μM H₂O₂, or media alone (control) for 48 h in the presence or absence of 10 mM NAC. Apoptosis was assessed using an ELISA to detect endonucleosomes exposed by DNA fragmentation. Values depicted are means ± SEM (n = 9 to 15). *, P < 0.05 compared to cells without NAC.