IFN-gamma synergizes with TNF-alpha but not with viable H. pylori in up-regulating CXC chemokine secretion in gastric epithelial cells

Abstract

Helicobacter pylori colonizes the gastric epithelial surface and induces epithelial cells to increase production of the neutrophil attractant IL-8. Little is known about the role of the gastric epithelium in regulating mucosal T cell trafficking. We therefore characterized constitutive and regulated epithelial expression of the CXC chemokines IP-10, I-TAC and Mig, which specifically attract CXCR3 expressing CD4(+) T cells. Human gastric epithelial cell lines (AGS, Kato III, NCI) were used to characterize the constitutive and regulated expression of three CXC chemokines in response to IFN-gamma, TNF-alpha and different H. pylori preparations. Chemokine mRNA and protein production were measured by RT-PCR and ELISA. Gastric epithelial cells constitutively expressed mRNA for IP-10, Mig and I-TAC. IFN-gamma in combination with TNF-alpha strongly induced secretion of those chemokines. Soluble or membranous fractions of H. pylori significantly inhibited IFN-gamma/TNF-alpha induced epithelial cell IP-10 and Mig production. Gastric epithelial cells may contribute to mucosal T cell trafficking. The capacity of H. pylori products to inhibit IP-10 and Mig secretion may explain, at least in part, the failure to induce protective immunity against this bacterium and the ability of H. pylori to affect the presentation of the local inflammation.

Fig. 1

Constitutive and regulated expression of IP-10, I-TAC and Mig in the gastric epithelial cell lines Kato III. Total RNA was isolated from stimulated and unstimulated confluent monolayers of cell lines after 3, 6 and 12 h as indicated. RT-PCR cDNA transcripts were generated and amplified using oligonucleotide primers specific for IP-10, Mig and I-TAC. RT-PCR reactions were also performed in the absence of RNA as a negative control. Amplification products were run on 4% polyacrylamide-gel electrophoresis, visualized by silver-staining and photographed. Representative results from one of three experiments are shown.

Fig. 2

(a) Dose-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of NCI cell lines were stimulated with increasing concentrations of either TNF-α or IFN-γ or both in combination (• IFN-γ; ○ TNF-α; ▾ TNF-α + IFN-γ). Supernatants were harvested after 18 h and chemokine-secretion was measured by ELISA. Similar results were obtained using AGS and NCI cell lines. Values are expressed as mean ± CI. Representative results from one of four independent experiments are shown. (b) Time-dependent secretion of IP-10 and Mig by gastric epithelial cells. Confluent monolayers of AGS and Kato III cells were stimulated for different times (as indicated) with either IFN-γ 50 ng/ml or TNF-α 50 ng/ml alone or both cytokines in combination. Supernatants were harvested after 6, 12 and 18 h and chemokine concentrations were measured by ELISA. Values are expressed as mean ± CI. Representative results from one of four experiments are shown.

Fig. 3

Dose-dependent increase in IL-8 but neither IP-10 nor Mig secretion by living H. pylori (▪ IL-8 secretion; □ IP-10 secretion; Mig secretion). Confluent monolayers of AGS cell line (2 × 10⁶ cells) were coincubated with different concentrations of living H. pylori for 20 h. Supernatants were harvested, centrifuged and analysed for chemokine-secretion by ELISA. Values are expressed as mean ± CI. Representative results from one of four experiments are shown.

Fig. 4

Determination of necrosis and apoptosis by propidium iodide (PI) and annexin V staining. Necrotic membrane damage as well as apoptosis of gastric epithelial cells cultured for 20 h in medium or in the presence of TNF-α and IFN-γ alone or in combination with membranous or soluble fractions (50 µg/ml) as well as living H. pylori (1 × 10⁸) was excluded by double staining with PI and annexin V. Gastric epithelial cells showed an increase in annexin V and PI staining after incubation with living H. pylori for 20 h, but remained negative for PI and annexin V concerning soluble or membranous fractions. Gastric epithelial cells permeabilized with 0·1% saponin served as positive control. Data are presented as percentage of gated cells.