MECHANISMS INVOLVED IN HELICOBACTER PYLORI-INDUCED INTERLEUKIN-8 PRODUCTION BY A GASTRIC-CANCER CELL-LINE, MKN45

Accumulating evidence suggests an important role of interleukin-8 (IL- 8) in Helicobacter pylori infection-associated chronic atrophic gastri tis and peptic ulcer, We observed in this study that a gastric cancer- derived cell line, MKN45, produced a massive amount of IL-8 upon cocul ture with live H. pylori but not with killed H. pylori, H. pylori cult ure supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herb imycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 ce lls cocultured with live bacteria, suggesting the involvement of a tyr osine kinase(s) in H, pylori-induced IL-8 production, In addition, coc ulture of H, pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 pro duction occurred at the transcriptional level, This region contain thr ee cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-91 to -81 bp), and NF-kappa B (-80 to -70 bp) binding sites, Mutation of the NF-kappa B binding site abrogated c ompletely the induction of luciferase activity, whereas that of the AP -1 site partially reduced the induction, However mutation of the NF-IL 6 binding site resulted in no decrease in the induction of luciferase activity, Moreover, specific NF-kappa B complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-kappa B as well as AP-1, leading to IL-8 gene tra nscription.