Prevalence of Virulence Factors in Helicobacter pylori Isolated from Koreans and Proinflammatory Cytokine Gene Expression in Human Gastric Epithelial Cells Induced by H. pylori with Virulence Factors

Kim, Jung Mogg; Jung, Hyun Chae; Lee, Joo Young; Lee, Kyung Mee; Chin, Young Joo; Kim, Young Jeon; Song, In Sung; Kim, Chung Yong

Abstract

OBJECTIVES
CagA or cytotoxin-positive H. pylori may be associated with gastroduodenal diseases. However, controversies about this association also exist. Moreover, there could be geographic differences in the prevalence of virulence factors such as cagA or cytotoxin. In H. pylori infection, the gastric mucosa shows acute and chronic inflammation. However, the pathogenesis of such as an inflammation by H. pylori is not well elucidated. We performed this study 1) to determine prevalence of the genes of virulence factor such as cagA and cytotoxin in H. pylori, 2) to assess the correlation of their presence with clinical findings, and 3) to test whether the vacuolating cytotoxin of H. pylori could evoke proinflammatory cytokine gene expression in gastric epithelial cells.
METHODS
1) The prevalence of the cagA, vacA and adhesin genes in H. pylori strains isolated from Koreans was determined by PCR analysis. 2) H. pylori was cultured in Brucella broth containing 10% fetal bovine serum for 3 days using a shaker in a microaerophilic condition. Cytotoxin assay was performed by determining whether addition of the concentrated culture supernatants is able to cause vacuolization of HeLa cells. 3) After human gastric epithelial cells, Hs746T and AGS were incubated with the culture supernatants containing vacuolating cytotoxin, each RNAs were extracted from the gastric epithelial cells. And then various cytokine gene expression were assessed using RT-PCR. The expressed cytokine transcripts were quantified by RT-PCR and standard synthetic RNA. Among cytokines, IL-8 proteins were also measured by ELISA.
RESULTS
1) More than 95% of H. pylori isolates from Korean adults possessed cagA, vacA and adhesin genes. And 80.6% of H. pylori strains have expressed vacuolating cytotoxicity against HeLa cells within 24 hours. 2) There was no correlation between the virulence factors of H. pylori strains and clinical findings. 3) Cytotoxin-positive culture supernatants also caused vacuolization in gastric epithelial cells, both Hs746T and AGS. 4) Expression of mRNA for proinflammatory cytokines such as IL-1alpha: IL-8, MCP-1 and GM-CSF was much more upregulated by vacuolating cytotoxin-positive culture supernatants than cytotoxin-negative ones in both Hs746T and AGS cells. Number of molecules of the expressed IL-8 transcripts was parallel to the amounts of IL-8 protein secreted from gastric epithelial cells.
CONCLUSION
These results suggest that virulence factors of H. pylori may not be factors determining disease entitiy in Korean patients infected with H. pylori. In addition, vacuolating cytotoxin secreted from H. pylori could give rise to vacuolization in gastric epithelial cells as well as induce proinflammatory cytokines from the cells.