Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability.
OBJECTIVE
To determine whether Helicobacter pylori (H pylori) vacuolating cytotoxin (VacA) regulates release of pro-inflammatory cytokines (IL-1beta, IL-8, TNF-alpha, and IL-6) or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes.


METHODS
Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial (AGS) cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays (ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR) were performed to examine the effects of H pylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacA-induced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response.


RESULTS
Addition of VacA alone reduced AGS cell viability (P < 0.05), and this reduction was enhanced by high doses of NaCl (P < 0.05). VacA alone induced expression of TNF-alpha, IL-8 and IL-1beta, while NaCl alone induced expression of TNF-alpha and IL-1beta. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-alpha, expression was dose-dependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by ELISA. Positive immunohistochemical staining of IL-1, IL-6, and TNF-alpha was found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However, the staining of these three cytokines was stronger in H pylori-infected animals fed with a 5 g/kg NaCl diet.


CONCLUSIONS
VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines induced by VacA, suggesting that NaCl plays an important role in H pylori-induced gastric epithelial cell cytotoxicity.
Sun
Juan
J
Department of Public Health and Hygiene(II), Faculty of Medicine, Oita University Oita 879-5593, Japan.
Aoki
Kazuo
K
Zheng
Jin-Xu
JX
Su
Bing-Zhong
BZ
Ouyang
Xiao-Hui
XH
Misumi
Junichi
J
D000818:Animals; D001426:Bacterial Proteins / Q000633:toxicity*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D005849:Gerbillinae; D006801:Humans; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D012965:Sodium Chloride / Q000494:pharmacology*; D014617:Vacuoles / Q000187:drug effects
D001426:Bacterial Proteins; D016207:Cytokines; D012333:RNA, Messenger; C074689:VacA protein, Helicobacter pylori; D012965:Sodium Chloride