Cytotoxicity of Helicobacter pylori on human gastric epithelial cells in vitro: Role of cytotoxin(s) and ammonia

Objective: To evaluate the respective roles of vacuolating cytotoxin(s) and urease-mediated ammonia production in Helicobacter pylori-induced cytotoxicity on cultured human gastric epithelial cells. Methods: Monolayers of MKN 28 cell line were incubated with broth culture filtrates of two urease-positive H. pylori strains: CCUG 17874, cytotoxin-producing and C21, non-cytotoxin-producing. After incubation, highly sensitive quantitative assays were used to measure the ammonia levels in the cell medium, cell vacuolation and cell death. In order to evaluate the cytotoxic role of ammonia more accurately, we performed an 'ammonia simulation' by measuring cell vacuolation and cytolethality induced by incubation with definite concentrations of ammonium chloride. Cell damage was also studied morphologically. Results: Both H. pylori strains produced ammonia and induced cell vacuolation and cytolethality. With the G21 strain, both cell vacuolation and cytolethality appeared virtually identical to ammonia simulation. With the CCUG 17874 strain, which induced a higher degree of cell vacuolation and cell death compared with the G21 strain, cell vacuolation and cytolethality appeared largely ammonia-independent. The vacuoles induced by each H. pylori strain and by ammonia simulation were similar to each other. In less severely affected cells, vacuole formation and ammonia simulation were regularly limited by smooth membranes and morphologically and topographically resembled dilated endosomes or trans Golgi vesicles. In more severely affected cells, large autophagic-like vacuoles containing cell debris were also prominent. Conclusions: The vacuolating cytotoxin(s) and ammonia are major H. pylori virulence factors. The cytotoxin(s) seem to play a more prominent role than ammonia in cell vacuolation. Nevertheless, ammonia seems to damage cells by other mechanisms in addition to vacuolation. A common pathway for cytotoxin- and ammonia-induced cell vacuolation seems likely.