In vitro studies on intestinal epithelial cell proliferation : effects of cytokines, Helicobacter pylori, serotonin and neuroendocrine peptides

Abstract

Previous studies suggest that bacteria and bacterial metabolites, proinflammatory cytokines, neuroendocrine peptides and amines may participate in the regulation of the gastrointestinal-epithelial cell kinetics. The complexity of interactions that take place in vivo makes it difficult to define the primary actions of these and other agents on gastrointestinal cell proliferation. Thus, in the present study we examine in vitro the actions on intestinal epithelial cell proliferation of: 1) water extracts and sonicates of cytotoxic and non-cytotoxic (lacking VacA toxin) strains of Helicobacter pylori, 2) ammonium compounds and urea, 3) selected neuroendocrine peptides and serotonin, 4) proinflammatory and other cytokines. For this purpose, DNA synthesis was estimated in autoradiographs (labeling index) of small intestinal epithelial cell lines IEC-6 and FHs 74 cells) and the total number of arrested metaphases was determined in human duodenal explants incubated with TNF-alpha and IL-8 in combination with vicristine. The effect of cytokines on apoptosis was evaluated by determination of the caspase activity and flow cytometrically using annexin V binding to externalised phosphatidylserine.

Ammonium compounds and urea have no effect on DNA synthesis in IEC-6 cells. However, water extracts and sonicates of both cytotoxic and non-cytotoxic strains of Helicobacter pylori significantly increase DNA synthesis in IEC-6 cells (p<0.001). At the higher dose range, the labeling index reached a plateau, particularly in the cells incubated with the sonicates of Helicobacter pylori.

Insulin-like growth factor, glucagon and substance P increase the labeling index in a dose-related fashion in both IEC-6 and FHs 74 cells (p<0.001). In contrast, serotonin and neurokinin A inhibit DNA synthesis (P<0.0001).

Tumor necrosis factor-[alpha], interferon-[gamma], interleukin-1-[alpha], interleukin-1-ß and interleukin-8 markedly increase the labeling index in both IEC-6 and FHs 74 cells (p<0.0001). No signs of apoptosis were observed. Transforming growth factor-ß significantly inhibit DNA synthesis (p<0.0001). Tumor necrosis factor-[alpha] and interleukin-8 increase the number of arrested metaphases in human duodenal crypts (p<0.0001). Tumor necrosis factor-[alpha] induces release of interleukin-8 and interleukin-6 into the culture media but the mitogenic action of tumor necrosis factor-[alpha] is mainly an effect its own and, only to a lesser extent is due to an increased production of interleukin-8.

It is concluded that Helicobacter pylori possess hitherto unknown mitogens to intestinal epithelial cells and that proinflammatory cytokines as well as selected neuroendocrine peptides and serotonin affect cell proliferation in intestinal epithelial cells even at low concentrations. Our findings indicate that these agents may contribute to the regulation of gastrointestinal cell proliferation in health and disease.