Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa.

Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal crypts. The mitogenic action of TNF-alpha is primarily a direct effect of the cytokine and only to a minor extent mediated by a secondary production of IL-8 in the duodenal explant. Our findings indicate that TNF-alpha and IL-8 may participate in the regulation of cell proliferation in the human small intestinal epithelium.

Interleukin-1, interleukin-8, tumour necrosis factor alpha and interferon gamma stimulate DNA synthesis but have no effect on apoptosis in small-intestinal cell lines.

OBJECTIVES: Cytokines stimulate lymphocyte cell proliferation and affect cell division in several other cell types. Helicobacter pylori-induced gastritis and coeliac disease are characterized by an increased cell proliferation in association with an increased production of proinflammatory cytokines, which could contribute to these cell kinetic changes. Our aim is to examine in vitro whether cytokines usually present in the gastrointestinal mucosa affect DNA synthesis and apoptosis in a rat and a human small-intestinal cell line. METHODS: IEC-6 and FHs-74 cells were incubated for 24 h with 10(-13)-10(-9) M of tumour necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta) and interferon gamma (IFN-gamma). IEC-6 cells were also incubated with 10(-13)-10(-9) M of interleukin-1alpha (IL-1alpha) and 10(-8) M of interleukin-1 receptor antagonist (IL-1ra). The cells were labelled with 3H-methyl thymidine for the final 4 hours, and then processed for autoradiography. DNA synthesis was evaluated by the labelling index (LI%). Apoptosis was evaluated in IEC-6 cells by changes in membrane lipid asymmetry using annexin-V binding to externalized phosphatidylserine (flow cytometry) and by estimating the caspase activity. RESULTS: TNF-alpha, IL-1beta, IL-8 and IFN-gamma significantly and markedly increased the LI, even at low concentrations (P< 0.0001), in both IEC-6 and FHs-74 cells, as did IL-1alpha in IEC-6 cells. TGF-beta significantly reduced the LI in both cell lines (P< 0.0001), whereas IL-2, IL-6 and IL-1ra did not affect DNA synthesis significantly. None of IL-1beta, IL-8, TNF-alpha or IFN-gamma affected apoptosis in IEC-6 cells. CONCLUSION: TNF-alpha, IL-1alpha, IL-1beta, IL-8 and IFN-gamma stimulated DNA synthesis in a human and a rat small-intestinal cell line. The cytokines exert their mitogenic action directly on the intestinal cells via specific receptors. Our findings indicate that pro-inflammatory cytokines may participate in the regulation of the gastrointestinal epithelial cell proliferation in health and disease.

Serotonin and neuroendocrine peptides influence DNA synthesis in rat and human small intestinal cells in vitro.

Animal studies suggest a mediator role for neuroendocrine peptides and amines in regulating cell proliferation in the gastrointestinal epithelium. Our aim was to examine the effect of serotonin and selected gastrointestinal peptides on DNA synthesis in a rat and human small intestinal cell line in vitro. IEC-6 and FHs-74 cells were incubated with epidermal growth factor (EGF), insulin-like growth factor II, glucagon, substance P, neurokinin A, calcitonin gene-related peptide (GRP, CCGRP), neurotensin and serotonin. The cells were labelled with [methyl-3H] thymidine and processed for autoradiography. DNA synthesis was evaluated by the labelling index. Epidermal growth factor, insulin-like growth factor II, glucagon, and substance P increased the labelling index in a dose-related manner (P < 0.003). In contrast, a significant dose-dependent reduction of the labelling index was observed after administration of serotonin and neurokinin A (P < 0.0001). Neurotensin and CGRP did not affect the labelling index. EGF, insulin-like growth factor II, glucagon, substance P, serotonin and neurokinin A may be important physiological regulators of proliferation, of gastrointestinal cells.

Helicobacter pylori stimulates DNA synthesis in a small intestinal cell line in vitro.

BACKGROUND: Helicobacter pylori, which causes gastritis and peptic ulcer, seems to be an important factor in the pathogenesis of gastric cancer and MALT lymphoma. Thus our aim was to examine whether H. pylori influences DNA synthesis in epithelial cells in vitro. METHODS: Sonicated and water extracts of H. pylori (cytotoxic strains NCTC 11637, 88-23 and A5, and a noncytotoxic isogenic mutant of A5, A5 vac A) were diluted to a final concentration of 1/1,000, 1/100, 1/50 and 1/10. Water extracts of Escherichia coli were used as reference. IEC-6 cells were incubated during 24 h with fragments of H. pylori or extracts of the concentrations described above. The cells were labeled with 3H-methylthymidine for 4 h and processed for autoradiography. DNA synthesis was evaluated by the labeling index (LI). RESULTS: The LI% of controls was 15.6 +/- 5.1%. All the water extracts and sonicated strains of H. pylori increased the LI% in a dose-dependent manner (p < 0.001). The highest concentrations of the sonicated strains tended to reduce the LI%, although these values were still higher than those of the control group. The water extracts of E. coli increased the LI% in a dose-dependent manner (p < 0.0001). CONCLUSION: H. pylori stimulates DNA synthesis in epithelial cells in vitro, but no association was found with the presence of cytotoxin production. Our results suggest that hitherto unknown components of H. pylori may contribute to the increase in cell proliferation observed in gastritis and to the development of MALT lymphoma and gastric cancer.