Hiroki Takaya

Counter‐regulatory effect of sodium butyrate on tumour necrosis factor‐alpha (TNF‐α)‐induced complement C3 and factor B biosynthesis in human intestinal epithelial cells

The various biological activities of butyrate have been well documented. In this study, we tested the effects of butyrate on TNF‐α‐induced complement C3 and factor B biosynthesis in human intestinal epithelial cells. The biosynthesis of C3, factor B and IL‐8 was evaluated at the protein and mRNA levels. To evaluate transcriptional activation, the nuclear run‐on assay was performed. The transcription factor–DNA binding activity was assessed by an electrophoretic gel mobility shift assay (EMSA). In the intestinal epithelial cell lines HT‐29, T84 and Caco‐2, sodium butyrate enhanced TNF‐α‐induced C3 secretion, but suppressed TNF‐α‐induced factor B and IL‐8 secretion. Nuclear run‐on assay revealed that transcriptional regulatory mechanisms are involved in the effects of sodium butyrate. The EMSAs indicated that sodium butyrate suppressed TNF‐α‐induced nuclear factor (NF)‐κB– and activation protein (AP)‐1–DNA binding activity, but enhanced TNF‐α‐induced activation of CCAAT/enhancer‐binding protein (C/EBP)β (NF‐IL‐6)–DNA binding activity. Sodium butyrate induced a counter‐regulatory effect on TNF‐α‐induced C3 and factor B biosynthesis in human intestinal epithelial cells. Butyrate action has been discussed with its activity to induce histone hyperacetylation, but its counter‐regulatory effect on complement biosynthesis may be closely associated with the modulation of transcription factor activation.

Cooperation of interleukin-17 and interferon-γ on chemokine secretion in human fetal intestinal epithelial cells

Interleukin (IL)‐17 is a newly identified T cell‐derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated the effects of IL‐17 and interferon (IFN)‐γ on chemokine secretion in human fetal intestinal epithelial cells. IL‐8 and monocyte chemoattractant protein (MCP)‐1 secretion by the human fetal intestinal epithelial cell line, intestine‐407, was evaluated by ELISA and Northern blot. The expression of IL‐17 receptor (R) was analysed by a binding assay using [125I]‐labelled IL‐17. The activation of nuclear factor‐κB (NF‐κB), NF‐IL6 and AP‐1 was assessed by an electrophoretic gel mobility shift assay (EMSA). IL‐17 induced a dose‐dependent increase in IL‐8 and MCP‐1 secretion. The inducing effects of IL‐17 on IL‐8 and MCP‐1 mRNA abundance reached a maximum as early as 3 h, and then gradually decreased. IL‐17 and IFN‐γ synergistically increased IL‐8 and MCP‐1 secretion and mRNA abundance. IFN‐γ induced a weak increase in IL‐17 R mRNA abundance, and incubation with IFN‐γ for 24 h enhanced [125I]‐labelled IL‐17‐binding by 2·4‐fold. IL‐17 rapidly induced the phosphorylation and degradation of IκBα molecules, and the combination of IL‐17 and IFN‐γ induced a marked increase in NF‐κB DNA‐binding activity as early as 1·5 h after the stimulation. Furthermore, this combination induced an increase in NF‐IL‐6 and AP‐1 DNA‐binding activity. In conclusion, it becomes clear that IL‐17 is an inducer of IL‐8 and MCP‐1 secretion by human fetal intestinal epithelial cells. The combination of IL‐17 with IFN‐γ synergistically enhanced chemokine secretion. These effects of IL‐17 and IFN‐γ might play an important role in the inflammatory responses in the intestinal mucosa.

The expression of chemokine genes correlates with nuclear factor-kappaB activation in human pancreatic cancer cell lines.

Chemokines may regulate the process of immune cell infiltration that is often found in pancreatic cancer. In this study, we investigated the secretion of the chemokines [interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, and RANTES (regulated on activation, normal T cell expressed and secreted)] in human pancreatic cancer cell lines. The chemokine secretion in three pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Northern blot, and the activation of nuclear factor-&kgr;B (NF-&kgr;B) and NF-IL6 was assessed by an electrophoretic gel mobility shift assay (EMSA). Without any stimulation, IL-8 secretion was detected in all cell lines, and MCP-1 secretion was detected in PANC-1 and MIA PaCa-2 cells. However, RANTES secretion was not detected in all cells. The addition of IL-1&bgr; and tumor necrosis factor (TNF)-&agr; strongly enhanced IL-8, MCP-1, and RANTES secretion; these responses were observed at the mRNA level as well as at the protein level. IL-1&bgr; and TNF-&agr; induced a rapid activation of nuclear factor (NF)-&kgr;B in PANC-1 cells, and the increase in chemokine mRNA expression correlated with NF-&kgr;B activation. The activation of NF-IL6 was modest. A blockade of NF-&kgr;B activation by TPCK markedly reduced the IL-1&bgr;- and TNF-&agr;–induced chemokine gene expression. Our findings indicate that chemokines are produced by pancreatic cancer cells, and suggest that these factors may contribute to the accumulation of tumor-associated immune cells. In addition, the transcriptional activation of chemokine genes in pancreatic cancer cells may be closely associated with NF-&kgr;B activation.

Co-operation of interleukin-17 and interferon-γ on chemokine secretion in human fetal intestinal epithelial cells

SUMMARY Interleukin (IL)-17 is a newly identified T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated the effects of IL-17 and interferon (IFN)-g on chemokine secretion in human fetal intestinal epithelial cells. IL-8 and monocyte chemoattractant protein (MCP)-1 secretion by the human fetal intestinal epithelial cell line, intestine-407, was evaluated by ELISA and Northern blot. The expression of IL-17 receptor (R) was analysed by a binding assay using [ 125 I]-labelled IL-17. The activation of nuclear factor-kB (NF-kB), NF-IL6 and AP-1 was assessed by an electrophoretic gel mobility shift assay (EMSA). IL-17 induced a dose-dependent increase in IL-8 and MCP-1 secretion. The inducing effects of IL-17 on IL-8 and MCP-1 mRNA abundance reached a maximum as early as 3 h, and then gradually decreased. IL-17 and IFN-g synergistically increased IL-8 and MCP-1 secretion and mRNA abundance. IFN-g induced a weak increase in IL-17 R mRNA abundance, and incubation with IFN-g for 24 h enhanced [ 125 I]-labelled IL-17-binding by 2·4-fold. IL-17 rapidly induced the phosphorylation and degradation of IkBa molecules, and the combination of IL-17 and IFN-g induced a marked increase in NF-kB DNAbinding activity as early as 1·5 h after the stimulation. Furthermore, this combination induced an increase in NF-IL-6 and AP-1 DNA-binding activity. In conclusion, it becomes clear that IL-17 is an inducer of IL-8 and MCP-1 secretion by human fetal intestinal epithelial cells. The combination of IL-17 with IFN-g synergistically enhanced chemokine secretion. These effects of IL-17 and IFN-g might play an important role in the inflammatory responses in the intestinal mucosa.