Mitsue Shimada

IL-6 Secretion by Human Pancreatic Periacinar Myofibroblasts in Response to Inflammatory Mediators

There is increasing evidence that IL-6 plays an important role in the pathophysiology of acute pancreatitis via its broad proinflammatory actions. To identify the local biosynthetic site for IL-6 in human pancreas, we investigated IL-6 secretion in human pancreatic periacinar myofibroblasts. IL-6 secretion was determined by ELISA and Northern blotting. The activation of NF-κB was assessed by EMSA. The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6 secretion was rapidly induced by IL-17, IL-1β, and TNF-α. EMSAs demonstrated that IL-17, IL-1β, and TNF-α induced NF-κB activation within 1.5 h after stimulation, and a blockade of NF-κB activation by the pyrrolidine derivative of dithiocarbamate and tosyl-phe-chloromethylketone markedly reduced the IL-17-, IL-1β-, or TNF-α-induced IL-6 gene expression. Furthermore, IL-17, IL-1β, and TNF-α induced a rapid activation of extracellular signal-related kinase p42/44 and p38 MAPKs, and specific MAPK inhibitors (SB203580, PD98059, and U0216) significantly reduced IL-17-, IL-1β-, or TNF-α-induced IL-6 secretion, indicating the role of MAPKs in the induction of IL-6. The combination of either IL-17 plus IL-1β or IL-17 plus TNF-α enhanced IL-6 secretion and IL-6 mRNA expression; in particular, the effects of IL-17 plus TNF-α were much stronger than those induced by IL-17 plus IL-1β. TNF-α-induced IL-6 mRNA degraded rapidly at any concentrations, and the combination of IL-17 and TNF-α markedly enhanced IL-6 mRNA stability. This indicates that the effects of IL-17 plus TNF-α were regulated at the post-transcriptional level. In conclusion, pancreatic periacinar myofibroblasts secreted a large amount of IL-6 in response to proinflammatory cytokines. These cells might play an important role in the pathogenesis of acute pancreatitis via IL-6 secretion.

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.

Extracellular signal-regulated kinases 1 and 2 participate in interleukin-17 plus tumor necrosis factor-α-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts

In human pancreatic myofibroblasts, interleukin (IL)-17 markedly enhances tumor necrosis factor (TNF)-alpha-induced IL-6 secretion through the induction of IL-6 mRNA stabilization. Induced stability of IL-6 mRNA was markedly decreased by the inhibitors of extracellular signal-regulated kinase (ERKs), PD98059 and U0216. This indicates that activation of the ERK pathway is involved in the induction of IL-6 mRNA stabilization by IL-17 plus TNF-alpha.

Sodium butyrate enhances complement-mediated cell injury via down-regulation of decay-accelerating factor expression in colonic cancer cells

Abstract. Decay-accelerating factor (DAF) expressed on the surface of colonic cancer cells presents a barrier to complement-mediated clearance by contributing to the ineffectiveness of the humoral immune response. In this study, to investigate the mechanisms responsible for the anti-tumor effects of butyrate, we evaluated how butyrate modulates DAF expression in colonic cancer cells. Three colonic cancer cell lines (HT-29, Caco-2, and T84 cells) were studied. DAF protein expression was assessed by western blot, and DAF mRNA expression was evaluated by northern blot. Complement C3 deposition on the surface of colonic cancer cells was determined by enzyme-linked immunosorbent assay (ELISA). The promoter activity of the DAF gene was assessed by a reporter gene-luciferase assay. Butyrate reduced the basal and interleukin-4 (IL-4)- and tumor necrosis factor-α (TNF-α)-induced expression of DAF protein and mRNA in HT-29 cells. It increased the susceptibility to complement attack and enhanced C3 deposition on HT-29 cells. The inhibitory effect of butyrate on DAF mRNA expression was also observed in T84 and Caco-2 cells. Butyrate decreased basal DAF expression at both transcriptional and post-transcriptional levels. The inhibitory effect of butyrate on IL-4-induced DAF expression was closely associated with a blockade of IL-4-induced DAF mRNA stability. TNF-α-induced transcriptional activation and the increased stability of the DAF gene were also blocked by butyrate. Similar but weak effects were induced by trichostatin A, a potent histone deacetylase inhibitor, suggesting that histone acetylation might participate in butyrate activity. These observations indicate that both a down-regulation of DAF expression and the induction of susceptibility to complement attack contribute to the anti-tumor effects of butyrate in colonic cancer.

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.

Ligation of the Fas antigen stimulates chemokine secretion in pancreatic cancer cell line PANC-11

Background and Aim: The role of chemokines in the process of immune cell infiltration into pancreatic cancer tissue has been reported. In this study, we investigated the induction of chemokines (interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1) by Fas antigen (Ag)‐stimulation in a human pancreatic cancer cell line, PANC‐1.

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.

Ligation of fas antigen stimulates chemoline secretion in pancreatic cancer cell line PANC-1

BACKGROUND AND AIM The role of chemokines in the process of immune cell infiltration into pancreatic cancer tissue has been reported. In this study, we investigated the induction of chemokines (interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1) by Fas antigen (Ag)-stimulation in a human pancreatic cancer cell line, PANC-1. METHODS The chemokine secretion was evaluated by using an ELISA and a northern blot, and the activation of nuclear factor-kappa B (NF-kappa B) was assessed by using an electrophoretic gel mobility shift assay (EMSA). RESULTS The Fas antigen (Ag) stimulation clearly induced an increase in IL-8 and MCP-1 secretion in PANC-1 cells. This effect was also observed at the mRNA level. The induction of chemokine secretion by Fas Ag stimulation required de novo gene expression and protein synthesis. The pretreatment with interferon (IFN)-gamma markedly enhanced the effects of Fas Ag stimulation; IFN-gamma pretreatment and Fas Ag stimulation synergistically induced not only apoptosis but also IL-8 and MCP-1 secretion. Flow cytometric analysis demonstrated that IFN-gamma significantly enhanced Fas Ag expression. In addition, Fas Ag stimulation actually evoked NF-kappa B activation in this cell line. CONCLUSION Our results indicate that Fas Ag stimulation can induce chemokine secretion in PANC-1 cells, suggesting the contribution of Fas stimulation to the accumulation of immune cells in pancreatic cancer tissue.