Kazunori Hata

Increased expression of interleukin 17 in inflammatory bowel disease

Background and aim: Interleukin (IL) 17 is a cytokine which exerts strong proinflammatory activities. In this study we evaluated changes in IL-17 expression in the inflamed mucosa and in the serum of patients with inflammatory bowel disease (IBD). Methods: Tissue samples were obtained endoscopically or surgically from patients with ulcerative colitis (UC) (n=20), Crohn’s disease (CD) (n=20), infectious colitis (n=5), ischaemic colitis (n=8), and normal colorectal tissues (n=15). IL-17 expression was evaluated by a standard immunohistochemical procedure. Serum IL-17 levels were determined by ELISA. IL-17 mRNA expression was analysed by reverse transcriptase-polymerase chain reaction. Results: IL-17 expression was not detected in samples from normal colonic mucosa, infectious colitis, or ischaemic colitis. In the inflamed mucosa of active UC and CD patients, IL-17 expression was clearly detectable in CD3+ T cells or CD68+ monocytes/macrophages. The average number of IL-17+ cells was significantly increased in active UC and CD patients compared with inactive patients. IL-17 mRNA expression was not detected in normal mucosa but was detectable in the mucosa from active UC and CD patients. IL-17 was not detected in the sera from normal individuals, infectious colitis, or ischaemic colitis patients but IL-17 levels were significantly elevated in IBD patients. Conclusions: IL-17 expression in the mucosa and serum was increased in IBD patients. It is likely that IL-17 expression in IBD may be associated with altered immune and inflammatory responses in the intestinal mucosa.

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.

Curcumin Prevents the Development of Dextran Sulfate Sodium (DSS)-Induced Experimental Colitis

Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric). We evaluated the effects of curcumin on the development of dextran sulfate sodium (DSS)-induced experimental colitis. BALB/c mice were fed a chow containing either 3.5% (wt/wt) DSS or 3.5% DSS + 2.0% (wt/wt) curcumin. The body weight loss was more apparent in DSS-treated mice than in DSS + curcumin-treated mice. The disease activity index, histological colitis score, and MPO activity were all significantly higher in DSS-treated mice than in DSS plus curcumin-treated mice. Microscopically, mucosal edema, cellular infiltration, and epithelial disruption were much more severe in DSS-treated mice than in DSS + curcumin-treated mice. In DSS + curcumin-treated mice, NF-κB activation was blocked in the mucosa. In conclusion, the development of DSS-induced colitis was significantly attenuated by curcumin. Being a nontoxic natural dietary product, curcumin could be useful in treatment of IBD patients.

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.

Elevated Circulating Platelet-Derived Microparticles in Patients with Active Inflammatory Bowel Disease

OBJECTIVES:Platelet-derived microparticles (PDMPs) are active molecules involved in the hemostatic and inflammatory responses. To evaluate the changes in the platelet function in patients with inflammatory bowel disease (IBD), we measured circulating PDMP levels.METHODS:Twenty-five healthy controls, 44 patients with ulcerative colitis (UC), and 43 patients with Crohns Disease (CD) were studied. The PDMP and soluble P-selectin (sP-selectin) levels were measured by enzyme-linked immunosorbent assay (ELISA).RESULTS:In the healthy controls, the PDMP levels were 17.2 ± 6.2 U/mL. Significant differences were not observed between the healthy controls and inactive UC patients (20.8 ± 9.5 U/mL, n = 25) or between the healthy controls and inactive CD patients (17.6 ± 7.8 U/mL, n = 24). In contrast, the PDMP levels were significantly higher in both active UC (49.2 ± 33.6 U/mL, n = 19) and active CD (48.6 ± 42.8 U/mL, n = 19) patients than in the healthy controls. A significant correlation was found between the PDMP levels and the clinical activity indexes (CAI) of UC patients (r = 0.65, p < 0.01, n = 44), and between the PDMP levels and Crohns disease activity indexes (CDAI) (r = 0.72, p < 0.01, n = 43). Elevated PDMP levels in active patients were significantly reduced after remission. A significant correlation was observed between the PDMP levels and the sP-selectin levels (r = 0.60, p < 0.01, n = 122).CONCLUSION:Elevated circulating PDMPs in active IBD patients suggest a role for platelets in the pathogenesis of IBD.

Interleukin-17 augments tumor necrosis factor-α-induced granulocyte and granulocyte/macrophage colony-stimulating factor release from human colonic myofibroblasts

BackgroundInterleukin (IL)-17 is a newly identified T-cell-specific cytokine. In this study, we investigated the effects of IL-17 on colony-stimulating factor (CSF) release in human colonic subepithelial myofibroblasts (SEMFs).MethodsCSF release and mRNA expression were determined by enzyme-linked immunosorbent assay (ELISA) and Northern blotting, respectively. Nuclear factor (NF)-κB- and activating protein (AP-1)-DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSAs).ResultsUnstimulated cells secreted a small amount of granulocyte G- and granulocyte/macrophage (GM)-CSF, and a considerable amount of M-CSF. IL-17 weakly enhanced G-CSF release, but did not affect GM- and M-CSF release. IL-17 selectively enhanced tumor necrosis factor (TNF)-α-induced G- and GM-CSF release. The combination of IL-17 plus TNF-α induced a marked increase in NF-κB- and AP-1-DNA binding activities. The adenovirus-mediated transfer of a stable form of IκBα and/or a dominant negative mutant of c-Jun markedly inhibited the IL-17 plus TNF-α-induced G- and GM-CSF mRNA expression. Furthermore, a stability study showed that IL-17 plus TNF-α markedly enhanced the stability of G- and GM-CSF mRNA.ConclusionsIL-17 augments TNF-α-induced G- and GM-CSF release via transcriptional and posttranscriptional mechanisms.

Increased aggregation response of platelets in patients with inflammatory bowel disease.

BackgroundPlatelets play an important role in hemostatic and inflammatory responses. To evaluate any potential enhancement of platelet activity in patients with inflammatory bowel disease (IBD), we measured the platelet aggregation responses to various stimuli.MethodsTwenty-two healthy controls, 24 patients with ulcerative colitis (UC) and 25 patients with Crohns Disease (CD) were studied. The aggregation responses induced by three agonists (epinephrine, collagen, and ADP) were measured by an 8-channel aggregometer. The platelet-derived microparticles (PDMP) levels were measured by an enzyme-linked immunosorbent assay.ResultsTwenty-one out of the 22 healthy controls did not respond to epinephrine (0.1 µg/ml), collagen (0.2 µg/ml), or ADP (1.0 µM). Eight out of the 12 active UC patients were sensitive to all agonists, and 4 patients showed increased sensitivity to epinephrine/collagen or epinephrine/ADP. Three out of the 12 inactive UC patients were normal, but 9 of these patients showed increased sensitivity, mainly to epinephrine. Ten out of the 12 active CD patients were sensitive to all agonists, and 2 active CD patients were sensitive to epinephrine/collagen or epinephrine/ADP. Eight out of the 13 inactive CD patients were sensitive to two or all agonists. Even after remission, almost all of the UC and CD patients showed some increased sensitivity to the agonists. The platelet number and the plasma PDMP levels were significantly higher in the active IBD patients than in the control group.ConclusionsPlatelet aggregation responses are enhanced in IBD, even in inactive-phase patients. This increased sensitivity of the platelets may play an important role in the pathophysiology of IBD.

Application of 2-aminopyridine fluorescence labeling in the analysis of in vivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography

The present study describes a size-exclusion high-performance liquid chromatographic method for the separation and quantification of sulfated polysaccharides, such as dextran sulfate sodium (DSS). Pyridylamination of DSS was achieved without difficulty using 2-aminopyridine as a fluorometric label. In addition, 0.1-0.2 M phosphate buffer (pH 3.0) was found to be the mobile phase which produced the best separation. In vitro enzymatic degradation of the pyridylamino-DSS (PA-DSS5000, Mr 5000) using alpha-amylase and the in vivo metabolism in the rat feces after oral administration of PA-DSS5000 were then evaluated. Two small peaks of approximately Mr 380 and 600 appeared after co-incubation with alpha-amylase, indicating PA-DSS5000 may be considerably depolymerized. In vivo, however, PA-DSS5000 excreted in the feces was mainly of PA-DSS5000 polymer. No peaks of less than Mr 5000 were not clearly detectable in the feces because of background fluorescence attributable to gut lumen contents. This method of fluorometric analysis allows fairly selective detection of sulfated polysaccharides in biological materials.

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.