Takafumi Okuno

IL-17 Selectively Down-Regulates TNF-α-Induced RANTES Gene Expression in Human Colonic Subepithelial Myofibroblasts

IL-17 enhances the TNF-α-induced IL-6 and IL-8 secretion in human colonic subepithelial myofibroblasts. In this study, we investigated how IL-17 modulates RANTES secretion in these cells. TNF-α potently induced RANTES secretion, but IL-17 dose-dependently inhibited the TNF-α-induced RANTES secretion. This was also observed at the mRNA level. Even after pretreatment with TNF-α for 12 h, the inhibitory effect of IL-17 was detectable. IL-17 did not affect the TNF-α-induced stability of the RANTES gene. IL-17 significantly decreased the TNF-α-induced increase in RANTES promoter activity, and IL-17 actually blocked the TNF-α-induced RANTES gene transcription. EMSAs demonstrated that IL-17 did not modulate the TNF-α-induced NF-κB DNA-binding activity, but markedly decreased TNF-α-induced IFN regulatory factor-1 (IRF-1) DNA-binding activity. Because cooperation between NF-κB and IRF-1 is important in the TNF-α-induced RANTES gene expression, the major mechanism mediating the inhibitory effect of IL-17 may be achieved by the inhibition of IRF-1 DNA-binding activity.

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.

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.

Intestinal subepithelial myofibroblasts in inflammatory bowel diseases

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the regulation of a number of epithelial cell functions and in the mucosal repair process. In this study, we evaluated the changes in α-smooth muscle actin (SMA)- and vimentin-positive SEMFs in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Tissue samples were surgically obtained from patients with active ulcerative colitis (UC) (n = 5) and active Crohn’s disease (CD) (n = 5). Normal intestinal tissues were also obtained (n = 5). The SMA and vimentin expression was evaluated by standard immunohistochemical procedures. In normal intestinal mucosa, SMA- and vimentin-positive SEMFs were located immediately subjacent to the basement membrane, juxtaposed against the bottom site of the epithelial cells. In the inflamed mucosa of active UC patients, there were relatively more SMA-positive cells compared with normal mucosa. In particular, the increase in SMA-positive cells was greatest at the marginal area of deep ulcers of UC patients. In active CD mucosa, SMA-positive cells were increased in all samples, and a marked increase was observed in two samples. The number of SMA-positive SEMFs was relatively higher in CD mucosa than in UC mucosa. An [3H]thymidine incorporation study demonstrated that platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF)-I significantly increased the uptake of [3H]thymidine into isolated SEMFs. In particular, PDGF had a strong stimulatory effect. We concluded that colonic SEMFs may play an important role in the repair process of IBD.

Primary bone marrow diffuse large B‑cell lymphoma accompanying cold agglutinin disease: A case report with review of the literature

Cold agglutinin disease (CAD) is a well-recognized complication of lymphoproliferative disorders. It has been previously recognized that cases of primary CAD frequently exhibit underlying malignant lymphoma in the bone marrow. Lymphoplasmacytic lymphoma is the most common subtype of malignant lymphoma; however, diffuse large B-cell lymphoma (DLBCL) has also been documented, albeit extremely rare. The current report presents a case of primary bone marrow DLBCL accompanying CAD. A 76-year-old male presented with fever and fatigue. Laboratory tests revealed anemia and elevated bilirubin and cold agglutinins with a titer of 8,192 at 4°C. Bone marrow biopsy demonstrated DLBCL and systemic surveillance failed to detect tumorous lesions or lymphadenopathy. Following R-THP-COP therapy, cold agglutinins titer was markedly decreased (by <4); however, malignant lymphoma relapsed and cold agglutinin levels increased again (4,096). This is the second documented case of primary bone marrow DLBCL accompanying CAD. Previously, malignant lymphoma exclusively involving the bone marrow, namely primary bone marrow lymphoma (PBML), has been recognized as a rare and aggressive subtype. The analyses of the present study revealed that the incidence of hemolytic anemia in primary bone marrow DLBCL may be high compared with conventional DLBCL. Therefore, additional analyses are required to clarify the clinicopathological features of PBML.