Jin Makino

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.

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.

Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling.

Polarized Th1 and Th2 cells expressing the same TCR produce distinct biochemical responses to ligand engagement. Compared to Th1 cells, Th2 cells show altered substrate tyrosine phosphorylation and a diminished or transient Ca2+ response. Here we demonstrate that agonist stimulation of Th1 cells leads to the predominant appearance of fully phosphorylated (p23) TCR ζ, substantial phosphorylation of zeta‐associated protein 70 (ZAP‐70), and strong elevation of intracellular Ca2+, whereas agonist stimulation of Th2 cells expressing an identical TCR results in an elevated p21:p23 TCR ζ ratio, little or no detectable ZAP‐70 phosphorylation, and a more limited elevation in intracellular Ca2+. Th2 cells consistently had twofold lower surface CD4 expression as compared to Th1 cells with the same TCR. When CD4 levels in Th2 cells were raised to Th1 levels using retroviral gene transfer, the transduced cells showed greater generation of p23 phospho‐ζ, measurable phosphorylation of ZAP‐70, and increased Ca2+ responses. These findings suggest that the apparent qualitative differences in TCR signaling characterizing Th1 versus Th2 cells are largely the result of modest quantitative variation in CD4 expression, with decreased CD4 expression playing a significant role in attenuating the proximal signaling responsiveness of Th2 cells to TCR ligands.

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.

Inhibitory effects of the new anti-inflammatory agent, IS-741, on spontaneous colitis in HLA-B27/beta2-microglobulin transgenic rats

Abstract Background: A novel anti‐inflammatory drug, IS‐741, blocked the adhesion of inflammatory cells to microvascular endothelial cells both in vivo and in vitro. Transgenic rats expressing human leukocyte antigen (HLA)‐B27 and human β2‐microglobulin (HLA‐B27 rats) spontaneously develop chronic colitis, which resembles human inflammatory bowel disease. In the present study, the authors examined the efficacy of IS‐741 against spontaneous colitis in HLA‐B27 rats.