Kenichi Sumiyoshi

Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

The increased expression of decay‐accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)‐α on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF‐α induced a marked increase in DAF mRNA and protein expression in HT‐29, T84 and Caco‐2 cells. In HT‐29 cells, the effects of TNF‐a on DAF mRNA accumulation were observed in a dose‐dependent manner; DAF mRNA accumulation reached a maximum at 3–6 hr, and then gradually decreased. These effects of TNF‐α required de novo protein synthesis. Messenger RNA stability studies suggested that TNF‐α partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF‐α and interleukin (IL)‐4 induced an additive increase in DAF mRNA accumulation in HT‐29 and T84 cells. In human intestinal epithelial cells, TNF‐α acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF‐α in the regulation of DAF expression at the inflamed mucosa.

Biosynthesis and secretion of MHC class III gene products (complement C4 and factor B) in the exocrine pancreas

We recently found that complement C3 is locally synthesized and secreted into the exocrine pancreas. In the present study, we attempted to demonstrate the secretion of complement C4 and factor B in the exocrine pancreas. In five samples of pancreatic fluid, both C4 and factor B proteins were detected by emzyme-linked immunosorbent assay (ELISA). Immunoblot analysis revealed the C4 and factor B molecules in pancreatic fluid to be identical with these molecules in serum. Reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis in pancreatic carcinoma cell lines suggested ductal epithelial cells to be the local production sites of these proteins in the pancreas. The secretion of C4 and factor B in ductal cell lines (PANC-1 and MIA PaCa-2) was independently regulated by interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ; C4 secretion was induced by IFN-γ, whereas factor B secretion was induced by IL-1β, TNF-α, or IFN-γ. These observations indicate that: (a) complement C4 and factor B are secreted into the exocrine pancreas, (b) ductal epithelial cells appear to be the site of C4 and factor B biosynthesis, and (c) local secretion of C4 and factor B in the pancreas is differentially regulated by IL-1β, TNF-α, and IFN-γ.

CHARACTERIZATION OF COMPLEMENT C3, C4, AND FACTOR B MOLECULES IN HUMAN BILE

We performed molecular analysis of complement components (C3, C4, and factor B) in human bile by sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa α-chain linked to a 70-kDa β-chain by disulfide bonds, and C3 levels ranged from 45 to 650μg/ml (n=15). C4 was detected as a triple chain (98-kDa α-chain, 73-kDa β-chain, and 33-kDa γ-chain) molecule linked by disulfide bonds, and C4 levels ranged from 2.5 to 60μg/ml. Factor B, a component of the alternative pathway, was also detected, as an intact form. Factor B levels ranged from 0.3 to 8.0μg/ml. The sizes and subunit structures of complement components in human bile were compatible with those reported in human serum. The results of a hemolytic assay indicated that complement molecules in human bile were functionally active. These molecules may participate in local immune and inflammatory responses in the biliary tract.

Modulation of complement C3, C4, and factor B production in human intestinal epithelial cells: differential effects of TNF-α, IFN-γ, and IL-4

Abstract Recent studies have suggested that intestinal epithelial cells (IEC) are local production sites of several complement (C) components. To clarify the regulatory relationships between C production and secretory component (SC)-mediated polymeric immunoglobulin (pIg) transport in IEC, we studied the effects of inducers of SC expression, such as TNF-α, IFN-γ and IL-4, on C production in the human intestinal epithelial cell line, HT-29. Unstimulated HT-29 cells secreted substantial amounts of factor B but not of C3 or C4. On the other hand, TNF-α induced C3 production and enhanced factor B production. IFN-γ induced C4 production and enhanced factor B production. In addition, the combination of TNF-α and IFN-γ synergistically induced both C3 and factor B production but did not affect C4 production. IL-4, by itself, had no effects, but this cytokine significantly decreased the IFN-γ-induced increase in C4 production by 72% and that in factor B production by 62%. These cytokines modulated C production at a pretranslational level. These responses to cytokines might serve a mechanism for the highly regulated interaction between the C system and secretory Ig system in the mucosa.

Regulation of complement C3 synthesis by interleukin-1 and transforming growth factor-β in rat non-transformed intestinal epithelial cell line, IEC-6

Intestinal epithelial cells are an important source of many biologically active molecules that modulate immune responses in the mucosa. The purpose of this study was to demonstrate the synthesis of complement C3 component in the rat non-transformed crypt-like intestinal epithelial cell line, IEC-6. Unstimulated IEC-6 cells secreted a low level of C3 protein and showed weak expression of C3 mRNA. The addition of interleukin (IL)-1β induced a dose- and time-dependent increase in C3 production. These effects of IL-1β were observed at a concentration as low as 0.01 ng/ml and reached a plateau at a concentration of 5 ng/ml. The effects were observed at the mRNA level as early as 6 h after the beginning of incubation. Transforming growth factor (TGF)-β alone had no effect. However, TGF-β at low concentrations (0.001–1 ng/ml) enhanced the effect of IL-1β in increasing C3 production; this enhancement was not observed at high concentrations (5–10 ng/ml). These effects of TGF-β were also observed at the mRNA level. The present findings indicate that intestinal epithelial cells are indeed capable of synthesizing complement C3 in response to IL-1β and TGF-β.

Evaluation of clinical features of ischemic colitis: Comparison between young and elderly

Background: It has been thought that ischemic colitis is caused by vascular and intestinal factors. Although elderly patients with arteriosclerosis are more susceptible to ischemic colitis, many young patients suffering ischemic colitis are also reported. The present study aimed to clarify the relationship between arteriosclerosis and ischemic colitis, and to evaluate various risk factors for ischemic colitis.