Hitoshi Sakumoto

Interleukin 4 Acts as an Inducer of Decay-Accelerating Factor Gene Expression in Human Intestinal Epithelial Cells

BACKGROUND & AIMS Decay-accelerating factor (DAF) protects host tissues from the attack of autologous complement activation. In this study, we attempted to define the cytokine regulation of DAF messenger RNA (mRNA) expression in human intestinal epithelial cells. METHODS The effects of cytokines on DAF mRNA accumulation were evaluated by Northern blot analysis. The DAF protein expression was analyzed by both immunoprecipitation and immunoblotting. RESULTS Interleukin (IL)-4 induced a marked increase in DAF mRNA accumulation in HT-29 cells. In this line, IL-1 beta evoked only weak induction, and IL-6, IL-8, IL-10, and interferon gamma had no effect. The effect of IL-4 was observed in a dose-dependent manner and confirmed at the protein level. The increase in DAF mRNA accumulation reached a maximum at 3-6 hours and then gradually decreased. These effects of IL-4 on DAF mRNA and protein expression were also observed in T84 cells. The mRNA stability studies suggested that IL-4 regulates DAF gene expression mainly at the transcriptional level. CONCLUSIONS In human intestinal epithelial cells, IL-4 acts as a potent inducer of DAF mRNA expression, suggesting a cytoprotective role for IL-4 against autologous complement activation.

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.

Detection of complement C3 and factor B gene expression in normal colorectal mucosa, adenomas and carcinomas.

Local secretion of complement components in the human intestine has been previously reported. However, the cellular source has not been identified. In this study, we demonstrate complement C3 and factor B mRNA expression in the normal colonic mucosa by in situ hybridization analysis. C3 and factor B genes were found to be expressed at high levels in the epithelial cells of the lower parts of the crypts in colonic mucosa, and this expression decreased gradually from the crypt base to the luminal surface. At the upper crypt and the luminal surface, these genes almost disappeared. C3 and factor B genes were expressed in all crypts at the same level. Furthermore, C3 and factor B gene expression was also identified in adenomas and carcinomas. In these neoplastic tissues, C3 and factor B genes were expressed uniformly, and the polarized distribution observed in the normal crypts was not detected. It is likely that complement components are locally synthesized in the intestine, and that these complement components may actively participate in normal immune and inflammatory responses over the enormous surface area of the intestinal 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.

Molecular characterization of complement components (C3, C4, and factor B) in human saliva.

A molecular analysis of complement components (C3, C4, and factor B) in human saliva was performed by 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 0.52 to 15.0 /μg/ml (n = 15). C4 was detected as a triple-chain molecule (98-kDa α chain, 73-kDa β chain, and 33-kDa γ chain) linked by disulfide bonds, and C4 levels ranged from 0.086 to 4.8 μg/ml. Factor B was detected as a 100-kDa single chain, and factor B levels ranged from 0.042 to 0.62/μg/ml. The sizes and subunit structures of the complement components in human saliva were compatible with those reported in human serum. The results of a hemolytic assay indicated that the complement molecules in human saliva were functionally active. These complement components may participate in the local immune and inflammatory responses in the oral cavity.

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.

Ketone body ratios of the superior and inferior vena cava and of pulmonary arterial blood compared to that of arterial blood: central venous ketone body ratio as a substitute for the arterial ketone body ratio.

To investigate the ketone body ratio (acetoacetate/3-hydroxybutyrate) of central venous blood compared to that of peripheral arterial blood, the acetoacetate and 3-hydroxybutyrate concentrations in paired peripheral arterial and central venous or pulmonary arterial blood were measured. The ketone body concentrations in superior and inferior vena cava blood were significantly (P < 0.0001) lower than those in peripheral arterial blood, whereas those in pulmonary arterial blood were almost the same as those in peripheral arterial blood. These results indicate that ketone bodies were metabolized in the muscles, which reduced their levels in vena cava blood, but ketone bodies newly produced by the liver were transported to the right side of the heart via the hepatic vein, giving concentrations in pulmonary arterial blood that were almost the same as those in peripheral arterial blood. On the other hand, the correlation coefficients (r2) of the arterial blood ketone body ratio to the ratio of superior and inferior vena cava and pulmonary arterial blood were 0.897, 0.767 and 0.882, respectively. The ratios of central venous ketone body ratio/arterial blood ketone body ratio were 0.89 +/- 0.15 in the superior vena cava, 0.64 +/- 0.18 in the inferior vena cava and 1.01 +/- 0.15 in the pulmonary artery.

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.