Ken-ichi Manaka

PPARα activators upregulate eNOS activity and inhibit cytokine-induced NF-κB activation through AMP-activated protein kinase activation

Endothelium-derived NO is an important mediator of vascular protection and adhesion molecule expression on the endothelial cell surface is critical for leukocyte recruitment to atherosclerotic lesions. We hypothesized that AMP-activated protein kinase (AMPK) activity is a down-stream mediator of the beneficial effects of PPARalpha activators on vascular endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with fenofibrate or WY14643 resulted in transient activation of AMPK, as monitored by phosphorylation of AMPK and its down-stream target, acetyl-CoA carboxylase. Fenofibrate caused phosphorylation of Akt and eNOS, leading to increased production of NO, and also caused inhibition of cytokine-induced NF-kappaB activation, leading to suppression of expression of adhesion molecule genes. Significant decreases in eNOS activity and NO production in response to fenofibrate were observed in cells treated with AMPK siRNA or with AraA, a pharmacological inhibitor of AMPK. The attenuation of fenofibrate-induced inhibition of NF-kappaB activation was observed in mouse endothelial (SVEC4) cells treated with AMPK siRNA or with AraA. We demonstrated that TNFalpha stimulates IkappaB-alpha phosphorylation through induction of IKK activity, and that fenofibrate inhibits IKK activity and TNFalpha-induced IkappaB-alpha phosphorylation. Our findings suggest that the beneficial effects of PPARalpha activators on endothelial cells such as inhibition of diabetic microangiopathy might be attributed to the induction of AMPK activation beyond its lipid-lowering actions.

Expression of monocyte chemoattractant protein-1 mRNA and protein in cultured human thyrocytes

Monocytes as well as lymphocytes infiltrate in the stroma of thyroid tissue in autoimmune and destructive thyroiditis. Monocyte chemoattractant protein‐1 (MCP‐1) is a cytokine that attracts T‐lymphocytes as well as monocytes. Using human thyrocytes in primary cultures, we show that expression of MCP‐1 mRNA and protein is remarkably stimulated by both interleukin‐1 (IL‐1) and tumor necrosis factor‐α (TNF‐α), and also that interferon‐γ (IFN‐γ) by itself is a weak stimulant but has a synergistic activity with either IL‐1 or TNF‐α. The finding indicates that MCP‐1 can be produced by thyrocytes themselves, suggesting a possible role of thyrocytes on accumulation of monocytes and T‐lymphocytes to the tissue from the blood in autoimmune and destructive thyroiditis.

Eosinophil peroxidase stimulates the release of granulocyte-macrophage colony-stimulating factor from bronchial epithelial cells

BACKGROUND Asthma is characterized by an accumulation of activated eosinophils in the airway. Eosinophil viability-enhancing activity is present in the sputum of patients with asthma, largely because of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bronchial epithelial cells have been shown to release cytokines including GM-CSF when stimulated with IL-1 beta or tumor necrosis factor-alpha. OBJECTIVE The study was designed to determine whether eosinophil peroxidase (EPO) stimulates the release of GM-CSF from bronchial epithelial cells. METHODS Epithelial cells (BEAS-2B) were cultured in serum free HD-F12 medium in a 24-well tissue culture plate until they became confluent. The cells were then exposed to EPO (5.9 x 10(-8) to 5.9 x 10(-7) mol/L) for 15 minutes, washed twice, and cultured in 1 ml of HD-F12. The supernatants were harvested at 3, 6, or 24 hours, and GM-CSF concentration was measured by ELISA. BEAS-2B cells were also treated with a system comprising EPO (1.9 x 10(-9) to 5.9 x 10(-8) mol/L) + 10(-5) mol/L H2O2 + 10(-4) mol/L Br for 24 hours. RESULTS The GM-CSF concentration in the supernatant pretreated with EPO increased in a time- and concentration-dependent manner compared with control. The release of GM-CSF was not inhibited by catalase but was inhibited by cyclohexamide and by mixing of EPO with heparin, suggesting that the action is due to the cationic property of EPO. When EPO was combined with H2O2 and Br, 5.9 x 10(-9) mol/L EPO + 10(-5) mol/L H2O2 released two times more GM-CSF into the supernatants compared with control. CONCLUSION These results suggest that EPO stimulates epithelial cells to release GM-CSF and forms a self-stimulatory cycle.

Microsatellite polymorphisms of the MICA gene among Japanese patients with Behçet's disease

BACKGROUND Behçets disease (BD) is a multisystemic inflammatory disease of unknown origin. Because some researchers have recently suggested a primary association of BD with the A6 allele of the human major histocompatibility complex class I chain-related A (MICA) gene, we investigated microsatellite polymorphisms of the MICA gene in subjects with and without BD. METHODS This was a case-control study of 23 Japanese patients with BD and 23 Japanese volunteers without BD who were compared for MICA microsatellite polymorphisms using the polymerase chain reaction (PCR). We also analysed associations between 5 MICA alleles and the clinical features of patients. RESULTS There was no significant difference between case patients and control subjects in phenotype frequencies. The MICA-A6 allele showed the strongest positive correlation with the human leukocyte antigen allele HLA-B51. Allele A5 showed a strong positive correlation with age at onset and a strong negative correlation with iridocyclitis and HLA-B51. A4 showed a strong negative correlation with ocular lesions and HLA-B51. Patients with the MICA-A6 allele had significantly higher HLA positivity than patients without the allele. INTERPRETATION While the MICA-A6 allele had no significant association with BD, it showed a strong association with HLA-B51. This finding suggests that an association between MICA-A6 and BD may be a secondary phenomenon related to HLA-B51. As several associations with MICA alleles and clinical features have been found, further investigation is expected to elucidate the biological mechanism of action of the MICA protein relative to disease onset.

Clinical Features of Japanese Patients with Behçet's Disease and MICA Polymorphism

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Detailed Characterization of a High-molecular-weight Glycoprotein Secreted by Lung Cancer Cells

A cancer‐associated, high‐molecular‐weight glycoprotein antigen (6B3·Ag) recognized by monoclonal antibody 6B3 was purified from culture medium of human large cell lung carcinoma cell line (HLC‐2) and characterized biochemically and immunochemically. The 6B3·Ag was purified more than 1,200‐fold with a yield of 30% by salting out, precipitation by acidification at pH 4.5, and chromatographies on Sepharose 4B and concanavalin A‐Sepharose. The molecular weight of 6B3·Ag is approximately 1,000,000 and the molecule is a homodecamer of 94,000 subnnits. The 6B3·Ag is a glycoprotein containing 22.9% sugars, consisting of both N‐ and O‐glycoside chains. The N‐terminal 19 amino acids were determined and only 4 out of 19 amino acid residues were different from those of an antigen, L3, secreted by lung carcinoma cell line Calu‐1. The serum level of 6B3·Ag was determined in normal adults as well as patients with various diseases by enzyme‐linked immunosorbent assay. The mean serum level of 6B3‐Ag was 3.1 μg/ml, ranging from 1.6 to 6.2 μg/ml in 131 healthy adults. When the cut‐off value was set at 6.2 μg/ml, the incidence of positive values in the sera was elevated not only in malignant diseases such as hepatoma (73%) and leukemia (62%), but also in benign diseases such as chronic hepatitis (42%) and liver cirrhosis (63%). While the incidence of positive values was elevated in advanced liver diseases, namely, chronic hepatitis, liver cirrhosis and hepatoma, the cancer specificity of 6B3·Ag did not appear to be high.

Role of Platelet-Activating Factor in the Release of Neutrophil and Eosinophil Chemotactic Attractants from Cultured Guinea-Pig Tracheal Epithelial Cells

Platelet-activating factor (PAF) has been implicated in the pathogenesis of allergic disease, particularly bronchial asthma, by recruitment and activation of inflammatory cells. In an effort to further elucidate this function of PAF, guinea-pig tracheal mucosa was cultured in the presence of PAF in vitro, and culture supernatants were injected intradermally into normal guinea-pigs. After 6 h, recruitment of the inflammatory cells in the tissue was evaluated as a marker of chemotactic activity. Neutrophil and eosinophil recruitment increased significantly 1 h after PAF stimulation, the latter more than the former. After fractionation of the culture supernatant using molecular sieve filters, the fraction of 10-30 kDa showed greater chemotactic activity than the fractions of below 10 kDA or greater than 30 kDA. This activity was inhibited in a dose-dependent manner (over about 1-100 mg/ml) by treatment with anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody. These results suggest that PAF induces the release of chemotactic factors for neutrophils and eosinophils from guinea-pig tracheal epithelial cells, and one of these factors may be GM-CSF.