|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1994 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1993 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1992 : ¥800,000 (Direct Cost : ¥800,000)
To explore the role of glomerular endothelial cells (GEN) in the pathogenesis of glomerulonephritis, the in vitro production of monocyte chemoattractant protein-1 (MCP-1) by bovine GEN was determined by chemotaxis assay, Northern blot analysis, and immunocytochemistry. Monocyte chemotactic activity of GEN-conditioned media was detectable by a chemotaxis assay using human peripheral blood monocytes. Exposure to human recombinant interleukin-1beta (IL-1beta) and phorbol myristate acetate (PMA) significantly increased the chemotactic activity of GEN-conditioned media. A checcurboard analysis showed that the response of monocytes to GEN-conditioned media was truly chemotactic. Immunoadsorption with a monoclonal antibody to human MCP-1 reduced the chemotactic activity of GEN-conditioned media by 85%. Northern blot analysis revealed that MCP-1 mRNA was constitutively expressed by GEN and that IL-1beta and tumor necrosis factor-alpha (TNF-alpha) increased MCP-1 mRNA levels in a dose- and time
-dependent manner. PMA also induced an increase in MCP-1 mRNA levels that was suppressed by the protein kinase C inhibitors staurosporine and H-7, whereas dibutyryl cyclic AMP and forskolin had minimal effects, indicating involvement of protein kinase C.However, MCP-1 mRNA expression induced by IL-1beta and TNF-alpha was suppressed by the tyrosine kinase inhibitor genistein, not by staurosporine, H-7, or the protein kinase A inhibitor H-89, suggesting importance of the tyrosine kinase activation on the cytokine-induced MCP-1 gene expression. Dexamethasone had a small inhibitory effect on constitutive and PMA-induced MCP-1 mRNA expression, but no effect on the induction by TNF-alpha.By immunoperoxidase staining with an anti-MCP-l monoclonal antibody, MCP-1 protein was detected in untreated GEN and increased by exposure to PMA,correspondeng to mRNA expression. These results demonstrate the production of MCP-1 by GEN at gene and protein levels as well as bioactivity, and suggest that GEN may participate in the development of glomerulonephritis through the production of MCP-1.
2.ウシ糸球体内皮細胞培養上清中に明らかな単求遊送活性を認め、これはIL-1β,phorbol ester刺激により増強した.培養上清の単求遊走活性はmonocyte chemoattractant protein-1(MCP-1)に対する特異抗体で吸収除去され,糸球体内皮細胞によるMCP-1産生が考えられた.
3.糸球体内皮細胞のMCP-1mRNA発現はIL-1β,TNF-αにより容量および時間依存性に増強した。MCP-1mRNA発現の細胞内情報伝達経路について,cAMPやforskolinは効果なくphorbol esterにより誘導され,その効果はprotein kinase C inhibitorで抑制されたことからprotein kinase C活性化の関与が考えられた.一方,IL-1β,TNF-αによる発現誘導はtyrosine kinase inhibitorで抑制され,炎症性サイトカインによるMCP-1mRNA発現におけるtyrosine kinase活性化の重要性が示された.また,dexamethasoneはMCP-1mRNA発現をわずかに抑制した.