Analysis on the physiological role histamine in airway inflammation using knock-out mice

1)HDC基因细胞特异性表达机制分析L-组氨酸脱羧酶（HDC）催化L-组氨酸转化为组胺，在造血谱系中仅在肥大细胞和嗜碱性粒细胞中表达。我们试图发现HDC基因表达在这些细胞中是如何受到限制的。在所测试的培养细胞系中，只有肥大细胞（HMC-1）和嗜碱性粒细胞（KU-812-F）强烈地转录HDC基因，而K562或Hela则没有。HDC基因的启动子区域被证明是敏感的，在表达HDC的细胞中的DNA酶。这种敏感性被证明取决于甲基化的程度。在Hela和K562中，HDC基因启动子区转录点附近的CpG几乎都发生了甲基化，而在HMC-1和KU-812-F中则没有甲基化。这些结果提示DNA甲基化改变是调节HDC基因细胞特异性表达的机制之一。2）HDC基因敲除小鼠过敏性炎症的分析为了阐明组胺在过敏反应中的作用，我们建立了HDC基因敲除小鼠模型。这些小鼠不能产生组胺，可用作缺乏组胺的实验模型。使用这些小鼠，我们分析了过敏反应，并将其与野生型小鼠进行了比较。我们用卵清蛋白检测了致敏的HDC基因敲除小鼠暴露于抗原后皮肤毛细血管的渗出。根据血管内注射伊文思蓝后组织中伊文思蓝的量计算渗出量。结果表明，敲除小鼠的速发型变态反应无渗出。给予含组胺的食物后，基因敲除小鼠在即刻过敏反应中出现渗出。这些结果清楚地表明，过敏反应中的血管渗出是由组胺引起的。

1) Analysis on the mechanism of cell-specific expression of HDC gene L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine, and in hematopoletic lineages the gene is expressed only in mast cells and basophils. We attempted to discover how HDC gene expression is restricted in these cells. In cultured cell lines tested, only the mast cells (HMC-1) and basophils (KU-812-F) strongly transcribed the HDC gene but K562 or Hela did not. The promotor region of the HDC gene proved to be sensitive to Dnase in HDC-expressing cells. This sensitivity was proved to depend on the extent of methylation. CpG near transcriptional point in the promoter region of HDC gene in Hela and K562 was almost methylated, in contrast, that was not in HMC-1 or KU-812-F. These results imply that alteration of DNA methylation is one of the mechanisms regulating cell-specific expression of the HDC gene.2) Analysis on allergic inflammation in HDC gene knock-out mice To elucidate the role of histamine in allergic reactions, we made knock-out mice which HDC gene was destructed. These mice are not capable to make histamine and are useful as the experimental model lacking histamine. Using these mice, we analyzed allergic responses and compared them to those in wild type mice. We measured exudation from capillaries in skin after exposure to antigen in sensitized HDC knock-out mice with ovalbumin. The exudation was calculated from amount Evans Blue in tissues, which was injected into vessels before. Result demonstrated that no exudation occurred in immediate allergic reaction in knock out mouse. After administrating food containing histamine, knock-out mice showed exudation in immediate allergic reaction. These results clearly indicated that exudation from vessels in allergic reaction was caused by histamine.