Spin-trapping detection of nitric oxide (NO) production from macrophage during matuation

为了研究巨噬细胞成熟过程中一氧化氮(NO)的产生，采用电子自旋共振波谱(ESR)和自旋捕捉相结合的方法，从硫代乙酸乙酯刺激的小鼠心包内分离出巨噬细胞，用干扰素-γ(INF-γ)、脂多糖(LPS)和乳铁蛋白(LF)刺激后测定NO的产生。用Fe~(2+)~(2+)-Gt~(2+)与N-(二硫代甲酰基)-N-甲基葡萄糖胺的络合物(MGD-Fe)作为自旋捕捉剂。在LPS+InF-γ刺激的巨噬细胞中，观察到由NO和MGD-Fe反应产生的MGD-Fe-NO产生的三重态ESR信号。用Griess试剂比色法也证实了NO的代谢产物NO2~-+NO2~(-3)对NO产生的增加反应。Western blotting检测到的合酶不受这些刺激的诱导。此外，LF可促进干扰素-γ刺激的巨噬细胞产生NO和诱导型一氧化氮合酶，而其本身并不产生任何NO。提示巨噬细胞的成熟是由炎症部位的中性粒细胞产生的LF和多种淋巴细胞产生的多种细胞因子共同作用的结果。

In order to investigate nitric oxide (NO) production from macrophage during maturation, peneatrial macrophage was isolated from thioglycolate-stimulated mouse and the production of NO after stimulation by interferon-gamma (INF-gamma), lipopolysaccharide (LPS) and lactoferrin (LF) was measured by the method combining electron-spin-resonance spectroscopy (ESR) and spin-trapping. The complex (MGD-Fe) of Fe^<2+> and N- (Dithiocarbonyl) -N-methylglucamine was used as spin-trapping reagent. Triplet ESR signal due to MGD-Fe-NO,which was produced by the reaction of NO and MGD-Fe, was time-dependently observed in LPS+Inf-gamma-stimulated macrophage. The NO production was also confirmed the increased-responses of NO_2^-+NO_3^-, metabolites of NO,by colorimetric method using Griess reagents. It was also shown that NO synthase detected by Western blotting was induced by these stimulation. Furthermore, LF promoted the NO production and iNOS induction in INF-gamma-stimulated macrophage, whereas LF itself did not produce any NO production in macrophage. These results was suggested that the maturation of macrophage was induced by LF which was produced from neutrophil in inflammation site as well as various cytokines which was produced from various lymphocytes.