Fujihara M, Ito N, Pace J L, Watanabe Y, Russell S W, Suzuki T
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160-7420.
J Biol Chem. 1994 Apr 29;269(17):12773-8.
The role of endogenous tumor necrosis factor alpha (TNF-alpha) and interferon-beta (IFN-beta) in lipopolysaccharide (LPS)-induced activation of the inducible nitric-oxide synthase (i-NOS) gene was investigated. By Northern analysis or reverse-transcription polymerase chain reaction, the mouse macrophage cell line (J774) was found to respond to LPS treatment by increased expression of mRNAs specific for TNF-alpha, IFN-beta, and i-NOS with the kinetics unique for each gene. Bioassay of the culture supernatants showed that TNF-alpha and IFN-beta secreted by J774 cells increased from an undetectable level to about 300 and 340 units/ml, respectively, 3-6 h after LPS stimulation. Nitrite concentration was found to increase from 0 to 7.8 and 28.5 microM by 12 and 24 h, respectively, in the culture supernatant of LPS-treated J774 cells. The presence of a neutralizing dose of antibodies against IFN-beta, but not against TNF-alpha, during treatment with either 10 ng or 1 microgram of LPS/ml significantly, but not completely decreased the level of i-NOS-specific mRNA expression and NO production. The incubation of J774 cells with mouse natural IFN-beta itself (up to the level of 1,200 units/ml) did not induce i-NOS-specific mRNA and therefore did not stimulate J774 cells to produce NO. However, natural IFN-beta synergistically augmented the expression of i-NOS mRNA and the production of NO by J774 cells triggered by suboptimal concentrations of LPS (1 to 5 ng/ml). These data thus suggest that endogenous IFN-beta, but not TNF-alpha, produced by LPS-stimulated J774 cells specifically contributes, probably in an auto/paracrine fashion, to the activation of the i-NOS gene expression by LPS.
研究了内源性肿瘤坏死因子α（TNF-α）和干扰素β（IFN-β）在脂多糖（LPS）诱导的诱导型一氧化氮合酶（i-NOS）基因激活中的作用。通过Northern分析或逆转录聚合酶链反应发现，小鼠巨噬细胞系（J774）对LPS处理的反应是，TNF-α、IFN-β和i-NOS特异性mRNA的表达增加，且每个基因的动力学独特。培养上清液的生物测定表明，LPS刺激后3-6小时，J774细胞分泌的TNF-α和IFN-β分别从不可检测水平增加到约300和340单位/毫升。在LPS处理的J774细胞培养上清液中，亚硝酸盐浓度在12小时和24小时分别从0增加到7.8和28.5微摩尔。在用10纳克或1微克/毫升LPS处理期间，存在中和剂量的抗IFN-β抗体（而非抗TNF-α抗体）可显著但未完全降低i-NOS特异性mRNA表达水平和NO产生。用小鼠天然IFN-β本身孵育J774细胞（浓度高达1200单位/毫升）不会诱导i-NOS特异性mRNA，因此不会刺激J774细胞产生NO。然而，天然IFN-β可协同增强由次优浓度LPS（1至5纳克/毫升）触发的J774细胞中i-NOS mRNA的表达和NO的产生。因此，这些数据表明，LPS刺激的J774细胞产生的内源性IFN-β而非TNF-α可能以自分泌/旁分泌方式特异性地促进LPS对i-NOS基因表达的激活。
