Lin J Y, Seguin R, Keller K, Chadee K
Institute of Parasitology of McGill University, Ste.-Anne de Bellevue, Québec, Canada.
Infect Immun. 1994 May;62(5):1534-41. doi: 10.1128/iai.62.5.1534-1541.1994.
Nitric oxide (NO measured as nitrite, NO2-) is the major effector molecule produced by activated macrophages for in vitro cytotoxicity against Entamoeba histolytica trophozoites. In this study, we determine whether tumor necrosis factor alpha (TNF-alpha) produced by activated bone marrow-derived macrophages (BMM) is involved in the induction of the inducible NO synthase gene (mac-NOS) for NO-dependent amebicidal activity. TNF-alpha alone did not directly induce macrophage NO2- production to kill amebae; however, in combination with increasing concentrations of TNF-alpha and gamma interferon (IFN-gamma), BMM amebicidal activity and NO2- production progressively increased and showed a significant linear correlation. Antiserum to TNF-alpha and the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) inhibited the synergistic effects of TNF-alpha and IFN-gamma. BMM activated with increasing concentrations of lipopolysaccharide (LPS) and IFN-gamma showed a significant linear correlation between TNF-alpha release and NO2- production. Antiserum to TNF-alpha suppressed TNF-alpha release, NO2- production, and amebicidal activity by 93, 53, and 86%, respectively. L-NMMA diminished NO2- production by 74% and macrophage amebicidal activity by 83% but had no effect on TNF-alpha release. Quantification by Northern (RNA) blot analyses demonstrated that IFN-gamma in combination with TNF-alpha or LPS increased markedly the accumulation of mac-NOS and TNF-alpha mRNAs in a time-dependent manner with a concomitant increase in NO and TNF-alpha production. Peak induction of mac-NOS occurred after 24 h, whereas TNF-alpha mRNA was rapidly expressed after 4 h and remained stable for 48 h. Taken together, these data argue that TNF-alpha augments NO-dependent macrophage cytotoxicity against E. histolytica via elevated levels of mac-NOS mRNA expression which may be associated with the accumulation of TNF-alpha mRNA.
一氧化氮（以亚硝酸盐，即NO₂⁻形式测量）是活化巨噬细胞产生的主要效应分子，用于体外对溶组织内阿米巴滋养体的细胞毒性作用。在本研究中，我们确定活化的骨髓来源巨噬细胞（BMM）产生的肿瘤坏死因子α（TNF-α）是否参与诱导诱导型一氧化氮合酶基因（mac-NOS）以产生依赖一氧化氮的杀阿米巴活性。单独的TNF-α并不能直接诱导巨噬细胞产生NO₂⁻来杀死阿米巴；然而，与浓度不断增加的TNF-α和γ干扰素（IFN-γ）联合使用时，BMM的杀阿米巴活性和NO₂⁻产生量逐渐增加，并呈现出显著的线性相关性。抗TNF-α血清和一氧化氮合酶抑制剂NG-单甲基L-精氨酸（L-NMMA）抑制了TNF-α和IFN-γ的协同作用。用浓度不断增加的脂多糖（LPS）和IFN-γ激活的BMM在TNF-α释放和NO₂⁻产生之间呈现出显著的线性相关性。抗TNF-α血清分别抑制TNF-α释放、NO₂⁻产生和杀阿米巴活性达93%、53%和86%。L-NMMA使NO₂⁻产生量减少74%，巨噬细胞杀阿米巴活性降低83%，但对TNF-α释放没有影响。通过Northern（RNA）印迹分析进行定量显示，IFN-γ与TNF-α或LPS联合使用时，以时间依赖性方式显著增加了mac-NOS和TNF-α mRNA的积累，同时伴随着NO和TNF-α产生量的增加。mac-NOS的峰值诱导在24小时后出现，而TNF-α mRNA在4小时后迅速表达并在48小时内保持稳定。综上所述，这些数据表明TNF-α通过提高mac-NOS mRNA表达水平增强了巨噬细胞对溶组织内阿米巴的依赖一氧化氮的细胞毒性，这可能与TNF-α mRNA的积累有关。
