Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis
Induction of nitric oxide in human monocytes and monocyte cell lines by Mycobacterium tuberculosis
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DOI：
10.1006/niox.1998.9999
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发表时间：
1998-01-01
影响因子：
3.9
通讯作者：
Hunter, RL
中科院分区：
文献类型：
--
作者：
Jagannath, C;Actor, JK;Hunter, RL
结核分枝杆菌感染时单核细胞中一氧化氮的诱导一直是一个有争议的问题。本研究描述了一个比较评估的比色法和荧光法检测NO结核分枝杆菌（MTB）感染的人外周血来源的单核细胞（PBM）和U937，人单核细胞来源的细胞系。MTB在体外单核细胞培养物中生长7或10天。使用不含抗生素并补充有L-精氨酸、Hepes、5%人AB血清和四氢生物蝶呤的RPMI 1640培养基来支持单核细胞生长。早在感染后72 h，使用二氨基萘（DAN）荧光测定法在培养基中可检测到可溶性亚硝酸盐。通过RT-PCR定量，NO的早期诱导与iNOSmRNA水平的增加相关，NO水平进行性增加，直至第10天（PBM）或第7天（U937），当150-200 nM/10（6）细胞的可溶性亚硝酸盐在培养物中积累时，如通过DAN测量的。此外，单核细胞染色阳性的人iNOS蛋白和过氧亚硝酸盐感染结核杆菌后。MTB对NO的诱导作用可被包括N-单甲基精氨酸在内的四种不同的iNOS酶抑制剂所抑制。NO的抑制导致MTB的五个临床分离株中的两个的细胞内生长的增强。从供体（S-亚硝基-N-青霉胺）释放的NO也对肉汤培养物中的相同菌株具有直接的抑菌作用。因此，MTB菌株显示出对细胞内和细胞外NO的不同敏感性。在大多数这些测定中，Greiss试剂受到其灵敏度的限制，并且在整个7-10天的孵育期间对可溶性亚硝酸盐保持阴性。因此，广泛使用的比色法在NO测定中可能给出假阴性结果。该报告还首次证明了MTB在人单核细胞/巨噬细胞培养物中诱导iNOS、iNOS蛋白、NO和过氧亚硝酸盐的mRNA，（C）1998 Academic Press。
The induction of nitric oxide in human monocytes during mycobacterial infection has been a controversial issue. This study describes a comparative evaluation of the colorimetric and fluorometric methods for the detection of NO in response to Mycobacterium tuberculosis (MTB) infection in human peripheral blood-derived monocytes (PBM) and in U937, a human monocyte-derived cell line. MTB was grown in monocyte cultures in vitro for 7 or 10 days. RPMI 1640 medium, without antibiotics and supplemented with L-arginine, Hepes, 5% human AB serum, and tetrahydrobiopterin was used to support monocyte growth. As early as 72 h after infection, soluble nitrite was detectable in the medium using the fluorometric assay with diaminonaphthalene (DAN), Early induction of NO correlated with an increase in the levels of iNOS mRNA as quantitated by RT-PCR, NO levels increased progressively up to day 10 (PBM) or day 7 (U937), when 150-200 nM/10(6) cells of soluble nitrite accumulated in cultures, as measured by DAN. Furthermore, monocytes stained positively for human iNOS protein and peroxynitrite after infection with MTB. The induction of NO by MTB was inhibited by four different inhibitors of iNOS enzyme including N-monomethylarginine. Inhibition of NO resulted in the enhancement of the intracellular growth of two of five clinical isolates of MTB. NO released from a donor (S-nitroso-N-penicillamine) also had a direct bacteriostatic effect on the same isolates in broth cultures. MTB strains thus showed a differential susceptibility to intracellular and extracellular NO, In most of these assays, the Greiss reagent was limited by its sensitivity and remained negative for soluble nitrite throughout the 7-10 days of incubation. Thus, the colorimetric method, which is widely used, may give false-negative results in NO assays. This report also demonstrates for the first time that MTB induces mRNA for iNOS, iNOS protein, NO, and peroxynitrite in human monocyte/macrophage cultures, (C) 1998 Academic Press.