Saito S, Nakano M
Department of Microbiology, Jichi Medical School, Tochigi-ken, Japan.
J Leukoc Biol. 1996 Jun;59(6):908-15. doi: 10.1002/jlb.59.6.908.
Infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) confers mice with strong abilities to produce nitric oxide (NO) and cytokines. Because the peritoneal macrophages taken from the mice immunized with live or heat-killed BCG can produce NO without any accessory cells and stimulants, it is difficult to clarify the immune regulation on NO production by manipulating the macrophages. Therefore, we investigated the participation of immune T cells and cytokines in NO production by using in vitro co-cultures of macrophages from non-immune mice with T cells prepared from BCG-infected mice in the presence or absence of a mycobacterial antigen, purified protein derivative (PPD). Although the non-immune thioglycollate (TGB)-elicited macrophages could not produce any detectable NO in the presence of PPD, supplementation of the macrophage cultures with CD4+ T cells prepared from BCG-infected mice enabled the macrophages to produce NO. Immunocytostaining showed that the macrophages, hut not the immune T cells, expressed inducible NO synthase (iNOS), indicating that they were NO producers. PPD could only induce NO production if there was cell-cell contact of the CD4+ T cells in the immune cells and antigen-presenting macrophages were required for the NO production in response to PPD; this interaction led to the production of soluble mediators that induced NO production by the TGB macrophages. NO production by the co-cultured cells was abrogated by adding either anti-interferon-gamma(IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibody. Furthermore, the roles of immune T cells and PPD could be replaced by adding recombinant IFN-gamma together with TNF-alpha to the macrophage cultures, but neither alone was sufficient to induce NO production by the macrophages. Our present data indicate that TNF-alpha produced by PPD-stimulated macrophages and IFN-gamma produced by cell-cell interaction of BCG-immune T cells and antigen-engulfed macrophages together activate the macrophages to produce NO.
用卡介苗（BCG）感染牛分枝杆菌可使小鼠产生一氧化氮（NO）和细胞因子的能力增强。由于从用活的或热灭活的BCG免疫的小鼠中获取的腹膜巨噬细胞在没有任何辅助细胞和刺激物的情况下就能产生NO，因此很难通过操纵巨噬细胞来阐明对NO产生的免疫调节。因此，我们通过在有或没有分枝杆菌抗原纯化蛋白衍生物（PPD）存在的情况下，将非免疫小鼠的巨噬细胞与从BCG感染小鼠制备的T细胞进行体外共培养，来研究免疫T细胞和细胞因子在NO产生中的作用。尽管在PPD存在的情况下，非免疫的巯基乙酸盐（TGB）诱导的巨噬细胞不能产生任何可检测到的NO，但用从BCG感染小鼠制备的CD4 + T细胞补充巨噬细胞培养物能使巨噬细胞产生NO。免疫细胞化学染色显示，巨噬细胞而非免疫T细胞表达诱导型NO合酶（iNOS），表明它们是NO的产生者。只有当免疫细胞中的CD4 + T细胞发生细胞间接触且抗原呈递巨噬细胞参与对PPD的反应时，PPD才能诱导NO产生；这种相互作用导致产生可溶性介质，进而诱导TGB巨噬细胞产生NO。通过添加抗干扰素-γ（IFN-γ）或抗肿瘤坏死因子-α（TNF-α）抗体，可消除共培养细胞产生的NO。此外，通过向巨噬细胞培养物中添加重组IFN-γ和TNF-α，可替代免疫T细胞和PPD的作用，但单独添加任何一种都不足以诱导巨噬细胞产生NO。我们目前的数据表明，PPD刺激的巨噬细胞产生的TNF-α以及BCG免疫T细胞与吞噬抗原的巨噬细胞之间的细胞间相互作用产生的IFN-γ共同激活巨噬细胞以产生NO。
