Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.
Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.
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DOI：
10.1182/blood.v86.3.1184.bloodjournal8631184
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发表时间：
1995-08
期刊：
影响因子：
20.3
通讯作者：
J. Weinberg;M. Misukonis;P. Shami;S. Mason;D. Sauls;W. Dittman;ER Wood;GK Smith;B. McDonald;K. Bachus
中科院分区：
文献类型：
--
作者：
J. Weinberg;M. Misukonis;P. Shami;S. Mason;D. Sauls;W. Dittman;ER Wood;GK Smith;B. McDonald;K. Bachus
一氧化氮（NO）由许多不同的细胞类型产生，并且它是许多过程的重要调节剂和介质，包括平滑肌松弛、神经传递和鼠巨噬细胞介导的对微生物和肿瘤细胞的细胞毒性。尽管小鼠巨噬细胞在活化后容易产生NO，但已报道人单核细胞和组织巨噬细胞在体外仅产生低水平的NO。本研究的目的是确定是否刺激人单核吞噬细胞产生诱导型一氧化氮合酶（iNOS）的mRNA，蛋白质和酶活性。通过逆转录-聚合酶链反应（RT-PCR）分析，我们表明，人单核细胞可以诱导表达iNOS mRNA后处理与脂多糖（LPS）和/或干扰素-γ（IFN-γ）。通过免疫荧光和免疫印迹分析，我们发现单核细胞和腹腔巨噬细胞含有可检测水平的诱导型一氧化氮合酶抗原与细胞因子在体外刺激后。对照单核细胞或与LPS和/或各种细胞因子一起培养的那些单核细胞具有低水平的NOS功能活性，如通过细胞提取物将L-精氨酸转化为L-瓜氨酸的能力所测量的，并且它们产生低水平的NO催化剂亚硝酸盐和硝酸盐。腹膜巨噬细胞在用LPS和/或IFN-γ处理后具有显著增强的亚硝酸盐/硝酸盐产生和NOS活性，而单核细胞亚硝酸盐/硝酸盐产生和NOS活性不被处理改变。与各种活的或热灭活的细菌、真菌或人类免疫缺陷病毒（HIV）-1一起培养的单核细胞不会产生高水平的亚硝酸盐/硝酸盐。针对转化生长因子-β（TGF-β）的抗体（一种已知在小鼠巨噬细胞中抑制iNOS表达和NO产生的因子）不增强人单核细胞或巨噬细胞中的NO产生。在新鲜分离或培养的人单核细胞和腹腔巨噬细胞中检测不到生物蝶呤（iNOS酶活性的专性辅因子）。然而，通过与细胞可渗透的、无毒的前体sepiapterin一起培养来补充细胞内四氢生物蝶呤水平并不能增强人单核吞噬细胞在体外产生NO的能力。混合实验表明，在人单核吞噬细胞中没有功能性NOS抑制剂的证据。因此，我们证明，人单核吞噬细胞可以产生iNOS mRNA和蛋白质，和（尽管如此）他们的能力，产生NO是非常低的。
Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage-mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell-permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.