内源性产生的一氧化氮可增加转染的人U937细胞中肿瘤坏死因子-α的产生。

Endogenously produced nitric oxide increases tumor necrosis factor-alpha production in transfected human U937 cells.

作者信息

Yan L, Wang S, Rafferty S P, Wesley R A, Danner R L

机构信息

Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.

出版信息

Blood. 1997 Aug 1;90(3):1160-7.

PMID:9242548

Abstract

Various functions of human phagocytes are modulated by nitric oxide (NO). We transfected the human U937 monoblastoid cell line with an expression vector containing human endothelial NO synthase (eNOS) or murine inducible NOS (iNOS) cDNA to study the regulatory role of NO without the nonspecific effects associated with exogenous NO sources. Western blot confirmed expression of eNOS or iNOS in respectively transfected cells, but not in naive or empty-vector transfected cells. Transfectants expressing iNOS, a calcium-independent enzyme, but not eNOS, a calcium-dependent enzyme, spontaneously produced NO (P < .001). The NO release from iNOS-transfected cells, as measured by nitrite and nitrate accumulation and by cyclic guanosine monophosphate (cGMP) increases in rat reporter cells, was inhibitable (P < .01 for both) with N(omega)-methyl-L-arginine (L-NMA), a NOS inhibitor. The eNOS transfectants were shown to contain functional enzyme by the conversion of L-arginine to L-citrulline in fractionated cells (P = .0001) and by exposing intact cells to calcium ionophore using the cGMP reporter cell assay (P = .0001). After differentiation with phorbol-12-myristate-13-acetate (PMA), iNOS transfectants produced more tumor necrosis factor-alpha (TNF-alpha) (124.9 +/- 25.4 pg/5 x 10(5) cells per 24 hours) than did empty-vector transfected cells (21.9 +/- 1.9 pg/5 x 10(5) cells per 24 hours; P = .02). This effect was inhibited by 500 micromol/L L-NMA (54.4 +/- 3.1 pg/5 x 10(5) cells per 24 hours; P = .05). However, in the presence of high concentrations of lipopolysaccharide (1 microg/mL), which further increased NO production in iNOS transfected cells (P = .044), TNF-alpha production was similar comparing PMA-differentiated iNOS and empty-vector transfectants (12.2 +/- 0.8 and 13.1 +/- 1.7 ng/5 x 10(5) cells per 24 hours, respectively; P = .5). The results show that under certain conditions endogenously produced NO can upregulate TNF-alpha production in human phagocytes.

摘要

一氧化氮（NO）可调节人类吞噬细胞的多种功能。我们用含有人类内皮型一氧化氮合酶（eNOS）或小鼠诱导型一氧化氮合酶（iNOS）cDNA的表达载体转染人类U937单核细胞系，以研究NO的调节作用，而不涉及外源性NO来源的非特异性影响。蛋白质免疫印迹法证实分别转染的细胞中eNOS或iNOS表达，但未转染的细胞或空载体转染的细胞中未表达。表达iNOS（一种不依赖钙的酶）而非eNOS（一种依赖钙的酶）的转染细胞自发产生NO（P <.001）。通过亚硝酸盐和硝酸盐积累以及大鼠报告细胞中环磷酸鸟苷（cGMP）增加来测量，iNOS转染细胞释放的NO可被NOS抑制剂N（ω）-甲基-L-精氨酸（L-NMA）抑制（两者P均 <.01）。通过在分离的细胞中将L-精氨酸转化为L-瓜氨酸（P =.0001）以及使用cGMP报告细胞测定法将完整细胞暴露于钙离子载体（P =.0001），显示eNOS转染细胞含有功能性酶。在用佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）诱导分化后，iNOS转染细胞产生的肿瘤坏死因子-α（TNF-α）（每24小时124.9±25.4 pg/5×10⁵个细胞）比空载体转染细胞（每24小时21.9±1.9 pg/5×10⁵个细胞；P =.02）更多。这种作用被500 μmol/L L-NMA抑制（每24小时54.4±3.1 pg/5×10⁵个细胞；P =.05）。然而，在高浓度脂多糖（1 μg/mL）存在下，脂多糖进一步增加了iNOS转染细胞中NO的产生（P =.044），比较PMA诱导分化的iNOS和空载体转染细胞，TNF-α的产生相似（每24小时分别为12.2±0.8和13.1±1.7 ng/5×10⁵个细胞；P =.5）。结果表明，在某些条件下，内源性产生的NO可上调人类吞噬细胞中TNF-α的产生。