Kolios G, Brown Z, Robson R L, Robertson D A, Westwick J
Department of Pharmacology, University of Bath, Claverton Down.
Br J Pharmacol. 1995 Dec;116(7):2866-72. doi: 10.1111/j.1476-5381.1995.tb15938.x.
1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic-epithelial cell line HT-29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin-1 alpha (IL-1 alpha), tumour necrosisfactor-alpha (TNF-alpha), interferon gamma (IFN-gamma) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time-dependent increase in nitrite generation of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 micrograms ml-1) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL-1 alpha/IFN-gamma produced a highly significant (P < 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL-1 alpha/IFN-gamma combination revealed significant (P < 0.001) increases in nitrite at 24 h (153 +/- 7), 48 h (306 +/- 24), and 72 h (384 +/- 15) compared to basal values of 50 +/- 4, 75 +/- 8, and 103 +/- 8 nM per 10(6) cells respectively. 6 Studies with IL-1 alpha/IFN-gamma combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL-1 alpha (0.3-10 ng ml-1) and IFN-gamma (10-300 u ml-1) in combination induced a concentration-dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL-1 alpha/IFN-gamma stimulation reduced nitrite levels to basal values. These data suggest that the IL-1 alpha/IFN-gamma-induced nitrite production by HT-29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF-alpha produced a significant (P < 0.001) 3 fold increase of IL-1 alpha/IFN-gamma-induced nitrite generation. In marked contrast the presence of TNF-alpha had no effect on IL-1 alpha/IFN-gamma-induced iNOS mRNA expression by HT-29 cells. These findings suggest that the up-regulation by TNF-alpha of IL-1 alpha/IFN-gamma-induced nitrite generation is at the post-transcriptional level. 9 These data suggest that pro-inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
我们已经确定了哪些细胞因子调节人结肠上皮细胞系HT - 29中人类诱导型一氧化氮合酶（iNOS）mRNA的表达以及亚硝酸盐的生成。
对生长停滞的细胞培养物用人类重组细胞因子白细胞介素 - 1α（IL - 1α）、肿瘤坏死因子 - α（TNF - α）、干扰素γ（IFN - γ）进行刺激，或单独添加载体或联合添加。通过Northern印迹分析测定人类iNOS mRNA，使用荧光测定法测定亚硝酸盐的生成。
未刺激的细胞在24小时、48小时和72小时时，每10⁶个细胞的亚硝酸盐生成量分别有小的时间依赖性增加，分别为50±4、75±8和103±8 nM。这种亚硝酸盐生成不受环己酰亚胺（5微克/毫升）预处理的影响，且未检测到iNOS mRNA。
单独的细胞因子均未诱导iNOS mRNA表达或亚硝酸盐生成增加。与基础值相比，IL - 1α/IFN - γ组合在48小时时使亚硝酸盐生成量显著增加（P < 0.001），增加了4倍，而其他细胞因子对未产生效果。
对IL - 1α/IFN - γ组合进行的时间进程研究显示，与每10⁶个细胞分别为50±4、75±8和103±8 nM的基础值相比，在24小时（153±7）、48小时（306±24）和72小时（384±15）时亚硝酸盐显著增加（P < 0.001）。
对IL - 1α/IFN - γ组合的研究表明iNOS mRNA存在时间依赖性表达，首先在6小时观察到，在24小时达到峰值，72小时时未检测到。IL - 1α（0.3 - 10纳克/毫升）和IFN - γ（10 - 300单位/毫升）联合在24小时时诱导iNOS mRNA的浓度依赖性表达。
在IL - 1α/IFN - γ刺激前用环己酰亚胺预处理可将亚硝酸盐水平降低至基础值。这些数据表明，HT - 29细胞中IL - 1α/IFN - γ诱导的亚硝酸盐生成依赖于从头合成蛋白质，可能是iNOS酶。
添加TNF - α使IL - 1α/IFN - γ诱导的亚硝酸盐生成显著增加（P < 0.001），增加了3倍。与之形成鲜明对比的是，TNF - α的存在对HT - 29细胞中IL - α/IFN - γ诱导的iNOS mRNA表达没有影响。这些发现表明，TNF - α对IL - 1α/IFN - γ诱导的亚硝酸盐生成的上调作用是在转录后水平。
这些数据表明，促炎细胞因子可能通过诱导iNOS在结肠上皮细胞中诱导NO生成，并且这些细胞可能是炎症性肠病中NO生成的主要来源。
