Olsen Nancy J, Spurlock Charles F, Aune Thomas M
Arthritis Res Ther. 2014 Jan 20;16(1):R17. doi: 10.1186/ar4444.
Methotrexate (MTX) has been for decades a standard treatment in a wide range of conditions, from malignancies to rheumatoid arthritis (RA). Despite this long experience, the mechanisms of action of MTX remain incompletely understood. Reported immunologic effects of MTX include induction of increased production of some cytokines, an effect that seems to be at odds with the generally anti-inflammatory effects of this drug in diseases like RA. To further elucidate these immune activities, we examined effects of MTX on the human monocytic cell line U937.
The U937 cell line was treated in vitro with pharmacologic-range concentrations of MTX and effects on production of interleukin (IL)-1, IL-6 and TNF alpha were measured. Changes in gene expression for IL-1 and IL-6 and specificities in the Jun-N-terminal kinase (JNK) signaling pathway including JNK 1, JNK2, JUN and FOS were also determined. The contribution of NF-kB, folate and adenosine pathways to the observed effects was determined by adding appropriate inhibitors to the MTX cultures.
MTX mediated a dose-dependent increase in IL-1 and IL-6 in U937 cells, as measured by secreted proteins and levels of gene expression. The increased cytokine expression was inhibited by addition of parthenolide and folinic acid, but not by caffeine and theophylline, suggesting that NF-kB and folates, but not adenosine, were involved in mediating the observed effects. When U937 cells were cultured with MTX, upregulated expression of JUN and FOS, but not JNK 1 or 2, also was observed.
MTX induces expression of proinflammatory cytokines in U937 monocytic cells. These effects might mediate the known toxicities of MTX including pneumonitis, mucositis and decreased bone mineral density.
几十年来，甲氨蝶呤（MTX）一直是从恶性肿瘤到类风湿性关节炎（RA）等多种病症的标准治疗药物。尽管有如此长期的应用经验，但MTX的作用机制仍未完全明确。据报道，MTX的免疫效应包括诱导某些细胞因子产生增加，而这种效应似乎与该药物在RA等疾病中通常的抗炎作用不一致。为了进一步阐明这些免疫活性，我们研究了MTX对人单核细胞系U937的影响。
用药理浓度范围的MTX体外处理U937细胞系，并检测其对白细胞介素（IL）-1、IL-6和肿瘤坏死因子α（TNFα）产生的影响。还确定了IL-1和IL-6基因表达的变化以及包括JNK 1、JNK2、JUN和FOS在内的Jun-N端激酶（JNK）信号通路的特异性。通过向MTX培养物中添加适当的抑制剂，确定NF-κB、叶酸和腺苷途径对观察到的效应的作用。
通过分泌蛋白和基因表达水平检测发现，MTX介导U937细胞中IL-1和IL-6呈剂量依赖性增加。添加小白菊内酯和亚叶酸可抑制细胞因子表达的增加，但咖啡因和茶碱则无此作用，这表明NF-κB和叶酸参与介导了观察到的效应，而腺苷未参与。当U937细胞与MTX一起培养时，还观察到JUN和FOS的表达上调，但JNK 1或JNK2未上调。
MTX诱导U937单核细胞中促炎细胞因子的表达。这些效应可能介导了MTX已知的毒性，包括肺炎、粘膜炎和骨矿物质密度降低。
