García J E, López A M, de Cabo M R, Rodríguez F M, Losada J P, Sarmiento R G, López A J, Arellano J L
Area de Medicina, Fundación Hospital de Alcorcón, Madrid, Spain.
Mediators Inflamm. 1999;8(4-5):253-9. doi: 10.1080/09629359990423.
In addition to its well-established effect on T cells, cyclosporin A (CsA) also inhibits inflammatory cytokine production by macrophages. However, little is known about the mechanism of action of CsA on macrophage cytokine production. We measured the effect of CsA on basal and phorbol-myristate-acetate (PMA)-stimulated production of interleukin-6 using the human monocyte cell line U937 differentiated with dimethylsulfoxide (DMSO). Interleukin-6 levels were measured in supernatant and cell lysates using specific enzyme-linked immunosorbent assays. We found that CsA decreases not only IL-6 release but also cytokine synthesis. The concentration of CsA used did not affect either cell viability or proliferation. Three possibilities may be advanced to explain the CsA-due decrease in IL-6 production by macrophages: (a) inhibition of the synthesis of an early common regulatory protein, (b) inhibition of cytokine gene transcription, or (c) modulation of post-transcriptional events. The first possibility was tested by measuring the effect of cycloheximide on the experimental system during the first 3 hours of culture. Although cycloheximide decreased total cytokine synthesis, the pattern of cytokine modulation by CsA persisted. These data suggest that CsA-mediated macrophage cytokine inhibition is not mediated by an early common regulatory protein. To further explore the inhibition mechanism, we measured IL-6 mRNA levels by Northern blot. IL-6 mRNA levels were unaffected by CsA both in resting and PMA-stimulated cells. We conclude that in human macrophages CsA diminishes IL-6 production at post-transcriptional level.
除了对T细胞有公认的作用外，环孢素A（CsA）还能抑制巨噬细胞产生炎性细胞因子。然而，关于CsA对巨噬细胞细胞因子产生的作用机制知之甚少。我们使用经二甲基亚砜（DMSO）分化的人单核细胞系U937，测量了CsA对基础状态和佛波酯-肉豆蔻酸酯-乙酸酯（PMA）刺激的白细胞介素-6产生的影响。使用特异性酶联免疫吸附测定法测量上清液和细胞裂解物中的白细胞介素-6水平。我们发现CsA不仅降低白细胞介素-6的释放，还降低细胞因子的合成。所用CsA的浓度既不影响细胞活力也不影响细胞增殖。可以提出三种可能性来解释CsA导致巨噬细胞产生白细胞介素-6减少的现象：（a）抑制早期共同调节蛋白的合成，（b）抑制细胞因子基因转录，或（c）调节转录后事件。通过在培养的前3小时测量放线菌酮对实验系统的影响来检验第一种可能性。尽管放线菌酮降低了总的细胞因子合成，但CsA对细胞因子的调节模式仍然存在。这些数据表明，CsA介导的巨噬细胞细胞因子抑制作用不是由早期共同调节蛋白介导的。为了进一步探索抑制机制，我们通过Northern印迹法测量白细胞介素-6 mRNA水平。在静息和PMA刺激的细胞中，白细胞介素-6 mRNA水平均不受CsA影响。我们得出结论，在人类巨噬细胞中，CsA在转录后水平降低白细胞介素-6的产生。
