Molecular mechanisms in down-regulation of tumor necrosis factor expression.

Excessive production of tumor necrosis factor (TNF) after stimulation by lipopolysaccharide (LPS) may result in fever, intravascular coagulation, and lethal shock. An efficient way of preventing the excessive TNF production is desensitization of monocytes/macrophages to LPS. We have analyzed the molecular mechanisms involved in the induction of desensitization and the mechanisms operative in the desensitized, LPS-refractory cells by employing the human monocytic cell line Mono-Mac-6. Similar to human blood monocytes, treatment of Mono-Mac-6 cells with LPS (1 microgram/ml) results in a rapid and transient expression of TNF. When Mono-Mac-6 cells are precultured in medium containing low levels of LPS, they become refractory to subsequent LPS stimulation and show no or little secretion of TNF protein. Desensitization can be blocked by the inhibition of cyclooxygenase and protein kinase C; both prostaglandin E2 (together with a second signal) and phorbol 12-myristate 13-acetate can mimic desensitization. By employing prostaglandin E2 and low concentrations of phorbol 12-myristate 13-acetate, a synergism in the induction of desensitization can be demonstrated. Hence, our studies show that two distinct pathways are involved in the induction of hyporesponsiveness. In both LPS-responsive and LPS-desensitized Mono-Mac-6 cells, LPS was able to induce the transcription factor NF-kappa B in the nucleus. Still, the prevalence of TNF-specific mRNA was dramatically reduced in the desensitized cells. These data indicate that LPS-desensitized Mono-Mac-6 cells are able to activate initial steps of signal transduction up to the level of the NF-kappa B transcription factor. The absence of TNF transcripts, however, indicates that additional nuclear factors may be missing or that silencers may be active such that transcription of the TNF gene is prevented. IMAGES:

脂多糖（lipopolysaccharide, LPS）刺激后过量产生的肿瘤坏死因子（tumor necrosis factor, TNF）可引发发热、血管内凝血及致死性休克。预防TNF过量产生的有效策略之一，是使单核细胞/巨噬细胞（monocytes/macrophages）对LPS产生脱敏现象。本研究以人单核细胞系Mono-Mac-6为模型，分析了脱敏诱导的分子机制，以及脱敏后LPS不应性细胞内的调控机制。与人体血液单核细胞类似，以1 μg/mL LPS处理Mono-Mac-6细胞后，可快速且一过性地诱导TNF表达。若将Mono-Mac-6细胞于含低浓度LPS的培养基中预培养，细胞会对后续LPS刺激产生不应性，几乎无法分泌或仅能检测到极少量TNF蛋白。脱敏过程可被环氧合酶（cyclooxygenase）与蛋白激酶C（protein kinase C）的抑制剂阻断；前列腺素E2（prostaglandin E2，需配合第二信号）与佛波醇12-肉豆蔻酸酯13-乙酸酯（phorbol 12-myristate 13-acetate）均可模拟脱敏效应。借助前列腺素E2与低浓度佛波醇12-肉豆蔻酸酯13-乙酸酯，可证实二者在脱敏诱导过程中存在协同作用。因此，本研究结果表明，低反应性的诱导涉及两条不同的信号通路。无论在LPS应答型还是LPS脱敏型Mono-Mac-6细胞中，LPS均可诱导细胞核内转录因子NF-κB（transcription factor NF-kappa B）的激活。但脱敏细胞中TNF特异性mRNA的丰度却显著降低。上述结果提示，LPS脱敏的Mono-Mac-6细胞可激活信号转导的初始步骤，直至转录因子NF-κB层面。然而TNF转录本的缺失表明，可能存在其他核因子的缺失，或是沉默子的激活，从而阻断了TNF基因的转录。图像：