Pelletier C, Guérin-Marchand C, Iannascoli B, Marchand F, David B, Weyer A, Blank U
Institut Pasteur, Unité d'Immuno-Allergie, Paris, France.
Inflamm Res. 1998 Dec;47(12):493-500. doi: 10.1007/s000110050364.
In the present study, we investigated signal transduction pathways involved in TNF-alpha gene expression and TNF-alpha secretion by mast cells stimulated through the high affinity IgE receptor (FcepsilonRI).
TNF-alpha mRNA steady state levels and TNF-alpha secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcepsilonRI. Relative amounts of TNF-alpha mRNA versus beta-actin levels were quantified by RNase protection and RT-PCR assays. TNF-alpha secretion was measured by a current ELISA test.
We show that EGTA (5 mM) prevented TNF-alpha mRNA expression and TNF-alpha secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-alpha secretion at 2 microM but had only a marginal effect on TNF-alpha mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-alpha secretion at low doses (EC50= 13 nM), but only partially affected mRNA expression.
Our results show that in FcepsilonRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-alpha secretion while for the increased TNF-alpha mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.
在本研究中，我们调查了通过高亲和力IgE受体（FcepsilonRI）刺激肥大细胞时，参与肿瘤坏死因子-α（TNF-α）基因表达和TNF-α分泌的信号转导途径。
使用通过FcepsilonRI刺激的大鼠嗜碱性白血病细胞（RBL-2H3），监测在存在特定药理剂的情况下TNF-α mRNA的稳态水平和TNF-α分泌。通过核糖核酸酶保护和逆转录-聚合酶链反应（RT-PCR）测定法对TNF-α mRNA与β-肌动蛋白水平的相对量进行定量。通过电流酶联免疫吸附测定（ELISA）试验测量TNF-α分泌。
我们发现乙二醇双四乙酸（EGTA，5 mM）可阻止抗原刺激细胞中TNF-α mRNA的表达和TNF-α分泌。蛋白激酶C（PKC）抑制剂双吲哚基马来酰亚胺I在2 microM时可显著阻断TNF-α分泌，但对TNF-α mRNA表达仅有轻微影响。当长期暴露于100 nM佛波酯（PMA）使PKC亚型耗竭时，结果相似。磷脂酰肌醇3-激酶（PI 3-激酶）抑制剂渥曼青霉素在低剂量时（半数有效浓度[EC50]= 13 nM）可阻断TNF-α分泌，但仅部分影响mRNA表达。
我们的结果表明，在FcepsilonRI刺激的RBL-2H3细胞中，钙动员、PKC和PI 3-激酶的激活对于TNF-α分泌是必需的，而对于TNF-α mRNA表达增加，PKC活性是可有可无的，PI 3-激酶活性仅部分需要。
