REGULATION OF GENE EXPRESSION BY INTERLEUKIN10 IN ENDOTOXIN-STIMULATED HUMAN MONO

细菌内毒素(LPS)刺激人单核细胞可诱导多种细胞因子的表达，包括肿瘤坏死因子(TNF)、白介素1(IL-1)、白介素6(IL-6)和白介素10。IL-10的表达相对于肿瘤坏死因子、IL-1和IL-6的表达延迟。此外，IL-10反馈抑制了肿瘤坏死因子、IL-1和IL-6的表达，从而为控制单核细胞促炎细胞因子的产生提供了有效的自分泌机制。在这个项目中，我们正在研究IL-10下调内毒素刺激的单核细胞产生肿瘤坏死因子和IL-1等细胞因子的机制。我们还在评估IL-10对单核细胞中由细胞因子如干扰素-γ和IL-4激活的信号转导事件的影响。我们已经发现IL-10抑制IL-4和干扰素-γ诱导的激活和基因表达(DickenSheets&Donnelly(1997)J.159：6226)。我们还确定，IL-10抑制IL-4诱导的基因表达的能力是由于IL-4诱导的转录因子STAT6的酪氨酸磷酸化和核转位减少的结果(DickenSheets&Donnelly(1999)J.Leukoc。比奥尔。65：307)。我们现在正在研究一个新的JAK/STAT抑制基因家族(SoCS基因)在介导这些IL-10诱导的抑制效应中的作用。为了进一步明确IL-10对单核细胞功能活动的作用，我们还检测了该细胞因子对某些可溶性细胞因子受体的合成和释放的影响，特别是IL-1和IL-1受体(IL-1RI和IL-1RII)以及1型和2型肿瘤坏死因子受体。单核细胞在受到内毒素刺激后释放出肿瘤坏死因子受体，通过与膜相关的肿瘤坏死因子受体竞争有效的肿瘤坏死因子受体，发挥肿瘤坏死因子拮抗剂的作用。我们已经发现，干扰素-g下调脂多糖刺激的单核细胞膜上肿瘤坏死因子-R2和可溶性肿瘤坏死因子-R2(sTNF-R2)的表达。(1997)血液90：4162)。经干扰素-g处理的单核细胞培养上清液中sTNF-R2的减少与其表达水平呈正相关，与其表达水平呈负相关。相反，IL-10上调sTNF-R2的产生，并显著抑制TNF-a的产生。IL-10还可拮抗干扰素-g抑制sTNF-R2的产生和增强TNF-a的产生。这些结果表明，IL-10协同下调单核细胞产生肿瘤坏死因子-α(一种肿瘤坏死因子受体激动剂)，并上调单核细胞产生s肿瘤坏死因子-R2(一种肿瘤坏死因子受体拮抗剂)。重组人IL-10目前正在作为一种潜在的治疗剂进行测试，用于治疗某些炎症性疾病，包括类风湿性关节炎和克罗恩病。我们的研究结果将增加我们对IL-10生物学作用的了解，从而提高该机构规范这种生物制剂临床使用的能力。

Stimulation of human monocytes with bacterial endotoxin, lipopolysaccharide (LPS), induces expression of multiple cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and IL-10. IL-10 expression is delayed relative to that of TNF, IL-1 and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF, IL-1 and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. In this project, we are examining the mechanism by which IL-10 down-regulates production of cytokines such as TNF and IL-1 in endotoxin-stimulated monocytes. We are also evaluating the effects of IL-10 on signal transduction events that are activated by cytokines such as IFN-gamma (IFN-g) and IL-4 in monocytes. We have found that IL-10 inhibits activation and gene expression induced by IL-4 and IFN-gamma (Dickensheets & Donnelly (1997) J. Immunol. 159:6226). We have also determined that the ability of IL-10 to inhibit IL-4-inducible gene expression is a consequence of decreased tyrosine phosphorylation and nuclear translocation of the IL-4-inducible transcription factor, STAT6 (Dickensheets & Donnelly (1999) J. Leukoc. Biol. 65:307). We are now examining the role of a novel family of JAK/STAT inhibitory genes (the SOCS genes) in mediating these IL-10-inducible inhibitory effects. To further define the actions of IL-10 on monocyte functional activities, we also examined the effects of this cytokine on synthesis and release of certain soluble cytokine receptors, particularly the type-I and type-II IL-1 receptors (IL-1RI and IL-1RII) and the type-1 and type-2 TNF receptors. TNF-Rs are shed from monocytes after stimulation by LPS, and can function as TNF antagonists by competing with membrane-associated TNF-R for available TNF. We have found that IFN-g down-regulates expression of both membrane TNF-R2 and soluble TNF-R2 (sTNF-R2) by LPS-stimulated monocytes (Dickensheets et al. (1997) Blood 90:4162). The decreased production of sTNF-R2 in cultures of IFN-g-treated monocytes correlated directly with decreased levels of TNF-R2 mRNA and inversely with the levels of TNF-a mRNA. In contrast, IL-10 up-regulated production of sTNF-R2 and markedly inhibited production of TNF-a. IL-10 also antagonized the ability of IFN-g to suppress production of sTNF-R2 and to potentiate production of TNF-a. These findings demonstrate that IL-10 coordinately down-regulates production of TNF-a (a TNF-R agonist), and up-regulates production of sTNF-R2 (a TNF-R antagonist) in monocytes. Recombinant human IL-10 is currently being tested as a potential therapeutic agent for the treatment of certain inflammatory diseases, including rheumatoid arthritis and Crohn's disease. The results of our studies will increase our knowledge of the biological actions of IL-10, and thereby improve the agency's ability to regulate the clinical use of this biologic agent.