Characterization of the Effects of Recombinant Human Int

重组人类基因效应的表征

• 批准号: 6679779
• 负责人: R. P DONNELLY
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6679779
• 关键词: CD3 molecule; RNase protection assay; T lymphocyte; cell population study; cytokine; cytokine receptors; gene expression; genetic regulation; human tissue; immunogenetics; immunoregulation; interferon gamma; interleukin 1; interleukin 10; interleukin 12; interleukin 13; interleukin 2; interleukin 4; leukocyte activation /transformation; protein structure function; recombinant proteins; tumor necrosis factor alpha

Summary: Interleukin-12 (IL-12) is known to upregulate production of interferon-gamma (IFN-g) by activated T cells; however, the effects of IL-12 on expression of other cytokines are less well defined. In this project, we are examining the effects of recombinant human IL-12 (rHuIL-12) on production of multiple cytokines, including IFN-g, IL-2, IL-4, IL-10 and IL-13, by purified normal human CD3+ T cells. Although resting T cells are largely nonresponsive to IL-12, anti-CD3-activated T cell blasts are strongly responsive, as demonstrated by the ability of IL-12 to induce STAT4-mediated DNA-binding activity. We found that activation of purified human T lymphocytes on immobilized anti-CD3 mAb induces rapid expression of TNF-alpha and IL-2 mRNA, and more gradual increases in mRNA levels for IFN-g and IL-10. Changes in mRNA expression levels were measured by RNase protection assay using primer template sets that enable simultaneous analysis of multiple cytokine genes. We found that IL-12 markedly upregulates expression of IFN-g and IL-10, and down-regulates production of IL-2. Inhibition of IL-2 production by IL-12 correlates directly with increased production of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production in IL-12-treated T cells, demonstrating that the inhibitory effects of IL-12 are IL-10-mediated. Thus, we have found that IL-12 simultaneously upregulates production of IFN-g and IL-10, and, by a direct IL-10-dependent pathway, feedback inhibits production of IL-2. The fact that IL-12 differentially regulates synthesis of IFN-g and IL-2 in T cells demonstrates that IL-12 does not globally enhance expression of all Th1-type lymphokines. Furthermore, the ability of IL-12 to upregulate production of IL-10 provides a mechanism for limiting the IL-2-dependent clonal expansion of activated T cells, and defines a novel cytokine regulatory pathway that is inducible by IL-12. In future studies, we plan to further define the effects of IL-12 on production of other cytokines, particularly the Th2-type cytokines IL-4 and IL-13 by activated T cells, and to compare the effects of IL-12 with that of other macrophage-derived immunoregulatory cytokines, including IL-1, IL-18 and TNF. We will also further explore the functional consequences of increased IL-10 production in IL-12-treated T cells. The results of these studies will expand our current understanding of the actions of IL-12 on immune effector cells. This information may be useful in interpreting any therapeutic activity induced by IL-12 in clinical trials that are now underway. It may also provide insight to the physiological basis for the systemic toxicity associated with high dose IL-12 therapy in cancer patients. Furthermore, analysis of the effects of IL-12 on cytokine and cytokine receptor gene expression in vitro may facilitate identification of useful markers of IL-12 bioactivity that could be useful in monitoring the potency of recombinant IL-12 in human recipients.

总结：已知白细胞介素-12（IL-12）通过活化的T细胞上调干扰素-γ（IFN-g）的产生;然而，IL-12对其他细胞因子表达的影响不太明确。 本课题研究了重组人IL-12（rHuIL-12）对纯化的正常人CD 3 + T细胞产生多种细胞因子（包括IFN-γ、IL-2、IL-4、IL-10和IL-13）的影响。 尽管静息T细胞在很大程度上对IL-12无应答，但抗CD 3激活的T细胞母细胞具有强烈的应答性，如IL-12诱导STAT 4介导的DNA结合活性的能力所证明的。 我们发现，活化纯化的人T淋巴细胞上固定的抗CD 3单克隆抗体诱导TNF-α和IL-2 mRNA的快速表达，以及IFN-γ和IL-10 mRNA水平的逐渐增加。 mRNA表达水平的变化通过使用引物模板组的RNA酶保护测定来测量，所述引物模板组能够同时分析多种细胞因子基因。 我们发现IL-12显著上调IFN-γ和IL-10的表达，并下调IL-2的产生。 IL-12对IL-2产生的抑制与IL-10产生的增加直接相关。 此外，用抗IL-10抗体中和IL-10活性使IL-12处理的T细胞中的IL-2产生正常化，表明IL-12的抑制作用是IL-10介导的。 因此，我们发现IL-12同时上调IFN-γ和IL-10的产生，并且通过直接的IL-10依赖性途径，反馈抑制IL-2的产生。 IL-12差异调节T细胞中IFN-γ和IL-2的合成的事实证明IL-12并不全面增强所有Th 1型淋巴因子的表达。 此外，IL-12上调IL-10产生的能力提供了限制活化T细胞的IL-2依赖性克隆扩增的机制，并定义了可由IL-12诱导的新型细胞因子调节途径。 在未来的研究中，我们计划进一步确定IL-12对其他细胞因子，特别是活化T细胞产生的Th 2型细胞因子IL-4和IL-13的影响，并比较IL-12与其他巨噬细胞衍生的免疫调节细胞因子，包括IL-1，IL-18和TNF的作用。 我们还将进一步探索IL-12处理的T细胞中IL-10产生增加的功能后果。 这些研究的结果将扩大我们目前对IL-12对免疫效应细胞作用的理解。 这些信息可能有助于解释目前正在进行的临床试验中IL-12诱导的任何治疗活性。 它还可以提供对癌症患者中与高剂量IL-12治疗相关的全身毒性的生理基础的了解。 此外，IL-12对细胞因子和细胞因子受体基因表达的影响的体外分析可能有助于鉴定IL-12生物活性的有用标志物，其可用于监测重组IL-12在人受体中的效力。