EFFECTS OF RECOMBINANT IL12 ON CYTOKINE GENE EXPESSION

已知白介素12(IL-12)可上调活化的T细胞产生干扰素-γ(IFN-g)；然而，IL-12对其他细胞因子表达的影响还不是很清楚。在本项目中，我们检测了重组人IL-12(rHuIL-12)对正常人CD3+T细胞产生多种细胞因子的影响，包括干扰素-g、IL-2、IL-4、IL-10和IL-13。尽管静息T细胞对IL-12基本无反应，但抗CD3激活的T细胞对IL-12有很强的反应，IL-12诱导STAT4介导的DNA结合活性的能力证明了这一点。我们发现，纯化的人T淋巴细胞在固定化的抗CD3单抗上激活后，可以快速表达肿瘤坏死因子-α和白介素2，并逐渐增加干扰素-g和白介素10的表达水平。通过使用能够同时分析多个细胞因子基因的引物模板集的核糖核酸酶保护试验来测量mRNA表达水平的变化。我们发现，IL-12显著上调干扰素-g和IL-10的表达，下调IL-2的产生。IL-12抑制IL-2的产生与IL-10的增加直接相关。此外，IL-10活性与抗IL-10抗体的中和使IL-12处理的T细胞中IL-2的产生正常化，表明IL-12的抑制作用是由IL-10介导的。因此，我们发现，IL-12同时上调干扰素-g和IL-10的产生，并通过直接依赖于IL-10的途径，反馈抑制IL-2的产生。IL-12对T细胞中干扰素-g和IL-2的合成有不同的调节作用，这一事实表明，IL-12并不能全面增强所有Th1型淋巴因子的表达。此外，IL-12上调IL-10产生的能力为限制IL-2依赖的T细胞克隆性增殖提供了一种机制，并定义了一种新的可被IL-12诱导的细胞因子调节途径。在未来的研究中，我们计划进一步确定IL-12对激活的T细胞产生其他细胞因子，特别是Th2型细胞因子IL-4和IL-13的影响，并比较IL-12与其他巨噬细胞来源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诱导的任何治疗活动。这也可能为大剂量IL-12治疗癌症患者的全身毒性提供生理学基础的洞察。此外，分析IL-12在体外对细胞因子和细胞因子受体基因表达的影响，可能有助于寻找有用的IL-12生物活性标志物，有助于监测重组IL-12在人类受体中的效力。

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.