Recombinant Human Interleukin 12 and Cytokine Expression

摘要：已知白细胞介素-12 （IL-12）可通过活化的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尚不明确。在这个项目中，我们正在研究重组人IL-12 （rHuIL-12）对纯化的正常人CD3+ T细胞产生多种细胞因子的影响，包括IFN-g、IL-2、IL-4、IL-10和IL-13。虽然静息T细胞对IL-12无应答，但抗cd3激活的T细胞母细胞对IL-12有强烈的应答，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，用固定的抗cd3单抗激活纯化的人T淋巴细胞，可诱导tnf - α和IL-2 mRNA的快速表达，IFN-g和IL-10 mRNA水平逐渐升高。通过RNase保护实验测量mRNA表达水平的变化，使用引物模板集，可以同时分析多个细胞因子基因。我们发现IL-12显著上调IFN-g和IL-10的表达，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IL-2的产生正常化，这表明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IFN-g和IL-10的产生，并且通过直接依赖IL-10的途径，反馈抑制IL-2的产生。IL-12在T细胞中调控IFN-g和IL-2合成的差异表明，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产生的能力为限制活化T细胞的il -2依赖性克隆扩增提供了一种机制，并定义了一种由IL-12诱导的新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对活化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，并将IL-12与其他巨噬细胞来源的免疫调节细胞因子IL-1、IL-18和TNF的作用进行比较。我们还将进一步探讨il -12处理的T细胞中IL-10产生增加的功能后果。这些研究结果将扩大我们目前对IL-12对免疫效应细胞作用的理解。这一信息可能有助于解释目前正在进行的临床试验中由IL-12诱导的任何治疗活性。它也可能为癌症患者高剂量IL-12治疗相关的全身毒性的生理基础提供见解。此外，分析IL-12对细胞因子和细胞因子受体基因在体外表达的影响可能有助于鉴定IL-12生物活性的有用标记物，这可能有助于监测重组IL-12在人受体中的效力。

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.