Regulation of Monocyte Gene Expression

单核细胞基因表达的调控

• 批准号: 6839785
• 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/6839785
• 关键词: CD3 molecule; T lymphocyte; cytokine; gene expression; genetic regulation; interferon gamma; interleukin 1; interleukin 10; interleukin 12; interleukin 13; interleukin 2; interleukin 4; macrophage; messenger RNA; monocyte

Research in this laboratory is focused on defining how T cell-derived cytokines such as interferon-gamma (IFN-g), interleukin-4 (IL-4) and IL-10 regulate the expression of specific genes in human monocytes and monocyte-derived macrophages. In this context, we are examining the repertoire of genes that are induced in monocytes by treatment with these cytokines. Particular attention is directed at defining how these cytokines regulate expression of the SOCS genes (Suppressors of Cytokine Signaling) in monocytes and macrophages. The proteins encoded by the SOCS genes play a critical role in regulating cytokine signaling. The second major area of research in this laboratory is focused on characterizing the expression and function of several novel IL-10-related cytokines. These include IL-19, IL-20, IL-22, and IL-24 (MDA-7). At present, very little is known regarding the cell types that produce these cytokines or the stimuli that induce their expression. Even less is known about the cell types that respond to these cytokines or the genes that they induce. Therefore, we are conducting studies to define what cell types express these cytokines and what genes are induced by these ligands in cells that express functional receptors. Although these cytokines exhibit approximately 25% sequence homology with IL-10, the target cells that they act upon and the functional activities that they induce are distinct from those of IL-10. Characterization of the Effects of Recombinant Human Interleukin-12 on Cytokine Gene Expression in Human T Cells. 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 to 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 novel markers of IL-12 bioactivity that could be useful in monitoring the potency of recombinant IL-12 in human recipients. Identification and Characterization of Novel Interleukin-10-Related Genes. Activation of human monocytes by bacterial endotoxin, lipopolysaccharide (LPS), induces expression of many 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. We have found that IL-10 inhibits activation and gene expression induced by IL-4 and IFN-gamma. 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. We are now examining the role of a novel family of JAK/STAT inhibitory genes, the Suppressors of Cytokine Signaling (SOCS) genes, in mediating these IL-10-inducible inhibitory effects. We have found that IL-10 selectively induces expression of at least one member of this newly identified gene family: SOCS-3. Forced expression of SOCS-3 in a macrophage cell line markedly inhibits induction of STAT activity by several cytokines, including IFN-gamma, GM-CSF and IL-4. Therefore, the ability of IL-10 to antagonize cytokine-inducible gene expression is associated with its ability to induce rapid expression of the SOCS-3 gene. In related studies, we are also examining the biological activities of several newly identified IL-10 homologues. One of these cytokines, IL-19, shares approximately 21% amino acid homology with IL-10, but signals through a receptor complex that is distinct from that used by IL-10. We have found that expression of the IL-19 gene is up-regulated by many of the same agents that induce IL-10 gene expression in monocytes, including many Toll-like receptor (TLR) agonists. However, unlike the IL-10 gene, IL-19 is not expressed in activated T cells. Another IL-10 homologue, IL-22, was also recently identified by several groups. In collaboration with scientists at the University of Medicine and Dentistry of New Jersey (UMDNJ), we identified and characterized the gene (CRF2-9) that encodes the ligand-binding chain of the receptor for this novel cytokine. This receptor chain heterodimerizes with the IL-10R-beta chain (IL-10R2) to form the functional IL-22 receptor complex. Therefore, IL-10R2 is an essential component of both the IL-10 and IL-22 receptors. We have found that the IL-22 receptor is expressed at high levels in liver and kidney, but not in hematopoietic tissues such as the spleen and thymus. This suggests a possible functional role for this receptor in regulating gene expression in these tissues. This project incorporates FY2002 projects 1Z01BN002021-07 and 1Z01BN002022-09.

该实验室的研究重点是确定 T 细胞衍生的细胞因子，如干扰素-γ (IFN-g)、白介素-4 (IL-4) 和 IL-10 如何调节人单核细胞和单核细胞衍生的巨噬细胞中特定基因的表达。在这种情况下，我们正在检查通过这些细胞因子处理在单核细胞中诱导的基因库。 特别关注的是定义这些细胞因子如何调节单核细胞和巨噬细胞中 SOCS 基因（细胞因子信号传导抑制因子）的表达。 SOCS 基因编码的蛋白质在调节细胞因子信号传导中发挥着关键作用。 该实验室的第二个主要研究领域集中于表征几种新型 IL-10 相关细胞因子的表达和功能。 这些包括 IL-19、IL-20、IL-22 和 IL-24 (MDA-7)。 目前，对于产生这些细胞因子的细胞类型或诱导其表达的刺激物知之甚少。 关于对这些细胞因子或它们诱导的基因做出反应的细胞类型知之甚少。 因此，我们正在进行研究，以确定哪些细胞类型表达这些细胞因子，以及哪些基因在表达功能性受体的细胞中由这些配体诱导。 尽管这些细胞因子与 IL-10 表现出大约 25% 的序列同源性，但它们作用的靶细胞和它们诱导的功能活性与 IL-10 不同。 重组人白细胞介素 12 对人 T 细胞细胞因子基因表达的影响的表征。已知白细胞介素 12 (IL-12) 可以上调激活的 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尚不清楚。 在该项目中，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（包括 IFN-g、IL-2、IL-4、IL-10 和 IL-13）的影响。 尽管静息 T 细胞对 IL-12 基本上没有反应，但抗 CD3 激活的 T 细胞母细胞具有强烈反应，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，固定化抗 CD3 mAb 上纯化的人 T 淋巴细胞的活化诱导 TNF-α 和 IL-2 mRNA 的快速表达，以及 IFN-g 和 IL-10 mRNA 水平的逐渐增加。 使用引物模板集通过 RNase 保护测定测量 mRNA 表达水平的变化，该引物模板集能够同时分析多个细胞因子基因。 我们发现IL-12显着上调IFN-g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IL-2的产生正常化，证明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-g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-g 和 IL-2 合成的事实​​表明，IL-12 并不全面增强所有 Th1 型淋巴因子的表达。 此外，IL-12 上调 IL-10 产生的能力提供了一种限制活化 T 细胞的 IL-2 依赖性克隆扩张的机制，并定义了一种可由 IL-12 诱导的新型细胞因子调节途径。 在未来的研究中，我们计划进一步明确IL-12对其他细胞因子产生的影响，特别是活化T细胞产生的Th2型细胞因子IL-4和IL-13，并将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）的影响进行比较。 我们还将进一步探讨 IL-12 处理的 T 细胞中 IL-10 产量增加的功能后果。 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。 该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。 它还可以为癌症患者高剂量 IL-12 治疗相关的全身毒性的生理学基础提供见解。 此外，体外分析IL-12对细胞因子和细胞因子受体基因表达的影响可能有助于鉴定IL-12生物活性的新标记，这些标记可用于监测重组IL-12在人类受体中的效力。 新型 IL-10 相关基因的鉴定和表征。 细菌内毒素脂多糖 (LPS) 激活人类单核细胞，诱导许多细胞因子的表达，包括肿瘤坏死因子 (TNF)、白介素-1 (IL-1)、IL-6 和 IL-10。 IL-10 的表达相对于 TNF、IL-1 和 IL-6 延迟。此外，IL-10反馈抑制TNF、IL-1和IL-6的表达，从而为控制单核细胞中促炎细胞因子的产生提供有效的自分泌机制。 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中细胞因子（例如 TNF 和 IL-1）产生的机制。我们还在评估 IL-10 对 IFN-gamma (IFN-g) 和 IL-4 等细胞因子激活的信号转导事件的影响。我们发现 IL-10 抑制 IL-4 和 IFN-γ 诱导的激活和基因表达。我们还确定，IL-10 抑制 IL-4 诱导型基因表达的能力是酪氨酸磷酸化减少和 IL-4 诱导型转录因子 STAT6 核易位的结果。我们现在正在研究一个新的 JAK/STAT 抑制基因家族，即细胞因子信号传导抑制因子 (SOCS) 基因，在介导这些 IL-10 诱导的抑制作用中的作用。我们发现 IL-10 选择性诱导这一新发现的基因家族中至少一个成员的表达：SOCS-3。 SOCS-3 在巨噬细胞系中的强制表达可显着抑制多种细胞因子（包括 IFN-γ、GM-CSF 和 IL-4）诱导的 STAT 活性。因此，IL-10拮抗细胞因子诱导基因表达的能力与其诱导SOCS-3基因快速表达的能力相关。在相关研究中，我们还研究了几种新鉴定的IL-10同系物的生物活性。其中一种细胞因子 IL-19 与 IL-10 具有大约 21% 的氨基酸同源性，但通过与 IL-10 所使用的受体复合物不同的受体复合物发出信号。我们发现，许多诱导单核细胞中 IL-10 基因表达的相同药物（包括许多 Toll 样受体 (TLR) 激动剂）都会上调 IL-19 基因的表达。然而，与 IL-10 基因不同，IL-19 在活化的 T 细胞中不表达。另一种 IL-10 同源物 IL-22 最近也被多个研究小组鉴定出。我们与新泽西医学和牙科大学 (UMDNJ) 的科学家合作，鉴定并表征了编码这种新型细胞因子受体配体结合链的基因 (CRF2-9)。该受体链与 IL-10R-β 链 (IL-10R2) 异二聚化，形成功能性 IL-22 受体复合物。 因此，IL-10R2是IL-10和IL-22受体的重要组成部分。 我们发现IL-22受体在肝脏和肾脏中高水平表达，但在脾脏和胸腺等造血组织中不表达。 这表明该受体在调节这些组织中的基因表达中可能发挥功能作用。 该项目包括 2002 财年项目 1Z01BN002021-07 和 1Z01BN002022-09。