Control of Cytokine Gene Expression in LymphoidMyeloid Cells

我们正在分析淋巴样细胞中细胞因子和趋化因子基因表达的调节。我们选择干扰素-γ基因表达作为分析自然杀伤细胞(NK细胞)和T细胞基因表达调控的模型系统。我们正在继续剖析人类干扰素-伽马基因的区域，以确定哪些区域增强/抑制基因转录，以响应细胞外信号。特别是，我们正在利用自然杀伤细胞系来阐明白介素2(IL-2)、白介素12(IL-12)、白介素15(IL-15)、白介素18(IL-18)或干扰素-α(干扰素-α)诱导或抑制干扰素-γ基因表达的转录和转录后机制。我们正在研究佛波酯(PMA)或Bryostatin+IL-12协同诱导干扰素-γ基因表达的生化途径。我们的初步结果表明，将Bryostatin和IL-12这两种目前正在进行临床试验的药物结合起来，在体外和体内小鼠模型系统中都能协同诱导干扰素-γ，从而表明这两种药物的结合可能代表着一种强大的癌症免疫治疗新方法。目前的研究表明，当IL-12与Bryostatin联合使用时，通过蛋白激酶C的信号转导可能延长。我们使用生物信息学的方法来确定干扰素-伽马基因3‘端非翻译部分的保守区。人们认为，这些保守区代表着基因结构中的重要调控元件，因为除非mRNA的非编码区提供了一些进化优势，否则在进化过程中不会有固有的保守区。基于这一分析，我们针对小鼠干扰素-γ3‘非翻译区的160bp区域进行了缺失，因为该区域含有丰富的AUA序列，并且这些区域在细胞因子基因表达的调节中具有重要作用。基因敲除(KO)小鼠已经成功创建，我们的数据表明，该小鼠在接受IL-12治疗后产生显著更多的干扰素-γ。此外，在基因敲除小鼠的血清中检测到低水平的干扰素-伽马，而不是野生型对照小鼠。此外，淋巴结、脾和胸腺的结构被破坏，肝脏显示出慢性炎症的迹象。T细胞动态平衡被破坏，因为存在增加的CD4+和CD8+T细胞，并且小鼠体内的T细胞具有更强的抑制活性。对抗原刺激的TH1反应也增加，而TH2反应降低。B细胞的数量也发生了变化，边缘带B细胞明显减少。除了表型后果外，B细胞对抗原的反应也被破坏，因为IgM和Ig2aab反应增加，而IgG1反应减少。同时也观察到了较强的抗DNA和抗核抗原抗体反应，提示慢性干扰素-γ表达可能在狼疮的发生发展中起一定作用。我们还试图鉴定一种可能与该区域相互作用的假定的RNA结合蛋白。综上所述，我们对阐明干扰素-γ调节的多种机制的方法表明，在免疫效应细胞中，干扰素-γ基因表达的调节是复杂的。此外，我们现在已经开发了一个小鼠模型，用于了解和阐明长期、慢性干扰素-γ基因表达的系统生物学效应。

We are analyzing the regulation of cytokine and chemokine gene expression in lymphoid cells. We have chosen interferon-gamma (IFN-gamma) gene expression as a model system for analysis of the control of gene expression in natural killer cells (NK cells) and T cells. We are continuing to dissect the regions of the human interferon-gamma gene to determine which regions enhance/repress gene transcription in response to extracellular signals. In particular, we are utilizing natural killer cell lines to elucidate the mechanisms, both transcriptional and post-transcriptional, by which interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-alpha (IFN-alpha) induce or inhibit interferon-gamma gene expression. We are characterizing the biochemical pathways involved in the synergistic induction of interferon-gamma gene expression in response to phorbol 12-myristate 13-acetate (PMA) or bryostatin + IL-12. Our initial results indicate that combining bryostatin and IL-12, two drugs currently being tested in clinical trials, results in synergistic induction of interferon-gamma both in vitro and in in vivo mouse model systems, thus suggesting that the combination of these drugs could represent a powerful new approach towards cancer immunotherapy. Current studies have revealed that the signaling through Protein Kinase C may be extended when IL-12 is combined with bryostatin. We have used a bioinformatics approach to identify conserved regions of the 3' untranslated portion of the interferon-gamma mRNA. It is believed that these conserved regions represent important regulatory elements in the gene structure as there would be no inherent region for conservation through evolution unless the non-coding regions of the mRNA provided some evolutionary advantage. Based on this analysis, we have targeted a 160-bp region of the murine interferon-gamma 3' untranslated region for deletion, as this region is rich in AUUA sequences and such regions have been previously shown to be important in the regulation of cytokine gene expression. The knockout (KO) mouse has been successfully created and our data indicates that this mouse produces significantly more interferon-gamma upon treatment with IL-12. In addition, low levels of interferon-gamma are detected in the serum of knockout mice but not wild type control mice. Furthermore, the architecture of lymph nodes, spleen and thymus is disrupted and the liver exhibits signs of chronic inflammation. T cell homeostasis has been disrupted as increased CD4+ and CD8+ T cells are present and the T reg cells in the mouse have more potent suppressor activity. There is also an increased TH1 response and a decreased TH2 response to antigenic stimulation. The B cell population is also altered with the apparently loss of marginal zone B cells. In addition to the phenotypic consequences, the B cell response to antigen is also disrupted as increased IgM and Ig2a ab responses are seen with a decrease in the IgG1 response. Strong anti-DNA and anti-nuclear antigen antibody responses are also observed, suggesting that chronic IFN-gamma expression may play a role in the development of lupus. We are also attempting to identify a putative RNA binding protein that may interact with this region. In summary, our approaches towards elucidating the multiple mechanisms involved in the regulation of interferon-gamma demonstrates the complexity by which interferon-gamma gene expression is regulated in immune effector cells. Furthermore we now have developed a mouse model for understanding and elucidating the systems biology effects of long term, chronic IFN-gamma gene expression.