VITAMIN A INFLUENCE ON INTERFERON-GAMMA GENE EXPRESSION

维生素 A 对干扰素-γ 基因表达的影响

• 批准号: 2146075
• 负责人: COLLEEN Elizabeth HAYES
• 金额: $ 11.09万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-20 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146075
• 关键词: T lymphocyte; antibody; cell growth regulation; clone cells; enzyme linked immunosorbent assay; gene induction /repression; genetic regulation; genetic transcription; genetic translation; hamsters; immunochemistry; interferon gamma; interferon inducers; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; northern blottings; nucleic acid probes; polymerase chain reaction; protein isoforms; retinoid binding proteins; retinoids

Surprisingly, vitamin A-deficient mice undergoing an infection, or immunized with an antigen, had T lymphocytes that secreted IFNgamma at a 10-fold higher rate than controls. The T cells from A-deficient mice functioned poorly as stimulators of B cell antibody responses; there was a low helper T cell frequency and the cells underproduced the lymphokines IL-4 and IL-5. Repletion with retinoic acid, a vitamin A metabolite, sharply downregulated IFNgamma, restored the helper T cell frequency, and returned antibody responses to normal levels in vitro. Retinoid repletion also restored antibody responses in vivo. Cloned mouse MD13-10 T cells mimic freshly explanted immune T cells with respect to IFNgamma production. When MD 13-10 T cells are retinoid depleted and stimulated, they overproduce IFNgamma, and retinoic acid directly downregulates the IFNgamma secretion rate. We found an unexpected retinoic acid receptor- alpha (RARalpha) transcript and a novel RARalpha-immunoreactive protein in MD13-10 T cells and other T cells. Together, our findings lead us to propose as a working hypothesis that retinoic acid, acting through an undefined mechanism possibly involving this novel RARalpha isoform, specifically downregulates T cell IFNgamma production. The experiments proposed here will test our hypothesis by studying the biochemistry of retinoid-mediated IFNgamma regulation in cloned MD 13- 10 T cells. We propose to define the retinoid-sensitive IFNgamma induction pathway, to explore whether retinoid-mediated control is at the IFNgamma transcript or protein level, and to analyze the dependence of regulation on macromolecular synthesis. We propose to characterize the T cell RARalpha transcripts by Northern blotting with exon-specific probes, and sequencing of novel 5' and/or 3' cDNA ends. We will investigate the putative RARalpha proteins using immunochemistry. We plan to express and purify the novel RARalpha N-terminal fragments, and raise antibodies. We will use the antibodies to develop RARalpha isoform-specific ELISA, retinoid-binding, and DNA-binding assays and apply them to MD13-10 T cell proteins. The proposed research is significant in that it will provide new information about a regulatory relationship between two potent controllers of immune function and cell growth. IFNgamma regulates immunity, autoimmunity, hemopoiesis, viral replication, and malignant cell growth. Retinoic acid also regulates immunity, and normal and malignant cell growth and differentiation. The powerful anticancer and immunoregulatory activities of both molecules emphasize the importance of investigating how retinoic acid downregulates IFNgamma gene expression.

令人惊讶的是，缺乏维生素 A 的小鼠受到感染，或者 经抗原免疫后，T淋巴细胞以一定的速度分泌IFNγ 比对照组高 10 倍。来自 A 缺陷小鼠的 T 细胞 作为 B 细胞抗体反应刺激剂的功能较差；有 辅助 T 细胞频率低且细胞产生的淋巴因子不足 IL-4 和 IL-5。富含视黄酸，一种维生素 A 代谢物， 大幅下调 IFNγ，恢复辅助 T 细胞频率，以及 使体外抗体反应恢复到正常水平。类维生素A补充 也恢复了体内的抗体反应。克隆小鼠 MD13-10 T 细胞 模拟新鲜移植的免疫 T 细胞的 IFNgamma 生产。当 MD 13-10 T 细胞被耗尽并受到刺激时， 它们过量产生 IFNgamma，而视黄酸直接下调 IFNγ分泌率。我们发现了一种意想不到的视黄酸受体—— α (RARalpha) 转录物和一种新型 RARalpha 免疫反应蛋白 MD13-10 T 细胞和其他 T 细胞。我们的发现共同引导我们 提出作为一个工作假设，视黄酸通过 未定义的机制可能涉及这种新型 RARalpha 亚型， 特异性下调 T 细胞 IFNgamma 的产生。实验 这里提出的将通过研究生物化学来检验我们的假设 克隆 MD 13-10 T 细胞中类视黄醇介导的 IFNγ 调节。我们 提议定义类维生素A敏感的IFNγ诱导途径， 探索类视黄醇介导的控制是否位于 IFNgamma 转录物或 蛋白质水平，并分析调节的依赖性 高分子合成。我们建议表征 T 细胞 RARalpha 使用外显子特异性探针进行 Northern blotting 并测序 新的 5' 和/或 3' cDNA 末端。我们将研究假定的 RARalpha 使用免疫化学的蛋白质。我们计划对小说进行表达和净化 RARalpha N 末端片段，并产生抗体。我们将使用 抗体用于开发 RARalpha 同种型特异性 ELISA、类维生素A 结合、 和 DNA 结合测定并将其应用于 MD13-10 T 细胞蛋白。这 拟议的研究意义重大，因为它将提供新的信息 关于两个有效的免疫控制器之间的调节关系 功能和细胞生长。 IFNgamma 调节免疫力、自身免疫、 造血、病毒复制和恶性细胞生长。视黄酸 还调节免疫力以及正常和恶性细胞的生长和 差异化。强大的抗癌和免疫调节活性 这两种分子的研究都强调了研究视黄酸如何 酸下调 IFNγ 基因表达。