CONTROL OF LYMPHOKINE PRODUCTION:EFFECT OF CYCLOSPORINE

控制淋巴因子的产生：环孢菌素的作用

• 批准号: 3293079
• 负责人: ANGELA GRANELLI-PIPERNO
• 金额: $ 13.77万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293079
• 关键词: B lymphocyte; T lymphocyte; cyclosporines; genetic transcription; human tissue; immunoregulation; interferons; interleukin 2; lymphokines; macrophage; messenger RNA; tissue /cell culture

The release of lymphokines like IL-2 and interferon-Gamma from T lymphocytes is controlled at the level of gene transcription. The induction of lymphokine mRNA has been monitored with an oocyte translation assay and with northern blotting. Primary populations of mouse and human T lymphoblasts, which release large amounts of lymphokine upon restimulation with lectin, have been used throughout. Application of cyclosporin A (CsA) blocks the production of lymphokine mRNA, but the induction of several other genes (c-fos, IL-2 receptor, heat shock protein) is not inhibited. Since these genes are induced by the same agents (lectin, phorbol esters, OKT3 antibody) as lymphokine genes, CSA probably does not inhibit signalling at the cell surface. This grant will continue to study the control of lymphokine release and the effects of CsA. The selectivity of CsA for lymphokine gene activity will be tested with: a large panel of DNA probes, unprimed and freshly sensitized T lymphocytes, and nuclear transcription and northern blotting assays. The function of accessory cells in lumphokine release will be monitored at the level of gene transcription. Different kinds of accessory cells (dendritic cells, macrophages, B lymphocytes) and different populations of responding T cells (resting vs. lymphoblasts; T4 vs. T8) will be tested. The possibility that accessory function can be replaced by phorbol esters, and that accessory cells can be directly inhibited by CsA, will be considered. The mechanism of action of CsA will be studied in two principal experiments. The preliminary observation that IL-2 can increase interferon mRNA will be pursued, since the increase is CsA-resistant. Conditions for the induction of interferon by IL-2 will be optimized and then evaluated on isolated nuclei to test if IL-2 stimulated cells exhibit CsA-insensitive, interferon gene transcription. Direct CsA-DNA interactions will be evaluated using footprinting assays on DNA segments obtained from the 5 flanking region of lymphokine genes. DNA binding regions should be protected from DNAase I and methidiumpropyl EDTA sensitivity. Nonimmunosuppressive analogues of CsA, provided by Sandoz, will be used as controls in this and all other experiments. Should these experiments indicate that CsA does not act directly on lymphokine genes, experiments will be initiated to identify proteins that are pertinent to lymphokine transcription.

T 细胞释放 IL-2 和干扰素-γ 等淋巴因子 淋巴细胞在基因转录水平上受到控制。 这 淋巴因子 mRNA 的诱导已通过卵母细胞翻译进行监测 测定和RNA印迹。 小鼠和人类 T 细胞的主要种群 淋巴母细胞，在再刺激时释放大量淋巴因子 与凝集素一起，一直被使用。 环孢素A（CsA）的应用 阻断淋巴因子 mRNA 的产生，但诱导几种 其他基因（c-fos、IL-2受体、热休克蛋白）不被抑制。 由于这些基因是由相同的试剂（凝集素、佛波酯、 OKT3抗体）作为淋巴因子基因，CSA可能不会抑制 细胞表面的信号传导。 这笔赠款将继续研究淋巴因子释放的控制和 CsA 的影响。 CsA 对淋巴因子基因活性的选择性 进行测试：一大组 DNA 探针，未引物且新鲜 致敏 T 淋巴细胞、核转录和 Northern 印迹 化验。 辅助细胞在淋巴因子释放中的功能是 在基因转录水平上进行监测。 不同种类的配件 细胞（树突状细胞、巨噬细胞、B 淋巴细胞）和不同 响应 T 细胞群（静息细胞与淋巴母细胞；T4 与 T8） 将受到测试。 附件功能可以被替换的可能性 佛波酯，并且辅助细胞可以被 CsA 直接抑制， 将被考虑。 CsA的作用机制将从两个方面进行研究 主要实验。 初步观察IL-2升高 将追踪干扰素 mRNA，因为这种增加具有 CsA 抗性。 IL-2诱导干扰素的条件将得到优化， 然后对分离的细胞核进行评估，以测试 IL-2 刺激的细胞是否表现出 CsA 不敏感的干扰素基因转录。 直接CsA-DNA 将使用 DNA 片段的足迹分析来评估相互作用 从淋巴因子基因的 5 个侧翼区域获得。 DNA结合 应保护区域免受 DNAase I 和甲啶丙基 EDTA 的影响 敏感性。 CsA 的非免疫抑制类似物，由 Sandoz 提供， 将用作本实验和所有其他实验的对照。 应该这些 实验表明CsA不直接作用于淋巴因子基因， 将启动实验来鉴定相关的蛋白质 淋巴因子转录。