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: 3293084
• 负责人: ANGELA GRANELLI-PIPERNO
• 金额: $ 17.27万
• 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/3293084
• 关键词: B lymphocyte; DNA footprinting; T lymphocyte; cyclosporines; genetic transcription; human tissue; immunoregulation; interferons; interleukin 2; laboratory mouse; 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、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。这个 通过卵母细胞翻译来监测淋巴因子mRNA的诱导 用Northern印迹法检测。小鼠和人T细胞的初级种群 淋巴母细胞，在重新刺激时释放大量淋巴因子 随着凝集素的使用，已经贯穿始终。环孢素A(CsA)的应用 阻断淋巴因子mRNA的产生，但对几种 其他基因(c-fos、IL-2受体、热休克蛋白)不受抑制。 由于这些基因是由相同的诱导剂(凝集素、佛波酯、 OKT3抗体)作为淋巴因子基因，CsA可能不抑制 在细胞表面发出信号。 这笔赠款将继续研究淋巴因子释放的控制和 CsA的作用。CsA对淋巴因子基因活性的选择性 用：一大堆未加底漆和新鲜的DNA探针进行测试 致敏T淋巴细胞、核转录和Northern印迹 化验。辅助细胞在Lumphokine释放过程中的作用 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 反应性T细胞群(静息与淋巴母细胞；T4与T8) 将会受到考验。辅助功能可以被替换为 佛波醇酯，辅助细胞可被CsA直接抑制， 将被考虑。CsA的作用机制将分两部分进行研究。 主要实验。白介素2升高的初步观察 由于增加的干扰素对CsA具有抵抗力，因此将继续追求干扰素mRNA。 优化IL-2诱导干扰素的条件， 然后在分离的细胞核上进行评估，以测试IL-2刺激的细胞是否表现出 CsA不敏感，干扰素基因转录。直接CsA-DNA 相互作用将使用DNA片段上的足迹分析进行评估 从淋巴因子基因的5个侧翼区获得。DNA结合 区域应受到DNAase I和甲基丙基EDTA的保护 敏感度。CsA的非免疫抑制类似物，由Sandoz提供， 将在这项实验和所有其他实验中用作对照。这些应该是 实验表明，CsA不直接作用于淋巴因子基因， 将开始进行实验，以确定与 淋巴因子转录。