CONTROL OF LYMPHOKINE PRODUCTION:EFFECT OF CYCLOSPORINE

T淋巴细胞释放的淋巴因子如IL-2和干扰素-γ 在基因转录水平控制淋巴细胞。 的 淋巴因子mRNA的诱导已经用卵母细胞翻译来监测 测定和用北方印迹。 小鼠和人T细胞的原代群体 淋巴母细胞，在再刺激时释放大量淋巴因子 与凝集素一起使用。 环孢素A（CsA）的应用 阻断淋巴因子mRNA的产生，但诱导几种 其它基因（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因子（凝集素、佛波酯， OKT 3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。 该基金将继续研究淋巴因子释放的控制以及 CsA的作用 CsA对淋巴因子基因活性的选择性将 可以用：一个大的DNA探针面板，未引发和新鲜 致敏T淋巴细胞，核转录和北方印迹 测定。 辅助细胞在凝血因子释放中的作用将被进一步研究。 在基因转录水平上进行监测。 各种配件 细胞（树突状细胞、巨噬细胞、B淋巴细胞）和不同的 应答T细胞群（静息与淋巴母细胞; T4与T8） 会得到考验 附件功能可被替换的可能性 佛波醇酯，并且辅助细胞可以被CsA直接抑制， 将被考虑。 CsA的作用机制将在两个 主要实验。 初步观察到IL-2可以增加 干扰素mRNA将被追踪，因为增加是CsA抗性的。 将优化IL-2诱导干扰素的条件， 然后在分离的细胞核上进行评价，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 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个侧翼区获得。 DNA结合 区域应免受DNA酶I和甲基丙基EDTA的影响 灵敏度 CsA的非免疫抑制类似物，由Sandoz提供， 将被用作本次实验和其他所有实验的对照。 如果这些 实验表明CsA不直接作用于淋巴因子基因， 将启动实验，以确定与 淋巴因子转录

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.