HUMAN LYMPHOCYTE PRODUCTION OF TUMOR NECROSIS FACTOR

肿瘤坏死因子(肿瘤坏死因子)在诱导许多 不正常的生理状况，如休克和恶病质。在……里面 免疫系统，它促进B和T细胞的增殖，抑制 造血祖细胞的集落形成和 刺激粒细胞活动。其中一种细胞类型 产生肿瘤坏死因子的是淋巴细胞。不同信号的作用 控制淋巴细胞中干扰素的产生将被研究。 特别是，蛋白激酶C之间的关系 活化、钙动员和cAMP增加及肿瘤坏死因子mRNA和 我们将研究B和T淋巴细胞中的细胞因子合成。肿瘤坏死因子 DRNA的积累和肿瘤坏死因子的产生将被量化 分别用RNA斑点印迹法和放射免疫法测定。为 区分INF mRNA的合成和积累， 将使用针对前者的核径流分析。后者 将通过S1-核酸酶或核糖核酸酶作图分析进行定量。B类 或者淋巴细胞将被用各种药物治疗以探测 不同的第二信使的角色。蛋白质的作用 将用它的刺激物PMA、二酰甘油来检测激酶C 和SC-9及其抑制剂，如H7。钙调蛋白抑制剂 W7和CGS93438将用于研究钙调蛋白的作用 在B和T细胞激活过程中。用EGTA或EGTA处理的细胞 在离子霉素存在的情况下，细胞内的耗竭将是 检查以确定刺激细胞中的钙需求 肿瘤坏死因子基因和细胞因子的合成。IP3将加载到单元格中 用三磷酸腺苷渗透检测瞬时胞浆的影响 游离钙离子增加。CAMP的作用将与 CAMP合成诱导霍乱毒素、百日咳毒素和 福斯科林。二丁酰环也将被使用。重要的是 CAMP依赖性激酶活性将用 抑制剂H8或HA1004。荷载渗透性的影响 具有GTP类似物GTPYS的淋巴细胞也将被检测。 这些实验将阐明蛋白激酶C的作用， 胞内游离钙和cAMP在控制淋巴细胞中的作用 肿瘤坏死因子mRNA转录和肿瘤坏死因子合成。 淋巴细胞产生肿瘤坏死因子在免疫功能中的重要性 也将接受检查。26kD膜结合态的发生 形式将通过免疫沉淀法与单克隆或 多克隆抗肿瘤坏死因子抗体。肿瘤坏死因子在B细胞中的作用 将通过处理扁桃体B细胞来研究增殖 抗肿瘤坏死因子和抗LT联合应用的促增殖作用 抗体，或肿瘤坏死因子加LT反义寡核苷酸。

Tumor necrosis factor (TNF) is important in inducing a number of abnormal physiological conditions such as shock and cachexia. In the immune system, it promotes B and T cell proliferation, inhibits the colony formation of hematopoietic precursor cells and stimulates granulocyte activities. One of the cell types that produce TNF is the lymphocytes. The role of different signals controlling the production of INF in lymphocytes will be examined. In particular, the relationship between protein kinase C activation, Ca2+ mobilization and cAMP increases and TNF mRNA and cytokine syntheses in B and T lymphocytes will be studied. TNF DRNA accumulation and TNF cytokine production will be quantitated by RNA dot-blot assays and radioimmunoassays, respectively. For differentiating between INF mRNA synthesis and accumulation, nuclear run-off assays for the former will be used. The latter will be quantitated by Sl-nuclease or RNase mapping analyses. B or a lymphocytes will be treated with various drugs to probe for the role of different second messengers. The role of protein kinase C will be examined with its stimulators PMA, diacylglycerols and SC-9 and its inhibitors such as H7. The calmodulin inhibitors W7 and CGS93438 will be used to examine the role of calmodulin during B and T cell activation. Cells treated with EGTA, or EGTA depletion of intracellular in the presence of ionomycin, will be examined to determine the Ca2+ requirement in the stimulation of TNF mRNA and cytokine syntheses. IP3 will be loaded into cells permealized with ATP to examine the effects of transient cytosolic free Ca2+ increases. The role of cAMP will be examined with the cAMP synthesis inducers cholera toxin, pertussis toxin and forskolin. Dibutyryl cAMP will also be used. The importance of cAMP dependent kinase activities will be assessed with the inhibitors H8 or HA1004. The effects of loading permealized lymphocytes with the GTP analogue GTPYS will also be examined. These experiments will clarify the role of protein kinase C, cytosolic free Ca2+ and cAMP in controlling lymphocyte transcription of TNF mRNA and TNF synthesis. The importance of a lymphocyte TNF production in immune function will be also examined. The occurrence of the 26 Kd membrane-bound form will be examined by immunoprecipitation with monoclonal or polyclonal anti-TNF-antibodies. The role of TNF in B cell proliferation will be studied by treating tonsillar B cells with proliferation stimulants in the presence of anti TNF plus anti LT antibodies, or TNF plus LT anti-sense oligonucleotides.