HUMAN LYMPHOCYTE PRODUCTION OF TUMOR NECROSIS FACTOR

肿瘤坏死因子（TNF）在诱导许多肿瘤细胞凋亡中起重要作用。 异常的生理状况，如休克和恶病质。 在 免疫系统促进B和T细胞增殖，抑制 造血前体细胞的殖民地形成， 刺激粒细胞活性。 其中一种细胞类型， 产生TNF的是淋巴细胞。 不同信号的作用 将检查控制淋巴细胞中INF的产生。 特别是蛋白激酶C与 激活，Ca 2+动员和cAMP增加，TNF mRNA和 将研究B和T淋巴细胞中的细胞因子合成。 TNF 将定量DRNA蓄积和TNF细胞因子产生 RNA斑点杂交和放射免疫分析。 为 区分INF mRNA的合成和积累， 将对前者进行核径流分析。 后者 将通过S1-核酸酶或RNA酶图谱分析进行定量。 B 或者淋巴细胞将被各种药物处理以探测 不同的第二信使的作用。 蛋白质的作用 激酶C将与其刺激剂PMA、二酰基甘油 和SC-9及其抑制剂如H7。 钙调蛋白抑制剂 W7和CGS 93438将被用来检查钙调素的作用 在B和T细胞活化期间。 用EGTA或EGTA处理的细胞 在离子霉素的存在下，细胞内的消耗，将是 检查以确定刺激中的Ca 2+需求， TNF mRNA和细胞因子合成。 IP 3将被加载到细胞中 用ATP渗透，以检查瞬时细胞溶质 游离Ca 2+增加。 cAMP的作用将用 cAMP合成诱导霍乱毒素、百日咳毒素和 毛喉素 还将使用二丁酰cAMP。 的重要性 cAMP依赖性激酶活性将用 抑制剂H8或HA 1004。 加载的影响渗透 还将检查具有GTP类似物GTPYS的淋巴细胞。 这些实验将阐明蛋白激酶C的作用， 细胞内游离Ca ~（2+）和cAMP对淋巴细胞的调控作用 TNF mRNA转录和TNF合成。 淋巴细胞产生TNF在免疫功能中的重要性 也将被审查。 26 Kd膜结合的发生 形式将通过免疫沉淀与单克隆或 多克隆抗TNF抗体。 肿瘤坏死因子在B细胞中的作用 将通过用以下物质处理扁桃体B细胞来研究增殖 在抗TNF+抗LT存在下的增殖刺激剂 抗体或TNF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.