MOLECULAR MECHANISM OF IMMUNOTOXIC ACTION OF TCDD & DTT

TCDD免疫毒作用的分子机制

• 批准号: 3252919
• 负责人: FUSAO HIRATA
• 金额: $ 20.73万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-01 至 1994-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3252919
• 关键词: arachidonate; autoradiography; binding proteins; complementary DNA; dioxins; dithiol; environmental toxicology; enzyme inhibitors; fatty acid metabolism; fluorescence microscopy; gel electrophoresis; gene expression; genetic regulation; halobiphenyl /halotriphenyl compound; high performance liquid chromatography; histocompatibility antigens; hormone receptor; human subject; immunoregulation; immunotoxicity; interleukin 1; laboratory mouse; macrophage; monocyte; nucleic acid hybridization; nucleic acid probes; protein biosynthesis; radioimmunoassay; receptor binding; second messengers; steroids; tissue /cell culture; tumor necrosis factor alpha

Some environmental contaminants such as 2.3.7.8 tetrachlorodibenzoparadioxin (TCDD) and 1.1.1-trichloro-2-(2- chlorophenyl)-2-(4-chlorophenyl)ethane (o,p'DTT), are known to have steroidal actions as well as immunotoxic actions in animals and humans. These compounds bind to intracellular receptor proteins similar to those for glucocorticoids and estrogens. Since the immune system plays a crucial role in host resistance and homeostasis, the immunomodulation induced by the xenobiotics may be the basis for adversed health effects. Monocytes/macrophages have various key functions in initiating the immune regulation, mediating through antigen presentation, monokine production, and formation of arachidonate metabolites, and these functions are known to be affected by glucocorticoids and estrogens. In the projects proposed herein, we aim to investigate the molecular mechanism of actions of steroids and xenobiotics on function of monocytes/macrophages and subsequently, of cell mediated immunity. The specific aims are to examine (a) whether these xenobiotics and steroids bind to the same or different receptors, (b) whether they induce or block the synthesis of second messenger proteins of steroids, (c) what are their actions on the gene expression of IL 1, TNF, HLA-DR and lipocortin and (d) whether these effects on IL 1, TNF, HLA-DR and lipocortin are attributed to alteration of arachidonate metabolism or of gene regulation by the xenobiotic- receptor complex or both. Xenobiotics and steroids will be incubated in vitro with fractionated human peripheral monocytes/macrophages in the absence and presence of specific and nonspecific activators such as gamma-lFN, LPS and phorbol esters. Metabolites of arachidonate will be quantitatively analyzed by high performance liquid chromatography. The binding analysis will be performed by competitive ligand binding assays. Identification of newly induced proteins will be carried out by two dimensional electrophoresis using (35S)methionine and their biological actions on monocytes and T lymphocytes will be examined. The regulation of IL 1, TNF, lipocortin (putative second messenger of glucocorticoids) and HLA-DR gene expression will be investigated by the Northern Blot using their cDNA clones as probes. To exploit not only the mechanism of immunotoxicity of xenobiotics but also the mechanism of immunoregulation leading to the immunosuppression (acquired immune deficiency), we propose to establish the mechanism of action of xenobiotics on the biochemical and cellular functions of monocytes/macrophages.

一些环境污染物，如2.3.7.8 四氯二苯并对二恶英（TCDD）和1.1.1-三氯-2-（2- 氯苯基）-2-（4-氯苯基）乙烷（o，p 'DTT）已知具有 类固醇作用以及动物免疫毒性作用， 人类 这些化合物与细胞内受体蛋白结合 类似于糖皮质激素和雌激素。 以来 免疫系统在宿主抗性中起着至关重要的作用， 内稳态，由外源性物质诱导的免疫调节可 是对健康不利影响的基础。 单核细胞/巨噬细胞 在启动免疫调节中具有各种关键功能， 通过抗原提呈、单核因子产生和 形成花生四烯酸代谢物，这些功能是 已知受糖皮质激素和雌激素影响。 在 本文提出的项目，我们的目标是研究分子 甾体激素和外源性物质对神经元功能的作用机制 单核细胞/巨噬细胞以及随后的细胞介导的免疫。 具体目的是审查（a）这些外源性物质和 类固醇与相同或不同的受体结合，（B）它们是否 诱导或阻断第二信使蛋白的合成， 类固醇，（c）它们对IL基因表达的作用 1、TNF、HLA-DR和脂皮质素的影响，以及（d）这些对IL-1、TNF、HLA-DR和脂皮质素的影响是否 1，TNF，HLA-DR和脂皮质素的改变， 花生四烯酸代谢或外源性生物质的基因调控- 受体复合物或两者。 异生物素和类固醇 体外与分离的人外周血细胞孵育 单核细胞/巨噬细胞在不存在和存在特异性和 非特异性活化剂，如γ-IFN、LPS和佛波酯。 花生四烯酸的代谢产物将通过高效液相色谱法进行定量分析。 高效液相色谱法 结合分析将是 通过竞争性配体结合测定进行。 鉴定 新诱导的蛋白质将通过二维 （35 S）蛋氨酸电泳及其生物学作用 对单核细胞和T淋巴细胞的影响。 调控 IL-1、TNF、脂皮质素（ 糖皮质激素）和HLA-DR基因表达将进行研究 以它们的cDNA克隆为探针进行北方印迹。 利用 不仅外源性物质的免疫毒性机制， 导致免疫抑制的免疫调节机制 （获得性免疫缺陷），我们建议建立机制， 外源性物质对生物化学和细胞功能的作用 单核细胞/巨噬细胞。