Immunotoxicology of Cigarette Smoke

香烟烟雾的免疫毒理学

• 批准号: 6830241
• 负责人: BRIAN M FREED
• 金额: $ 30.8万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6830241
• 关键词: AP1 protein; T lymphocyte; acrolein; catechols; cell cycle; chemical carcinogenesis; clinical research; environmental toxicology; flow cytometry; gel mobility shift assay; gene expression; human subject; hydroquinones; immunotoxicity; leukocyte activation /transformation; nuclear factor kappa beta; polymerase chain reaction; protooncogene; smoking; tobacco abuse; western blottings

DESCRIPTION (provided by applicant): Cigarette smoking induces profound suppression of T cell responses in the lungs, but peripheral responses are not affected. We have identified four compounds in cigarette smoke that, at doses found in a single cigarette, block discrete steps in T lymphocyte activation and cell cycle progression. Acrolein and crotonaldehyde are volatile alpha, beta-unsaturated carbonyl compounds in the gas phase of cigarette smoke; they inhibit production of several proinflammatory cytokines, including IL-2, TNF-alpha and IFN-gamma, which are critical for T cell responses. Acrolein and crotonaldehyde suppress cytokine gene expression by inhibiting induction of NF-kappaB and AP-1 DNA binding activities. Hydroquinone (HQ) and catechol are present in the particulate fraction. They do not have significant effects on cytokine production and allow partial progression from G0 to G1 following T cell activation. However, they inhibit normal entry and progression through the G1 phase of the cell cycle. The localized nature of smoking-induced immune suppression is due to the fact that only the lungs and adjacent lymph nodes are subjected to immunosuppressive levels of these compounds. The long-term objective of this project is to identify the molecular mechanisms by which cigarette smoke suppresses T cell responses. Towards this goal, we propose the following specific aims: (1) to determine the effects of acrolein and crotonaldehyde on induction of NF-kappaB and AP-1 complexes and their ability to interact with their respective target promoters; (2) to determine the mechanism by which HQ and catechol regulate E2F-mediated gene expression and thereby control entry and progression through the G1 phase of the cell cycle; and (3) to assess the effects of HQ and catechol on c-myc transcription initiation and elongation. Human peripheral blood or lymph node lymphocytes (from non-smoking cadaveric organ donors) will be isolated and treated with cigarette smoke extracts, HQ, catechol, acrolein or crotonaldehyde, then stimulated with anti-CD3 + anti-CD28 or PMA. Whole cell, cytoplasmic or nuclear extracts of these cells will be analyzed for NF-kappaB and AP-1 by Western blot and mobility shift assays. Analysis of cell cycle proteins (Myc, Cdk4, Cdk6, cyclins, E2F4, p130) will be performed by flow cytometry, immunoprecipitation and western blot techniques. Myc RNA will be measured by real time PCR and nuclear run-on assays.

描述（由申请人提供）：吸烟吸烟会引起对肺中T细胞反应的深刻抑制，但外周反应不受影响。我们已经确定了四种香烟中的化合物，这些化合物在单个香烟中发现的剂量，在T淋巴细胞激活和细胞周期进程中的嵌段离散步骤。丙烯醛和克罗替醛是挥发性α，在香烟烟气中的气相中β-不饱和羰基化合物；它们抑制了几种促炎细胞因子的产生，包括IL-2，TNF-Alpha和IFN-Gamma，这对于T细胞反应至关重要。丙烯醛和克罗替醛通过抑制NF-kappab和AP-1 DNA结合活性来抑制细胞因子基因表达。羟基级别（HQ）和儿茶酚存在于颗粒部分中。它们对细胞因子的产生没有显着影响，并允许在T细胞激活后从G0到G1的部分进展。但是，它们抑制了正常的进入和通过细胞周期的G1阶段的进展。吸烟引起的免疫抑制的局部性质是由于以下事实：仅肺和相邻的淋巴结受到这些化合物的免疫抑制水平。该项目的长期目标是确定香烟烟雾抑制T细胞反应的分子机制。为了实现这一目标，我们提出了以下特定目的：（1）确定丙烯醛和克罗替醛对NF-kappab和AP-1复合物诱导的影响及其与各自目标启动子相互作用的能力； （2）确定总部和儿茶酚调节E2F介导的基因表达的机制，从而控制和通过细胞周期的G1相进入和进展； （3）评估总部和儿茶酚对C-MYC转录启动和伸长的影响。人类外周血或淋巴结淋巴细胞（来自非吸烟的尸体器官供体）将被隔离并用香烟烟雾提取物，HQ，Catechol，Acrolelin或crotonaldeyde进行处理，然后用抗CD3 +抗CD3 +抗CD28或PMA刺激。这些细胞的全细胞，细胞质或核提取物将通过Western blot和迁移性转移测定法分析NF-kappab和AP-1。细胞周期蛋白（MYC，CDK4，CDK6，细胞周期蛋白，E2F4，P130）的分析将通过流式细胞仪，免疫沉淀和蛋白质印迹技术进行。 MYC RNA将通过实时PCR和核跑步测定法进行测量。