MECHANISM OF CIGARETTE SMOKE-INDUCED IMMUNO SUPRESSION

吸烟与上呼吸道疾病的风险增加有关 肠道感染、多种器官的癌症和心血管疾病。 为了确定感染易感性的增加是否与 香烟烟雾引起的免疫系统变化，我们研究了 香烟烟雾对啮齿动物的影响 我们的研究结果表明，慢性 大鼠/小鼠暴露于来自R1香烟的香烟烟雾（高焦油、高焦油、高焦油） 尼古丁，肯塔基州大学参考香烟）主要影响 B淋巴细胞功能 此外，最易受感染的B细胞反应， 香烟烟雾，是那些通过抗原受体激活 （表面免疫球蛋白的交联）。 本提案的目标是 （a）了解免疫抑制的潜在机制 香烟烟雾的影响，以及（B）识别香烟烟雾的特定成分 香烟烟雾冷凝物，负责观察到的 免疫抑制 本文提出的实验旨在评估以下因素的影响： 香烟烟雾对B细胞增殖的已知阶段的影响， 分化 来自烟雾暴露和假对照动物的B细胞将 检查它们激活的能力，受体交联依赖性， 磷脂酰肌醇代谢，包括酪氨酸活化 磷酸激酶（使用磷酸酪氨酸的mAb的蛋白质印迹分析）， 磷脂酶C（肌醇磷酸代谢），增加 细胞内钙含量（indo-1荧光的变化），以及 蛋白激酶C的活化（32 P标记）。 流式细胞术，使用 吖啶橙子染色用于确定细胞周期阶段。 已知与淋巴细胞活化相关的原癌基因活化， 如c-fos和c-myc，将通过北方印迹使用32 P- 标记的cDNA探针。 此外，还将检查B细胞的 产生免疫球蛋白的能力（通过ELISA测定） 抗原/促有丝分裂活化，并且在存在或不存在 淋巴因子如IL 4、IL 5、IL 6和tau-IFN。 将采用既定方案获得馏分和亚馏分 香烟烟雾冷凝物。 这些部分将被合并到 恒定释放惰性丸粒（含有特定级分的丸粒将 由Innovative Research of America，Toledo，OH制造），植入 皮内注射到动物体内，并监测对免疫系统的影响， 植入后的不同时间。 有效部位将进一步 评价它们在体外影响淋巴细胞功能的能力。 这些实验的结果可能会提供关于 吸烟会影响免疫系统 的适用性 最近的研究结果加强了这些结果对人类吸烟者的影响。 来自Weissman等人（见附录1），其中香烟烟雾影响 人类免疫系统以类似于我们在实验中观察到的方式， 实验室，在老鼠。

Cigarette smoking is associated with elevated risks of upper respiratory tract infections, cancers of several organs, and cardiovascular disease. To determine whether this increased susceptibility to infections correlated with cigarette smoke-induced changes in the immune system, we studied the effects of cigarette smoke in rodents. Our results indicated that chronic exposure of rats/mice to cigarette smoke from R1 cigarettes (high tar, high nicotine, University of Kentucky Reference Cigarette) affects primarily the B lymphocyte function. Moreover, the B cell responses, most susceptible to cigarette smoke, are those which are activated via the antigen receptors (cross-linking of surface immunoglobulins). The goals of this proposal are to: (a) understand the underlying mechanism(s) for the immunosuppressive effects of cigarette smoke, and (b) to identify specific components of cigarette smoke condensate that are responsible for the observed immunosuppression. Experiments proposed herein are directed at evaluating the effects of cigarette smoke on the known stages of B cell proliferation and differentiation. B cells from smoke-exposed and sham control animals will be examined for their ability to activate, receptor crosslinking-dependent, metabolism of phosphatidylinositol, including activation of tyrosine phosphokinase (western blot analysis using mAb to phosphotyrosines), phospholipase C (metabolism of inositol phosphates), increase in intracellular calcium content (changes in indo-1 fluorescence), and the activation of protein kinase C (32P-labeling). Flow cytometry, using acridine orange staining will be employed to determine cell cycle phases. Activation of protooncogenes known to correlate with lymphocyte activation, like c-fos and c-myc, will be assayed by northern blotting using 32P- labeled cDNA probes. In addition, B cells will be examined for their ability to produce immunoglobulin (assayed by ELISA) in response to antigenic/mitogenic activation, and in the presence or absence of lymphokines such as IL4, IL5, IL6, and tau-IFN. Established protocols will be employed to obtain fractions and subfractions of cigarette smoke condensate. These fractions will be incorporated into constant-release inert pellets (pellets containing specific fractions will be made by Innovative Research of America, Toledo, OH), implanted intradermally into animals, and effects on the immune system monitored at various times after implantation. The effective fractions will be further evaluated for their ability to influence lymphocyte functions in vitro. Results from these experiments are likely to provide ideas abut the manner in which cigarette smoke affects the immune system. The applicability of these results to human smokers has been strengthened by the recent results from Weissman et al. (see Appendix 1) in which cigarette smoke affects the human immune system in a manner similar to those observed, in our laboratory, in the rat.