PULMONARY LYMPHOCYTE APOPTOSIS AND CELL CYCLE ARREST

已知或怀疑与哮喘和结节病等肺部疾病有关 对无处不在的吸入抗原的不适当的局部免疫反应。至 改进治疗方法和制定早期干预策略， 必须确定下调肺免疫反应的机制。 生理性肺免疫反应减弱，即使重复抗原也是如此 暴露，但负责任的机制尚未确定。实质肺 细胞在体外通过多种机制抑制淋巴细胞增殖； 然而，无论淋巴细胞是暂时性的生长抑制还是 在活体的肺中永久存在还不确定。研究肺脏 免疫调节，我们开发了一个实验模型系统，在其中 抗原致敏的C57BL/6小鼠气管内注射T细胞 细胞依赖抗原绵羊红细胞(SRBC)，诱导应答 这是充满活力的，但重要的是，它会自我终止。我们发现， 在这些小鼠中，肺淋巴细胞似乎被细胞周期停滞；他们 在体外和体内弱自发地增殖，并 尽管CD25的表达比例很高，但对IL-2无效。多肺 淋巴细胞发生凋亡。肺内淋巴细胞数量增加。 环孢素-A体内治疗，已知可抑制激活- 诱导细胞死亡(AICD)。细胞的凋亡仅限于 CD_4~-、CD_8~-、B220~-，但以CD3~+为主。另一个相当大的肺 淋巴细胞亚群也有CD3+、CD4-、CD8-、B220-，但未显示 DNA片段化。后一类人群的命运被指定为CD3+ 双阴性(CD3+dN)细胞及其与其他淋巴细胞的关系 人口，是未知的。因此，我们的发现证明了一种新的方法，即 哪些肺部免疫反应可以受到限制。我们假设 活化的T细胞面临Fas介导的凋亡风险，因为细胞- 周期停滞是由不充分的协同刺激和 肺细胞产生的抑制因子。这些T细胞向下- 调节CD4(成为CD3=DN细胞)，并最终调节其他表面 受体，否则可能会传递抗凋亡信号。 相比之下，尽管Fas表达，但接受足够共刺激的T细胞- 刺激和抑制死亡的信号避免因持续的细胞凋亡 抗细胞凋亡基因Bcl2家族成员的表达此应用程序将 探讨细胞周期停滞与肺细胞凋亡的关系 淋巴细胞的体外检测和体内免疫活性分析， 突变和转基因小鼠。具体目标是：(1)确定是否 肺淋巴细胞的凋亡依赖于Fas。(2)决定是否- 抗-HBs过表达阻断肺淋巴细胞的凋亡 Bcl2家族的凋亡基因(3)。确定肺T细胞是否 细胞周期受阻(4)确定哪些肺APC被触发 或防止淋巴细胞凋亡。(5)确定哪个肺淋巴细胞 亚群致力于细胞凋亡。我们的长期目标是将这一点用于 活体模型系统将体外观察转化为全面的 了解生理免疫调节机制。理解 这些机制应导致采用新的方法来控制 免疫性肺部疾病与同种异体肺移植排斥。

Lung diseases as asthma and sarcoidosis are known or suspected to involve inappropriate local immune responses to ubiquitous inhaled antigens. To mprove therapies and develop early intervention strategies, the mechanisms down-regulating pulmonary immune responses must be defined. Physiologic pulmonary immune responses wane, even with repeated antigen exposure, but responsible mechanisms are undefined. Parenchymal pulmonary cells inhibit lymphocyte proliferation in vitro by multiple mechanisms; owever, whether lymphocytes are growth-inhibited transiently or permanently in the lungs in vivo is uncertain. To study pulmonary immunoregulation, we have developed an experimental model system in which antigen-primed C57BL/6 mice are intratracheally challenged with the T cell-dependent antigen sheep red blood cells (SRBC), inducing a response which is vigorous but, importantly, self-terminating. We have found that in these mice, lung lymphocytes appeared to be cell-cycle arrested; they proliferated meagerly spontaneously in vitro and in vivo and were refractory to IL-2 despite a high percentage of CD25 expression. Many lung lymphocytes underwent apoptosis. Lung lymphocyte numbers were increased by cyclosporine-A treatment in vivo, which is known to inhibit activation- induced cell death (AICD). Apoptosis was restricted to cells which were CD4-, CD8-, B220-, but most of which were CD3+. Another sizeable lung lymphocyte population was also CD3+, CD4-, CD8-, B220-, but did not show DNA fragmentation. The fate of this latter population, designated CD3+ double negative (CD3+ DN) cells, and its relationship to other lymphocyte populations, is unknown. Our findings, thus, demonstrate a novel method by which pulmonary immune responses can be limited. We hypothesize that activated T cells become at risk for Fas-mediated apoptosis due to cell- cycle arrest which is triggered by inadequate co-stimulation and by suppressive factors produced by pulmonary cells. These T cells down- regulate CD4 (becoming CD3= DN cells) and eventually other surface receptors, which might otherwise have delivered anti-apoptotic signals. By contrast, despite Fas expression, T cells which receive adequate co- stimulation & death-repressing signals avoid apoptosis due to sustained expression of anti-apoptotic Bcl-2 family members. This application will explore the relationship between cell-cycle arrest and apoptosis of lung lymphocytes using in vitro assays and in vivo analysis of immunocompetent, mutant, and transgenic mice. The Specific Aims are: (1) Determine whether pulmonary lymphocyte apoptosis is Fas-dependent. (2) Determine whether - pulmonary lymphocyte apoptosis is blocked by over-expression of anti- apoptosis genes of the Bcl-2 family (3). Determine whether pulmonary T cells are cell-cycle arrested (4) Determine which pulmonary APCs trigger or prevent lymphocyte apoptosis. (5) Determine which pulmonary lymphocyte subsets are committed to apoptosis. Our long-term goal is to use this in vivo model system to translate in vitro observations into a comprehensive understanding of physiologic immunoregulatory mechanisms. Understanding these mechanisms should lead to novel approaches to the control of immunologic lungs diseases and lung allograft rejection.