PULMONARY LYMPHOCYTE APOPTOSIS AND CELL CYCLE ARREST

肺部疾病如哮喘和结节病是已知或怀疑涉及 对普遍存在的吸入抗原的不适当的局部免疫应答。到 改进治疗方法并制定早期干预策略， 必须确定下调肺免疫应答的机制。 生理性肺免疫应答减弱，即使有重复抗原 暴露，但责任机制尚不明确。肺实质 细胞通过多种机制抑制体外淋巴细胞增殖; 无论淋巴细胞是暂时性生长抑制还是 在体内是否能在肺中永久存在尚不确定。为了研究肺 免疫调节，我们已经开发了一个实验模型系统， 抗原致敏的C57 BL/6小鼠用T 细胞依赖性抗原绵羊红细胞（SRBC），诱导应答 这是充满活力的，但重要的是，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阻滞;它们 在体外和体内自发增殖， 尽管CD 25表达百分比高，但IL-2难治。许多肺部 淋巴细胞发生凋亡。肺淋巴细胞数量增加， 环孢菌素A治疗在体内，这是已知的抑制活化- 诱导细胞死亡（AICD）。细胞凋亡仅限于那些 CD 4-、CD 8-、B220-，但以CD 3+为主。另一个相当大的肺 淋巴细胞群也是CD 3+，CD 4-，CD 8-，B220-，但没有显示 DNA片段化。后一个群体的命运，称为CD 3 + 双阴性（CD 3 + DN）细胞及其与其它淋巴细胞的关系 人口是未知的。因此，我们的研究结果证明了一种新的方法， 肺部免疫反应可能受到限制。我们假设 活化的T细胞由于细胞- 周期停滞，由不充分的共刺激和 肺细胞产生的抑制因子。 这些T细胞下降- 调节CD 4（成为CD 3 = 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， 刺激和死亡抑制信号避免细胞凋亡， 抗凋亡Bcl-2家族成员的表达。此应用程序将 探讨细胞周期阻滞与肺细胞凋亡的关系 使用免疫活性的体外测定和体内分析， 突变体和转基因小鼠。具体目标是：（1）确定是否 肺淋巴细胞凋亡是Fas依赖的。(2)决定是否- 肺淋巴细胞凋亡被抗- Bcl-2家族的凋亡基因（3）。确定肺T 细胞周期停滞（4）确定哪些肺APC触发 或阻止淋巴细胞凋亡。(5)确定哪些肺淋巴细胞 亚群致力于细胞凋亡。我们的长期目标是将其用于 体内模型系统，将体外观察转化为全面的 了解生理免疫调节机制。理解 这些机制应该导致新的方法来控制 免疫性肺部疾病和肺移植排斥反应。

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.