Mediators of Acute Lung Injury

我们对依赖细胞因子和趋化因子的啮齿动物急性肺损伤的研究表明，补体系统和过敏性毒素C5a的作用，促进了促炎介质的产生。建议的研究将评估由免疫球蛋白免疫复合物或细菌脂多糖(LPS)的呼吸道沉积在体内诱导的急性炎症性肺损伤。我们将确定两个C5a受体，C5aR和C5L2的作用。C5aR是一种促炎受体，而C5L2被认为是一种C5a“默认”或清道夫受体，在这种受体中信号事件无法发生。在两种肺损伤模型中，我们将测量血管和呼吸道中C5aR和C5L2的定量变化。肺内C5aR和C5L2受体将通过抗体和siRNA技术被选择性地阻断。反之，通过腺病毒载体技术，肺内C5aR和C5L2水平将得到提高。我们将在两种肺损伤模型中确定，在siRNA方法的情况下，肺中C5aR水平的升高是否会加剧肺损伤，而C5L2的表达增强是否会抑制肺损伤，反之亦然。肺损伤的指标包括125I-白蛋白漏出、肺髓过氧化物酶(MPO)积聚、支气管肺泡灌洗液(BAL)中细胞因子和趋化因子含量。我们将进行平行的体外研究，使用血中性粒细胞和肺泡巨噬细胞，这将被内毒素，C5a或其组合刺激。将使用其他介体来观察我们是否能实现巨噬细胞上C5aR和C5L2的介体特异性变化。将腺病毒载体导入巨噬细胞，以增加C5aR或C5L2的受体含量。然后将细胞暴露在内毒素或C5a或两者的组合中，并测量TNFpha、IL-1β以及特定的CXC和CC趋化因子的产生。这项拟议的研究可能对我们理解人类炎症性肺部疾病有所启示。如申请书所述，项目一将与项目二(孔克尔)和项目四(卢卡奇)保持密切联系。

Our studies of cytokine and chemokine-dependent acute lung injury in rodents have implicated the role of the complement system and the anaphylatoxin, C5a, which enhances production of pro-inflammatory mediators. The proposed studies will evaluate acute inflammatory lung injury induced in vivo by airway deposition of IgG immune complexes or bacterial lipopolysaccharide (LPS). We will determine the roles of the two C5a receptors, C5aR and C5L2. C5aR is a pro-inflammatory receptor while C5L2 is postulated to be a C5a "default" or scavenger receptor in which signaling events fail to occur. In the two models of lung injury, we will measure quantitative changes in C5aR and C5L2 in both the vasculature and in the airways. C5aR and C5L2 receptors in lung will be selectively blocked by the use of antibodies and by the use of siRNA technology. Conversely, C5aR and C5L2 levels will be enhanced in lung by the use of adenoviral vector technology. We will determine in the two models of lung injury if enhanced levels of C5aR in lung intensifies lung injury, while enhanced C5L2 expression depresses lung injury, and vice-versa in the case of siRNA approach. The parameters of lung injury will be 125I-albumin leak, buildup of lung myeloperoxidase (MPO), and content of cytokines and chemokines in bronchoalveolar lavage (BAL) fluids. We will conduct parallel in vitro studies using blood neutrophils and alveolar macrophages which will be stimulated by LPS, C5a or the combination. Other mediators will be employed to see if we can achieve mediator specific changes in C5aR and C5L2 on macrophages. Macrophages will be transfected with adenovirus vectors to enhance receptor content of C5aR or C5L2. Cells will then be exposed to LPS or C5a, or the combination, and the production of TNFalpha, IL-1beta and specific CXC and CC chemokines will be measured. The proposed studies may have implications for our understanding of inflammatory lung diseases of humans. Project I will have close ties with Project II (Kunkel) and Project IV (Lukacs) as described in the application.