Mediators of Acute Lung Injury

我们对啮齿类动物中细胞因子和趋化因子依赖性急性肺损伤的研究涉及补体系统和过敏毒素C5 a的作用，C5 a可增强促炎介质的产生。拟开展的研究将评价IgG免疫复合物或细菌脂多糖（LPS）在气道沉积体内诱导的急性炎性肺损伤。我们将确定两种C5 a受体C5 aR和C5 L2的作用。C5 aR是促炎受体，而C5 L2被假定为C5 a“默认”或清道夫受体，其中信号传导事件未能发生。在两种肺损伤模型中，我们将测量血管系统和气道中C5 aR和C5 L2的定量变化。肺中的C5 aR和C5 L2受体将通过使用抗体和通过使用siRNA技术被选择性地阻断。相反，通过使用腺病毒载体技术，肺中的C5 aR和C5 L2水平将提高。我们将在两种肺损伤模型中确定肺中C5 aR水平的提高是否加重肺损伤，而C5 L2表达的提高是否抑制肺损伤，并且在siRNA方法的情况下反之亦然。肺损伤的参数包括125 I-白蛋白泄漏、肺髓过氧化物酶（MPO）的积聚以及支气管肺泡灌洗（BAL）液中细胞因子和趋化因子的含量。我们将使用血液中性粒细胞和肺泡巨噬细胞进行平行的体外研究，这些细胞将被LPS、C5 a或其组合刺激。将采用其他介质来观察我们是否可以实现巨噬细胞上C5 aR和C5 L2的介质特异性变化。将用腺病毒载体转染巨噬细胞以增强C5 aR或C5 L2的受体含量。然后将细胞暴露于LPS或C5 a或其组合，并测量TNF α、IL-1 β和特异性CXC和CC趋化因子的产生。拟议的研究可能对我们理解人类炎症性肺部疾病有影响。项目I将与申请中描述的项目II（Kunkel）和项目IV（Lukacs）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.