OXIDANT AND PROTEASE INTERACTIONS IN ACUTE LUNG INJURY

这份修订后的竞争对手续订申请是我们 白细胞氧化剂和蛋白水解酶作用机制研究进展 大鼠急性肺损伤模型中的相互作用。在我们的原版中 我们假设氧化剂-蛋白水解酶相互作用在 大鼠中性粒细胞依赖性肺损伤模型的建立 免疫球蛋白免疫复合物。事实上，我们发现情况就是这样。氧化剂 似乎对伤害的开始负有责任，但他们的影响 时间似乎有限。相反，丝氨酸和金属蛋白酶存在于 损伤的肺和添加蛋白酶抑制剂SLPI和SLPI TIMP-2具有明显的保护作用。在相关的体外研究中，蛋白酶- 氧化剂的相互作用可增强中性粒细胞介导的细胞毒性。 和基质降解。因此，体内和体外研究表明 氧化剂和蛋白水解酶在中性粒细胞介导的急性白血病中都很重要 组织损伤。鉴于这些先前的发现，我们提出了一系列 确定氧化剂与蛋白酶相互作用机理的研究(S) 从而导致蛋白水解酶的激活和组织损伤。将此模型用作 作为一个依赖巨噬细胞的免疫球蛋白A模型，我们将做一个完整的时间过程 肺和支气管肺泡灌洗液中蛋白水解酶的鉴定及其意义 活性以及对SLPI和TIMP-2调节的敏感性。我们会 预测巨噬细胞中激活的金属蛋白酶参与其中 在组织损伤中的免疫球蛋白A模型。事实上，情况似乎就是这样 正如初步研究所显示的那样。我们还认为，它是高度 体内的蛋白酶活性可能受氧化剂和蛋白水解酶的调节 互动。这些交互作用可以通过以下三种可能的方式之一发生。 氧化剂通过激活体外调节蛋白水解酶活性 金属蛋白酶和丝氨酸蛋白酶抑制剂的失活。如果 这发生在体内，然后添加抗氧化剂，如过氧化氢酶 应减少蛋白酶的活化量。第二种可能性 氧化剂和蛋白水解酶是独立激活的，但 协同作用诱导肺损伤。在本例中，添加 抗氧化剂不应影响蛋白水解酶的激活和添加 酶抑制剂对体内氧化剂的生成没有影响。 第三，体外蛋白水解酶通过以下机制调节氧化剂的活性 引发白细胞氧化剂的爆发和细胞因子的产生。如果这个 在体内发生，那么SLPI和TIMP-2的加入应该会减少氧化剂 肺部的浓度。通过这些方法，我们希望能够精确地绘制出 了解氧化剂和蛋白酶如何在中性粒细胞和巨噬细胞中相互作用 介导性急性肺损伤模型。

This revised competing renewal application represents a continuation of our ongoing studies on the mechanisms of leukocyte oxidant and protease interactions in models of acute lung injury in the rat. In our original proposal we hypothesized that oxidant-protease interactions are critical in the development of a model of neutrophil dependent lung injury induced by IgG immune complexes. In fact, we found this to be the case. Oxidants appear responsible for the initiation of the injury but their effects over time appear limited. Rather, serine and metalloproteinases are present in the injured lungs and the addition of the protease inhibitors SLPI and TIMP-2 is markedly protective. In correlative in vitro studies protease- oxidant interactions were found to enhance neutrophil-mediated cytotoxicity and matrix degradation. Thus, the in vivo and in vitro studies suggest that both oxidants and proteases are important in neutrophil-mediated acute tissue injury. Given these previous findings, we propose a series of studies to determine the mechanism(s) of oxidant and protease interactions which result in protease activation and tissue injury. Using this model as well as a macrophage-dependent IgA model we will do a complete time course study to identify proteases in lung and bronchoalveolar lavage, their activity, and susceptibility to regulation by SLPI and TIMP-2. We would predict that activated metalloproteinases from the macrophage are involved in the tissue injury in the IgA model. In fact, this seems to be the case as shown in the preliminary studies. We also believe that it is highly likely that protease activity in vivo is regulated by oxidant and protease interactions. These interactions can occur in one of three possible ways. Oxidants regulate protease activity in vitro by activating metalloproteinases and inactivating the serpin protease inhibitors. If this occurs in vivo then the addition of antioxidants such as catalase should decrease the amount of protease activation. The second possibility is that oxidants and proteases are activated independently but work synergistically to induce lung injury. In this case, the addition of the antioxidants should have no effect on protease activation and the addition of the protease inhibitors no effect on oxidant generation in vivo. Thirdly, proteases in vitro regulate oxidant activity by mechanisms such as priming of the leukocyte oxidant burst and cytokine generation. If this occurs in vivo then the addition of SLPI and TIMP-2 should decrease oxidant levels in the lung. By these methods, we hope to be able to precisely map out how oxidants and proteases interact in neutrophil and macrophage mediated models of acute lung injury.