OXIDANT AND PROTEASE INTERACTIONS IN ACUTE LUNG INJURY

这一修订后的竞争性续期申请代表了我们的延续 白细胞氧化剂和蛋白酶机制的研究进展 在大鼠急性肺损伤模型中的相互作用。 在我们最初的 我们假设，氧化剂-蛋白酶相互作用是关键， 致中性粒细胞依赖性肺损伤模型的建立 IgG免疫复合物。 事实上，我们发现情况确实如此。 氧化剂 似乎是造成伤害的原因，但它们的影响超过了 时间似乎有限。 相反，丝氨酸和金属蛋白酶存在于 以及蛋白酶抑制剂SLPI和 TIMP-2具有明显的保护作用。 在相关的体外研究中， 发现氧化剂相互作用增强嗜铬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， 氧化剂和蛋白酶在嗜中性粒细胞介导的急性 组织损伤 鉴于这些先前的发现，我们提出了一系列 确定氧化剂和蛋白酶相互作用机制的研究 其导致蛋白酶活化和组织损伤。 使用该模型作为 作为巨噬细胞依赖性伊加模型，我们将做一个完整的时间过程， 鉴定肺和支气管肺泡灌洗液中蛋白酶的研究， 活性以及对SLPI和TIMP-2调节的敏感性。 我们 预测来自巨噬细胞的激活的金属蛋白酶参与 在伊加模型的组织损伤中。 事实上，似乎是这样的 如初步研究所示。 我们还认为， 可能体内蛋白酶活性受氧化剂和蛋白酶调节 交互. 这些相互作用可以以三种可能的方式之一发生。 氧化剂通过激活蛋白酶活性来调节体外蛋白酶活性 金属蛋白酶和失活丝氨酸蛋白酶抑制剂。 如果 这发生在体内，然后加入抗氧化剂如过氧化氢酶 会降低蛋白酶的激活量。 第二种可能 氧化剂和蛋白酶是独立激活的， 协同地诱导肺损伤。 在这种情况下，添加 抗氧化剂应该对蛋白酶活化没有影响， 蛋白酶抑制剂对体内氧化剂的产生没有影响。 第三，蛋白酶在体外通过以下机制调节氧化剂活性： 引发白细胞氧化剂爆发和细胞因子产生。 如果这 那么SLPI和TIMP-2的加入应该减少氧化剂的产生， 肺中的水平。 通过这些方法，我们希望能够精确地绘制出 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 介导的急性肺损伤模型。

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.