Novel Roles for MMP-8 in Acute Lung Injury

描述（由申请人提供）：金属蛋白酶-8（MMP-8）在急性肺损伤（ALI）中的作用尚未确定。 PMN是急性肺损伤急性渗出期肺内主要的炎性细胞，其颗粒内含有MMP-8，并在其降解时以可溶性蛋白酶的形式释放。 可溶性MMP-8被认为介导该酶的活性。然而，我们的数据表明，激活的PMN表达一种新形式的MMP-8在其细胞表面，在体外和在小鼠急性淋巴细胞白血病。 膜结合MMP-8具有与可溶性MMP-8相似的催化活性，但与可溶性MMP-8不同，膜结合MMP-8对MMP的组织抑制剂（TIMP）的抑制具有抗性。 这表明它可能是蛋白酶在体内的一种重要的生物活性形式。新的数据表明，令人惊讶的是，MMP-8的抑制剂TIMP-1也在PMN表面上表达，在那里它作为PMN表面上活性MMP-8的受体发挥作用。 为了评估MMP-8在ALI中的作用，我们通过基因靶向产生了MMP-8缺陷的小鼠（MMP-8 -/-小鼠），并在具有ALI的急性渗出期和纤维增生期特征的小鼠模型中研究了它们。 与野生型小鼠相比，MMP-8-/-小鼠在AL 1的急性渗出期具有增加的PMN流入肺中。 然而，MMP-8-/-小鼠在亚急性期被保护免于进展为纤维增殖。 这表明MMP-8在ALI的急性渗出期具有意想不到的抗炎作用，并且在ALI的亚急性期具有违反直觉的促纤维化作用。 我们的中心假设是，PMN衍生的MMP-8在ALI的不同阶段起着不同的作用。作为推论，我们还将研究PMN上的膜结合MMP-8是蛋白酶的生物活性形式的假设，这可能有助于其在体内的活动的重要方式。 为了验证我们的假设，我们提出了三个具体的目的：具体的目的1：测试的假设，在PMN表面，MMP-8和TIMP-1形成复合物介导的两个分子的COOH-末端的相互作用，和氨端抑制结构域TIMP-1锚定复合物的PMN表面。具体目的2：检验MMP-8通过灭活PMN趋化因子下调AL 1期间急性肺部炎症的假设。 具体目标3：检验MMP-8通过以下方式促进AL 1中纤维增殖进展的假设：A）下调对损伤的肺部炎症反应;和/或B）蛋白水解调节促纤维化和/或抗纤维化介质的生物活性。 我们预期这些研究将提供关于MMP-8在AL 1中的生物活性以及MMP-8在体内保持其活性的机制的新信息。 AL 1将是第一种研究MMP-8活性的肺部疾病。 我们预期，在未来，我们的研究将促进新的治疗策略的开发，其可以降低ALL综合征的死亡率和发病率。

DESCRIPTION (provided by applicant): The roles of metalloproteinase-8 (MMP-8) have not been determined in acute lung injury (ALI). PMN, the predominant inflammatory cell in the lung during the acute-exudative phase of ALI contain MMP-8 within their granules, and release it as a soluble proteinase when they degranulate. Soluble MMP-8 is thought to mediate the activities of the enzyme. However, our data show that activated PMN express a novel form of MMP-8 on their cell surface, in vitro and during ALl in mice. Membrane-bound MMP-8 has similar catalytic activity as soluble MMP-8, but unlike soluble MMP-8, membrane-bound MMP-8 is resistant to inhibition by tissue inhibitors of MMPs (TIMPs). This indicates that it could be an important bioactive form of the proteinase in vivo. Novel data indicate that, surprisingly, TIMP-1 an inhibitor of MMP-8 is also expressed on the surface of PMN where it functions as the receptor for active MMP-8 on the PMN surface. To assess the roles of MMP-8 in ALI, we have generated mice deficient in MMP-8 by gene targeting (MMP-8 -/- mice) and studied them in murine models having features of the acute-exudative and fibro-proliferative phases of ALI. MMP-8-/- mice have increased influx of PMN into the lungs in the acute-exudative phase of ALl compared to wild type mice. However, MMP-8-/- mice are protected from progression to fibro-proliferation in the sub-acute phase. This indicates that MMP-8 has an unexpected anti-inflammatory role in the acute-exudative phase of ALI, and a counterintuitive, pro-fibrotic role in the sub-acute phase of ALl. Our central hypothesis is that PMN-derived MMP-8 plays distinct roles in different phases of ALI As a corollary to this, we will also examine the hypothesis that membrane-bound MMP-8 on PMN is a bioactive form of the proteinase which may contribute in important ways to its activities in vivo. To test our hypotheses, we propose three Specific Aims: Specific Aim 1: Test the hypothesis that on the PMN surface, MMP-8 and TIMP-1 form complexes mediated by the interaction of the COOH-terminals of both molecules, and the NH2-terminal inhibitory domain of TIMP-1 anchors the complex to the PMN surface. Specific Aim 2: Test the hypothesis that MMP-8 down-regulates acute lung inflammation during ALl by inactivating PMN chemokines. Specific Aim 3: Test the hypothesis that MMP-8 promotes progression to fibro-proliferation in ALl by: A) downregulating the lung inflammatory response to injury; and or B) proteolytically regulating the biologic activities of pro-, and/or anti-fibrotic mediators. We anticipate that these studies will provide novel information about the biologic activities of MMP-8 in ALl, and the mechanisms by which MMP-8 retains its activity in vivo. The ALl will be the first lung disease in which MMP-8's activities will be studied. We anticipate that in the future, our studies will facilitate the development of new treatment strategies that may reduce mortality and morbidity in ALl syndromes.