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。急性ALI急性渗出期肺中的主要炎症细胞PMN在其颗粒中含有MMP-8，并在脱粒时作为可溶性蛋白酶释放。可溶性MMP-8被认为介导酶的活性。然而，我们的数据显示，在体外和小鼠ALl期间，活化的PMN在其细胞表面表达一种新型的MMP-8。膜结合的MMP-8具有与可溶性MMP-8相似的催化活性，但与可溶性MMP-8不同的是，膜结合的MMP-8对组织抑制剂（TIMPs）的抑制具有抗性。这表明它可能是该蛋白酶在体内的一种重要生物活性形式。新的数据表明，令人惊讶的是，MMP-8的抑制剂TIMP-1也在PMN表面表达，它在PMN表面作为活性MMP-8的受体。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缺乏的小鼠（MMP-8 -/-小鼠），并在具有ALI急性渗出期和纤维增殖期特征的小鼠模型中进行了研究。与野生型小鼠相比，MMP-8-/-小鼠在ALl急性渗出期有更多的PMN流入肺部。然而，在亚急性期，MMP-8-/-小鼠被保护免于进展为纤维增殖。这表明MMP-8在ALI急性渗出期具有意想不到的抗炎作用，而在ALl亚急性期具有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的不同阶段发挥着不同的作用。作为推论，我们还将研究PMN上膜结合的MMP-8是蛋白酶的生物活性形式，可能以重要的方式促进其在体内的活性。为了验证我们的假设，我们提出了三个特定目的：特定目的1：验证假设，在PMN表面，MMP-8和TIMP-1通过两个分子的cooh末端相互作用介导形成复合物，TIMP-1的nh2末端抑制域将复合物锚定在PMN表面。特异性目的2：验证MMP-8通过灭活PMN趋化因子下调ALl急性肺炎症的假说。特异性目的3：验证MMP-8通过：A)下调肺损伤炎症反应促进ALl纤维化进程的假说；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。我们期望这些研究将提供关于MMP-8在ALl中的生物活性的新信息，以及MMP-8在体内保持活性的机制。这将是首个研究MMP-8活性的肺部疾病。我们预计，在未来，我们的研究将有助于开发新的治疗策略，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.