Novel Roles for MMP-8 in Acute Lung Injury

描述(申请人提供)：金属蛋白酶-8(MMP8)在急性肺损伤(ALI)中的作用尚未确定。中性粒细胞是ALI急性渗出期肺内的主要炎性细胞，其颗粒内含有基质金属蛋白酶-8，脱颗粒时以可溶性蛋白酶的形式释放出来。可溶性的基质金属蛋白酶-8被认为是该酶活性的中介。然而，我们的数据显示，在体外和在小鼠体内，激活的PMN在其细胞表面表达一种新的形式的基质金属蛋白酶-8。膜结合的基质金属蛋白酶-8具有与可溶性基质金属蛋白酶相似的催化活性，但与可溶性基质金属蛋白酶不同，膜结合的基质金属蛋白酶-8不受基质金属蛋白酶组织抑制物(TIMPs)的抑制。这表明它可能是该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，MMP8的抑制因子TIMP-1也在PMN表面表达，其功能是作为PMN表面活性MMP8的受体。为了评价基质金属蛋白酶-8在急性肺损伤中的作用，我们通过基因打靶技术建立了基质金属蛋白酶-8缺陷小鼠(MMP8-/-小鼠)，并在具有急性肺损伤渗出期和纤维增殖期特征的小鼠模型上进行了研究。与野生型小鼠相比，在ALL急性渗出期，MMP8-/-小鼠的PMN流入肺的量增加。然而，在亚急性期，MMP-8-/-小鼠受到保护，不会进展为纤维增殖。这表明，在ALI的急性渗出期，MMP-8具有意想不到的抗炎作用，而在ALL的亚急性期，则具有反直觉的促纤维化作用。我们的中心假设是PMN来源的MMP-8在ALI的不同阶段发挥着不同的作用，作为推论，我们还将检验PMN上结合的MMP-8是一种生物活性形式的蛋白酶的假说，这可能在体内对其活性起重要作用。为了验证我们的假设，我们提出了三个特定的目标：特定的目标1：验证这样的假设，即在PMN表面，MMP8和TIMP-1通过两个分子的COOH-末端的相互作用形成复合物，TIMP-1的NH2末端抑制结构域将该复合体锚定在PMN表面。具体目的2：验证基质金属蛋白酶-8通过灭活中性粒细胞趋化因子而下调急性肺炎症的假说。具体目标3：通过：A)下调肺损伤的炎症反应；或B)蛋白水解性调节促纤维化和/或抗纤维化介质的生物活性，来检验这一假说，即基质金属蛋白酶-8在所有方面都促进纤维增殖的进展。我们期望这些研究将提供关于基质金属蛋白酶-8的全部生物学活性的新信息，以及基质金属蛋白酶-8在体内保持其活性的机制。ALL将是第一个研究基质金属蛋白酶-8‘S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，从而降低所有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.