A New Pathway for Neutrophil-induced Airway Inflammation

描述(由申请人提供)：在这份续展申请中，我们描述了一种新的途径，信号转导中性粒细胞(PMN)的流入和对呼吸道的损害，这一途径在父母的资助中描述了，它可能在慢性肺部疾病(如阻塞性肺疾病(COPD)、囊性纤维化(CF)和慢性肺移植排斥反应)中发挥启动或辅助因素的作用。具体地说，化学或酶解胶原蛋白释放三肽、Pgp和/或相关的Pgp序列，该序列在体外对PMN具有趋化作用。我们证明，将PGP引入呼吸道会引起PMN的强劲流入，但不会导致单核细胞的流入。值得注意的是，PGP的PMN趋化活性可能是由于与ELR+CXC趋化因子的受体结合域(如IL-8)具有明显的结构相关性，这些趋化因子含有这个胶原蛋白序列或类似物。长时间的呼吸道暴露会导致肺泡扩大和右室肥厚，从而重述COPD的某些方面。我们开发了一种有效的拮抗剂Arg-Thr-Arg，称为RTR，它能在溶液中与Pgp结合，并阻断其体外趋化活性和体内病理生理效应。可能是由于与PGP具有相同的结构，RTR还可以抑制IL-8的活性。在脂多糖诱导的COPD小鼠模型中，rtr完全抑制肺泡扩大和右室肥厚。胶原的降解和Pgp的释放是一个分步骤的过程，最初涉及基质金属蛋白酶(MMP)-8和/或9，最后的反应是由Pro内肽酶(PE)催化的。我们发现一种高度特异的PE抑制剂--N-苄氧羰基(Z)-脯氨醛(ZPP)，可以阻断Pgp的产生和PMN对内毒素的内流。令人惊讶的是，ZPP钝化PMN内流不仅是由于PE的抑制，而且是由于ZPP对CXCR1/2的拮抗作用，这可能是ZPP与PGP结构相似的结果。这表明，在这项提议中要探索的耐人寻味的可能性是，可以开发出既是蛋白酶抑制剂又是受体拮抗剂的单一化合物。如果成功，这将代表着一种全新的嵌合类型的铅化合物，用于治疗中性粒细胞炎症，特别是肺部疾病。我们发现Pgp存在于几乎所有CF、COPD或慢性肺移植排斥反应患者的支气管肺泡灌洗液(BALF)和/或痰中，但不存在于对照组或哮喘患者。此外，COPD和CF患者的痰中含有从纯化的胶原蛋白体外产生PGP所需的所有酶机制，这种产生可以被PE抑制剂ZPP阻断。总之，这些发现使我们假设PGP和PE是COPD的新生物标记物，可能与疾病有关，PGP和PE在配体、受体和生成酶水平上是有吸引力的治疗靶点。 与公共健康相关：慢性阻塞性肺病现在是美国第四大死因，预计到2020年将上升到第三位。这种疾病不仅给人们造成了巨大的经济负担，还给人们带来了许多痛苦。由于缺乏诊断生物标记物并在临床试验中用作终点，以及缺乏对疾病过程本身的基本了解，对COPD的理解导致成功的治疗方法受到阻碍。如果成功，目前的提案将产生对疾病过程的基本见解和COPD的新的特定生物标记物，以及建议新的治疗靶点。

DESCRIPTION (provided by applicant): In this renewal application, we describe a new pathway signaling neutrophil (PMN) influx and damage to the airways that was delineated in the parent grant and that may play a role as an initiator or cofactor for chronic lung diseases such as obstructive pulmonary disease (COPD), cystic fibrosis (CF), and chronic lung transplant rejection. Specifically, chemical or enzymatic breakdown of collagen releases a tripeptide, PGP, and/or a related PGP-containing sequence that is chemotactic for PMN in vitro. We demonstrate that introduction of PGP into the airways causes a robust influx of PMN, but not monocytes. Remarkably, the PMN chemotactic activity of PGP may be due to a marked structural relatedness to a receptor binding domain of ELR+ CXC chemokines such as IL-8 which contain this collagen sequence or a close analog. Prolonged airway exposure to this peptide causes alveolar enlargement and right ventricular hypertrophy and thus recapitulates aspects of COPD. We have developed a potent antagonist, Arg-Thr-Arg, termed RTR, which binds PGP in solution and blocks its in vitro chemotactic activity and in vivo pathophysiologic effects. Likely as a result of shared structure with PGP, RTR also blocks IL-8 activity. In a mouse model of LPS-induced COPD, RTR completely inhibited alveolar enlargement and right ventricular hypertrophy. The degradation of collagen and release of PGP is shown to be a stepwise process initially involving matrix metalloproteases (MMP)-8 and/or 9 with prolyl endopeptidase (PE) catalyzing the final reaction. We have found that a highly specific PE inhibitor, N-benzyloxycarbonyl (Z) prolyl prolinal (ZPP), can block airway PGP production as well as PMN influx in response to LPS. Surprisingly, the very potent blunting of PMN influx by ZPP appears to be due not only to PE inhibition but also as a result of ZPP antagonism of CXCR 1/2 likely as a result of structural similarity between ZPP and PGP. This suggests the intriguing possibility, to be explored in this proposal, that a single compound can be developed that is both a protease inhibitor and receptor antagonist. If successful, this would represent an entirely new chimeric type of lead compound for the treatment of neutrophilic inflammation, in general, and lung diseases in particular. We have found that PGP is present in bronchoalveolar lavage fluids (BALF) and/or sputum from virtually all CF, COPD or chronic lung transplant rejection patients but not controls or asthmatics. Furthermore, sputum from COPD and CF patients but not control individuals contains all the enzymatic machinery necessary for the ex vivo generation of PGP from purified collagen and such PGP production can be blocked by the PE inhibitor, ZPP. Collectively, these findings lead us to hypothesize that PGP and PE represent novel biomarkers for COPD that may contribute to disease and that PGP and PE represent attractive therapeutic targets at the level of the ligand, receptor, and generating enzyme. PUBLIC HEALTH RELEVANCE: COPD is now the fourth leading cause of death in the U.S. and is expected to be the third by 2020. This disease causes much human suffering as well as a large monetary burden. An understanding of COPD that leads to successful therapeutics has been hampered by a lack of biomarkers to diagnose and use as endpoints in clinical trials as well as a lack of a fundamental understanding of the disease process itself. If successful, the current proposal will yield fundamental insights into the disease process and a new and specific biomarker for COPD as well as suggesting new therapeutic targets.