Delacourt C, Le Bourgeois M, D'Ortho M P, Doit C, Scheinmann P, Navarro J, Harf A, Hartmann D J, Lafuma C
Département de Physiologie, INSERM U 296, Faculté de Médecine, Créteil, France.
Am J Respir Crit Care Med. 1995 Aug;152(2):765-74. doi: 10.1164/ajrccm.152.2.7633740.
A growing body of evidence suggests that neutrophil-derived proteinases play a major role in lung tissue damage in cystic fibrosis (CF). Most previous studies have focused on serine proteinases such as neutrophil elastase, providing no information on the extent to which metalloproteinases participate in proteolytic processes in CF. To address this issue, we evaluated the contribution of one of the major neutrophil metalloproteinases, i.e., 95 kDa gelatinase (type IV collagenase), to the total gelatinolytic activity measured in sputum specimens from 27 patients with CF. Compared with asthmatic children (n = 9), CF patients had a 6.7 times greater level of total gelatinase activity in sputum revealed by zymography. The 95 kDa gelatinase was increased 3.7-fold in the CF subjects (2,441 +/- 411 [SEM] arbitrary units [AU] x 10(6) per ml of sputum versus 665 +/- 201 in asthmatics) and the 88-kDa active form 23.2-fold (2,272 +/- 372 AU x 10(6) per ml of sputum versus 98 +/- 43, respectively). Using radiolabeled 3H-gelatin as the substrate, we demonstrated uninhibited gelatinolytic activity in all CF patients; this activity was significantly correlated to disease severity as assessed by pulmonary function tests. Western blotting using anti-tissue inhibitor of metalloproteinase (anti-TIMP) and anti-95/88-kDa gelatinase antibodies demonstrated a more than 10-fold excess of 95/88 kDa gelatinase over TIMP. Bacterial proteinases from Pseudomonas aeruginosa were shown to contribute little to the gelatinolytic activity measured in sputum supernatants from patients with CF, although culture supernatants from various P. aeruginosa strains expressed gelatinolytic activity in vitro. Finally, lung damage, as assessed by increased type IV collagen degradation products in sputum, was significantly correlated to concentrations of active 88 kDa gelatinase. These data argue for a significant role of 95/88 kDa gelatinase in airway damage in CF.
越来越多的证据表明，中性粒细胞衍生的蛋白酶在囊性纤维化（CF）的肺组织损伤中起主要作用。以前的大多数研究都集中在丝氨酸蛋白酶，如中性粒细胞弹性蛋白酶，而没有提供关于金属蛋白酶在CF的蛋白水解过程中参与程度的信息。为了解决这个问题，我们评估了主要的中性粒细胞金属蛋白酶之一，即95 kDa明胶酶（IV型胶原酶），对27例CF患者痰液标本中测得的总明胶分解活性的贡献。与哮喘儿童（n = 9）相比，CF患者通过酶谱法显示痰液中总明胶酶活性水平高6.7倍。CF受试者中95 kDa明胶酶增加了3.7倍（每毫升痰液2,441 +/- 411 [SEM]任意单位 [AU] x 10(6)，而哮喘患者为665 +/- 201），88 kDa活性形式增加了23.2倍（每毫升痰液分别为2,272 +/- 372 AU x 10(6)和98 +/- 43）。使用放射性标记的3H-明胶作为底物，我们在所有CF患者中都证明了不受抑制的明胶分解活性；该活性与通过肺功能测试评估的疾病严重程度显著相关。使用抗金属蛋白酶组织抑制剂（抗-TIMP）和抗95/88 kDa明胶酶抗体的蛋白质印迹显示，95/88 kDa明胶酶比TIMP过量10倍以上。尽管来自各种铜绿假单胞菌菌株的培养上清液在体外表现出明胶分解活性，但铜绿假单胞菌的细菌蛋白酶对CF患者痰液上清液中测得的明胶分解活性贡献很小。最后，通过痰液中IV型胶原降解产物增加评估的肺损伤与活性88 kDa明胶酶的浓度显著相关。这些数据表明95/88 kDa明胶酶在CF气道损伤中起重要作用。
