Daudi I, Gudewicz P W, Saba T M, Cho E, Vincent P
Department of Physiology and Cell Biology, Albany Medical College, New York 12208.
J Leukoc Biol. 1991 Oct;50(4):331-40. doi: 10.1002/jlb.50.4.331.
Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125I-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60-200 kD) were not labelled, but the smaller molecular weight fragments (less than 45 kD), derived from the artificial matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.
受刺激白细胞释放的蛋白酶对内皮细胞下基质结合的纤连蛋白的切割作用与肺血管损伤有关。我们研究了炎性多形核白细胞（PMNL）对结合于变性胶原的纤连蛋白的降解作用。用125I标记的大鼠血浆纤连蛋白预处理包被有变性胶原（明胶）的组织培养孔，以便在加入大鼠炎性PMNL之前使纤连蛋白结合。在加入经调理酵母聚糖吞噬作用刺激的PMNL以及白细胞弹性蛋白酶后，对125I标记的人工基质中完整和片段化纤连蛋白的释放进行定量。与未刺激的PMNL相比，刺激的PMNL在4小时孵育期间从变性胶原表面释放的放射性标记纤连蛋白多两倍。在没有PMNL的情况下，单独加入纯化的白细胞弹性蛋白酶也可引发这种125I-纤连蛋白释放模式。加入白细胞弹性蛋白酶特异性抑制剂甲氧基琥珀酰-丙氨酸-丙氨酸-缬氨酸氯甲基酮（AAPVCK）以及非特异性丝氨酸蛋白酶抑制剂苯甲基磺酰氟（PMSF），可剂量依赖性地阻断刺激的PMNL释放放射性标记的纤连蛋白。蛋白质印迹分析结合放射自显影证实，加入PMNL或白细胞弹性蛋白酶后培养基中存在纤连蛋白片段。大分子质量片段（60 - 200 kD）未被标记，但源自人工基质的小分子质量片段（小于45 kD）被标记。因此，与变性胶原复合的纤连蛋白易受刺激的炎性PMNL的蛋白水解降解作用影响。这样一个过程可能在活化血液白细胞微血管边缘化后急性血管损伤的发病机制中起作用。
