Influence of DNA on the Activities and Inhibition of Neutrophil Serine Proteases in Cystic Fibrosis Sputum
Influence of DNA on the Activities and Inhibition of Neutrophil Serine Proteases in Cystic Fibrosis Sputum
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DOI：
10.1165/rcmb.2011-0380oc
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发表时间：
2012-07-01
影响因子：
6.4
通讯作者：
Attucci, Sylvie
中科院分区：
文献类型：
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作者：
Dubois, Alice V.;Gauthier, Alexandre;Attucci, Sylvie
肺分泌物中中性粒细胞丝氨酸蛋白酶（NSP）的不受控制的蛋白水解是囊性纤维化（CF）的标志。我们已经证明CF痰液中活性中性粒细胞弹性蛋白酶、蛋白酶3和组织蛋白酶G部分抵抗外源性蛋白酶抑制剂的抑制。这种耐药性可能是由于它们与CF痰液中活化的中性粒细胞分泌的中性粒细胞胞外陷阱（NET）结合以及与衰老和死亡中性粒细胞释放的基因组DNA结合。用DNA酶处理CF痰显著增加其弹性蛋白酶活性，然后可以通过外源性弹性蛋白酶抑制剂化学计量地抑制弹性蛋白酶活性。然而，DNA酶处理不增加蛋白酶3和组织蛋白酶G的活性，表明它们在CF痰中的不同分布和/或结合。纯化的血液中性粒细胞在受到机会性CF细菌铜绿假单胞菌和金黄色葡萄球菌刺激时分泌NET。三种蛋白酶的活性在这些条件下没有变化，但随后的DNA酶处理产生了所有三种蛋白水解活性的显着增加。用钙离子载体激活的中性粒细胞不分泌NET，但释放大量活性蛋白酶，其活性不被DNA酶修饰。我们的结论是，NET是活性蛋白酶的水库，保护他们免受抑制，并保持他们在一个快速动员的状态。将蛋白酶抑制剂的作用与DNA降解剂的作用相结合可以对抗CF肺分泌物中NSP的有害蛋白水解作用。
Uncontrolled proteolysis by neutrophil serine proteases (NSPs) in lung secretions is a hallmark of cystic fibrosis (CF). We have shown that the active neutrophil elastase, protease 3, and cathepsin G in CF sputum resist inhibition in part by exogenous protease inhibitors. This resistance may be due to their binding to neutrophil extracellular traps (NETs) secreted by the activated neutrophils in CF sputum and to genomic DNA released from senescent and dead neutrophils. Treating CF sputum with DNase dramatically increases its elastase activity, which can then be stoichiometrically inhibited by exogenous elastase inhibitors. However, DNase treatment does not increase the activities of protease 3 and cathepsin G, indicating their different distribution and/or binding in CF sputum. Purified blood neutrophils secrete NETs when stimulated by the opportunistic CF bacteria Pseudomonas aeruginosa and Staphylococcus aureus. The activities of the three proteases were unchanged in these conditions, but subsequent DNase treatment produced a dramatic increase in all three proteolytic activities. Neutrophils activated with a calcium ionophore did not secrete NETs but released huge amounts of active proteases whose activities were not modified by DNase. We conclude that NETs are reservoirs of active proteases that protect them from inhibition and maintain them in a rapidly mobilizable status. Combining the effects of protease inhibitors with that of DNA-degrading agents could counter the deleterious proteolytic effects of NSPs in CF lung secretions.