Heck L W, Blackburn W D, Irwin M H, Abrahamson D R
Department of Medicine, Veterans Administration Medical Center, Birmingham, Alabama.
Am J Pathol. 1990 Jun;136(6):1267-74.
To determine the susceptibility of laminin to proteolytic degradation by inflammatory cells, soluble laminin was incubated with supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated human neutrophils. The appearance of laminin cleavage fragments was then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of supernatants with diisopropylfluorophosphate (DFP), anti-human neutrophil elastase (HNE), and anti-human neutrophil cathepsin G (HNCG) IgGs effectively blocked the degradation of laminin. In contrast, treatment of supernatants with EDTA failed to inhibit laminin digestion, indicating that neutrophil metalloproteinases had little laminin-degrading activity. In additional experiments, laminin was incubated with purified HNE and HNCG. Both enzymes extensively cleaved laminin in a dose- and time-dependent manner yielding similar products, but HNE was generally more potent. Immunofluorescence microscopy of cryostat sections of mouse kidney treated with HNE or HNCG also showed widespread loss of laminin epitopes from basement membranes. The proteolytic degradation of laminin by neutrophil elastase and cathepsin G indicates an important role for these enzymes in basement membrane damage during inflammation.
为了确定层粘连蛋白对炎症细胞蛋白水解降解的敏感性，将可溶性层粘连蛋白与佛波醇12 -肉豆蔻酸酯13 -乙酸酯（PMA）刺激的人中性粒细胞的上清液一起孵育。然后通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）检测层粘连蛋白裂解片段的出现。用二异丙基氟磷酸（DFP）、抗人中性粒细胞弹性蛋白酶（HNE）和抗人中性粒细胞组织蛋白酶G（HNCG）IgG处理上清液可有效阻断层粘连蛋白的降解。相反，用EDTA处理上清液未能抑制层粘连蛋白的消化，这表明中性粒细胞金属蛋白酶的层粘连蛋白降解活性很低。在其他实验中，将层粘连蛋白与纯化的HNE和HNCG一起孵育。两种酶都以剂量和时间依赖性方式广泛裂解层粘连蛋白，产生相似的产物，但HNE通常更有效。用HNE或HNCG处理的小鼠肾脏冰冻切片的免疫荧光显微镜检查也显示基底膜中层粘连蛋白表位广泛丢失。中性粒细胞弹性蛋白酶和组织蛋白酶G对层粘连蛋白的蛋白水解降解表明这些酶在炎症过程中对基底膜损伤起重要作用。
