A study on possible roles of granulocyte neutral proteinases and the control mechanism of their activities in the process of periodontal breakdown

为了阐明宿主源性溶酶体蛋白酶在牙周破裂机制中的作用，研究了两种主要的丝氨酸蛋白酶组织蛋白酶G和髓质酶。在牙周炎患者龈沟液（GCF）中，两种酶的活性几乎全部检测不到，而酶联免疫吸附法测定的两种酶的量随着龈沟液体积的增加而增加，表明两种酶参与了牙周炎的进展。它们在GCF中失去活性的原因可能是它们被其特异性的血清衍生蛋白酶抑制剂，特别是alpha_1蛋白酶抑制剂阻断，因为使用每种酶和alpha_1蛋白酶抑制剂对GCF进行免疫印迹分析显示，各自的酶和抑制剂单独具有弱蛋白带，而它们的复合物具有强蛋白带。在不采取任何口腔卫生措施21 d后引起的实验性牙龈炎实验中，在各种临床参数监测的炎症检测之前，GCF中这些酶显着增加，表明它们参与了炎症的发展。在发炎的牙龈组织中，这些酶在中性粒细胞和巨噬细胞中受到限制，特别是在袋上皮附近集中，部分细胞外有脱肉芽。这一发现表明，除了宿主对细菌病原体的防御机制外，这些酶还参与宿主组织的直接降解。此外，纯化酶注射到兔牙龈引起炎症反应，其特征是血管明显损伤，炎症细胞包括中性粒细胞和巨噬细胞轻度浸润。然而，用它们的特异性抑制剂alpha_1-蛋白酶抑制剂或alpha_1-抗凝乳胰蛋白酶处理后，这些酶的功能完全丧失。从本研究结果来看，组织蛋白酶G和髓质酶存在于GCF和牙龈组织中，参与了血清源性蛋白酶抑制剂调节的宿主防御机制。一旦酶抑制剂平衡失去，这两种酶反过来对牙周组织起破坏性作用。少

To clarify the role of host-derived lysosomal proteinases in the mechanism of periodontal breakdown, cathepsin G and medullasin, two major serine proteinases, were investigated. In gingival crevicular fluid (GCF) obtained from periodontitis patients, almost all activities of both enzymes were not detected, while the amounts of both enzymes determined by enzyme-linked immunosorbent assay increased with increasing GCF volume, indicating the participation of the enzymes in the progression of periodontitis. The reason why their activities were lost in GCF is suggested that they were blocked by their specific serum-derived proteinase inhibitor, especially alpha_1-proteinase inhibitor, since immunoblotting analyzes of GCF using each enzyme and alpha_1-proteinase inhibitor revealed a weak protein band of the respective enzyme and the inhibitor alone and a strong band of the complex of those. In experimental gingivitis of volunteers induced by refraining from all oral hygiene measures for 21 d … More ays, a remarkable increase of these enzymes in GCF were shown prior to the detection of inflammation monitored by various clinical parameters, suggesting the involvement in the development of inflammation. In inflamed gingival tissues, these enzymes were restricted in neutrophils and macrophages and concentrated especially in the vicinity of pocket epithelium partly with degranulation extracelluarly. This finding suggests the participation of these enzymes in direct degradation of host tissues besides the host-defensive mechanism against bacterial pathogens. Moreover, purified enzymes injected into rabbit gingivae caused inflammatory response characterized by noticeable injury to vessels and mild infiltration of inflammatory cells including neutrophils and macrophages. The function of the enzymes, however, were completely lost with the treatment by their specific inhibitor, alpha_1-proteinase inhibitor or alpha_1-antichymotrypsin. From the findings in this study, cathepsin G and medullasin are present in GCF and gingival tissue and participated in the host-defensive mechanism modulated by serum-derived proteinase inhibitors. Once the enzyme-inhibitor balance is lost, these two enzymes conversely play a destructive role against periodontal tissues. Less