Romanelli R, Mancini S, Laschinger C, Overall C M, Sodek J, McCulloch C A
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario.
Infect Immun. 1999 May;67(5):2319-26. doi: 10.1128/IAI.67.5.2319-2326.1999.
Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.
中性粒细胞胶原酶（基质金属蛋白酶8 [MMP - 8]）是炎症性疾病中组织破坏的重要介质。对厌氧性牙周感染的研究表明，龈沟液中的活性MMP - 8与进展期牙周炎中牙周组织的降解有关，而在牙龈炎中潜伏酶占主导。由于MMP - 8的激活似乎是牙周炎中的关键步骤，我们使用可溶性生物素化胶原底物检测了龈沟液样本中MMP - 8的激活情况。对牙周炎、牙龈炎和对照样本的龈沟液分析显示，与牙龈炎（n = 17）样本相比，牙周炎（n = 12）样本中的活性胶原酶水平高出六倍（P < 0.001），牙龈炎样本的活性水平较低，而对照（n = 25）样本无活性。收集龈沟液后，体外未发生潜伏胶原酶的进一步激活。虽然免疫印迹法可检测到MMP - 1和MMP - 8，但未检测到MMP - 13，针对MMP - 1的阻断抗体表明胶原酶活性主要由MMP - 8贡献，MMP - 8定位于病变组织的基质中。龈沟液中的MMP - 8主要以60 kDa的形式迁移，还有少量78 kDa的形式，而从外周中性粒细胞分离的MMP - 8分别以70 kDa和89 kDa迁移，分别对应于该酶的活性形式和潜伏形式。60 kDa和70 kDa条带中的大多数MMP - 8选择性地与金属蛋白酶组织抑制剂2和胶原结合，表明这些条带中的大多数但并非全部酶处于激活形式。然而，不同样本中龈沟液78 kDa和60 kDa形式的量与通过胶原酶测定法测得的潜伏酶和活性酶不相关（r2 = 0.028）。总体而言，这些研究在龈沟液中鉴定出了潜伏和活性MMP - 8的不同形式，这些形式似乎源于牙周炎中发生的独特激活机制。分别从龈沟液和中性粒细胞样本获得的60 kDa和70 kDa条带中存在MMP - 8的潜伏、激活和超激活形式，这进一步表明了MMP - 8激活的复杂性。
