Effects of tetracyclines on neutrophil, gingival, and salivary collagenases. A functional and western-blot assessment with special reference to their cellular sources in periodontal diseases

Abstract

The characterization and regulation of matrix metalloproteinases (MMPs) have been studied to determine their role(s) in periodontal tissue destruction. Progress in elucidating the roles of MMPs in periodontal tissue destruction has led to a new concept involving the chemotherapeutic inhibition on MMPs, a therapeutic strategy which less than a decade ago was considered "a difficult and perhaps impossible task." Tetracyclines/doxycycline (DOXY) and their chemically modified nonantimicrobial derivatives (CMTs) are known to inhibit the matrix metalloproteinases, especially preferring human neutrophil collagenase (MMP-8), and prevent the oxidative activation of procollagenases. We characterized by Western blotting the molecular forms and cellular sources of gingival tissue, dental plaque, gingival crevicular fluid (GCF), and salivary MMPs associated with periodontitis. Also the molecular forms of tissue inhibitors of matrix metalloproteinases (TIMP-1 and TIMP-2) in periodontitis were studied by Western blot. Neutrophil (PMN)-derived MMPs were found to predominate in periodontitis, and phospholipase C present in increased amounts in periodontitis sites was found to be a potential inducer of PMN degranulation. We further studied the effects of DOXY on molecular forms of different latent and active MMPs purified from different cellular sources (PMNs, fibroblasts, keratinocytes) and present in vivo in oral exudates (gingival extracts, GCF, and saliva). DOXY inhibition of activated (oxidatively or proteolytically) MMPs were not associated with MMP fragmentation. Michaelis-Menten plots of initial rates of degradation of soluble type I collagen revealed an apparent Km value of 0.3-0.6 microM for MMP-8, and 75 microM DOXY inhibited MMP-8 in a manner which did not result in changes in apparent Km value but did prevent the initial degradation reaching Vmax providing evidence for noncompetitive inhibition. Treatment of patients with long-term DOXY medication results in decreased MMP-8 activities/levels in gingival tissue, crevicular fluid, and saliva, but not fragmentation of MMP-8 in vivo. These data further support and extend the key role of PMN-MMPs in periodontitis, and the activities of these PMN MMPs can be inhibited directly by therapeutic levels of DOXY.