title: Activation of neutrophil collagenase in periodontitis.
abstract: 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.
authors.affiliation: Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario.
authors.forename: R
authors.initials: R
authors.lastname: Romanelli
authors.forename: S
authors.initials: S
authors.lastname: Mancini
authors.forename: C
authors.initials: C
authors.lastname: Laschinger
authors.forename: C M
authors.initials: CM
authors.lastname: Overall
authors.forename: J
authors.initials: J
authors.lastname: Sodek
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D016022:Case-Control Studies; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005883:Gingival Crevicular Fluid / Q000201:enzymology; D005891:Gingivitis / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D020781:Matrix Metalloproteinase 1; D053509:Matrix Metalloproteinase 13; D020784:Matrix Metalloproteinase 8; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010518:Periodontitis / Q000201:enzymology* / Q000209:etiology; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D019716:Tissue Inhibitor of Metalloproteinase-2; D003094:Collagen; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020784:Matrix Metalloproteinase 8; D020781:Matrix Metalloproteinase 1