Hypochlorous acid induced activation of human neutrophil and gingival crevicular fluid collagenase can be inhibited by ascorbate.

Interstitial collagenase either obtained from human neutrophils by phorbol myristate acetate (PMA) induced degranulation or isolated from human gingival crevicular fluid was found to be activated by addition of an oxidative agent, hypochlorous acid (HOCl). Collagenase released by PMA stimulated neutrophils was completely in latent form but underwent partial autoactivation during 16 h incubation at 22 degrees C in the presence of soy bean trypsin inhibitor. The partial autoactivation was potentiated to complete activation of released collagenase after addition of exogenous HOCl. Ascorbate prevented this activation of neutrophil collagenase. Isolated human gingival crevicular fluid collagenase represented an apparent Mr of 70 kD in completely latent form, whereas 70/54 kD enzyme species were detected for partially autoactive form of the enzyme. Western blot analysis of gingival crevicular fluid using a polyclonal antibody raised against purified human neutrophil collagenase revealed the same 70/54 kD molecular forms of the enzyme. The latent gingival crevicular fluid collagenase was also activated by HOCl and this activation could be prevented by ascorbate. Activation of the 70 kD latent collagenase by HOCl as well as by other non-proteolytic activators such as an organomercurial compound (phenylmercuric chloride) and a gold(I) compound (gold thioglucose) was not associated with detectable changes in apparent Mr, whereas trypsin activation resulted in fragmentation of 70 kD enzyme to 54 kD species. Our results provide further evidence for the neutrophil origin of gingival crevicular fluid collagenase and suggest that, in addition to proteolytic activation, oxidative and antioxidative agents seem to be able to regulate neutrophil collagenase activity.     

A trypsin-like protease from Bacteroides gingivalis: partial purification and characterization

Extracts of cell sonicates of Bacteroides gingivalis were shown to contain proteo-lytic enzymes capable of degrading connective tissue proteins. In this study, neutral proteolytic enzymes, i.e. collagenase and a trypsin-like protease, were isolated. The trypsin-like protease was readily separated from collagenase by affinity chromatography on Benzamidine-Sepharose. Proteases were further purified by gel filtration on Sephacryl S-200; apparent molecular weights of 35 kDa and 70 kDa were obtained for a trypsin-like protease and collagenase, respectively. Further characterization of the potent trypsin-like protease showed that the enzyme was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and benzamidine and by metalloprotease inhibitor EDTA, as well as ascorbic acid. Activation of the enzyme was observed with reducing agents and human serum. The trypsin-like protease was found to be capable of degrading native type IV collagen and denatured type I collagen but not native type I collagen. Thus, we conclude that in addition to collagenase a potent trypsin-like protease from Bacteroides gingivalis may be involved in the etiopathogenesis of periodontal disease. Since the trypsin-like protease is able to degrade the basement membrane collagen (type IV) in the presence of human serum, this enzyme may be a potent virulence factor of Bacteroides gingivalis in relation to invasiveness and connective tissue destruction.     

Matrix metalloproteinase-8 (MMP-8) in pulpal and periapical inflammation and periapical root-canal exudates

AIM: To study the presence, levels and molecular forms of matrix metalloproteinase (MMP) -8 (collage-nase-2) in pulpal and periapical inflammation, and the changes in MMP-8 levels in root-canal exudates during root-canal treatment. METHODOLOGY: Periapical exudate samples were collected from 11 necrotic teeth with radiographically verified periapical periodontitis during three root-canal treatment visits with interappointment calcium hydroxide (Ca(OH)2) medication. MMP-8 levels and molecular forms were analyzed with immunofluorescent assay (IFMA) and Western immunoblot. Inflamed pulp tissue and periapical granuloma tissue (n = 10 for both) were obtained from other patients and used for MMP-8 immunohistochemical (IHC) staining. RESULTS: The periapical exudate samples demonstrated marked differences in MMP-8 levels between the teeth in the first visit and significant decrease in MMP-8 levels during the root-canal treatment (P = 0.0107). One specimen failed to show a decrease in MMP-8 below 1000 ng mL(-1) a vertical root fracture was later diagnosed and the tooth extracted. IHC staining showed that in addition to PMN-leucocytes, macrophages and plasma cells produced MMP-8 in pulp and periapical granulomas. CONCLUSIONS: The findings demonstrate the presence of MMP-8 in the inflamed pulp and periapical tissue, indicating that MMP-8 has a role in pulpal and periapical inflammation, most likely participating in tissue extracellular matrix degradation. They further indicate that MMP analysis from periapical exudate could be used to indicate and monitor inflammatory activity and the success of treatment in teeth with periapical lesions.     

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

OBJECTIVE: Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatin-ases present in dental plaque were investigated. MATERIALS AND METHODS GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. RESULTS: PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml^-I) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.I-1.0 U ml^-1 PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-I. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. CONCLUSION: Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.     

Collagenases in different categories of peri-implant vertical bone loss

The loosening of dental implants is associated with peri-implant vertical bone loss. The mechanisms and mediators of this bone destruction are not known. To test the hypothesis that collagenase-2 and collagenase-3 might be markers or maybe even mediators in this process, we measured collagenase-2 (time-resolved immunofluorometric assay) and collagenase-3 (quantitative immunoblot) in peri-implant sulcus fluid in 49 implant sites in 13 patients. Vertical bone loss was graded as being < 1 mm, from 1 to 3 mm, or > 3 mm. The severity of inflammation, as rated according to Gingival Index, did not correlate with the category of bone loss (p > 0.05). Collagenase-2 and collagenase-3 were higher (p < 0.05) in the group which had lost > 3 mm of bone than in the two other groups. Gingival Index is not a clinically important marker for bone loss, but collagenase-2 and collagenase-3 in peri-implant sulcus fluid are. They might participate in peri-implant osteolysis.     

MMP-9 activation by tumor trypsin-2 enhances in vivo invasion of human tongue carcinoma cells

Various human cancer cells express tumor-associated trypsinogen-2 (TAT-2), which can efficiently activate matrix metalloproteinases (MMPs) in vitro. MMP-2 and MMP-9 are particularly associated with the invasive malignant potential of several tumors. To investigate the role of TAT-2 in tumor invasion, we overexpressed TAT-2 in two malignant human squamous cell carcinoma cell lines of tongue and in non-malignant human papilloma virus transformed gingival keratinocytes. The TAT-2 overexpression significantly increased the levels of active MMP-9 in the most malignant cell line. TAT-2-transfected cells intravasated (invaded blood vessels) up to 60% more efficiently than did the control cells in an in vivo chick embryo chorioallantoic membrane invasion model. This increased intravasation was almost completely abolished by a specific tumor-associated trypsin inhibitor (TATI). These results indicate that TAT-2 has a role in the invasive growth of tumors, either alone or in cascade with gelatinases, especially by generating active MMP-9.     

Gingival crevicular fluid matrix metalloproteinase-8 levels following adjunctive use of meloxicam and initial phase of periodontal therapy

BACKGROUND: The purpose of the present study was to assess the effects of adjunctive meloxicam on the matrix metalloproteinase-8 (MMP-8) levels of gingival crevicular fluid (GCF) in chronic periodontitis patients following the initial phase of periodontal therapy. METHODS: Twelve chronic periodontitis patients received 7.5 mg meloxicam, and 10 patients received placebo tablets together with scaling and root planing in a 1 x 1 regimen for 10 days. Scaling and root planing were performed on day 3 of drug intake. The MMP-8 levels in GCF samples obtained before and on day 10 of drug intake were determined by using the immunofluorescence assay. Plaque index (PI), papilla bleeding index (PBI), and GCF MMP-8 levels were compared within each patient group, between the 2 patient groups, and also with a clinically healthy control group using non-parametric statistical analyses. RESULTS: Both meloxicam and placebo groups showed statistically significant reductions in PBI, PI, and GCF MMP-8 levels on day 10 compared to baseline (P<0.01). The GCF MMP-8 level on day 10 in the meloxicam group was similar to the clinically healthy control group (P>0.05), while it was significantly higher in the placebo group (P<0.01). Positive correlations were found between MMP-8 total amounts and PBI scores at baseline and day 10 of drug intake in the patient groups. CONCLUSIONS: Meloxicam showed a tendency to reduce GCF MMP-8 levels in vivo within the first 10 days when used as an adjunct in the initial phase of periodontal treatment that consists of scaling and root planing. Verification of this effect on collagenase-2 downregulation, as well as on the clinical periodontal parameters in long-term studies using larger test and control groups, is needed to provide further support for the adjunctive use of selective cyclooxygenase (COX)-2 inhibitors in the treatment of chronic periodontitis.     

Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis

BACKGROUND: A rapid chair-side test based on the immunological detection of elevated levels of collagenase-2 (matrix metalloproteinase-8, MMP-8) in gingival crevicular fluid (GCF) was developed to identify and monitor the course and treatment of adult periodontitis. METHODS: MMP-8 was determined in GCF from periodontitis (11 patients, 90 sites), gingivitis (10 patients, 58 sites) and healthy control (8 patients, 59 sites) sites (i) by a test stick incorporating monoclonal antibodies to two epitopes on MMP-8 and (ii) by measuring MMP-8 concentration by a quantitative immunofluorometric assay. Patients with adult periodontitis were treated by scaling and root planing (SRP) and received oral hygiene instructions. GCF MMP-8 testing and clinical measurements were done before and after SRP. RESULTS: MMP-8 GCF levels and chair-side test differentiated periodontitis from gingivitis and healthy control sites. MMP-8 GCF levels > 1 mg/l and positive chair-side test identified especially severe periodontitis sites. A positive and negative test stick result, the outcome of which was rapidly detectable in 5 mins, in GCF correlated well with MMP-8 immunofluorometric assay analysis from the collected GCF samples and the severity of periodontitis. Scaling and root planing reduced the MMP-8 levels in severe periodontitis sites with positive MMP-8 test and gingival probing pocket depth (PD) > 5 mm before treatment. The test stick result and the quantitative assay were discrepant in only 18 of the 207 sites tested, thus agreement was very good (kappa = 0.81). With a threshold of 1 mg/l MMP-8 activity the chair-side test provided a sensitivity of 0.83 and specificity of 0.96 (n = 207). CONCLUSION: The MMP-8 test can be used to differentiate periodontitis from gingivitis and healthy sites as well as to monitor treatment of periodontitis. A reduction in GCF MMP-8 levels and a change in test stick result provide a means to optimize patient control during maintenance of periodontal treatment.     

Matrix metalloproteinases (MMP-8 and -9) and neutrophil elastase in gingival crevicular fluid of cyclosporin-treated patients

BACKGROUND: Gingival overgrowth (GO) is one of the most important side effects of cyclosporin A (CsA) medication, but its pathogenesis is not completely understood. The aim of this study was to identify and compare collagenase-2 (MMP-8), gelatinase-B (MMP-9), and neutrophil (PMN)-elastase levels in gingival crevicular fluid (GCF) from 15 renal transplant patients receiving CsA therapy and exhibiting CsA GO, 14 patients with gingivitis, and 10 periodontally healthy subjects. METHODS: Clinical data were obtained on plaque index, papilla bleeding index, and hyperplastic index from each site studied. GCF samples and clinical data were collected from: 2 sites exhibiting CsA GO (CsA GO+) and 2 sites not exhibiting CsA GO (CsA GO-) in each CsA-treated patient; 2 diseased sites in each patient with gingivitis; and 2 healthy sites in each subject with clinically healthy periodontium. CsA GO+ and CsA GO- sites were divided into 2 subgroups as clinically not inflamed (PBI = 0) and inflamed (PBI > or =1). GCF MMP-8, MMP-9, and PMN-elastase levels were analyzed by immunofluorometric assay. RESULTS: GCF MMP-8 and -9 levels and clinical degrees of gingival inflammation in CsA GO+ sites were similar to those in diseased sites. However, GCF elastase levels were significantly lower in CsA GO+ sites compared to those in diseased sites. GCF MMP-8, -9 and PMN-elastase levels were not different between CsA GO- sites and healthy sites. Additionally, GCF MMP-8 and -9 levels in inflamed CsA GO+ sites were higher but not statistically significantly than those in diseased sites. In contrast, GCF PMN-elastase levels in inflamed CsA GO+ sites were significantly lower than the levels in diseased sites. CONCLUSIONS: These results show that CsA therapy does not have a significant effect on GCF MMP-8 and MMP-9 levels, but the gingival inflammation seems to be the main reason for their elevations. However, low GCF PMN-elastase levels can be an important factor in the pathogenesis of CsA-induced gingival overgrowth. CsA therapy does not eliminate the potential use of GCF MMP-8 and -9 as future diagnostic markers of gingival inflammation.