Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases.

OBJECTIVE: Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. MATERIALS AND METHODS: Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. RESULTS: Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. CONCLUSIONS: Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.

Collagenase-2 (MMP-8) and collagenase-3 (MMP-13) in adult periodontitis: molecular forms and levels in gingival crevicular fluid and immunolocalisation in gingival tissue.

AIM: To determine the cellular and molecular forms of MMP-8 (collagenase-2) and MMP-13 (collagenase-3) associated with chronic adult periodontitis by examining the species present in gingival crevicular fluid (GCF) and enzyme distribution in gingival tissue. METHODS: 30-s GCF samples were collected directly from the periodontal pockets of 12 untreated patients using filter paper strips. After elution into buffer, the samples were examined by Western immunoblotting with polyclonal antibodies for MMP-8 and MMP-13 and quantification by scanning image analysis. Individual band intensities were expressed as a percentage of total sample absorbance and mean patient values were calculated. Gingival tissue from 6 patients was fixed in formalin and embedded in paraffin wax. MMP-8 and MMP-13 were localised using the same antibodies and an avidin-biotin-peroxidase detecting system. Double staining was performed with a contrasting substrate reaction. RESULTS: The majority of MMP-8 staining in pre-treatment GCF was present in 80, 75 and 60 kD bands corresponding to prepro-, pro- and active forms of PMN-type enzyme. 43 and 38 kD bands evidently represented active, fibroblast-type MMP-8. Immunoreactivities at >100 kD and < or =30 kD were probably enzyme-inhibitor complex and degraded fragments, respectively. MMP-13 was seen mainly as 60 kD proenzyme with some 40 kD active enzyme and a small proportion of >100 kD complex. The percentages of MMP-8 PMN-type enzyme and MMP-13 proenzyme bands correlated significantly with gingival and bleeding indices (p<0.05). Immunohistochemistry demonstrated MMP-8 in PMNs, sulcular epithelial and also plasma cells in inflamed gingival connective tissue. MMP-13 immunoreactivity was detected in the sulcular epithelium and in macrophage-like cells. CONCLUSION: Multiple species and elevated levels of both MMP-8 and MMP-13 from many rather than single cellular sources in the diseased periodontium are identified in untreated periodontitis GCF and active forms contribute to GCF collagenase activity.

Evaluation of collagenase activity, matrix metalloproteinase-8, and matrix metalloproteinase-13 in horses with chronic obstructive pulmonary disease.

OBJECTIVES: To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 12 horses with COPD and 12 healthy control horses. PROCEDURE: Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage fluid (BALF). RESULTS: Collagenase activity was approximately 7 times higher in samples obtained from horses with COPD, compared with control horses. During stabling, horses with COPD had significantly higher collagenase activity than after being maintained on summer pasture, when activity was similar to that of control horses. Immunoreactivity of MMP-8 and MMP-13 was significantly increased in TELF of horses with COPD, compared with healthy horses. In TELF, a positive correlation was detected between immunoreactivity of MMP-8 and MMP-13 and the amount of degradation of type-I collagen. Macrophages and epithelial cells were the major cellular sources of MMP-8 and MMP-13. CONCLUSIONS AND CLINICAL RELEVANCE: Increased collagenase activity in TELF indicates active ongoing disease and, thus, may reflect lung tissue changes in horses with COPD. Measurements of collagenase activity and MMP immunoreactivity may provide additional diagnostic tools to identify the active phase of chronic lung disease.