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RESEARCH REPORT |
1 Department of Medicine/Invärtes medicin, Helsinki University Hospital, Biomedicum Helsinki, PO Box 700 (Haartmaninkatu 8), FIN-00029 HUS, Helsinki, Finland;
2 Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland;
3 Medico-social, Dental Clinic, Bogazici University, Istanbul, Turkey;
4 Department of Oral and Maxillofacial Diseases, HUCH, Institute of Dentistry, University of Helsinki, Box 41 FIN-00014 Helsinki, Finland;
5 University of Gazi, Faculty of Dentistry, Department of Periodontology, Ankara, Turkey;
6 TNO-Prevention and Health, Leiden, The Netherlands;
7 ORTON Orthopaedic Hospital of the Invalid Foundation, Helsinki, Finland; and
8 COXA Hospital for Joint Replacement, Tampere, Finland
* corresponding author, yrjo.konttinen{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. However, Western blot revealed partially activated MMP-3, MMP-8, and MMP-9 in periodontitis GCF. Active MMP-8 (p < 0.05) and MMP-9 (p < 0.05) correlated with the presence of active MMP-3. It seems that resident gingival fibroblasts produce pro-MMP-3 in GCF, where it becomes activated, probably by cathepsin G or elastase released by neutrophils. Active MMP-3 then activates neutrophil-derived pro-MMP-8 and -9. Different tissue compartments/cells exert co-operative actions in mutual local MMP activation cascades.
KEY WORDS: stromelysin • collagenase • gelatinase • periodontitis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Pro-collagenase (pro-MMP-8) and pro-gelatinase (pro-MMP-9) in GCF mainly derive from fluid-phase neutrophils (Golub et al., 1995). Degranulating neutrophils activated by bacterial plaque release latent pro-enzymes (Ding et al., 1996, 1997). It is not clear how they become activated in the gingival crevice in adult periodontitis.
Stromelysin-1 (MMP-3) activates pro-MMP-8 (Ito and Nagase, 1988) and pro-MMP-9 (Ogata et al., 1992). Neutrophils do not contain stromelysin-1 (Weiss, 1989). Stromelysin-1 (MMP-3) is synthesized by fibroblasts and, to a lesser extent, by some other cells. Also, stromelysins are released as latent pro-enzymes (Okada and Nakanishi, 1989; Ingman et al., 1994, 1996). We speculated that GCF enzymes and gingival tissue stromelysin-1 may participate in reciprocal activation cascades, leading to activation of the crevicular fluid pro-collagenases and pro-gelatinases.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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2; Löe, 1967), the plaque index ( 2; Silness and Löe, 1964), and probing pocket depths ( 4 mm), combined with radiographic evidence of bone loss as detected from orthopantomograms. No study subjects had received periodontal treatment or medication during the preceding 6 mos. None of the patients smoked. GCF sampling and processing were done as has been described in detail elsewhere (Tervahartiala, 2003). Although the ages between the two study groups were different, there were no significant correlations between the age and any of the MMPs studied in adult periodontitis patients or controls, or in both groups combined, indicating that age was not a confounding factor in this context.
Western Blotting
Various molecular-weight forms of MMP-3, -8, and -9 from GCF samples were analyzed by Western blotting (Ingman et al., 1994). Blots were incubated with sheep anti-human MMP-3 IgG (2 µg/mL) (Calbiochem, Oncogene Research Products, San Diego, CA, USA), or rabbit anti-human MMP-8 or MMP-9 IgG at 1:500 and 1:1000 dilutions (antibodies were provided by Dr. Jurgen Michaelis, Department of Pathology, Christchurch Medical School, New Zealand, Michaelis et al., 1990, and Dr. Lars Kjeldsen, Granulocyte Research Laboratory, University Hospital Copenhagen, Kjeldsen et al., 1993, respectively). Evaluation was performed with a Bio-Rad Model GS-700 Imaging Densitometer and Molecular Analyst/PC program (Bio-Rad, Hercules, CA, USA).
Zymography
GCF samples were analyzed for gelatinolytic activities by the use of 7.5% and 11% SDS-polyacrylamide gels containing 1 mg/mL gelatin substrate (Ding et al., 1997). The destained gels were quantified by a Bio-Rad Model GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA, USA).
Immunohistochemical Staining of MMP-3 in Gingival Tissues
We stained 6-µm paraffin sections (Tervahartiala, 2003) with 2 µg/mL anti-human MMP-3 IgG (Oncogene Research Products, Calbiochem, San Diego, CA, USA).
Cell Culture
Human gingival fibroblasts isolated by the explant culture technique were maintained in RPMI-1640 containing 10% fetal calf serum and antibiotics. Confluent cells from the 4–7th passages were transferred to serum-free RPMI-1640 for 24 hrs before culture without or with TNF- (10 ng/mL, R&D Systems Inc., Minneapolis, MN, USA) in serum-free media for 48 hrs. Control and conditioned media were collected.
Activity Assay for MMP-3, MMP-8, and MMP-9
MMP activities were measured by a modified pro-urokinase substrate method (Biotrak activity assay systems, Amersham Biosciences, Buckinghamshire, England) (Hanemaaijer et al., 1998). Color production was measured in a Titertek Multiscan 8-channel photometer (Flow Laboratories, Irvine, Scotland).
Statistical Analysis
Statistical calculations were made by means of a Prism data analysis program (GraphPad Software Inc., San Diego, CA, USA). We used one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple-comparison test, to compare stimulated and non-stimulated fibroblasts. P values of less than 0.05 were considered statistically significant. The Mann-Whitney U Test was used to analyze the differences between the adult periodontitis patients and controls. Pearson’s correlation test was used to assess the correlations among MMP-3, -8, -9, and their different molecular forms. Correlation between plaque index and pro/active forms of MMP-3 was done by the Pearson correlation test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The ratio of the proMMP to active MMP was 0.5 ± 0.1 for MMP-3, 1.2 ± 0.1 for MMP-8, and 3.3 ± 0.7 for MMP-9 in adult periodontitis (mean ± SEM) (see Appendix Table 2). Zymographic analysis revealed a similar but non-significant tendency for total gelatinolytic activity, composed of both the pro-gelatinase and gelatinase bands (2.156 ± 1.152 vs. 0.473 ± 0.277, p > 0.05) (Appendix Table 1) (Fig. 1B). Western blot analysis revealed that the healthy control GCF samples produced only 55 kDa pro-MMP-3 (not any 45 kDa active MMP-3). In adult periodontitis, 55-kDa-molecular-weight pro-MMP-3 proteolytically processed 45-kDa MMP-3, corresponding to its active form, and small-molecular-weight degradation fragments (28 kDa) were observed (Okada et al., 1987; Housley et al., 1993). Some of the MMP-3 bands were in > 100 kDa complexes (Nagase et al., 1997). MMP-8 was either in its latent 75-kDa proform in controls, was not produced at all, or was produced in amounts too low to be detected. Both latent pro-MMP-8 and 60 kDa (active) MMP-8 species were observed in periodontitis. The 55-kDa mesenchymal/fibroblast-type isoform of MMP-8 was found in periodontitis, but not in controls. High-molecular-weight (> 100 kDa) MMP-8 species representing complexes or dimers were detected in periodontitis. MMP-9 was partially proteolytically degraded to 82 kDa, 72 kDa, 47 kDa, and 33 kDA fragments in periodontitis, but not in controls. A 130-kDa MMP-9 band was seen and probably represents MMP-9 complexed with neutrophil gelatinase-associated lipocalin (Kjeldsen et al., 1993) (Figs. 1A, 1B).
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Immunohistochemical Staining of the Gingival Tissues
The apparent number of MMP-3-positive cells and their intensity of staining were high in adult periodontitis compared with healthy control gingiva. MMP-3 immunoreactivity was observed in fibroblast- and macrophage-like cells in gingival granulation tissue in periodontitis (Fig. 2). There were 3.7 ± 0.7 cells per high-power field (x 400) in periodontitis (n = 7) compared with 1.3 ± 0.4 in controls (n = 5) (p = 0.0057). Controls showed negative immunoreactivity (Fig. 2). For morphometry, we examined 5 randomly selected fields per section and analyzed 2 sections for each patient.
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stimulation of fibroblasts (p < 0.001). MMP-8 and MMP-9 synthesis was increased (Fig. 3). We performed the MMP-activity assay with and without aminophenylmercuric acetate to assess separately the total (pro-form and active forms combined) and activated (enzyme only, activated without external aminophenylmercuric acetate activator) MMPs. Although TNF- significantly increased MMP-3, -8, and -9 synthesis, all MMP-3 in the stimulated fibroblast culture supernatant was in the latent pro-form, whereas MMP-8 and -9 were in their active forms (Fig. 3). Zymography of fibroblast supernatants confirmed that MMP-9 was all in the 82-kDa active form (Appendix, Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Healthy gingiva contains some MMP-3, probably necessary for normal tissue remodeling, whereas MMP-3 is pathologically increased in adult periodontitis gingiva. This conclusion was confirmed in in vitro cultures. Fibroblasts increased their MMP synthesis upon stimulation with TNF-, particularly that of MMP-3. TNF--stimulated gingival fibroblasts also produced neutrophil collagenase-2 (MMP-8), in accordance with TNF--stimulated synovial fibroblasts (Hanemaaijer et al., 1997). This enzyme represents, based on its 55-kDa weight, the mesenchymal isoform of MMP-8. All of the MMP-3 released into gingival fibroblast culture supernatant was in its 55-kDa latent pro-form. Since neutrophils, the major cells of the GCF in adult periodontitis, do not contain MMP-3, it can be concluded that the high GCF MMP-3 in adult periodontitis must derive from the gingival tissue compartment. Since TNF--stimulated gingival fibroblasts produce only the 55-kDa latent pro-enzyme MMP-3, but adult periodontitis GCF also contained proteolytically activated 40- to 45-kDa MMP-3, it can be concluded that pro-MMP-3 must, after secretion, undergo proteolytic conversion from its pro- to the corresponding active form. This may partly relate to TIMPs (see Appendix).
Based on the proteolytic activation of MMP-3, MMP-8, and MMP-9, and their proteolytic degradation products in the neutrophil-rich adult periodontitis crevicular fluid, we hypothesized that gingival tissue and crevicular fluid compartments co-operate in mutual proteinase activation cascades. It was found that all MMP-3 released by the stimulated fibroblasts was in its latent form, which is compatible with our hypothesis that neutrophil enzymes like cathepsin G and elastase are needed for its activation. In contrast, some of the MMP-8 and -9 was active. This suggests auto-activation or plasmin-driven activation (Birkedal-Hansen et al., 1993). High levels of t-PA, an effective pro-MMP activator (Ueda and Matsushima, 2001), have been described in TNF-stimulated gingival fibroblasts. Adult periodontitis crevicular fluid contains elevated levels of neutrophil-derived cathepsin G (Tervahartiala et al., 1996) and elastase (Ingman et al., 1994). Cathepsin G and elastase, derived from the primary, azurophilic neutrophil granules, are able to activate pro-MMP-3 (Okada and Nakanishi, 1989; Jenne, 1994). It is therefore proposed that they activate gingival tissue fibroblast-derived pro-MMP-3 in GCF. Pro-MMP-8 and pro-MMP-9 are also released from activated, degranulating neutrophils (from secondary/specific granules; Weiss, 1989). Active MMP-3 then cleaves off the 10-kDa activation peptide blocking the active-site zinc in pro-MMP-8 and pro-MMP-9. This would guarantee co-ordinated activation of pro-collagenase-8 (MMP-8) and pro-gelatinase B (MMP-9), which exert consecutive actions upon degradation of type I collagen and gelatin, respectively. This is supported by the highly significant correlation between the presence of active MMP-3 and the degree of collagenase (MMP-8) and gelatinase (MMP-9) activation in adult periodontitis. This cascade was not activated in controls. Our results demonstrate that this mutual activation cascade between gingival tissue MMP-3 and neutrophil MMP-8 and -9 plays a role only in periodontal tissue destruction, not in normal gingival tissue remodeling, perhaps according to Fig. 4, although the hypothesis has not been fully substantiated. MMPs are also activated by tissue kallikreins, leukocyte elastase, trypsin, and cathepsin G. We and others have shown that bovine and human trypsins are effective activators of pro-MMP-9 (Morodomi et al., 1992; Sorsa et al., 1997). Proteases from Porphyromonas gingivalis are capable of activating MMP-2 (Grayson et al., 2003). When one considers the complexity of pathogenic mechanisms associated with periodontal disease, this Fig., although probably depicting one important activation cascade, is naturally a little simplistic.
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Non-neutrophil cells, such as gingival and periodontal ligament fibroblasts, release MMP-8, whereas monocytes and macrophages form a potentially important source of MMP-9 (Page, 1991; Birkedal-Hansen et al., 1993).
The proposed role of MMP-3 is emphasized by two findings. Fibroblast-mediated synthesis of MMP-3 was stimulated with TNF-, indicating this as a key process in gingival soft tissue inflammation. Second, somewhat surprisingly, the largest difference between healthy controls and periodontitis patients was seen not in levels of collagenase-2 (MMP-8) or gelatinase B (MMP-9), but in stromelysin-1 (MMP-3). This further indicates that "ignition", i.e., the potential to activate pro-collagenases and pro-gelatinases, is the key. This mutual activation cascade (Fig. 4
) is based on cooperative actions between not only gingival tissues and crevicular fluid, but also between fibroblasts and neutrophils, and between serine proteinases and MMPs. This may be important to prevent inadvertent and potentially harmful accidental activation of potent collagenolytic MMP machinery, which occurs only when all the circumstances for mutual and reciprocal co-operation are fulfilled.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received November 28, 2004; Last revision September 12, 2005; Accepted September 29, 2005
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