Matrix metalloproteinase-7 in periodontitis with type 2 diabetes mellitus.

BACKGROUND AND OBJECTIVE: Elevated levels of matrix metalloproteinase-7 (MMP7) have been observed in serum samples of subjects with type 2 diabetes mellitus (T2DM) and in gingival tissues of subjects with periodontitis. The aim of the present study was to collect in vivo and in silico evidence on the role of MMP7 in the interplay between T2DM and generalized periodontitis (GP). MATERIAL AND METHODS: The extent of MMP7 expression and localization were immunohistochemically analyzed in gingival tissues of patients with GP with T2DM (T2DM/GP, n = 11), systemically healthy patients with GP (n = 7), and systemically and periodontally healthy controls (n = 11). An in silico network model was built to determine the interactions between MMP7 and T2DM pathways. Regulation of neutrophil transmigration by MMP7 was analyzed in a knock-out mice model. RESULTS: In human gingival tissues, the proportion of cells with robust MMP7 expression was elevated in patients with T2DM/GP in comparison to controls (P = .014). According to the in silico analysis, "hydroxyl radical" and "hydrogen peroxide" compounds were among the most central nodes of the network, and were within the shortest paths connecting "glucose" to "MMP7." In MMP7 knock-out mice, an intense accumulation of neutrophils was observed in the gingival epithelium as compared to wild-type mice (P = .0001). CONCLUSION: Elevated MMP7 expression in gingival tissues of patients with T2DM/GP is related to the activation of reactive oxygen species by hyperglycemia. Suppression of MMP7 expression results in impaired neutrophil transmigration in gingiva.

Periodontal health knowledge and smoking are associated with periodontal treatment need according to tooth brushing levels.

OBJECTIVE: The aim of this study is to determine whether periodontal health knowledge is associated with frequency of tooth brushing and periodontal treatment need. METHODS: Four hundred and two subjects participated in the study. Data on sociodemographic variables (age, gender, marital status, income, and education), general health, smoking behaviour tooth cleaning habits and knowledge on periodontal health/disease were collected with a questionnaire. Periodontal treatment need was examined using the Community Periodontal Index of Treatment Needs (CPITN). According to the CPITN scores, the treatment needs were grouped as minimum (CPITN = 0), low-level (CPITN = 1-2), or high-level (CPITN = 3-4). RESULTS: Statistical differences were found between the frequency of tooth brushing and smoking status, marital status, periodontal health knowledge and periodontal treatment needs. Gender (females), place of residence (urban areas), education and periodontal health knowledge had positive relationship with tooth brushing frequency, while smoking and periodontal treatment need had negative relationship. When multivariate logistic regression analysis was applied, age, marriage and poor periodontal knowledge were associated with increased low-level periodontal treatment needs, and age, marriage and smoking were associated with increased high-level periodontal treatment need. CONCLUSION: In the limits of this study, we suggest that gender, smoking habits, marital status, place of residence, education and periodontal health knowledge are determining factors related to tooth brushing frequency. Periodontal knowledge and smoking are associated with periodontal treatment needs.

Oral rinse MMP-8 point-of-care immuno test identifies patients with strong periodontal inflammatory burden.

OBJECTIVE: To determine whether oral rinse matrix metalloproteinase (MMP)-8 levels, measured by three different methods, tissue inhibitor of matrix metalloprotease-1 (TIMP-1) levels and elastase activity differentiate subjects with different periodontal condition; and second, to find out if MMP-8 levels were comparable among the methods used. METHODS: MMP-8 levels were analysed with an immunofluorometric method (IFMA), dentoELISA and commercial ELISA. Also TIMP-1 levels and elastase activity were measured. For statistical analysis 214 study subjects were categorized into four groups, specified by the presence and number of moderate (4-5mm) and deep (≥6mm) periodontal pockets, and bleeding on probing percentage. RESULTS: MMP-8 levels especially measured by dentoELISA and adjusted to the number of teeth per subject differentiated the study group with strong periodontal inflammatory burden from groups with lower levels. This was also verified with receiver operating characteristic (ROC) analysis. Elastase activity associated with higher IFMA and dentoELISA MMP-8 levels. IFMA MMP-8/TIMP and dentoELISA MMP-8/TIMP-1 tended to be higher with the increasing level of periodontal inflammatory burden. TIMP-1 levels decreased with increasing age. CONCLUSIONS: Oral rinse MMP-8 together with TIMP-1 analysis may have potential in complementary periodontal diagnostics. dentoELISA can be applied in quantitative oral rinse chair side biomarker diagnostics.

Prevotella intermedia ATCC 25611 targets host cell lamellipodia in epithelial cell adhesion and invasion.

INTRODUCTION: The Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens, are phylogenetically closely related and potentially connected with oral and gastrointestinal tract disease pathogenesis. The aim of the present study was to examine whether these species differ in their capabilities of adhesion to and invasion of epithelial cells. METHODS: Adhesion and invasion were assayed by standard antibiotic/culture assays and fluorescent microscopy techniques. The effect of Prevotella strains on epithelial cell viability was measured using a commercial cell proliferation assay. RESULTS: The strains P. intermedia ATCC 25611 and P. nigrescens ATCC 33263 adhered to epithelial cells, the adhesion numbers of P. intermedia being twice as high as those of P. nigrescens. These strains invaded epithelial cells but invasion was weak. The adhesion of P. intermedia was specifically targeted to epithelial cell lamellipodia. The number of adhered P. intermedia cells increased or decreased when the formation of lamellipodia was stimulated or inhibited, respectively. None of the tested strains showed toxic effects on epithelial cells; a clinical P. intermedia strain even increased the number of viable cells by about 20%. CONCLUSION: The results suggest that among the P. intermedia group bacteria, P. intermedia and P. nigrescens type strains can adhere to and invade epithelial cells, the capability of P. intermedia ATCC 25611(T) being highest in this context. This strain proved to have a special affinity in binding to epithelial cell lamellipodia.

Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9, -13) and interleukin-8 secretion by fusobacteria.

BACKGROUND/AIMS: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin-8 (IL-8) secretion upon exposure to fusobacteria. METHODS: Eight different oral and non-oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), and IL-8 secretion by epithelial cells. RESULTS: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP-9 and MMP-13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP-2 secretion. F. nucleatum and F. necrophorum also increased IL-8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL-8 production, suggesting that their expression is differently regulated. CONCLUSION: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP-9, MMP-13, and IL-8 from epithelial cells.

Keratinocyte growth factor stimulation of gelatinase (matrix metalloproteinase-9) and plasminogen activator in histiotypic epithelial cell culture.

The purpose of this investigation was to examine the role that keratinocyte growth factor (KGF) plays in the control of matrix-degrading protease activity in epithelial cells. The culture conditions had a significant effect on cellular responses to the growth factor. In histiotypic culture on porous-polycarbonate membranes, porcine periodontal ligament epithelial cells responded to KGF with increased 92-kDa gelatinase (matrix metalloproteinase [MMP]-9) activity. No such response was observed in cells maintained on plastic plates. Epidermal growth factor and platelet-derived growth factor also increased MMP-9 activity in the histiotypic cultures of epithelial cells. Addition of heparin with KGF produced a further increase in MMP-9 activity, with heparin alone having no effect. Precoating of polycarbonate membranes with matrix components showed that fibronectin and an engineered poly-RGD molecule substrate were required for KGF plus heparin to increase MMP-9 activity. Precoating plastic culture plates with the same proteins did not generate the same response. Concomitant with gelatinase activity, KGF also increased urokinase-type plasminogen activator in the epithelial cells. Thus, KGF appears to be an important regulator of protease secretion in epithelial cells.

Keratinocytes in human wounds express alpha v beta 6 integrin.

Cell adhesion receptors of the integrin family play a major role during re-epithelialization of human wounds. We have previously documented that the expression of alpha v family integrins is induced in keratinocytes of mucosal wounds [1]. In the present investigation, we extended these studies to determine whether alpha v beta 6 integrin is expressed during wound healing in humans. Mucosal and epidermal wound sections from 1- to 7-day-old wounds were used for immunolocalization of integrins and their putative ligands. In addition, freshly isolated epidermal keratinocytes were used to study integrin expression in vitro. Expression of alpha v beta 6 integrin appeared relatively late during mucosal and dermal wound healing. Maximal expression was seen in 7-day-old wounds in which epithelial sheets had fused and granulation tissue was present. Fibronectin and tenascin, both possible ligands for alpha v beta 6 integrin, were found concentrated underneath the basal epithelial cells expressing this receptor, and the maximal expression of tenascin coincided with that of alpha v beta 6 integrin. Freshly isolated epidermal keratinocytes did not stain for alpha v beta 6 integrin but began to express this integrin after subculturing. Our results suggest that the expression of alpha v beta 6 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing.

Stimulation of collagenase (matrix metalloproteinase-1) synthesis in histiotypic epithelial cell culture by heparin is enhanced by keratinocyte growth factor.

The role of heparin and heparan sulfate in the control of epithelial collagenase production was investigated utilizing a histiotypic cell culture model. The effect of keratinocyte growth factor (KGF), a heparin-binding growth factor, on collagenase secretion was also examined. Heparin, and, to a lesser extent, heparan sulfate induced release of a 58-kDa, gelatin-degrading enzyme which was subsequently identified as the collagenase, matrix metalloproteinase-1. The increase in collagenase secretion by heparin was further enhanced by the addition of KGF. KGF alone did not have any effect. Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis. Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin. Heparin and heparan sulfate in combination with KGF may thus have important roles in the regulation of epithelial cell collagenase under conditions such as inflammation and wound healing.

Depletion of alphaV integrins from osteosarcoma cells by intracellular antibody expression induces bone differentiation marker genes and suppresses gelatinase (MMP-2) synthesis.

Integrin heterodimers sharing the common alphaV subunit are receptors for adhesion glycoproteins such as vitronectin and fibronectin. They are suggested to play an essential role in cell anchoring, differentiation, and survival. Here, we describe the construction of an expression plasmid coding for an intracellular single-chain antibody against alphaV integrin subunit. Saos-2 osteosarcoma cells transfected with this DNA construct showed an approximately 70-100% decrease in the cell surface expression of alphaVbeta3 and alphaVbeta5 integrins as shown by flow cytometry. Intracellular antibody expression had no effect on the mRNA levels of alphaV integrin. Pulse chase experiments of metabolically labeled integrins showed that the translation of precursor alphaV integrin subunit was not affected. However, the maturation of alphaV integrins as glycoproteins was slow suggesting that the transport from endoplasmic reticulum to Golgi complex was partially prevented. Depletion of alphaV integrins from Saos-2 cells led to a decreased ability to spread on fibronectin and vitronectin. Furthermore, the expression of osteoblast differentiation marker genes, alkaline phosphatase and osteopontin, was induced and concomitantly the expression of matrix metalloproteinase-2 decreased. Thus, alphaV integrins seem to be important regulators of osteosarcoma cell phenotypes. Our data also indicate that the expression of intracellular antibodies is an effective strategy to study the significance of specific integrins for cell phenotype and differentiation.

Doxycycline and chemically modified tetracyclines inhibit gelatinase A (MMP-2) gene expression in human skin keratinocytes.

The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.

Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation.

Actinobacillus actinomycetemcomitans is an important pathogen in periodontitis. In the present study we localized the GroEL- and DnaK-like heat shock proteins (Hsp) in subcellular fractions of 12 A. actinomycetemcomitans strains of various clinical origin and compared their effects on periodontal epithelial cell proliferation and viability. In all strains, GroEL-like protein was found in the membrane, cytoplasm, and periplasm, whereas DnaK-like protein was present in the cytoplasm and periplasm. No correlation was observed between the Hsp expression and the serotype or origin of A. actinomycetemcomitans strains. The bacterial membrane fractions that expressed the GroEL-like protein moderately or strongly induced epithelial cell proliferation more strongly than strains that expressed the protein weakly. The results suggest that GroEL-like Hsp may play a role in the virulence of A. actinomycetemcomitans by increasing epithelial proliferation.

Epithelial cell response to challenge of bacterial lipoteichoic acids and lipopolysaccharides in vitro.

Accumulating dental plaque at the gingival margin contains lipoteichoic acids (LTAs) from the cell walls of gram-positive bacteria. In subgingival plaque associated with periodontal disease the amount of lipopolysaccharides (LPSs) from gram-negative bacteria increases. As the gingival junctional epithelium (JE) is an important structural and functional tissue, participating in the first line defence against apical advancement of dental plaque, this study examined the direct effects of LTAs (from Streptococcus mutans and S. sanguis) and LPSs (from Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Escherichia coli) on two epithelial cell lines (HaCaT and ERM) and a culture model for human JE. The cells were exposed to the LTAs or LPSs (10-50 microg/ml) for variable periods of time. None of the bacterial surface components had any effect on primary adhesion or on the epithelial attachment of the JE cultures. However, cell growth and mitotic activity were consistently reduced in all cultures treated with LTAs. In contrast, LPSs showed only slight or no effects on cell growth and mitotic activity depending on the epithelial cells used. This suggests that LPSs, despite their established role as modulators of inflammation, do not have direct harmful effects - at the concentrations found in dental plaque and gingival crevicular fluid - which would explain the mechanism of epithelial degeneration and detachment from the tooth surface. However, the LTAs appear to inhibit the renewal of epithelium and may thus contribute to degeneration of coronal JE and subgingival colonisation by periodontal pathogens.

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains.

Non-serotypeable Actinobacillus actinomycetemcomitans strains may be derived from the serotypeable ones. In the present study, we compared the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of serotypeable and non-serotypeable A. actinomycetemcomitans strains (n=24) of the same genotype in the same subject (n=6) to find out if alterations on the cell-surface contribute to the non-serotypeability. Serotypeable and non-serotypeable A. actinomycetemcomitans strains showed great similarity in the OMP patterns both within and between subjects. Using serotype-specific antisera, clear immunoblotting LPS profiles in the O-antigenic region were seen in serotype b and c strains but not in non-serotypeable strains from the same subjects. The results suggest that changes in LPS lead to the altered antigenicity of non-serotypeable A. actinomycetemcomitans strains.

Activation of latent collagenase of human leukocytes and gingival fluid by bacterial plaque.

Collagenase activity was studied in human leukocytes, gingival crevicular fluid and bacterial plaque, with soluble radioactive collagen as substrate. Inflamed gingiva liberated vertebrate type collagenase into the crevicular fluid in active form. Healthy gingiva, in contrast, released collagenase in a latent form that could be activated by trypsin or plaque. Plaque also stimulated leukocytes to release collagenase, and activated the latent enzyme.

Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells.

The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)

Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: cellular origin and relationship to periodontal status.

Proteolytic enzymes released by the host cells are associated with the tissue destruction in periodontal diseases. Matrix metalloproteinases (MMPs) have the primary role in this process, since, in concert, they can degrade most of the extracellular matrix components. In the present study, we investigated MMP-2 and MMP-9 in oral fluids of healthy subjects and periodontitis patients and the contributions of different oral cells to the enzyme production. The enzymograms revealed that the main gelatinase in oral rinses, crevicular fluid, and whole saliva migrated at 92 kDa. Activity was also detected at 200 kDa and 130 kDa and minor activity at 86 kDa, 72 kDa, and 40 kDa. Traces of gelatinolytic activity were also detected in pure parotid secretions. The 92-kDa enzyme was identified to MMP-9 and the 200-kDa gelatinase to MMP-2, by means of specific anti-72-kDa antiserum. Gingival keratinocytes produced mainly MMP-9, while gingival and granulation tissue fibroblasts expressed MMP-2. Glandular tissue contained mainly MMP-9, and mRNA for MMP-9 was also found in acinar epithelial cells. Periodontitis patients had significantly higher levels of MMP-9 than healthy subjects. Also, MMP-2 was elevated in periodontitis patients. Periodontal treatment reduced the amount of gelatinases dramatically. This study shows that gelatinases are produced by various cells in the oral cavity. The amount of gelatinases is elevated during periodontal disease, while conventional periodontal treatment efficiently reduces the levels these enzymes. We suggest that MMP-2 and MMP-9 could participate in tissue destruction in periodontitis.

Expression of heparan sulphate and small dermatan/chondroitin sulphate proteoglycans in chronically inflamed human periodontium.

Proteoglycans (PGs) function in regulating aspects of cell behavior, such as proliferation, adhesion, and migration. In this report, we investigated the localization of three heparan sulphate PGs (basement membrane [BM] heparan sulphate PG, CD44, and syndecan-1) and two small dermatan/chondroitin sulphate PGs (decorin and biglycan) in chronically inflamed human periodontium. Frozen sections were analyzed by immunofluorescence microscopy. In inflamed tissue, BM heparan sulphate PG showed reduced immunostaining in subepithelial and subendothelial basement membrane. Loss of CD44 and syndecan-1 was common in epithelial cells of inflamed periodontal tissue. Suprabasal keratinocytes of epithelium expressed involucrin, a cornified envelope protein and marker for epithelial differentiation, while the expression of syndecan-1 was weak or absent. In contrast, expression of the mesenchymal variant of CD44 and syndecan-1 was strong in infiltrating lymphocytes. Small dermatan/chondroitin sulphate PGs, decorin and biglycan, were also present in markedly reduced amounts in the periodontal connective tissue in chronic inflammation. In addition, decorin localized in the connective tissue along short rod-like structures. The results suggest that proteoglycan-dependent intercellular adhesion of keratinocytes is decreased and that adhesion of lymphocytes to matrix molecules via cell surface PGs increased in chronic inflammation. Disappearance of adhesion-modulating small dermatan/chondroitin sulphate PGs may further regulate cell migration in inflamed periodontium.

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium.

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions.

Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.

Membrane components of Treponema denticola trigger proteinase release from human polymorphonuclear leukocytes.

Tissue destruction during periodontitis is believed to be primarily brought about by leukocyte proteinases. We postulate that oral spirochetes cause discharge of polymorphonuclear leukocyte (PMN) lysosomal enzymes. Effects of Treponema denticola 53-kDa outer membrane protein, lipopolysaccharide (LPS), and peptidoglycan on degranulation of matrix metalloproteinases (MMP)-8 (collagenase) and -9 (gelatinase), cathepsin G, and elastase by human peripheral blood PMNs were studied by specific enzyme assays and Western blot analysis. T. denticola 53-kDa kDa outer membrane protein was found to be a particularly efficient inducer of MMP-8 release. The induction was comparable with that of phorbol myristate acetate, a known inducer of PMN specific granule discharge. All of the treponemal substances, most notably the 53-kDa protein and LPS, induced release of MMP-9, a component of C-type granules. Both collagenase and gelatinase released from PMNs were mostly in active forms. Release of cathepsin G and elastase was also observed with the 53-kDa protein treatment. The other T. denticola substances did not induce release of these serine proteinases. Lactate dehydrogenase was not released from PMNs by the treatments, indicating that the degranulation was specific and not caused by toxic effects of the substances. This was confirmed by transmission electron microscopy of PMNs treated with the 53-kDa protein that showed rapid vacuole formation and cell shape changes but no disintegration of the cells. Thus, T. denticola may participate in the PMN-dependent extracellular matrix degradation during the course of periodontal inflammation by triggering the secretion and activation of matrix metalloproteinases.