Langerhans cells express matrix metalloproteinases 9 and 2 and tissue inhibitors of metalloproteinases 1 and 2 in healthy human gingival tissue and in periodontitis

BACKGROUND: As antigen-presenting cells, Langerhans cells may play an important role in the initiation and maintenance of periodontal disease. This study is the first report that extends our knowledge of the expression of matrix metalloproteinases and their endogenous tissue inhibitors by Langerhans cells in healthy and diseased gingival tissues. METHODS: Single and double immunolabeling procedures were carried out using monoclonal antibodies against CD1a, matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2, and analyzed by conventional and confocal microscopes. RESULTS: Langerhans cells expressed matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2 in healthy and diseased gingival tissues. The tissue inhibitors of matrix metalloproteinase-positive Langerhans cells were mainly observed in the upper epithelial layers. Matrix metalloproteinase 9-positive Langerhans cells were observed especially during periodontitis and in the basal epithelial layer or crossing the basement membrane. CONCLUSION: During periodontal disease, changes in the expression of matrix metalloproteinases and their tissue inhibitors by gingival Langerhans cells could be implicated in the migration of the cells towards the connective tissue.     

Tissue inhibitors of metalloproteinases level and collagenase activity in gingival crevicular fluid: the relevance to periodontal diseases

OBJECTIVES: To provide an overall assessment of levels of tissue inhibitors of metalloproteinases (TIMPs), collagenase activities, and of immuno-reactivities for matrix metalloproteinases (MMP)-1 and -8 in gingival crevicular fluid (GCF) obtained from healthy subjects, and gingivitis and periodontitis patients, and to analyse the relationships between periodontal tissue destruction and the GCF components in periodontal diseases by principal component analysis. MATERIALS AND METHODS: GCF was sampled with sterile paper strips from 10 gingivitis and 11 periodontitis patients. Ten volunteers served as clinically healthy controls. TIMP-1 and -2 protein amounts in GCF were measured by ELISA, and active and APMA-activatable collagenase activities were determined by functional assays using image-analysis after SDS-PAGE. RESULTS: GCF TIMP-1 level and both active and latent collagenase activities were significantly higher in the diseased groups than in the healthy group. TIMP-2 was detectable in only 29% of all subjects (mean: 2.06 ng). Western blot analysis showed that MMP-8 was the major interstitial collagenase in the GCF of the diseased groups. Principal component analysis using clinical parameters and the GCF components has indicated components one to three account for 87% of total variation when evaluating the relevance of their measurements to periodontal diseases. CONCLUSIONS: We conducted the functional and immunological characterization of MMPs and TIMPs in the GCF of periodontally diseased patients. Principal component analysis indicated components one to three explaining 87% of total variation, and further suggested that higher collagenase activity (especially in active collagenase) would be an important marker in evaluating the pathogenesis of periodontitis. Consequently, these observations may have significant therapeutic and diagnostic implications.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue?

BACKGROUND: Evidence of the role of matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in periodontal destruction is now well established. The purpose of this study was to quantify, in healthy and diseased upper gingival connective tissue, the area fraction (AA%) occupied by collagen fibers, the cell number belonging to inflammatory cell subsets, and the amounts of MMPs and TIMPs (tissue inhibitors of MMPs) in order to investigate the possible correlations, if any, between such molecules, collagen loss, and inflammatory cell subsets. METHODS: Gingival tissue specimens from 6 healthy controls (C) and 6 patients with severe periodontitis (P) were divided into 2 groups. The first group of specimens was frozen and used for the staining of collagen fibers by sirius red F3Ba and for immunohistochemistry with antibodies against CD8, CD4, CD22, CD68, and TIA-1 molecules. The second group was used for organ culture, zymography, Western blotting, and dot blotting. Morphometric and automated image analysis was performed for the evaluation of the area fraction occupied by collagen fibers, the number of inflammatory cell subsets and for enzymatic activities developed by MMPs, and the amounts of TIMPs expressed during periodontal disease. RESULTS: In group P, the area fraction of collagen fibers (33 +/- 10%) was significantly decreased (P < 0.0002) when compared to group C (60 +/- 7%), and was correlated with the number of all inflammatory cells and amounts of MMPs and TIMPs. In group P, there were significant increases of CD8+, CD22+, CD68+, and TIA-1+ cells, as well as increases in the amounts of MMP-1, MMP-2, MMP-3, MMP-9, and the active form of MMP-9. The active form of MMP-9 and the amount of TIMP-1 were positively correlated with the number of CD22+, CD68+, and TIA-1+ cells. CONCLUSIONS: The present study showed an imbalance between MMPs and TIMPs associated with the pathologic breakdown of the extracellular matrix during periodontitis. The active form of MMP-9 could be a marker for the clinical severity of periodontal disease.     

The systemic immune response is more prominent than the mucosal immune response in the pathogenesis of periodontal disease

BACKGROUND, AIM: The diseased periodontium appears to express features of a systemic and a mucosal immune response. Our aims were to determine differences in immunoglobulin expression between gingivitis and periodontitis lesions and to ascertain whether immune and inflammatory cells were recruited into the diseased periodontium by the mucosal addressin adhesion molecule (MAdCAM-1). METHODS: In situ hybridization and immunohistochemistry were used to detect the expression of chemokines, adhesion molecules and immunoglobulins in tissue sections of gingival and granulation tissues excised from periodontitis-affected sites and of healthy tissue and gingivitis-affected tissue excised during crown-lengthening procedures. RESULTS: Greater numbers of plasma cells were observed in periodontitis gingival/granulation tissue lesions compared with gingivitis lesions. While IgA1 were predominant in all lesions, IgA2 and J-chain expressing plasma cells were present in increased proportions in gingival tissues compared with granulation tissue. Intracellular adhesion molecule-1 (ICAM-1) was higher in periodontitis than in gingivitis and interleukin-8 mRNA was higher in lesions with a pronounced neutrophil infiltrate. Vascular cell adhesion molecule-1 (VCAM-1) localized to the deep connective tissue and indicated the presence of a systemic type of immune response in this region. Periodontal tissues (n=71 biopsies) did not appear to express MAdCAM-1, in positive control sections of small intestine where it was detected. CONCLUSION: Overall, the systemic-type immune response is predominant, and although the mucosal immune response is minor and limited to the superficial tissues it may have an important role in the host defense to periodontal pathogens.     

Differential gene and protein expression of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues from drug induced gingival overgrowth

Objectives

The purpose of this study was to analyse mRNA expression and protein localization of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues removed from drug (calcium-channel blocker) induced gingival overgrowth and periodontitis patients.

Design

Employing RT-PCR, we evaluated TIMP-3 and TIMP-4 mRNA levels of 20 human gingival tissue samples taken from patients suffering gingival overgrowth (GO) and periodontitis (P). Then, using immunohistochemistry we investigated the TIMP-3 and TIMP-4 protein localization of five sample tissues from each group.

Results

TIMP-4 mRNA levels in GO-gingiva tended to be lower than in P-gingiva but the results differed little (p = 0.22). Varying degrees of inflammation in the protein localization of TIMP-3 and TIMP-4 were found. TIMP-4 immunoreactivity (IR) was weak in the endothelial cells, fibroblasts, epithelial basal and parabasal cells while the degree of inflammation differed as well. TIMP-3 and TIMP-4 IR in inflammatory cells, including lymphocytes, plasma cells, and macrophages, were faint and intense respectively. For P-gingiva, both TIMP-3 and TIMP-4 IR expression was weak in the endothelial cells, fibroblasts, basal and parabasal epithelial layers. Expression of TIMP-3 was faint in the inflammatory cells, whereas TIMP-4 IR was strong.

Conclusion

Our findings suggest that TIMP-3 and TIMP-4 expression differs in GO and P-gingival tissues, both of which are potentially involved in pathogenesis.     

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.     

IL-4- and IL-6-producing cells in human periodontal disease tissue

IL-4- and IL-6-producing cells in human periodontal disease tissues were investigated using immunohistochemical and in situ hybridization techniques. Immunohistochemical analysis demonstrated the presence of IL-4-producing cells within the CD45RO⁺ subset and the percentage of IL-4+ cells was significantly higher in periodontal lesions than in gingivitis tissues (p<0.01). The percentage of IL-6-producing memory cells was higher in periodontal lesions compared with gingivitis tissues, although it was not statistically significant (p<0.05). A reverse tendency in IL-4- and IL-6-positive cells was observed in a few individual cases. No IL-4 mRNA could be detected using the in situ hybridization technique. However, high levels of IL-6 mRNA were present in clinically healthy tissues, with a further increase in both epithelium and connective tissues affected by gingivitis, although only the former was significant (p< 0.025). There was a significant decrease in IL-6 mRNA in both the connective tissue (p<0.025) and epithelium (p<0.01) in periodontitis tissues compared with levels in gingivitis tissues. However, the levels of IL-6 mRNA in periodontal tissues were high compared with those of IL-1 mRNA, which was used in this study as a positive control. These results suggest that Th2–type cells may accumulate in periodontal lesions.     

Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. I. Light, immunofluorescence and electron microscopic studies

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.     

硫酸软骨素蛋白多糖与牙周炎的相关性

本实验旨在研究硫酸软骨素蛋白多糖与牙周炎的相关性。采用免疫组织化学方法,对牙周炎引起的硫酸软骨素蛋白多糖变化进行研究。结果显示硫酸软骨素(DS、C4S、C6S)广泛分布于上皮下结缔组织、牙周韧带和血管周围,在牙槽骨局限于哈佛氏管和骨小腔内壁。牙周炎时随着组织溶解破坏,其染色反应减弱消失,说明硫酸软骨素是牙周组织的基本成分,与牙周炎关系密切。     

Use of hyaluronic acid binding protein for detection of hyaluronan in ligature-induced periodontitis tissue

This study was designed to demonstrate, by use of biotin-labeled hyaluronic acid binding protein (HABP) and an avidin-enzyme system, the localization of hyaluronan (HA) in periodontal tissue of beagle dogs during experimentally induced periodontitis. Experimental periodontitis was induced in the dogs by ligation of the gingival sulcus. Experimental tissue was collected at 0, 3, 7 and 21 days after ligation. HA was revealed by strong staining in the intercellular space around epithelial cells and periodontal ligament, and by light staining in the gingival connective tissue. According to the progression of periodontal tissue breakdown, HA was detected in a small number of leukocytes and monocytes, on the surface of osteoclasts, the surface of alveolar bone, thickened endothelium and in epithelial cells related to rete peg formation. Streptomyces hyaluronidase-treated specimens gave negative staining. This study suggests that HA may be associated with the inflammatory reaction in experimental periodontitis tissue.     

Use of hyaluronic acid binding protein for detection of hyaluronan in ligature-induced periodontitis tissue

This study was designed to demonstrate, by use of biotin-labeled hyaluronic acid binding protein (HABP) and an avidin–enzyme system, the localization of hyaluronan (HA) in periodontal tissue of beagle dogs during experimentally induced periodontitis. Experimental periodontitis was induced in the dogs by ligation of the gingival sulcus. Experimental tissue was collected at 0, 3, 7 and 21 days after ligation. HA was revealed by strong staining in the intercellular space around epithelial cells and periodontal ligament, and by light staining in the gingival connective tissue. According to the progression of periodontal tissue breakdown, HA was detected in a small number of leukocytes and monocytes, on the surface of osteoclasts, the surface of alveolar bone, thickened endothelium and in epithelial cells related to rete peg formation. Streptomyces hyaluronidase-treated specimens gave negative staining. This study suggests that HA may be associated with the inflammatory reaction in experimental periodontitis tissue.     

Hepatocyte growth factor (HGF) system in gingiva: HGF activator expression by gingival epithelial cells

Hepatocyte growth factor (HGF) acts as a mitogen, motogen, morphogen, and anti-apoptotic factor for various kinds of epithelial cells. We previously showed that periodontal ligament and gingival fibroblasts secreted an HGF-like chemoattractant for a gingival epithelial cell line and found that the HGF content of gingival crevicular fluid was well correlated with clinical parameters and interleukin-1beta level. Since HGF is secreted as an inactive form (proHGF), and converted to an active form by serine proteases such as HGF activator (HGFA), extracellular processing of proHGF is presumed to be critical in the regulation of HGF activity. To examine the role of the HGF system in epithelial invasion followed by loss of connective tissue attachment in periodontitis, mRNA expression of HGF, its receptor (c-met) and HGFA in gingival tissues was monitored. Ten gingival biopsies were obtained, and epithelium and connective tissues were separated by enzymatic digestion. The gene expression of HGF and keratinocyte growth factor (KGF) in gingival connective tissue, and c-met, HGFA and KGF receptor (KGFR) in gingival epithelial tissues was monitored using RT-PCR. Furthermore, HGFA protein in the conditioned medium of cultured primary gingival epithelial cells was examined using Western blotting. All the connective tissue samples expressed KGF, and 8 out of 10 samples expressed HGF. All the epithelial samples expressed KGFR and c-met, whereas 5 out of 10 samples expressed HGFA. Protein expression of HGFA by cultured primary gingival epithelial cells was also confirmed. In terms of local production and activation of HGF in gingival tissue, these results suggest that synergistic expression of HGF in connective tissue and HGFA expression in epithelium may contribute to disease progression in periodontitis.     

Hydrocortisone affects the expression of matrix metalloproteinases (MMP-1, -2, -3, -7, and -11) and tissue inhibitor of matrix metalloproteinases (TIMP-1) in human gingival fibroblasts

BACKGROUND: There is a positive correlation between the course of periodontal disease and psychosocial stress status. Stress leads to activation of the hypothalamic-pituitary-adrenal axis, resulting in increased cortisol release. The present study evaluates the effect of two different hydrocortisone concentrations on mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) in cultured, human gingival fibroblasts. METHODS: Gingival fibroblasts were stimulated with 10(-7) or 10(-9) M hydrocortisone for 24 hours; untreated cells served as controls. Alterations in the expression of MMP-1, -2, -3, -7, -11 and TIMP-1 and -2 were evaluated using real-time polymerase chain reaction and Western blotting. beta-actin mRNA expression was used as a reference to normalize gene expression. RESULTS: Although the higher hydrocortisone concentration upregulated MMP-1, -2, -7, -11, and TIMP-1 (P <0.05) expression, the lower concentration induced downregulation or diminished upregulation. The lower hydrocortisone concentration induced a 23-fold increase in MMP-3 gene expression, whereas the higher concentration induced less upregulation; however, protein expression was regulated similarly by both hydrocortisone concentrations. The effect of hydrocortisone on TIMP-2 expression was not significant (P >0.05). CONCLUSIONS: Hydrocortisone produced a dose-dependent regulation of MMP and TIMP expression. The higher hydrocortisone concentration significantly upregulated expression of MMP-1, -2, -7, and -11 and TIMP-1 in human gingival fibroblasts, which may constitute a mechanism underlying the increased periodontal breakdown associated with psychosocial stress status.     

Influence of phase I periodontal therapy on levels of matrix metalloproteinase 1 and tissue inhibitor of metalloproteinase 1

Background

Matrix metalloproteinase-1 (MMP-1) is a member of a family of enzymes that can degrade most extracellular matrix macromolecules. Extracellularly, MMPs are controlled by tissue inhibitors of metalloproteinases (TIMPs) and by mechanisms of pro-MMP activation. Levels of MMPs and TIMPs change during healing, inflammation, and normal tissue turnover. Herein we aimed to evaluate the levels of MMP-1 and TIMP-1 in gingival crevicular fluid (GCF) from periodontally healthy patients (control group) and chronic periodontitis patients before and after phase 1 therapy.

Methods

In this study we examined 30 patients who had chronic periodontitis with probing depth sites ⩾5 mm and a clinical attachment level (CAL) ⩾5 mm. We included 30 periodontally healthy patients as a control. Clinical measurements such as plaque (PI) and gingival (GI) indices, papillary bleeding index (PBI), probing depths (PD), and CAL were recorded both before treatment (BT) and after phase I periodontal treatment (AT). Assays for MMP-1 and TIMP-1 were performed with an enzyme-linked immunosorbent assay (ELISA) method.

Results

All clinical parameters were significantly reduced at the post-therapy visit. MMP-1 levels were significantly higher in patients BT than the controls; however, the patients AT were not statistically different than the controls. TIMP-1 levels in patients BT were significantly lower than in the controls and significantly lower than patients AT. We observed a significant positive correlation between GCF volume and MMP-1 levels. Furthermore, TIMP-1 levels were significantly negatively correlated with both GCF volume and all clinical parameters.

Conclusions

We observed that as the extent of periodontal destruction increases, MMP-1 concentration increases and TIMP-1 concentration decreases in GCF. When chronic periodontitis patients were treated by scaling and root planing (SRP), the average MMP-1 concentrations decreased and TIMP-1 concentrations increased in GCF.     

Immunohistochemical localization of Toll‐like receptors 1–10 in periodontitis

Background/aim: In periodontitis, bacteria and pathogen‐associated molecular patterns are sensed by Toll‐like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR‐1 to TLR‐10) were immunohistochemically detected in gingival epithelium and connective tissue. Methods: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR‐positive cells were determined. Results: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR‐10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. Conclusions: For the first time, the cellular expression and distribution of TLR‐1 to TLR‐10 have been studied in periodontitis, indicating that TLR‐1 to TLR‐9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR‐7 and TLR‐8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.