Effect of cytokine and antigen stimulation on peripheral blood lymphocyte syndecan-1 expression

INTRODUCTION: Cytokines are not only produced by activated lymphocytes but also interact with a number of cell-surface molecules on the same cells. Syndecan-1 is one such cell-surface molecule, which has the capacity to bind a variety of growth factors as well as cytokines. The aim of this study was to examine the effects of transforming growth factor beta (TGF-beta), interleukin-1 (IL-1), IL-2, IL-4, lipopolysaccharide (LPS) from Porphyromonas gingivalis and tetanus toxoid on syndecan-1 expression by B and T lymphocytes. METHODS: B and T lymphocytes were obtained from the peripheral blood of healthy donors. Following exposure to the above growth factors, cytokines and antigens, syndecan-1 expression was determined by flow cytometry. RESULTS: Subjects could be categorized as high or low expressors of syndecan-1. In the high-responder group TGF-beta1 alone resulted in a significant increase in syndecan-1 expression by both B and T cells. None of the other cytokines and antigens produced a significant response. When analysed in combination, TGF-beta1 in combination with IL-2, IL-4, P. gingivalis LPS and tetanus toxoid all produced significant increases in syndecan-1 expression by B cells. For T cells, combinations of TGF-beta1 with IL-2 and tetanus toxoid resulted in increased syndecan-1 expression. CONCLUSIONS: Both B and T lymphocytes synthesize the cell-surface proteoglycan syndecan-1 and its expression can be modulated by TGF-beta1, either alone or in combination with IL-2, IL-4 and LPS from P. gingivalis and tetanus toxoid. While these may reflect general responses under inflammatory conditions their biological significance requires further investigation.     

Porphyromonas gingivalis gingipains mediate the shedding of syndecan‐1 from the surface of gingival epithelial cells

Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan‐1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan‐1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan‐1 detected in the culture medium increased significantly in a time‐dependent manner. However, neither a heat‐inactivated supernatant nor a supernatant from a gingipain‐deficient mutant had a significant effect on syndecan‐1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.     

Effect of initial treatment of chronic inflammatory periodontal disease in adults on spontaneous peripheral blood lymphocyte proliferation

The spontaneous proliferative response (SPR) of peripheral blood lymphocytes, as a measure of the autologous mixed lymphocyte reaction (AMLR), has been found to be depressed in adults with chronic inflammatory periodontal disease (CIPD). The aim of the present study was to test the hypothesis that initial treatment of CIPD in adults restores the SPR to normal levels. 10 periodontal disease subjects (mean probing attachment loss of 4.2 mm and a mean bleeding index of 0.65) and 10 age- and sex-matched healthy control subjects were studied. The SPR for each patient was evaluated on days 3, 5, 7, 9 and 11 in culture, before and after initial treatment for CIPD. The peak SPR, which occurred at day 5, was depressed in the untreated periodontal disease subjects compared to the healthy control subjects (p less than 0.01). In addition, the kinetics of the SPR were found to be significantly different in 4 of the 10 parameters compared with the untreated periodontal disease patients and the healthy control subjects. After treatment, there was a significant reduction in probing attachment loss and bleeding indexes (p less than 0.001). In addition, the magnitude of the peak SPR was not significantly different from that of the healthy control subjects. Nevertheless, a difference in 1 of the 10 kinetic parameters persisted, which suggested that complete restoration of the SPR to normal had not occurred so soon after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin‐1α stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis

Periodontal pathogenic bacteria are associated with elevated levels of interleukin‐1α (IL‐1α) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL‐1α induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac‐6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL‐1α protein levels were measured after 6 h of incubation. In addition, monocytes were co‐stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg‐X and Lys‐X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL‐1α production, but P. gingivalis was the weakest. Co‐stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL‐1α production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis‐associated bacterial species stimulate IL‐1α production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro‐inflammatory cytokine levels may impair the ability of the host to tackle infection.     

Syndecan‐1 immunohistochemical expression in gingival tissues of chronic periodontitis patients correlated with various putative factors

Kotsovilis S, Tseleni‐Balafouta S, Charonis A, Fourmousis I, Nikolidakis D, Vrotsos JA. Syndecan‐1 immunohistochemical expression in gingival tissues of chronic periodontitis patients correlated with various putative factors. J Periodont Res 2010; 45: 520–531. © 2010 John Wiley & Sons A/S Background and Objective: Limited information is available on the expression and distribution of syndecan‐1 within human gingival tissues/cells and on putative factors that might affect its expression. Therefore, the objective of the present study was to determine immunohistochemically the expression and distribution of syndecan‐1 in the gingival tissues of patients with chronic periodontitis and to examine the correlation of syndecan‐1 expression with various putative factors (environmental, patient/systemic and local factors). Material and Methods: Gingival specimens were surgically excised from the area of the junctional/pocket epithelium (study group 1, including 30 chronic periodontitis patients) or the gingival oral epithelium (study group 2, comprising another 30 chronic periodontitis patients), adjacent to teeth with poor prognosis. Standard two‐step immunohistochemistry and semi‐quantitative evaluation of immunohistochemical staining were used to determine syndecan‐1 expression. Statistical analyses on the impact of various putative factors were performed. Results: In the junctional/pocket epithelium or the oral epithelium, syndecan‐1 expression was weak to moderate in the suprabasal and basal epithelial cells and absent to weak in the internal basal lamina, external basal lamina and gingival connective tissue matrix. Syndecan‐1 expression in the junctional/pocket epithelium was statistically significantly stronger than in the oral epithelium in inflammatory cells within the underlying gingival connective tissue (primarily plasma cells and lymphocytes) and in scattered fibroblast‐like cells. Conclusions: Syndecan‐1 expression in the junctional/pocket epithelium or the oral epithelium can exhibit a significant positive correlation with the severity/degree of histologically evaluated local gingival inflammation, but in general is not significantly correlated with age, smoking, full‐mouth and local clinical (probing pocket depth and clinical attachment level) and radiographical parameters (radiographical bone loss) of periodontal status.     

Involvement of MT1‐MMP and TIMP‐2 in human periodontal disease

Oral Diseases (2010) 16 , 388–395 Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. Methods: MT1‐MMP and TIMP‐2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis‐affected human subjects. MT1‐MMP and TIMP‐2 expression were assessed through Western‐blot of tissue homogenates. The main cell types involved in MT1‐MMP and TIMP‐2 production were evaluated by means of immunohistochemistry. Results: Both MT1‐MMP and TIMP‐2 were significantly increased in periodontitis‐affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1‐MMP and its inhibitor TIMP‐2 was altered in periodontitis‐affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1‐MMP in fibroblasts and macrophages in gingival tissues. MT1‐MMP was detected in cells in close association with the gingival collagen matrix. TIMP‐2 expression was identified in fibroblasts, macrophages and epithelial cells. Conclusions: Our observations show an increased expression of MT1‐MMP and TIMP‐2 in periodontitis‐affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.     

Flow-cytometric analysis of lymphocyte subsets in patients with advanced periodontitis

Peripheral blood lymphocytes from 36 patients with advanced periodontitis and from 34 healthy subjects were examined using a panel of monoclonal antibodies and fluorescence flow cytometry. The absolute and relative counts of B-cells, T-cells, T-helper (TH), T-suppressor/cytotoxic (TS) cells, activated T-cells and natural killer cells were assessed and the TH/S ratio was calculated. In the periodontitis patients, the TH/S ratio was 1.56 +/- 0.62 and in the controls 1.35 +/- 0.55. This was not statistically significant. A classification on the basis of sex also did not show significant differences in the TH/S ratio. B-cells and activated T-cells were slightly increased in the periodontitis group. However, all determined lymphocyte subpopulations revealed no significant differences between the groups. These results indicate that local inflammatory reactions and immunoregulatory dysfunction are limited to the periodontium in the patients with advanced periodontitis, with no significant quantitative effects on the peripheral lymphocyte subpopulations.     

Establishment of peripheral blood and gingival T lymphocyte clones responsive to Porphyromonas gingivalis

T cells are central to the immune response to infection and studies have indicated a local immunoregulatory imbalance may exist in human periodontal disease. Since Porphyromonas gingivalis is generally recognized as a major periodontopathogen, the aim of this study was to establish T cell lines and clones specific to P. gingivalis from the gingival tissues and peripheral blood of P. gingivalis — infected subjects. Two subjects were selected from two groups of individuals (one from each group) established on the basis of P. gingivalis in their plaque and the presence of serum antibodies which react with P. gingivalis antigens. The two groups differed however in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. The mean ages ± standard error of the mean of the two groups were 47.9 ± 2.2 and 49.6 ± 3.7, respectively, so that resistance in the gingivitis group was related to the age of the subjects. T cell lines and clones were established from the peripheral blood of one patient from each of the two groups and also from the gingival tissues of the same periodontitis subject. This study has demonstrated the capability of establishing P. gingivalis -specific T cell lines and clones from P. gingivalis -infected subjects and FACS analysis of the T cell receptor variable regions demonstrated that the clones were indeed monoclonal. The CD4:CD8 ratios of the peripheral blood-derived T cell lines were 1.2 and 0.4 for the gingivitis-derived line and the periodontitis-derived line, respectively, thus supporting the clinical differences displayed by the two subjects.     

Effect of the interleukin-1 genotype on monocyte IL-1beta expression in subjects with adult periodontitis

An association has been reported between polymorphisms in the genes encoding IL‐1α(−889) and IL‐1β(+3953)(periodontitis susceptibility trait, PST), and an increased severity of periodontitis (18). The IL‐1β polymorphism was reported to correlate with increased IL‐1β expression by monocytes in response to bacterial stimulants. In the present study, we determined if PST positive subjects with periodontitis exhibit elevated production of IL‐1β, compared to PST negative periodontitis patients. Peripheral blood monocytes were obtained from 10 PST+ and 10 PST− age‐ and disease‐balanced subjects with adult forms of periodontitis. Monocytes were cultured with a panel of bacterial stimulants, including Escherichia coli and Porphyromonas gingivalis LPS, and whole formalinized periodontal pathogens P. gingivalis , Bacteroides forsythus and Prevotella intermedia , and health‐associated organisms Veillonella parvula and Streptococcus sanguis . Our results demonstrate that monocytes from PST+ and PST− patients showed no significant differences in IL‐1β production in response to any stimulant tested. In addition, the periodontal pathogens P. gingivalis , B. forsythus and P. intermedia failed to stimulate higher IL‐1β responses compared to health‐associated species V. parvula and S. sanguis . A marked interindividual variation in production of IL‐1β was seen, with high, low and intermediate responders present in both PST+ and PST− groups. We conclude that genetic loci other than the PST polymorphisms are also important regulators of monocyte IL‐1 responses.     

Immunomodulatory effects of Bacteroides products on in vitro human lymphocyte functions

Bacteroides spp. have been implicated in the pathogenesis of several diseases, including periodontal diseases. In this study sonic extracts of 6 Bacteroides spp. were examined for their abilities to alter human lymphocyte function. We found that soluble extracts from Bacteroides intermedius, Bacteroides endodontalis, Bacteroides asaccharolyticus, Bacteroides melaninogenicus, and to a lesser degree Bacteroides loescheii, caused dose-dependent inhibition of human lymphocyte responsiveness to both mitogens and antigens. Suppression involved altered DNA, RNA and protein synthesis as well as immunoglobulin production. In contrast, Bacteroides gingivalis did not suppress these responses; instead, it stimulated lymphocyte proliferation and enhanced immunoglobulin production. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many infections. The data presented in this paper suggest that microbial mediated immunosuppression may conceivably alter the nature and consequences of host-parasite interactions in periodontal disease.     

Down‐regulation of interleukin‐1α‐induced matrix metalloproteinase‐13 expression via EP1 receptors by prostaglandin E2 in human periodontal ligament cells

In the present study, we investigated the effect of prostaglandin (PG) E₂ on matrix metalloproteinase (MMP)‐13 production in human periodontal ligament cells stimulated with interleukin (IL)‐1α. IL‐1α enhanced both MMP‐13 and PGE₂ production. Indomethacin, a nonselective cyclooxygenase inhibitor, and NS‐398, a specific cyclooxygenase‐2 (COX‐2) inhibitor, significantly enhanced IL‐1α‐induced MMP‐13 production in periodontal ligament cells, although both the agents completely inhibited IL‐1α‐induced PGE₂ production. Exogenous PGE₂ reduced IL‐1α‐induced MMP‐13 mRNA and protein production in a dose‐dependent manner. 17‐phenyl‐ω‐trinor PGE₂, a selective EP₁ receptor agonist, mimicked the inhibitory effect of PGE₂ on IL‐1α‐induced MMP‐13 mRNA and protein production. On the basis of these data, we suggest that COX‐2‐dependent PGE₂ down‐regulates IL‐1α‐elicited MMP‐13 production via EP₁ receptors in human periodontal ligament cells. PGE₂ may be involved in the regulation of destruction of extracellular matrix components in periodontal lesions.     

Understanding the pathophysiology behind chairside diagnostics and genetic testing for IL‐1 and IL‐6

In order for chairside diagnostic testing to make an impact on dental therapy, practitioners require a better understanding of genetic mutations contributing to the pathophysiology of periodontal disease. Commensal and pathogenic bacterial colonization in oral cavity tissues produces a cascade of proinflammatory signaling pathways ultimately detrimental to host tissues. Resolving inflammation is a multifactorial process involving the downregulation of proinflammatory cytokines while allowing commensal bacterial levels to return to normal. Because of the complicated nature of commensal bacteria and oral health homeostasis, the emphasis of dental therapy should place renewed focus on limiting destructive inflammation rather than solely eliminating bacteria. Salivary diagnostics are an easy, non‐invasive way to assess inflammatory markers. Inflammatory cytokine levels can help determine the subclinical health of a patient, showing the transition from health to gingivitis, or periodontitis, prior to clinical presentation. Single nucleotide polymorphism mutations can aid in determining increased risk of developing periodontitis. Taken together, and alongside regular clinical evaluations, chairside diagnostics help individualize treatment plans to slow, or halt, the progression of disease—before tissue destruction can take place. While more studies are needed analyzing specific mutations across periodontal categories, chairside diagnostics present an exciting adjunct to improve patient care.     

Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells

Pi S‐H, Jeong G‐S, Oh H‐W, Kim Y‐S, Pae H‐O, Chung H‐T, Lee S‐K, Kim E‐C. Heme oxygenase‐1 mediates nicotine‐ and lipopolysaccharide‐induced expression of cyclooxygenase‐2 and inducible nitric oxide synthase in human periodontal ligament cells. J Periodont Res 2010; 45: 177–183. © 2010 John Wiley & Sons A/S Background and Objective: Although heme oxygenase‐1 (HO‐1) plays a key role in inflammation, its anti‐inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of HO‐1 on the proinflammatory mediators activated by nicotine and lipopolysaccharide (LPS) stimulation in human periodontal ligament (PDL) cells. Material and Methods: The production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and HO‐1 proteins was evaluated by Western blot analysis. Results: Lipopolysaccharide and nicotine synergistically induced the production of NO and PGE₂ and increased the protein expression of iNOS, COX‐2 and HO‐1. Treatment with an HO‐1 inhibitor and HO‐1 small interfering RNAs blocked the LPS‐ and nicotine‐stimulated NO and PGE₂ release as well as the expression of iNOS and COX‐2. Conclusion: Our data suggest that the nicotine‐ and LPS‐induced inflammatory effects on PDL cells may act through a novel mechanism involving the action of HO‐1. Thus, HO‐1 may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.     

牙龈卟啉菌表面相关物质诱导外周血中淋巴细胞凋亡的体外研究

目的 :将牙龈卟啉菌表面相关物质和外周血中淋巴细胞共同培养 ,观察淋巴细胞的凋亡情况。方法 :通过流式细胞仪对 2 0名健康人外周血中淋巴细胞培养组 (对照组 )与牙龈卟啉菌表面相关物质和淋巴细胞共同培养组 (实验组 )进行比较研究 ,观察淋巴细胞的凋亡情况。结果 :实验组与对照组相比 ,淋巴细胞凋亡数明显增多 (P <0 .0 5 )。结论 :牙龈卟啉菌表面相关物质诱导外周血中淋巴细胞凋亡 ,细菌诱导细胞凋亡在牙周可疑致病菌的致病机制中可能具有作用。