Adenosine regulates the IL-1 beta-induced cellular functions of human gingival fibroblasts.
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In this study we examined the influence of adenosine on the cellular functions of human gingival fibroblasts (HGF), such as the production of inflammatory cytokines and extracellular matrices (ECM), and the expression and function of adhesion molecules. Concerning the expression of adenosine receptors, RT-PCR analysis revealed that HGF expressed adenosine receptor A1, A2a and A2b, but not A3 mRNA. Ligation of adenosine receptors by adenosine or its related analogue, 2-chloroadenosine (2-CADO), N(6)-cyclopentyladenosine (CPA) or CGS21680 synergistically increased IL-1beta-induced IL-6 and IL-8 production. In terms of ECM expression, adenosine and the adenosine receptor agonists, 2-CADO and CPA, enhanced constitutive and IL-1beta-induced expression of hyaluronate synthase mRNA, but not the mRNA levels of other ECM, such as collagen type I, III and fibronectin. Moreover, the adherence of IL-1beta-stimulated HGF to activated lymphocytes was also inhibited by adenosine, which is in part explained by the fact that adenosine down-regulated the IL-1beta-induced expression of ICAM-1 on HGF. These results provide new evidence for the possible involvement of adenosine in the regulation of inflammatory responses in periodontal tissues. (+info)
Inflammatory reactions in extraoral tissues in mice after intragingival injection of lipopolysaccharide.
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Intragingival (ig) injection into mice of lipopolysaccharide (LPS) from Prevotella intermedia or Escherichia coli elevated the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), in the mandible, liver, lung, and spleen, with a time course similar to that seen with intravenous (iv) injection. The effect of i.g. injection was less than that of i.v. injection but similar to that of intraperitoneal (ip) injection. The i.g. injection also increased hepatic serotonin, reflecting platelet accumulation. In galactosamine-treated mice, the minimum ig dose of LPS needed to induce lethal hepatitis was very small (less than that needed by ip injection). These results support the idea that the LPS produced in oral tissues may be transported easily to extraoral tissues and, in some cases, may cause inflammatory or immune responses. It also may influence the pathogenesis of some systemic diseases. (+info)
Biological characterization of lipopolysaccharide from Treponema pectinovorum.
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This study investigated the endotoxic and biological properties of purified lipopolysaccharide (LPS) isolated from an oral spirochete, Treponema pectinovorum. Endotoxicity, measured by Limulus amoebocyte lysate kinetic assay, showed that the LPS contained 1.28 endotoxin units per microg of purified LPS, which was approximately 4,000 times less than Escherichia coli O55:B5 LPS. To determine in vivo endotoxicity, LPS responder mice were administered LPS following galactosamine (GalN) sensitization. The LPS induced neither endotoxic symptoms nor lethality for 96 h, suggesting negligible or very low endotoxicity. In contrast, infection with live T. pectinovorum induced 100% lethality within 12 h in GalN-sensitized LPS responder mice, indicating an endotoxin-like property of this treponeme. Heat-killed microorganisms exhibited no lethality in GalN-sensitized mice, suggesting that the endotoxicity was associated with heat-labile components. To determine cytokine and chemokine induction by LPS, human gingival fibroblasts were stimulated and secretion of interleukin 1beta (IL-1beta), granulocyte-macrophage colony-stimulating factor, gamma interferon, IL-6, IL-8, and monocyte chemoattractant protein 1 (MCP-1) was assessed. The purified LPS induced significant amounts of only IL-6, IL-8, and MCP-1, although they were substantially lower than levels after challenge with live T. pectinovorum. After injection of LPS or live or heat-killed T. pectinovorum, serum was collected from mice and analyzed for proinflammatory cytokines IL-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6. LPS induced only IL-6 consistently. Both live and heat-killed T. pectinovorum induced serum IL-6, which was higher than the level detected following LPS administration. Importantly, live bacteria elicited systemic TNF-alpha and IL-1beta levels similar to those induced by a lethal dose of live E. coli O111. The results indicated that T. pectinovorum LPS has very low or no endotoxicity, although it can elicit low levels of cytokines from host cells. In contrast to the LPS, live T. pectinovorum demonstrated in vivo toxicity, which was associated with serum IL-1beta, TNF-alpha, and IL-6, suggesting an endotoxin-like property of a heat-labile molecule(s) of the spirochete. (+info)
Lipopolysaccharides from periodontopathic bacteria Porphyromonas gingivalis and Capnocytophaga ochracea are antagonists for human toll-like receptor 4.
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Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-kappaB (NF-kappaB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Capnocytophaga ochracea, and Fusobacterium nucleatum, and a non-oral bacterium, Escherichia coli, all species of the bacteria induced NF-kappaB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-dried A. actinomycetemcomitans, F. nucleatum, and E. coli also induced CD25 expression in 7.7/huTLR4 cells, freeze-dried P. gingivalis did not. Similarly, lipopolysaccharides (LPS) extracted from A. actinomycetemcomitans, F. nucleatum, and E. coli induced CD25 expression in 7.7/huTLR4 cells, but LPS from P. gingivalis and C. ochracea did not. Furthermore, LPS from P. gingivalis and C. ochracea attenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS from E. coli. LPS from P. gingivalis and C. ochracea also inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1beta from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS from E. coli. These findings indicated that LPS from P. gingivalis and C. ochracea worked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases. (+info)
Functional differences among FimA variants of Porphyromonas gingivalis and their effects on adhesion to and invasion of human epithelial cells.
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Fimbriae of Porphyromonas gingivalis, a periodontopathogen, play an important role in its adhesion to and invasion of host cells. The fimA genes encoding fimbrillin (FimA), a subunit protein of fimbriae, have been classified into five types, types I to V, based on nucleotide sequences. We previously reported that P. gingivalis with type II fimA was strongly associated with adult periodontitis. In the present study, we compared the abilities of recombinant FimA (rFimA) types I to V to adhere to and invade human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells) by using rFimA-conjugated microspheres (rFimA-MS). There were no significant differences in the abilities of the rFimA-MS to adhere to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than those of other types of rFimA-MS. We also observed that type II rFimA-MS invaded epithelial cells and accumulated around the nuclei. These adhesion and invasion characteristics were eliminated by the addition of antibodies to type II rFimA and alpha5beta1-integrin. In contrast, Arg-Gly-Asp-Ser peptide and a synthetic peptide of proline-rich protein C had negligible inhibitory effects. Furthermore, P. gingivalis strain HW24D1 with type II fimA adhered to cells and invaded them more than strains with other fimA genotypes. These results suggest that type II FimA can bind to epithelial cells most efficiently through specific host receptors. (+info)
Porphyromonas gingivalis lipopolysaccharide activated bone resorption of osteoclasts by inducing IL-1, TNF, and PGE.
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AIM: To study the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on inducing interleukin-1 (IL-1), tumor necrosis factor (TNF), prostaglandin E (PGE), and activating osteoclasts, in order to understand mechanism of osteoclast activation. METHODS: Pg-LPS was prepared by phenol-water method. IL-1, TNF, and PGE induced by Pg-LPS were isolated by chromatography. Ca2+ concentration was detected by atomic absorption spectrophotometry. Acid phosphatase and carbonic anhydrase in periodontal membranes were examined by histochemistry. RESULTS: Pg-LPS was able to stimulate peripheral blood mononuclear cells (PBMC) or the cells from human periodontal tissue secreting IL-1, TNF, and PGE. The outputs of these cytokines were increased in pace with the enhancement of Pg-LPS at the dose range of 0.5 - 5.0 mg/L. All of the three cytokines showed activities of accelerating Ca2+ release from rat calvarial bones, and the activity of PGE was the strongest. The amounts of both the acid phosphatase and carbonic anhydrase in the periodontal membranes of Pg-LPS injected rats were obviously increased (P < 0.01). In the periodontal membranes of Pg-LPS injected rats, the amount of activated osteoclasts were obviously increased in pace with Pg-LPS injection times (P < 0.01). However, the activating rates of osteoclasts were stable to approximately 65 % because of the increase of inactivated osteoclasts. CONCLUSION: Pg-LPS possessed strong activities to induce human PBMC and the cells from human periodontal tissue to produce IL-1, TNF, and PGE in a dose-dependent m anner within a certain concentration range of the LPS. Pg-LPS could efficiently activate osteoclasts, and the mechanism of osteoclast activation was probably associated with the increase of acid phosphatase and carbonic anhydrase. (+info)
Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family.
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Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the beta-defensin family. Previous work has shown that multiple signaling pathways are involved in human beta-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-kappaB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-kappaB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-kappaB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-kappaB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH(2)-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-kappaB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue. (+info)
An investigation of immunocompetence substances in normal gingival and periodontitis tissue.
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OBJECTIVE: To investigate the effects of nitric oxide (NO), endothelin (ET), substance P (SP) and calcitonin gene-related peptide (CGRP) immunocompetence substances and their relationship to chronic periodontitis. METHODS: Immunohistochemical aod histochemical staining methods were used to detect the expression of the NO synthase (NOS), ET, SP and CGRP levels in 20 patients with chronic periodontitis and 20 healthy subjects as control. RESULTS: Quantitative analysis by Quantimat 970 showed that NOS and ET in periodontitis tissue increased significantly (P < 0.01), particularly the content of ET in comparison with healthy subjects. The intergroup expression of SP and CGRP showed no remarkable changes. CONCLUSION: Our results demonstrate that the level of NOS and ET were significantly increased in periodontic tissue, which may diminish the blood supply and influence the periodontal tissue causing tissue damage. Our study suggests that immunocompetence substances NO and ET are closely associated with periodontitis and may play an important role in the disease. (+info)