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RESEARCH REPORT |
B System
1 Department of Restorative Dentistry & Periodontology,
3 Department of Orthodontics,
4 Experimental Surgery & Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Goethe Street 70, 80336 Munich, Germany;
2 Institute of Clinical Chemistry & Pathobiochemistry, Klinikum rechts der Isar, Technische Universität München, Germany; and
5 Institute of Clinical Chemistry, Medizinische Hochschule Hannover, Germany
* corresponding author, khuth{at}dent.med.uni-muenchen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B system, a paradigm for inflammation-associated signaling/transcription. We showed that NF-B activity in oral cells stimulated with TNF, and in periodontal ligament tissue from root surfaces of periodontally damaged teeth, was inhibited following incubation with ozonized medium. Under this treatment, IB
proteolysis, cytokine expression, and B-dependent transcription were prevented. Specific ozonized amino acids were shown to represent major inhibitory components of ozonized medium. In summary, our study establishes a condition under which aqueous ozone exerts inhibitory effects on the NF-B system, suggesting that it has an anti-inflammatory capacity.
KEY WORDS: aqueous ozone • NF-B • signaling • inflammation • oral
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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For the manifestation of these disease entities, bacterial colonization of the dentogingival complex or the endodontic region, respectively, has been considered as the primary causative factor (Graves et al., 2000; Nair, 2004). However, it is generally accepted that periodontal disease and apical periodontitis do not result from direct tissue destruction by pathogenic bacteria. Rather, the destructive potential of the host immune response leads mainly to signs and symptoms of these diseases (Honda et al., 2006; Nair, 2004). Therefore, immune modulation effects of treatment strategies for these diseases may also be considered. Both disease entities are characterized by an inflammatory reaction involving different oral hard and soft tissues, e.g., the gingiva, periodontal attachment fibers, or alveolar bone (Nair, 2004; Bartold and Narayanan, 2006). The inflammatory process is primarily induced by pathogen-associated molecular patterns (PAMPs), particularly by bacterial lipopolysaccharides (Madianos et al., 2005). The subsequent activation of the inflammatory molecular cascade leads to the expression of several pro-inflammatory cytokines—e.g., interleukin-1, interleukin-8, and tumor necrosis factor (TNF)—that ultimately mediate the destruction of the alveolar bone and periodontal connective tissue (Gamonal et al., 2000; Graves et al., 2000; Márton and Kiss, 2000).
The transcription factor NF-B plays a pivotal role in inflammatory/immune processes and apoptosis (Bonizzi and Karin, 2004). NF-B is also thought to be of paramount importance in the regulation of periodontal/periapical inflammatory reactions and the pathogenesis of periodontal disease and apical periodontitis (Nichols TC et al., 2001; Sabeti et al., 2005; Bartold and Narayanan, 2006). This transcription factor exists as a dimer which is trapped in the cytosol by inhibitory proteins, e.g., IB (Bonizzi and Karin, 2004). The NF-B system is activated by numerous agents, including cytokines (e.g., TNF, interleukin-1) and microbial pathogens/products. The activation of NF-B is mediated by the IB kinase complex that phosphorylates IB, which is subsequently degraded by the proteasome. The thus-freed NF-B translocates to the nucleus, where it binds to B sequences in promoters/enhancers, thereby regulating the expression of various genes such as interleukin-1/-8 or TNF.
Ozone gas is known to activate NF-B under certain conditions (Haddad et al., 1996; Laskin et al., 2002). However, it is not known if aqueous ozone also interferes with the NF-B system. This is important, since an activation of NF-B might adversely affect the therapeutic benefit of aqueous ozone when used against periodontal disease and apical periodontitis. Hence, the aim of the present study was to investigate the effect of aqueous ozone on NF-B-associated signaling/transcription in oral cells.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Ozonation of Reagents
Aqueous ozone was applied to the cells in the form of ozonized medium without fetal calf serum (O3 medium), or ozonized phosphate-buffered saline (O3-phosphate-buffered saline) without/with 1 g/L amino acids or 2 g/L glucose. These solutions were treated with gaseous ozone (75 µg/mL, 15 min) in an ozone generator (Ozonosanphotonic, Dr. Hänsler, Iffezheim, Germany). This condition—which, when applied to water, would result in a final ozone concentration of 20 µg/mL (saturation point)—was defined as the 100% ozonation state. For dose-response experiments, the solutions were diluted accordingly. The ozone gas concentration was monitored by a photometer integrated into the ozone generator and confirmed by another ozone gas detector (GM-6000-NZL, Anseros, Tübingen, Germany). The ozone concentrations of the respective aqueous solutions were monitored photometrically (Palintest, Gateshead, England).
Electrophoretic Mobility Shift Assay
Nuclear extracts of cell lines or periodontal ligament tissue were prepared, and an electrophoretic mobility shift assay was performed (Page et al., 1999; Appendix A). The prototypic immunoglobulin -chain oligonucleotide was used as a probe and labeled with the Klenow fragment of DNA polymerase I (Roche, Penzberg, Germany), together with [-32P]dCTP (PerkinElmer-LifeSciences, Brussels, Belgium). The Sp-1 consensus oligonucleotide (Promega) was labeled with [
-32P]ATP (PerkinElmer-LifeSciences) and T4-polynucleotidekinase (Roche). Samples were run on non-denaturing 4% polyacrylamide gels and analyzed by autoradiography.
Western Blot Analysis
Cytosolic extracts were isolated, and electrophoresis was performed (Page et al., 1999; Appendix A). After transfer, the membranes were incubated with IB (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or actin antibodies (Sigma-Aldrich, Deisenhofen, Germany), followed by secondary peroxidase-conjugated antibodies (Dianova, Hamburg, Germany). Antibody binding was visualized on x-ray film with Chemiluminescent-Reagent-Plus (PerkinElmer-LifeSciences).
Determination of Cytokines
Interleukin-8 and -1ß concentrations in supernatants were measured by immunoassays (R&D-Systems, Wiesbaden, Germany; Bender-MedSystems, Vienna, Austria).
Luciferase Assay
For luciferase assays, HeLa cells were transiently transfected with a firefly-luciferase reporter plasmid (pGL2-3B-Luc, 3-B binding motifs), together with a constitutively active Renilla-luciferase plasmid, pRLtk (Promega) (Page et al., 1999; Appendix A), according to a Superfect-based protocol (Qiagen, Hilden, Germany). Subsequent to stimulation, cells underwent lysis, and luciferase activity was determined with the Dual-Luciferase-Reporter-Assay (Promega). Results were expressed as relative luciferase activity (firefly-relative light-units divided by Renilla-relative light-units).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B Activation is Inhibited by O3 MediumB. Dose-response and time-course experiments demonstrated that O3 medium alone did not modulate the NF-B system (data not shown). Next, we tested whether pre-incubation with O3 medium affected the activation of NF-B by other stimuli. Cells were pre-incubated with O3 medium followed by exposure to TNF, and optimal cellular conditions were again evaluated by dose-response/time-course experiments (data not shown). In the absence of ozone, a marked activation of NF-B by TNF was observed (Figs. 1A, 1B
). Incubation with O3 medium, however, inhibited this activation, whereas constitutive Sp-1 binding was unchanged. We excluded a potential toxicity of O3 medium by monitoring cell morphology, trypan-blue exclusion, and ATP levels (Cell Viability Assay) (Appendix B). As a positive control, pre-incubation with the proteasome inhibitor PSI (10 µM, 1 hr) markedly prevented TNF-induced NF-B activity (BHY, inhibition by 68 ± 7%; HeLa, inhibition by 75 ± 6%; n = 3). Furthermore, we investigated whether O3 medium also modulated NF-B activity in periodontal ligament tissue from the root surfaces of periodontally diseased teeth. These experiments revealed strong NF-B binding activity when this tissue was incubated in medium alone, which was markedly inhibited following treatment with ozonized medium (Fig. 1C).
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B Proteolysis is Inhibited in the Presence of O3 MediumB inhibitor IB was affected by O3 medium. Stimulation of cells with TNF led to proteolysis of IB (Fig. 2A). However, in O3-medium-pre-incubated cells, this TNF-induced IB degradation was inhibited, whereas constitutive actin levels were unchanged.
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B-dependent Transcription Were Prevented
) and interleukin-1ß (Fig. 2C). In addition, we transfected cells with a B-dependent luciferase construct and treated them as described previously, to monitor a direct impact on B-dependent transcription. TNF induced a marked increase in transcriptional activity, which again was strongly inhibited by O3 medium (Fig. 2D).
O3 Amino Acids Selectively Inhibited NF-B
The inhibitory effect of O3 medium could be due to ozone itself or to ozonized medium constituents. Therefore, cells were pre-incubated with O3-phosphate-buffered saline without further ingredients. In contrast to the results obtained above, the NF-B activation by TNF was not inhibited in all 3 cell lines by O3-phosphate-buffered saline (Fig. 3A, data not shown). This indicated that the inhibitory effect of O3 medium was not directly caused by ozone, but rather was mediated by the formation of medium-ozonation products. To identify these inhibitory ozonation products, we added various amino acids (medium concentration) of the different groups (Cataldo, 2003), as major medium components, to phosphate-buffered saline prior to ozonation. After pre-incubation of cells with ozonized and non-ozonized amino acids, TNF was added. Dependent on the different ozonized amino acids, NF-B activity was variously affected: The sulfhydryl group containing cysteine and the aromatic tryptophan blocked the NF-B signal almost completely (Figs. 3B, 3C); the sulfur-containing methionine resulted in clear inhibition, the basic arginine in a modest, and the non-polar alanine in a slight reduction of NF-B activity, whereas the small, non-polar glycine or non-ozonized amino acids had no effect. Similarly, dose-dependent effects were observed in all 3 cell lines (data not shown). To determine whether the inhibition of TNF signaling by O3 medium was also mediated by glucose, we added glucose (medium concentration) to phosphate-buffered saline, followed by ozonation, and treated the cells as described above. These experiments showed that the NF-B activity was not inhibited by O3-glucose (Fig. 3D). Taken together, these results signify that ozonized amino acids, to various extents, are an important component responsible for the inhibitory effect of O3 medium.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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B system, which is involved in the pathogenesis of periodontal disease and apical periodontitis (Nichols TC et al., 2001; Sabeti et al., 2005; Bartold and Narayanan, 2006). Analysis of our data revealed that activation of NF-B induced by the potent cytokine TNF (Bonizzi and Karin, 2004) was dose-dependently inhibited in oral cells cultured in O3 medium, while no toxic effects were observed. Supporting this finding, incubation with O3 medium also inhibited NF-B activity in periodontal ligament tissue of periodontally damaged teeth. This suggests that aqueous ozone under certain conditions displays anti-inflammatory effects.
Furthermore, our study demonstrated that TNF-stimulated proteolysis of the inhibitor IB (Bonizzi and Karin, 2004) was prevented in the presence of O3 medium, which indicates that incubation with O3 medium affects signaling at the level and/or upstream of IB (Fig. 4). In addition, our experiments showed that B-dependent transcription and the expression of the NF-B target genes interleukin-8 and -1ß (Pahl, 1999) were prevented by O3 medium. Both cytokines have been correlated with the biological and clinical severity of periodontal disease (Gamonal et al., 2000) and are secreted by various cells, e.g., epithelial cells, fibroblasts, and monocytes/macrophages. Interleukin-8 expression shows a rapid onset in an early inflammatory stage and exerts chemotactic/activation effects on neutrophils (Okada and Murakami, 1998; Bartold and Narayanan, 2006). Interleukin-1ß, when inadequately expressed, is known to mediate tissue-destructive effects, e.g., bone resorption and loss of periodontal attachment (Gamonal et al., 2000; Graves et al., 2000).
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B. Therefore, the question was which ozonation-product in the O3 medium was responsible for this effect. To this end, we added various amino acids from the different groups to phosphate-buffered saline (medium concentration), and found that TNF-induced NF-B activation was affected differently. The ozonized sulfhydryl group containing cysteine, as well as the aromatic tryptophan, were found to be most effective in inhibiting NF-B, which agreed with earlier reports showing that the ozone attack is essentially directed toward the aromatic amino acids, and that there is a high oxidation reactivity of cysteine and methionine (Cataldo, 2003).
Earlier reports of the use of gaseous ozone throughout showed an activation of NF-B (Haddad et al., 1996; Laskin et al., 2002). This discrepancy with our results may be explained by the fact that direct exposure of cells to gaseous ozone initiates an immediate oxidative stress by the increase of reactive oxygen species (Chen and Qu, 1997; Nichols BG et al., 2001), a condition which directly activates NF-B (Pahl, 1999). In contrast, the ozonation of amino acids in an aqueous milieu may lead to the production of modified amino acid molecules (Cataldo, 2003), which may interact with cellular signaling systems, thus preventing subsequent stimulation, as has been shown for lipid oxidation products such as 4-hydroxynonenal (Page et al., 1999).
The clinical consequences of the inhibitory effects of aqueous ozone on NF-B found herein remain to be elucidated. The attenuated activation of the host immune system may reduce periodontitis-associated tissue destruction (Bartold and Narayanan, 2006), which, as already mentioned, has been suggested to be caused by an inadequate and exaggerated inflammatory/immune response against a bacterial stimulus (Honda et al., 2006). However, a prolonged inhibition of NF-B should be avoided, to prevent a disequilibrium between the immune response and the bacterial challenge (Yamamoto and Gaynor, 2001).
In summary, our study establishes a condition under which the aqueous ozone exerts inhibitory effects on the NF-B system, suggesting that it has anti-inflammatory and immune-modulatory capacities. Thereby, the data presented build an experimental basis for a comprehensive clinical study on the use of aqueous ozone in the oral cavity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received March 19, 2006; Last revision December 2, 2006; Accepted January 1, 2007
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