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Keywords:

  • Prevotella intermedia;
  • lipopolysaccharide;
  • TNF-α;
  • THP-1 cells;
  • mitogen-activated protein kinases

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The purpose of this study was to investigate the effects of lipopolysaccharide from Prevotella intermedia, a major cause of inflammatory periodontal disease, on the production of tumor necrosis factor (TNF)-α and the expression of TNF-α mRNA in differentiated THP-1 cells, a human monocytic cell line. The potential involvement of the three main mitogen-activated protein kinase (MAPK) signaling pathways in the induction of TNF-α production was also investigated. Lipopolysaccharide from P. intermedia ATCC 25611 was prepared by the standard hot phenol–water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. It was found that P. intermedia lipopolysaccharide can induce TNF-α mRNA expression and stimulate the release of TNF-α in differentiated THP-1 cells without additional stimuli. Treatment of the cells with P. intermedia lipopolysaccharide resulted in a simultaneous activation of three MAPKs [extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38]. Pretreatment of the cells with MAPK inhibitors effectively suppressed P. intermedia lipopolysaccharide-induced TNF-α production without affecting the expression of TNF-α mRNA. These data thus provided good evidence that the MAPK signaling pathways are required for the regulation of P. intermedia lipopolysaccharide-induced TNF-α synthesis at the level of translation more than at the transcriptional level.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Periodontal disease is a chronic inflammatory process accompanied by destruction of surrounding connective tissue and alveolar bone, and sometimes loss of teeth (Williams, 1990). The primary causative agents of periodontal disease are particular gram-negative anaerobic bacteria that accumulate in the gingival sulcus. Prevotella intermedia is a major periodontal pathogen that is dominant in the periodontal pockets of patients with adult periodontitis (Slots et al., 1986). This bacterium has also been frequently recovered from subgingival flora in patients with acute necrotizing ulcerative gingivitis (Chung et al., 1983) and pregnancy gingivitis (Kornman & Loesche, 1980).

Lipopolysaccharide is a major constituent of the outer membrane of gram-negative bacteria, including P. intermedia. It has the ability to trigger a number of host cells, especially mononuclear phagocytes, to produce and release a wide variety of pharmacologically active mediators, including IL-1β, IL-6, IL-8, and, most importantly, tumor necrosis factor α (TNF-α) (Morrison & Ryan, 1987).

TNF-α is a potent immunologic mediator of proinflammatory responses contributing significantly to the pathophysiology of different infections (Birkedal-Hansen, 1993). TNF-α is produced mainly by activated monocytes/macrophages in response to various stimuli, including bacterial lipopolysaccharide (Shapira et al., 1998; Jansky et al., 2003). There is evidence to suggest that TNF-α plays a central role in the pathogenesis of periodontal disease. TNF-α has been found at high levels in gingival crevicular fluids and in gingival tissues from periodontally diseased sites over those in healthy sites (Rossomando et al., 1990; Stashenko et al., 1991). Moreover, it was shown that TNF-α has a strong potential to induce connective tissue degradation and alveolar bone resorption (Abu-Amer et al., 1997; Kobayashi et al., 2000). Furthermore, blockade of the activity of TNF-α was found to inhibit the inflammatory response and bone loss in a primate model of experimental periodontitis (Assuma et al., 1998).

Although P. intermedia lipopolysaccharide induced expression of the IL-10 receptor gene (Tokuda et al., 2003) and of genes encoding proinflammatory cytokines (Nagaoka et al., 1996; Tokuda et al., 2001), such as IL-6 and IL-8, in human dental pulp cell cultures, there are few publications related to the interactions between macrophages and lipopolysaccharide from this periodontopathic bacterium. The purpose of this study was to investigate the effects of purified P. intermedia lipopolysaccharide on the production of TNF-α and the expression of TNF-α mRNA in differentiated THP-1 cells, a human monocytic cell line. The potential involvement of the three main mitogen-activated protein kinase (MAPK) signaling pathways in the induction of TNF-α production was also investigated.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacteria and culture conditions

Prevotella intermedia ATCC 25611 was used throughout. It was grown anaerobically on the surface of enriched trypticase soy agar containing 5% (v/v) sheep blood, or in general anaerobic medium (GAM) broth (Nissui, Tokyo, Japan) supplemented with 1 μg mL−1 menadione and 5 μg mL−1 hemin.

Lipopolysaccharide isolation

Lipopolysaccharide was prepared from lyophilized P. intermedia cells by the standard hot phenol–water method as described previously (Kim et al., 2004). The protein content of the purified lipopolysaccharide, determined by the method of Markwell et al. (1978), was <0.1%. Coomassie blue staining of overloaded sodium dodecyl sulfate (SDS)-polyacrylamide gels did not reveal any visible protein bands in the purified lipopolysaccharide, confirming the purity of the preparation (data not shown). Salmonella typhimurium lipopolysaccharide (phenol extract) was purchased from Sigma.

Cell cultures

The human monocytic cell line THP-1 (American Type Culture Collection, Rockville, MD) was grown routinely in Nunc flasks in RPMI 1640 medium supplemented with 10% [v/v] heat-inactivated fetal bovine serum, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 25 mM HEPES, 100 U mL−1 of penicillin, and 100 μg mL−1 of streptomycin in a humidified chamber with 5% CO2/95% air at 37°C. The cells (5 × 105 cells per well in 24-well culture plates) were incubated in the medium supplemented with 50 ng mL−1 of phorbol myristate acetate to induce differentiation into macrophage-like cells. The cells were allowed to differentiate and adhere to plastic for 72 h and washed three times with medium. Various concentrations of lipopolysaccharide were then added and the cells were cultured for the indicated times, after which culture supernatants were collected and assayed for TNF-α.

Measurement of TNF-α production

The amount of TNF-α secreted into the culture medium was determined by an enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (OptEIA; BD Pharmingen, San Diego, CA) according to the protocols recommended by the manufacturer. The sensitivity of the assay was 7.8 pg mL−1, according to the manufacturer.

Western blot analysis of activated MAPKs

Cells were plated in 60 mm tissue culture dishes, at a density of 2 × 106 cells per dish, and treated with various concentrations of P. intermedia lipopolysaccharide for the indicated periods of time. After incubation, they were washed two times with phosphate-buffered saline (PBS) and lysed by adding 1 × SDS sample buffer containing 62.5 mM Tris/Cl (pH 6.8), 2% SDS, 50 mM dithiothreitol, and 10% glycerol. Twenty-five microliters of each sample was heated to 100°C for 5 min and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels with 3% stacking gels. The resolved proteins were transferred onto a nitrocellulose membrane by electroblotting, and the blots were blocked for 1 h in PBST (PBS with 0.1% Tween-20) containing 3% nonfat dry milk, followed by incubation with antibodies directed against three activated members of the MAPK family, phospho-ERK (ERK, extracellular signal-related kinase), -JNK (JNK, c-Jun N-terminal kinase), and -p38, from Cell Signaling Technology (Beverly, MA) following the manufacturer's protocols. Immunoreactive bands on the nitrocellulose sheet were visualized on an X-ray film using a Phototope-HRP Western Blot Detection System (Cell Signaling) utilizing a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling). To assess the total amount of kinases, blots were probed with antibodies directed against total-MAPK (ERK, JNK, and p38) obtained from Cell Signaling Technology. Binding of antibodies was detected as described above.

Reverse transcription (RT)-PCR and analysis of PCR products

Cells were plated in 100 mm tissue culture dishes, at a density of 5 × 106 cells per dish, and treated with various concentrations of P. intermedia lipopolysaccharide for the indicated periods of time. Following incubation, they were washed twice with PBS and collected by centrifugation. Total RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. Synthesis of cDNA from the extracted RNA and subsequent amplification of the cDNA by RT-PCR were carried out with an AccuPower RT/PCR Premix kit (Bioneer, Korea) and a thermal cycler (GeneAmp PCR system 2400; PE Applied Biosystems, Foster City, CA). β-actin served as the internal control. PCR amplification of TNF-α was carried out for 35 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The oligonucleotide primers were as follows: TNF-α, 5′-TCAGCCTCTTCTCCTTCCTG-3′ (sense) and 5′-TGAAGAGGACCTGGGAGTAG-3′ (antisense) (corresponding to positions 183–202 and 506–487, respectively, of the published human TNF-α mRNA sequence), yielding a 324-bp product; β-actin, 5′-AAGAGAGGCATCCTCACCCT-3′ (sense) and 5′-TACATGGCTGGGGTGTTGAA-3′ (antisense) (corresponding to positions 222–241 and 439–420, respectively, of the published human actin mRNA sequence), yielding a 218-bp product. The PCR-amplified products were run on a 1.5% agarose gel containing ethidium bromide and visualized with UV light.

Statistical analysis

Statistical analysis was performed using Student's paired t-test with P<0.05 considered to be statistically significant. Data are expressed as means±SD of four independent experiments.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

TNF-α induction by P. intermedia lipopolysaccharide

The concentrations of TNF-α were measured 24 h after adding various concentrations of purified P. intermedia ATCC 25611 lipopolysaccharide to differentiated THP-1 cells. Prevotella intermedia lipopolysaccharide induced TNF-α release from the THP-1 cells over the range 0.1 ng mL−1–10 μg mL−1 (Fig. 1a). Basal TNF-α release was about 0.01 ng mL−1. It was effective at a concentration as low as 0.1 μg mL−1, and maximum TNF-α production (about 3.63 ng mL−1) was achieved at a concentration of 10 μg mL−1. Salmonella typhimurium lipopolysaccharide, as a control, also stimulated TNF-α production to a maximum of 3.04 ng mL−1. Its activity was similar to that of P. intermedia lipopolysaccharide with respect to the minimum stimulatory dose, although TNF-α induction by P. intermedia lipopolysaccharide was significantly greater than with S. typhimurium lipopolysaccharide.

image

Figure 1.  Dose response (a) and time course (b) of TNF-α production by differentiated THP-1 cells stimulated with Prevotella intermedia lipopolysaccharide. Salmonella typhimurium lipopolysaccharide was used as a control. The results are means±SD of four experiments. (a) Cells were incubated with increasing concentrations of lipopolysaccharide, and the supernatants were removed after 24 h and assayed for TNF-α. (b) Cells were incubated with 10 μg mL−1 of lipopolysaccharide for different periods of time.

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THP-1 cells were challenged with 10 μg mL−1 of P. intermedia lipopolysaccharide, and the production of TNF-α in the culture supernatant was measured at various time points thereafter. TNF-α secretion increased linearly from 0 to 48 h. TNF-α accumulation reached 6.55 ng mL−1 (Fig. 1b). Salmonella typhimurium lipopolysaccharide also caused a marked elevation in TNF-α secretion from 0 to 48 h.

Expression of TNF-α mRNA by P. intermedia lipopolysaccharide

Exposure of cells to P. intermedia lipopolysaccharide enhanced TNF-α mRNA expression (Fig. 2). When differentiated THP-1 cells were exposed to increasing concentrations of P. intermedia lipopolysaccharide, there was a concentration-dependent accumulation of TNF-α mRNA (Fig. 2a). TNF-α mRNA was detectable with a concentration of P. intermedia lipopolysaccharide as low as 0.001 μg mL−1, and reached a maximum at a concentration of 10 μg mL−1. Figure 2b shows the time course of changes in TNF-α mRNA expression induced by 10 μg mL−1 of P. intermedia lipopolysaccharide. The maximum expression of TNF-α mRNA was achieved at 2 h.

image

Figure 2.  Dose response (a) and time course (b) of TNF-α mRNA expression in differentiated THP-1 cells stimulated with Prevotella intermedia lipopolysaccharide. See ‘Materials and methods’ for further details. The PCR bands on a gel photograph in one of two separate experiments yielding similar results are shown. (a) Cells were incubated with different concentrations of P. intermedia lipopolysaccharide for 2 h. (b) Cells were incubated in the presence of 10 μg mL−1 of P. intermedia lipopolysaccharide for different periods of time.

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Phosphorylation of MAPKs by P. intermedia lipopolysaccharide

To investigate whether the MAPK-related signaling pathways are involved in P. intermedia lipopolysaccharide-induced TNF-α production in differentiated THP-1 cells, the activation of MAPKs (ERK1/2, JNK1/2, and p38) in cells exposed to P. intermedia lipopolysaccharide was analyzed. As shown in Fig. 3, immunoblot analysis revealed that P. intermedia lipopolysaccharide activated MAPKs in differentiated THP-1 cells. When differentiated THP-1 cells were exposed to increasing concentrations of P. intermedia lipopolysaccharide, there was a concentration-dependent increase in the level of phosphorylation of all three MAPKs (Fig. 3a). An increase in phosphorylated forms of each kinase by P. intermedia lipopolysaccharide treatment was first detected after 10 min for ERK and p38 kinase and after 15 min for JNK, and the phosphorylation of three MAPKs markedly increased to the maximum level between 30 and 60 min after beginning the treatment and declined thereafter (Fig. 3b). The relative expression levels of total MAPKs were not significantly affected by P. intermedia lipopolysaccharide treatment.

image

Figure 3.  Dose response (a) and time course (b) of MAPK phosphorylation in differentiated THP-1 cells stimulated with Prevotella intermedia lipopolysaccharide. See ‘Materials and methods’ for further details. A representative immunoblot from two separate experiments with similar results is shown. (a) Cells were incubated with different concentrations of P. intermedia lipopolysaccharide for 30 min. (b) Cells were incubated in the presence of 10 μg mL−1 of P. intermedia lipopolysaccharide for different periods of time.

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Effects of MAPK inhibitors on TNF-α protein production and mRNA expression

In order to analyze whether the observed P. intermedia lipopolysaccharide-induced phosphorylation of MAPKs could account for the increase in TNF-α protein and mRNA expression in P. intermedia lipopolysaccharide-stimulated THP-1 cells, cells were pretreated with specific inhibitors of MAPKs for 30 min before incubation with 10 μg mL−1 of P. intermedia lipopolysaccharide. Pretreatment with PD98059 and SP600125, specific inhibitors of ERK and JNK pathways, respectively, significantly attenuated the stimulation of TNF-α secretion by P. intermedia lipopolysaccharide in the range between 0.1 and 50 μM (Fig. 4a and b). TNF-α production was completely blocked when PD98059 was used at a 10 μM concentration. However, the p38 MAPK inhibitor SB203580 was less potent in the suppression of P. intermedia lipopolysaccharide-induced TNF-α production than PD98059 and SP600125 as shown in Fig. 4c. Treatment of cells with PD98059, SP600125, and SB203580 at the concentrations used did not significantly affect cell viability up to 24 h of incubation (data not shown).

image

Figure 4.  Effects of MAPK inhibitors on TNF-α protein production induced by Prevotella intermedia lipopolysaccharide. Differentiated THP-1 cells were pretreated with the indicated concentrations of each inhibitor before a 24-h incubation with P. intermedia lipopolysaccharide (10 μg mL−1). Other details as in Fig. 1.

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The effects of MAPK inhibitors on TNF-α mRNA accumulation were assessed by RT-PCR. THP-1 cells were pretreated with specific inhibitors of MAPKs for 30 min and then treated with 10 μg mL−1 of P. intermedia lipopolysaccharide for 2 h. As shown in Fig. 5, pretreatment of the cells with MAPK inhibitors did not affect TNF-α mRNA accumulation induced by P. intermedia lipopolysaccharide.

image

Figure 5.  Effects of MAPK inhibitors on TNF-α mRNA expression induced by Prevotella intermedia lipopolysaccharide. Cells were pretreated with the indicated concentrations of each inhibitor before a 2-h incubation with P. intermedia lipopolysaccharide (10 μg mL−1). Total RNA was extracted, and the expression of TNF-α mRNA was evaluated by RT-PCR. See ‘Materials and methods’ for further details. The PCR bands on a gel photograph in one of two separate experiments yielding similar results are shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Because the production of TNF-α has been recognized to be a marker in a variety of human diseases associated with inflammation (Birkedal-Hansen, 1993), the effects of the lipopolysaccharide of P. intermedia on the production of TNF-α and the expression of TNF-α mRNA in differentiated THP-1 cells, a human monocytic cell line, were studied, and the role of MAPK signaling pathways in the process was assessed. Macrophages are known to be the main producers of TNF-α and a dense infiltration of inflammatory cells, including macrophages, occurs in the gingival connective tissues of patients with periodontal disease (Stoufi et al., 1987). To minimize the effects of contaminating protein, the P. intermedia lipopolysaccharide preparation was treated with proteinase K.

Salmonellatyphimurium lipopolysaccharide was used as a control and the TNF-α induction was compared with P. intermedia lipopolysaccharide. Lipopolysaccharide preparations extracted from oral black-pigmented bacteria including P. intermedia have been reported to possess unique chemical and immunobiological properties quite different from those of the classical lipopolysaccharides from the family Enterobacteriaceae such as Escherichia coli and Salmonella species (Hamada et al., 1990). Kirikae et al. (1999) also indicated that the active molecule(s) and mode of action of P. intermedia lipopolysaccharide are quite different from those of lipopolysaccharide from Salmonella. Hashimoto et al. (2003) demonstrated the structure of lipid A from P. intermedia ATCC 25611 lipopolysaccharide to be composed of a diglucosamine backbone with a phosphate at the 4-position of the nonreducing side sugar, as well as five fatty acids containing branched long chains. Moreover, they also found that the lipid A activates murine cells through a TLR4-mediated signaling pathway.

TNF-α induction by P. intermedia lipopolysaccharide in differentiated THP-1 cells proved to be significantly greater than with S. typhimurium lipopolysaccharide. TNF-α is controlled mainly at the transcriptional level and it was confirmed in this study that P. intermedia lipopolysaccharide induces TNF-α expression predominantly at the transcriptional level. It is of interest to note that there was some delay between TNF-α mRNA expression and TNF-α production. Evidently, in P. intermedia lipopolysaccharide-stimulated THP-1 cells, TNF-α mRNA is produced at high levels, but the decrease before TNF-α secretion reaches its maximum, and high levels of TNF-α are found long after expression of TNF-α mRNA had started to decline.

MAPKs, including ERK, JNK, and p38, are important components of many intracellular signaling pathways and are activated by phosphorylation. Various members of the MAPK family are considered to play roles in inflammatory responses (Hambleton et al., 1996; Carter et al., 1999) and may modulate the production of TNF-α in stimulated monocytes/macrophages. The results of this study indicate that P. intermedia lipopolysaccharide is capable of simultaneous activation of three MAPKs, ERK, JNK, and p38, in differentiated THP-1 cells.

To determine the involvement of MAPKs in transcriptional and translational regulation of P. intermedia lipopolysaccharide-induced TNF-α production, blocking studies were also performed using specific inhibitors of the MAPK pathways. In the present study, all of the MAPK inhibitors effectively suppressed P. intermedia lipopolysaccharide-induced TNF-α production in THP-1 cells without affecting the expression of TNF-α mRNA, suggesting a posttranscriptional action. These data thus provided good evidence that the MAPK signaling pathways, including ERK, JNK, and p38, are required for regulation of P. intermedia lipopolysaccharide-induced TNF-α synthesis at the level of translation more than at a transcriptional level. The p38 MAPK inhibitor SB203580 had a much smaller effect on the P. intermedia lipopolysaccharide-induced TNF-α protein production than did the ERK inhibitor PD98059 and JNK inhibitor SP600125, suggesting that ERK and JNK may more strongly be involved in the production of TNF-α than p38 MAPK. It would be interesting to find out how these pathways coordinate for TNF-α production.

Understanding the intracellular signaling events triggered by P. intermedia lipopolysaccharide in THP-1 cells may be important in designing a novel therapeutic strategy for periodontal disease. The inhibition of the MAPK pathways could be advocated as a therapeutic strategy for inflammatory diseases such as periodontitis, and the findings from this study may justify the use of specific inhibitors of MAPKs as a new approach to the treatment and prevention of inflammatory periodontal disease. It is noteworthy that ERK inhibitor PD98059 showed potent inhibition of TNF-α production without affecting cell viability (about 100% inhibition at the test concentration of 10 μM). The inhibition of TNF-α production by PD98059 may be useful in the therapy of inflammatory diseases such as periodontitis. This hypothesis, however, remains to be tested.

The periodontium is consistently in contact with lipopolysaccharide produced by gram-negative periodontopathogenic bacteria. TNF-α synthesis is increased in periodontal disease, as a result of macrophage infiltration in the periodontal tissues (Rossomando et al., 1990; Stashenko et al., 1991). TNF-α might play a role in the pathogenesis of both periodontitis and subsequent bone loss, either directly, or indirectly by promoting the release of tissue-derived enzymes, the matrix metalloproteinases (Brenner et al., 1989; Kobayashi et al., 2000). The ability of P. intermedia lipopolysaccharide to promote the production of TNF-α may be important in the establishment of the chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease. However, it is unclear whether the activation of the MAPK signaling pathway by P. intermedia lipopolysaccharide is sufficient for the induction of TNF-α expression in THP-1 cells. The precise mechanism by which P. intermedia lipopolysaccharide induces TNF-α production remains to be elucidated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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