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RESEARCH REPORT |
1 Department of Periodontology, Faculty of Dentistry, Chulalongkorn University, Henry Dunant Rd., Bangkok 10330, Thailand;
2 Immunology Lab, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand;
3 Department of Oral Medicine, Faculty of Dentistry, Mahidol University, Bangkok, Thailand;
4 Department of Periodontics & Endodontics, State University of New York at Buffalo, Buffalo, NY, USA;
5 Department of Immunology and Medicine, US Army Medical Component, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand;
* corresponding author, mrangsin{at}chula.ac.th
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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T-cells were analyzed by flow cytometry, and the production of interleukin-1ß and prostaglandin E2 was monitored by ELISA. LPS stimulation resulted in a dose-dependent up-regulation of CD40, CD80, and CD86 on monocytes, and up-regulation of CD69 on NK cells and T-cells in both the periodontitis and non-periodontitis groups. The levels of activation markers and the mediator production after LPS stimulation were quite similar for both groups. In conclusion, we did not observe hyper-responsiveness of monocytes to P. gingivalis LPS challenge in Thai patients with aggressive periodontitis.
KEY WORDS: monocyte activation • Porphyromonas gingivalis • co-stimulatory molecule expression • whole-blood cultures
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Early studies suggested that certain individuals are more susceptible to severe forms of periodontitis, including aggressive periodontitis, and this may be due to genetic heterogeneity of the host defense system against microbial challenge (Garrison and Nichols, 1989; Shapira et al., 1994; Offenbacher and Salvi, 1999). Hyper-responsiveness of monocytes to bacterial LPS, e.g., Escherichia coli and P. gingivalis, has been hypothesized as the immunological profile of the aggressive periodontitis group. The release of high levels of monocytic inflammatory mediators such as prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-
) in these patients has been thought to be associated with the disease pathogenesis (Offenbacher and Salvi, 1999). Most of these studies were performed with the use of peripheral blood mononuclear cells (PBMC) or monocytes isolated from patients having active periodontal infection. It is known that during active bacterial, viral, or parasitic infections, significant immunological abnormalities, including increased levels of cytokines, are commonly observed in peripheral blood (Hober et al., 1993; May et al., 2000; Groeneveld et al., 2003). The cytokine-rich environment could indeed ‘prime’ peripheral blood cells to become over-reactive or poorly reactive. Therefore, analysis of the patient’s immune cell activity or function in response to different stimulants in vitro during active disease may not be appropriate for studying genetic-associated hyper-responsiveness. In this study, we avoided the untreated aggressive periodontitis patients and selected those who had completed the hygienic phase of periodontal therapy.
Previous studies performed on PBMC or monocytes involved mononuclear cell separation from peripheral blood, which may introduce problems associated with the pre-activation of monocytes. The introduction of simple whole-blood cultures which permit the measurement of activation markers on monocytes and cytokines released from stimulated immune cells (Petrovsky and Harrison, 1995; Pichyangkul et al., 2001; Hussain et al., 2002) has opened up new avenues for re-visiting the issue of monocyte hyper-responsiveness in patients susceptible to periodontitis. In this study, we investigated P. gingivalis LPS-stimulated monocytes in un-manipulated whole blood from aggressive periodontitis patients and compared results with those from age- and sex-matched non-periodontitis subjects. Expression of co-stimulatory molecules (CD40, CD80, and CD86) on monocytes was analyzed by flow cytometry, and the production of inflammatory mediators (IL-1ß and PGE2) was monitored by ELISA.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Monoclonal antibodies against CD14 (FITC), CD56 (PE), TCR- (PE), and CD69 (FITC) were obtained from Becton Dickinson (San Jose, CA, USA). Monoclonal antibodies against CD40 (PE), CD80 (PE), and CD86 (PE), as well as mouse isotype control monoclonal antibodies (FITC, PE), were obtained from PharMingen (San Jose, CA, USA).
Highly purified LPS from P. gingivalis strain 381 was prepared as previously described (Schifferle et al., 1998; Preshaw et al., 1999). Briefly, the LPS was purified by phenol-water extraction and subsequent treatment with DNase I, RNase A, and proteinase K, followed by chromatographic purification. The purity of the preparation was confirmed by immunodiffusion analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining.
Subjects
Under a protocol approved by the Thailand Research Fund review board, subjects signed a consent form after being advised of the nature of the study. Seventeen pairs of periodontitis patients and non-periodontitis control subjects (age- and sex-matched) were recruited from the Department of Periodontology, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand. There were four males and 13 females in each group (aged 20 to 36 yrs). The average ages of males and females were 28.5 ± 1.78 yrs and 29.0 ± 1.34 years, respectively. Periodontitis patients were clinically diagnosed as having generalized aggressive periodontitis based on the Consensus Report for aggressive periodontitis of the 1999 International Workshop for a Classification of Periodontal Diseases and Conditions. In brief, the patients had at least 20 natural teeth and showed pronounced gingival inflammation as detected by bleeding upon probing. They exhibited generalized severe periodontal destruction with interproximal attachment loss (
5 mm) affecting at least 3 permanent teeth other than first molars and incisors. To minimize the stage of active periodontal infection, we took heparinized blood from the patients after completion of their hygienic periodontal treatment. Clinical improvement was generally observed with significant reduction in gingival inflammation and the amounts of microbial deposits. The non-periodontitis control subjects consisted of individuals with clinically healthy gingiva or mild marginal gingivitis with no teeth showing more than 3 mm attachment loss. The controls were matched to the patients by age and gender. All subjects were free of systemic disease. No subjects had received antibiotics or systemic steroid therapy within the preceding 3 mos.
Whole-blood Cultures and PBMC Assays
Peripheral blood was obtained by venipuncture from generalized aggressive periodontitis patients and non-periodontitis control subjects. For whole-blood assay, heparinized whole blood was diluted (1:1) with RPMI 1640 medium. For PBMC assays, PBMCs were isolated by a standard density gradient method. Cells were re-suspended in RPMI 1640 medium containing 10% heat-inactivated autologous serum. Various concentrations of P. gingivalis LPS at 0, 1, 3, and 10 ng/mL were added to 1 mL whole-blood cultures or PBMC cultures (2 x 106 cells/mL) and incubated at 37°C with 5% CO2 for 48 hrs. Cells were then stained and analyzed by flow cytometry, and supernatant mediator levels were measured.
Flow Cytometry Analysis
Whole-blood or PBMC cultures incubated with various concentrations of P. gingivalis LPS were separated into aliquot portions and then stained for 30 min at room temperature (RT) with 1 of 5 monoclonal antibody combinations: (1) anti-CD14 (FITC) plus anti-CD40 (PE), (2) anti-CD14 (FITC) plus anti-CD80 (PE), (3) anti-CD14 (FITC) plus anti-CD86 (PE), (4) anti-CD56 (PE) plus anti-CD69 (FITC), or (5) anti-TCR- (PE) plus anti-CD69 (FITC). Mouse isotype control antibodies conjugated with PE or FITC were used as controls.
The stained blood cells were treated with red blood cell lysing solution (FACs Lysing Solution, Becton Dickinson) for 10 min at RT in the dark. The cells were washed in phosphate-buffered saline (PBS) and then reconstituted in 1% paraformaldehyde. For analysis, 100,000 cells were analyzed by two-color flow cytometry (FACSCalibur, Becton Dickinson, Mountain View, CA, USA). CD14+, CD56+, and TCR-+ cells were gated as monocytes, NK cells, and T-cells, respectively. CD14+ monocytes were analyzed for expression of CD40, CD80, and CD86, whereas CD56+ NK cells and T-cells were analyzed for expression of CD69. Results were expressed as either mean fluorescence intensity (MFI) (monocytes) or % positive cells (NK cells and T-cells).
Mediator Measurement
Supernatant mediator levels were measured with the use of commercially available ELISA kits for IL-1ß and PGE2 (R&D Systems, Minneapolis, MN, USA). The detection limits for IL-1ß and PGE2 were 1 pg/mL and < 36.5 pg/mL, respectively.
Statistical Analysis
Data were analyzed by SigmaStat (Jandel Scientific, San Rafael, CA, USA). The Student’s t test was used for parametric data, and the Mann-Whitney rank-sum test was used for non-parametric data. P values of 0.05 or less were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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shows representative histograms (n = 3) comparing whole blood with PBMC cultures regarding CD40 and CD80 expression on monocytes. In unstimulated cultures, PBMC monocytes consistently expressed detectable levels of CD40 and CD80, whereas the expression in whole-blood monocytes was minimal. Unlike the PBMC cultures, P. gingivalis LPS induced up-regulation of monocyte CD40 and CD80 in whole-blood cultures. Fold increase in mean fluorescence intensity (MFI) was used for comparison between the two assays (Fig. 1B) and was calculated as MFI of cell-surface molecule expression of interest in stimulated cultures divided by MFI of cell-surface molecule expression in unstimulated cultures. The mean MFI-fold increase of monocyte CD40 and CD80 expression in whole-blood cultures was markedly higher than in the PBMC cultures (CD40, 4.1 ± 2.58 vs. 0.4 ± 0.05; and CD80, 6.6 ± 2.99 vs. 1.1 ± 0.09).
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. Generally, P. gingivalis LPS induced up-regulation of monocyte CD40, CD80, and CD86 expression in a dose-dependent manner. The response expressed as an MFI-fold increase between aggressive periodontitis and non-periodontitis subjects was not significant.
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T-cells in P. gingivalis LPS-stimulated whole-blood cultures. The fold increase of % positive cells of CD56+CD69+ and TCR-+CD69+ cells was comparable between the two study groups (Fig. 2B
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Assessment of IL-1ß and PGE2 Production in Aggressive Periodontitis Patients and Non-periodontitis Subjects
The production of IL-1ß and PGE2 from aggressive periodontitis patients and non-periodontitis subjects (n = 17, each group) was measured from supernatants of whole-blood cultures following 48 hrs of P. gingivalis LPS stimulation. Fig. 3 illustrates IL-1ß and PGE2 production in both study groups in response to increasing concentrations of P. gingivalis LPS (0, 1, 3, and 10 ng/mL). Culture supernatants of the unstimulated whole-blood cultures demonstrate some level of PGE2 (238 ± 15 pg/mL) production. Comparison between the two study groups revealed no statistical significance in either IL-1ß or PGE2 production at all LPS concentrations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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is important in enhancing the levels of cytokine secretion (Galbraith et al., 1999). The genotypic polymorphism of IL-1ß (IL-1ß +3953 loci) has been reported to associate with a four-fold increase in IL-1ß production (Kornman and di Giovine, 1998). The idea of using a genotypic polymorphism to determine the risk of developing chronic periodontitis is very attractive; however, it is far from straightforward. Studies in a Caucasian population link the IL-1 genotype with severity of periodontitis (Kornman et al., 1997; Cullinan et al., 2001). However, recent studies reported that Chinese (Armitage et al., 2000), Thai (Anusaksathien et al., 2003), and Japanese (Tai et al., 2002) populations who were susceptible to periodontitis are IL-1 genotype-negative. Only 2.3% of the Chinese, 1.6% of the Thai, and 9.3% of the Japanese subjects were IL-1 genotype-positive (Armitage et al., 2000; Tai et al., 2002; Anusaksathien et al., 2003). These findings suggest that IL-1 gene polymorphism may not be appropriate as a method for determining disease susceptibility.
In this study, we re-evaluated the issue of monocyte hyper-responsiveness associated with an increased severity of periodontal disease. We used a whole-blood culture, which provides a simple tool and is more relevant for assessing immune response, to investigate LPS-induced monocyte activation. In our initial experiments, we compared whole-blood and PBMC cultures for the measurement of P. gingivalis LPS-induced monocyte response, specifically focused on up-regulation of CD40 and CD80. Analysis of the data indicated that the whole-blood culture is more sensitive than the PBMC culture with regard to the enhanced expression of monocyte CD40 and CD80. Analysis of data from the whole-blood culture in both non-periodontitis and aggressive periodontitis patients demonstrated that P. gingivalis LPS induced the up-regulation of monocyte CD40, CD80, and CD86 in a dose-dependent manner. Up-regulation of CD69 on NK cells and T-cells was also observed. Statistical analysis revealed that the fold increase of monocyte CD40, CD80, and CD86 as well as the fold increase of CD69 expression on NK cells and T-cells was not significant between the two study groups. In addition, the production of IL-1ß and PGE2 revealed no differences between the non-periodontitis and the aggressive periodontitis groups.
Early studies pointed out the predominant role of Actinobacillus actinomycetemcomitans in aggressive periodontitis (Fives-Taylor et al., 1999; Slots and Ting, 1999). Using molecular-based approaches, recent groups of investigators have studied Korean and Japanese aggressive periodontitis subjects and have demonstrated that P. gingivalis, Tannerella forsythensis, and Treponema species had a high prevalence as compared with A. actinomycetemcomitans (Lee et al., 2003; Takeuchi et al., 2003). This discrepancy could be due to racial differences in the populations studied and/or to the methodology used for bacterial analysis. P. gingivalis was suspected to be involved in Thai aggressive periodontitis; however bacterial analysis was not evaluated in this study. Future investigation involving monocyte response to LPS from different periodontopathic bacteria in conjunction with bacterial identification is warranted.
Our inability to detect hyper-responsiveness (co-stimulatory molecule expression and mediator production) in our study (Thai population) does not support the earlier hypothesis. The study of the inappropriate response of only one cell type, such as monocytes/macrophages, may not allow for the explanation of the mechanism of disease susceptibility. Several cell types, including immune cells and non-immune cells, actively participate in the pathologic process. Non-immune cells—especially epithelial cells, fibroblasts, and endothelial cells—have recently received special attention, since they can interact with bacterial components through Toll-like receptors (TLRs), leading to cytokine production (Tabeta et al., 2000; Asai et al., 2001; Bulut et al., 2002). In addition to IL-1, TNF-, and PGE2, other mediators that are released from activated T-cells deserve attention as well. IFN- up-regulates CD40 on gingival fibroblasts (Sempowski et al., 1997) and induces expression of TLR4 on epithelial cells (Wolfs et al., 2002). IL-17 stimulates epithelial cells, endothelial cells, and fibroblasts to secrete IL-6, IL-8, granulocyte macrophage-colony stimulating factor (GM-CSF), as well as PGE2 (Fossiez et al., 1998). IL-17 may also be involved in bone resorption (van Bezooijen et al., 1999). The development of periodontitis may be more complex than we have thought and thus may require additional detailed analysis of the inappropriate responses of a variety of cell types and cytokines.
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ACKNOWLEDGMENTS |
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Received September 2, 2003; Last revision April 4, 2004; Accepted May 6, 2004
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