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P. gingivalis-specific T-cell Lines Produce Th1 and Th2 Cytokines

Immunopathology Laboratory, Oral Biology and Pathology, School of Dentistry, The University of Queensland, Brisbane 4072, Australia;

*corresponding author, e.gemmell{at}mailbox.uq.edu.au

	ABSTRACT

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Cytokines produced by T-cells in periodontal lesions may determine the nature of the adaptive immune response. Since different antigen-presenting cells (APC) may direct the Th1/Th2 response, P. gingivalis-specific T-cell lines were established by different APC subpopulations, and their cytokine profiles were determined. Peripheral blood mononuclear cells induced similar percentages of IL-4+ and IFN-gamma+ T-cells and lower percentages of IL-10+ T-cells. Epstein-Barr virus-transformed B-cells (LCL) induced higher percentages of IL-4+ cells than IFN-gamma+ cells, with lower percentages of IL-10+ cells. Peripheral blood mononuclear cells induced a higher percent of IFN-gamma+ CD8 cells than LCL (p = 0.004). Purified B-cells, monocytes, and dendritic cells induced similar percentages of IL-4+ and IFN-gamma+ cells, although again, the percentage of IL-10+ cells was lower. The results of the present study have demonstrated that, as measured by FACS analysis of intracytoplasmic cytokines, P. gingivalis-specific T-cells produce both Th1 and Th2 cytokines, regardless of the APC population.

KEY WORDS: P. gingivalis • T-cells • periodontal disease • cytokines • antigen-presenting cells

	INTRODUCTION

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Periodontal disease is a chronic inflammatory response to plaque bacteria. The type of immune response that occurs in the periodontal lesion is vital in determining whether or not a protective outcome occurs. T-cells are the dominant cell type, and the cytokines they produce determine the nature of specific antibody production and the outcome of the disease (Gemmell et al., 1997). The transition from a stable gingivitis to a progressive periodontitis lesion is accompanied by a shift in lymphocyte populations in the inflammatory infiltrate from a T-cell-dominant lesion with few B-cells to one with an increased proportion of B-cells and plasma cells (Seymour, 1991). The increased proportions of B-cells in periodontitis suggest a role for Th2-type cytokines such as IL-4, which are required for B-cell proliferation and differentiation. The Th1 cytokine IFN-gamma is found in sites of delayed-type hypersensitivity (DTH) reactions (Tsicopoulos et al., 1992), and the developing gingival lesion has been shown to follow a pattern similar to that of a controlled DTH response (Seymour et al., 1988), suggestive of a Th1 response. IL-10, which is produced by both Th1 and Th2 cells in the human, plays a major role in suppressing immune and inflammatory responses and is a potent inhibitor of IL-1 production (De Waal Malefyt et al., 1993). At this stage, the role of Th1 and Th2 cytokines in periodontal disease is not clear.

Several factors influence the development of T-cell subsets, including APC, different subpopulations of which have been suggested to direct T-cells to a Th1 or Th2 pathway (Bloom et al., 1992). P. gingivalis is a major periodontopathogen, and the cytokines induced in the host by this organism may influence the course of periodontal disease. The aim of the present study was to determine whether presentation of P. gingivalis antigens by different APC affected the cytokine responses of P. gingivalis-specific T-cell lines.

	MATERIALS & METHODS

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Subjects
Peripheral blood samples were collected from a total of 20 subjects. A written explanation of the purpose of the study was provided for each subject, and signed consent according to the Helsinki Declaration was obtained. Institutional ethics review committee approval for the study was obtained. Eleven of the subjects had periodontal disease and attended the Periodontal Clinic, Dental School, The University of Queensland. The other nine subjects were healthy volunteers from the staff of the School of Dentistry, the University of Queensland. While serum anti-P. gingivalis antibody levels were determined, no differences in cytokine responses were found between the antibody-positive and -negative groups, nor were differences found relative to clinical status. The subjects therefore were treated as one group. The clinical status, age, and gender parameters of the 20 subjects are outlined in the Table.

Subsequent Use of LCL as APC to Present P. gingivalis Antigens to P. gingivalis-specific T-cell Lines
Fifteen of the P. gingivalis-specific T-cell lines established above were subsequently re-stimulated with the use of autologous Epstein-Barr virus (EBV)-transformed B-cells (lymphoblastoid cell lines, LCL) as APC. These LCL were prepared as described previously (Gemmell et al., 1998, 1999) and used to stimulate the P. gingivalis-specific T-cell lines at least 3 times (5-6 wks in culture). We stained the T-cells again to determine the cytokine profiles of each line.

Generation of P. gingivalis-specific T-cell Lines with B-cells and Monocytes as APC
Further blood samples were obtained from ten subjects (subjects 7, 10, 11, 12, and 14-19 in the Table), and the B-cells and monocytes were extracted from each mononuclear cell sample by magnetic cell separation. B-cells were first labeled with CD19+ MACS Microbeads, after which the cells were separated with the use of an LS+ column on a MidiMACS magnetic separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Monocytes were then separated after being labeled with CD14+ MACS Microbeads. Analysis of the monocyte fractions showed 1.42 ± 0.55% contamination with B-cells and 3.51 ± 0.60% contamination by monocytes of the B-cell fractions. The resulting T-cell fraction demonstrated 1. 15 ± 0.42 and 2.11 ± 0.92% contamination by monocytes and B-cells, respectively.

Two T-cell lines from each blood sample were then established, T-cells being incubated with P.gingivalis outer membrane antigens together with irradiated B-cells to establish one line, and irradiated monocytes to establish the second T-cell line, for each subject. Re-stimulation was carried out every 2 wks as above with antigen and the irradiated APC of choice, and after approximately 5-6 wks, the T-cells were again stained for intracytoplasmic cytokines.

Generation of P. gingivalis-specific T-cell Lines with Dendritic Cells Generated with GM-CSF and IL-4 as APC
Further blood samples were taken from eight subjects (subjects 2, 10, 11, 14, and 16-19 in the Table). The method used for the generation of dendritic cells was based on that described by Häusser et al. (1997). Monocytes from each mononuclear cell sample were extracted by magnetic cell separation as described above, and then incubated at a concentration of 2 x 10⁶ cells/well in 24-well tissue culture plates (Nunc, Roskilde, Denmark) in RPMI-1640 containing human AB serum, glutamine, and antibiotics (Gemmell et al., 1998), together with recombinant human GM-CSF (500 U/mL) (PharMingen, San Diego, CA, USA) and recombinant human IL-4 (200 U/mL) (PharMingen). Immature dendritic cells were present after 6-7 days. P. gingivalis antigens (5 µg/mL) were then added for 24 hrs. We added recombinant human TNF-alpha (400 U/mL) (PharMingen) for another 24 hrs to ensure maturation of P. gingivalis-pulsed dendritic cells. The pure population of dendritic cells was identified by their typical morphology and confirmed by the expression of CD1a and CD83 as determined by flow cytometry. The dendritic cells were then added to purified populations of autologous T-cells for 2 wks. At wks 2 and 4, the T-cells were again re-stimulated with the use of mature P. gingivalis-pulsed dendritic cells, after which the T-cells were stained for the presence of intracytoplasmic cytokines.

Generation of P. gingivalis-specific T-cell Lines with Peripheral Blood Dendritic Cells
Further blood samples were obtained from eight subjects (subjects 7, 10, 11, 14, and 17-20 in the Table), and the monocytes and B-cells were extracted from the mononuclear cells by magnetic cell separation. The remaining fraction contained T-cells and peripheral blood dendritic cells, which were incubated with P. gingivalis antigens (5 ìg/mL) for 6 hrs, after which we added TNF-alpha to ensure maturation of P. gingivalis-pulsed dendritic cells. Two and 4 wks later, stored autologous T-cell/dendritic cell fractions were pulsed with P. gingivalis outer membrane antigens and treated with TNF-alpha as above. After gamma-irradiation, the cells were added to the T-cell lines. After 5-6 wks, the cytokine profiles of the T-cell lines were determined.

Statistics
We carried out multivariate analysis of variance using the general linear model to test for differences between the expression of each cytokine by CD4 and CD8 cells. Selected pairs of groups were then tested for significance by means of Student's t test. A significance level of 0.02 was determined to reduce the probability of significant differences occurring by chance. We used the Minitab statistical package (Minitab Inc., State College, PA, USA) to perform the analyses.

	RESULTS

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Presentation of P. gingivalis outer membrane antigens by autologous peripheral blood mononuclear cells resulted in profiles in which the mean percent IL-10+ CD4 cells was significantly lower than the percent IL-4+ (p = 0.014) and IFN-gamma+ CD4 cells (p = 0.001) (Fig. 1A). Presentation of P. gingivalis to these same lines by autologous LCL resulted in profiles in which IL-4+ CD4 and CD8 cells predominated, with lower percentages of IFN-gamma+ T-cells and very low IL-10+ cells (Figs. 1A, 1B). The mean percentages of IL-4+ CD4 and CD8 cells were significantly higher than the mean percentages of IFN-gamma+ CD4 and CD8 cells (p = 0.001 and 0.000, respectively) and IL-10+ CD4 and CD8 cells (p = 0.000 and 0.000, respectively). The mean percentages of IFN-gamma+ CD4 and CD8 cells were also significantly higher compared with those of IL-10+ CD4 and CD8 cells (p = 0.000 and 0.000, respectively).

View larger version (35K): [in this window] [in a new window]   Figure 1. Cytokine expression by P. gingivalis-specific T-cell lines in response to homologous outer membrane antigens presented by mononuclear cells. (A) Mean (± standard error of the mean) % IL-4+, IFN-gamma+, and IL-10+ CD4 cells in P. gingivalis-specific T-cell lines established following presentation of P. gingivalis outer membrane antigens by peripheral blood mononuclear cells (PBMC) (n = 16) and EBV-transformed B-cells (LCL) (n = 15), respectively. (B) Mean (± standard error of the mean) % IL-4+, IFN-gamma+, and IL-10+ CD8 cells in P. gingivalis-specific T-cell lines established following presentation of P. gingivalis outer membrane antigens by peripheral blood mononuclear cells (PBMC) (n = 16) and EBV-transformed B-cells (LCL) (n = 15), respectively.

Lines established from subjects in whom B-cells, monocytes, and dendritic cells generated from monocytes and peripheral blood dendritic cells as APC were used all displayed Th1 and Th2 patterns (Figs. 2A, 2B). In all lines, the percent IL-4+ CD4 cells were higher than the percent IL-10+ CD4 cells (p = 0.004, 0.003, 0.000, and 0.000, respectively) (Fig. 2A).

View larger version (79K): [in this window] [in a new window]   Figure 2. Cytokine expression by specific T-cell lines in response to P. gingivalis outer membrane antigens presented by other antigen-presenting cells. (A) Mean (± standard error of the mean) % IL-4+, IFN-gamma+, and IL-10+ CD4 cells in P. gingivalis-specific T-cell lines established following presentation of P. gingivalis outer membrane antigens by B-cells (n = 8), monocytes (n = 10), monocyte-derived dendritic cells (MoDC) (n = 8), and peripheral blood (PBDC) (n = 8) dendritic cells, respectively. (B) Mean (± standard error of the mean) % IL-4+, IFN-gamma+, and IL-10+ CD8 cells in P. gingivalis-specific T-cell lines established following presentation of P. gingivalis outer membrane antigens by B-cells (n = 8), monocytes (n = 10), monocyte-derived dendritic cells (MoDC) (n = 8), and peripheral blood (PBDC) (n = 8) dendritic cells, respectively.

In lines established with LCL as APC, the percentages of IL-4+ CD4 cells, IFN-gamma+ CD4 and CD8 cells, and IL-10+ CD4 and CD8 cells (Figs. 1A, 1B) were all reduced in comparison with those in lines established with the use of monocytes, B-cells, monocyte-derived, and peripheral blood dendritic cells (p < 0.006) (Figs. 2A, 2B).

	DISCUSSION

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The results of the present study have shown that P. gingivalis-specific T-cell lines established with the use of peripheral blood mononuclear cells as APC demonstrate an IFN-gamma/IL-4 response, while IL-4+ T-cells dominate when LCL are used as APC. The percent IFN-gamma+ CD8 cells was significantly higher in lines where peripheral blood mononuclear cells were used as APC in comparison with that of cells in lines established with the use of LCL. Peripheral blood mononuclear cells consist of several subpopulations, including monocytes, B-cells, dendritic cells, and, of course, T-cells. The use of purified populations of monocytes, B-cells, and either peripheral blood or monocyte-derived dendritic cells as APC resulted in a trend toward a predominant IL-4 or IL-4/IFN-gamma response and lower percentages of IL-10+ T-cells, regardless of the type of APC.

Previous reports have demonstrated decreased Th1 cytokines in peripheral blood mononuclear cell cultures (Gemmell and Seymour, 1994; Sigusch et al., 1998). However, a bias toward Th2-type cytokines in periodontal disease progression has also been demonstrated (Manhart et al., 1994; Yamazaki et al., 1994; Aoyagi et al., 1995; Tokoro et al., 1997). In contrast, Ebersole and Taubman (1994) found that IFN-gamma message was prominently expressed by diseased gingival tissue cells, while another study demonstrated reduced Th2 responses in periodontitis patients with so-called "terminal dentition" periodontitis (Salvi et al., 1998). The involvement of Th0 cells in periodontal disease has also been suggested (Fujihashi et al., 1996; Prabhu et al., 1996). In this context, T-cell lines and clones specific for P. gingivalis have been shown to resemble Th0 cells (Gemmell et al., 1996). However, 80% of the CD4 clones established by non-specific activation from the gingival tissues of four patients with chronic periodontitis have been reported to have Th2 phenotypes producing high levels of IL-4 and low levels of IFN-gamma, while the majority of CD8 clones demonstrated a Th0-like pattern producing equal amounts of IL-4 and IFN-gamma (Wassenaar et al., 1995).

The present study has demonstrated both Th1 and Th2 responses to P. gingivalis outer membrane antigens as measured by analysis of intracytoplasmic stained T-cells. However, cytokine determination by other means, such as measurement of secreted cytokines in the culture supernatants, may or may not provide valuable correlation of these data. The responses to whole P. gingivalis antigens or isolated immunologically dominant antigens may also show different results. P. gingivalis is one of several periodontopathic organisms, including Actinobacillus actinomycetemcomitans and Bacteroides forsythus, and the responses to each of these organisms have yet to be reported. The role of co-infection in determining the cytokine profile remains to be determined (Choi et al., 2000). Also, the CD4/CD8 ratio has been shown to decrease with periodontal disease progression (Gemmell and Seymour, 1994), indicating a role for CD8 cells as well as CD4 cells in periodontitis. Indeed, the present study has demonstrated that CD4 and CD8 cells were stimulated to produce cytokines by P. gingivalis, suggesting a role for both subsets in periodontitis. It is likely that different T-cell subsets predominate at different phases of disease, and investigators' inability to determine disease activity clinically in subjects from whom samples are taken is a major limitation in all these studies.

In conclusion, this study has demonstrated that when purified populations of monocytes, B-cells, or dendritic cells present P. gingivalis outer membrane antigens to P. gingivalis-specific T-cells, the resultant cytokine profile is consistent with both Th1 and Th2 responses with lower percentages of IL-10+ T-cells, both CD4 and CD8 cells being stimulated to similar degrees. Therefore, any shift in the Th1 or Th2 profiles in the periodontal lesion may be due not to the type of APC but rather to other factors, such as the nature of the antigen. Finally, the low percentages of IL-10+ T-cells induced by P. gingivalis have been suggested to indicate progressive disease such that this cytokine may be of fundamental importance in the control of periodontal disease progression (Gemmell et al., 1997).

	ACKNOWLEDGMENTS

 
This work was supported by the Mayne Bequest Fund and by the Government Employees Medical Research Fund.

Received September 11, 2001; Last revision February 19, 2002; Accepted February 28, 2002

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