Gingivalis Outer Membrane Antigens Research Articles

Gene Expression in Splenic CD4 and CD8 Cells From BALB/c Mice Immunized With Porphyromonas gingivalis

T cells are fundamental in the pathogenesis of periodontal disease. Suppression of cell-mediated responses is associated with disease progression together with the concomitant increase in plaque pathogens including Porphyromonas gingivalis. The aim of the present study was to examine gene expression in T cells in response to P. gingivalis in mice. BALB/c mice were given weekly intraperitoneal injections of P. gingivalis outer-membrane antigens with Freund's incomplete adjuvant for 3 weeks, whereas control mice received phosphate buffered saline (PBS) and adjuvant only. Splenic CD4 and CD8 subpopulations were isolated by magnetic cell separation and their responses investigated using microarray analysis. Most genes coded for enzymes concerned with metabolic pathways. Only five and 28 genes, respectively, were upregulated in CD4 and CD8 cells extracted from P. gingivalis-immunized mice, including immunoglobulin (Ig) heavy-chain genes for IgG1 and IgG2a in CD4 cells. In contrast, 1,141 and 1,175 genes, respectively, were downregulated. A total of 60 and 65 genes, respectively, coded for immune response proteins or those relevant to periodontal disease pathogenesis. The overlap of genes in the two subsets was 21%. One of the major effects, apart from T-cell function suppression, was the shift away from Th1 responses, although there was also a downregulation of two genes and upregulation of one Th2-response gene. Genes downregulated included those encoding cytokines, proteins involved in Ig binding, antigen presentation, innate immunity, extracellular matrix, and cell adhesion molecules that could result in dysregulation in the progressive periodontal lesion. Early findings in humans demonstrated that periodontopathic bacteria induce immunosuppressive effects on T cells. The present study has shown that P. gingivalis had a predominant downregulatory effect on gene expression in CD4 and CD8 T cells in mice.

Genetic dependence of the specific T-cell cytokine response to Porphyromonas gingivalis in mice.

Susceptibility to periodontal infections may, in part, be genetically determined. Porphyromonas gingivalis is a major periodontopathogen, and the immune response to this organism requires T-cell help. The aim of the present study was to examine the specific T-cell cytokine responses to P. gingivalis outer membrane antigens in a mouse model and their relationship with H-2 haplotype. BALB/c and DBA/2J (H-2d), CBACaH (H-2k), and C57BL6 (H-2b) mice were immunized with P. gingivalis outer membrane antigens weekly for 3 weeks. One week after the final injection, the spleens were removed, and 6 T-cell lines specific for P. gingivalis were established for each mouse strain. The percentage of CD4 and CD8 cells in the P. gingivalis-specific T-cell lines staining positive for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10 was determined by 2-color flow cytometry. The cytokine profiles of T-cell lines from BALB/c and DBA/2J mice showed no significant differences. Significantly fewer IL-4+, IFN-gamma+, and IL-10+ CD4 cells than IL-4+, IFN-gamma+, and IL-10+ CD8 cells, respectively, were demonstrated for both strains. P. gingivalis-specific T-cell lines generated from CBACaH mice were similar to those generated from BALB/c and DBA/2J mice; however, the mean percentage of IL-4+ CD4 cells in CBACaH mice was lower than the percentage of IFN-gamma+ CD4 cells. Also, the mean percentage of IFN-gamma+ CD4 cells in CBACaH mice was significantly increased compared to DBA/2J mice. Unlike the other 3 strains, T-cell lines established from C57BL6 mice contained similar percentages of cytokine-positive cells, although the percentage of IL-4+ CD4 cells was reduced in comparison to the percentage of CD8 cells. However, comparisons with the other 3 strains demonstrated a higher percentage of IL-4+ CD4 cells than in lines established from the spleens of DBA/2J mice, IFN-gamma+ CD4 cells than in lines established from BALB/c and CBACaH mice, and IL-10+ CD4 cells than in lines established from all 3 other strains. No significant differences in the percentage of positive CD8 cells were demonstrated between lines in the 4 strains of mice. The specific T-cell response to P. gingivalis in mice may, in the case of the CD4 response, depend on MHC genes. These findings are consistent with the concept that patient susceptibility is important to the outcome of periodontal infection and may, in part, be genetically determined.

The influence of genetic variation on the splenic T cell cytokine and specific serum antibody responses to Porphyromonas gingivalis in mice.

T cell cytokine profiles in the spleens and anti-Porphyromonas gingivalis antibodies in the sera of P. gingivalis-immunized BALB/c (H-2d), CBA/CaH (H-2k), C57BL6 (H-2b), and DBA/2J (H-2d, C5 deficient) mice were examined. Mice were immunized either by intraperitoneal injections of P. gingivalis outer membrane antigens and Freund's incomplete adjuvant weekly for 3 weeks or sham-immunized with PBS and adjuvant, followed by subcutaneous challenge with live organisms 1 week after the final immunization. Spleens were excised and blood samples collected by heart puncture at 0 and 7 days after challenge. Splenic CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IF)-gamma, and IL-10 and levels of anti-P. gingivalis antibodies in the serum samples determined by ELISA. Lesion sizes in immunized BALB/c mice remained stable for the 7-day experimental period. Immunized CBA/CaH and C57BL6 mice exhibited large lesions at day 1 reducing by day 7 particularly in the latter strain. Lesions in immunized DBA/2J mice were still larger than the other strains at day 7. With the exception of DBA/2J mice, sham-immunized mice demonstrated lesions which did not show signs of healing by day 7. T cell cytokine responses in sham-immunized mice at day 0 were low, increasing to a variable degree by day 7 after challenge in the 4 strains. Immunized BALB/c mice demonstrated intermediate T cell responses while generally exhibiting a stronger IFN-gamma response than IL-4 or IL-10. Immunized CBA/CaH and C57BL6 mice showed weak T cell cytokine responses while immunized DBA/2J displayed the strongest T cell responses particularly in regard to IL-4 positive cells. Sham-immunized mice had low levels of serum anti-P. gingivalis antibody levels at day 0 with levels increasing significantly by day 7 after challenge. Antibody levels in immunized mice seemed to correlate with lesion sizes. Immunized C57BL6 mice had the highest antibody levels followed by CBA/CaH, BALB/c with DBA/2J exhibiting low levels. The T cell and B cell antibody responses in each strain appeared to exhibit an inverse relationship. This study has shown that genetic differences at the level of H-2 haplotype induce variations in the local and T and B cell responses to P. gingivalis antigens. The responses of DBA/2J mice which have the same haplotype as BALB/c mice suggest that factors other than H-2 haplotype such as the C5 deficiency may influence this immune response. The significance of the specific antibody and T cell responses and of their inverse relationship to susceptibility to periodontal disease remains to be determined.

Chemokine expression in Porphyromonas gingivalis-specific T-cell lines.

Autologous non-T cells (monocytes and B cells) were added to Porphyromonas gingivalis-specific T cell lines established from 9 healthy adults together with P. gingivalis outer membrane antigens for 4-6, 16-18, 24 and 48 h. Flow cytometry was employed to analyze the CD4 and CD8 cells, monocytes and B cells for intracytoplasmic IP-10 (interferon-gamma inducible protein 10), MCP-1 (monocyte chemoattractant protein 1), MIP-1 alpha (macrophage inflammatory protein 1 alpha) and RANTES (regulated on activation normal T cell expressed and secreted) at the four time periods. All cell types were positive for each chemokine throughout the 48-h time period. There were significantly fewer MCP-1-positive cells compared with the other 3 chemokines. However, the percentages of MCP-1, MIP-1 alpha- and RANTES-positive CD8 cells were significantly higher than the percentages of positive CD4 cells in all cultures. IP-10-positive CD4, CD14-positive monocytes and CD19-positive B cells were predominant compared with MIP-1 alpha- and RANTES-positive cells at 24 h. In conclusion, the present study has shown that P. gingivalis-specific T cells, monocytes and B cells produce chemokines in response to P. gingivalis outer membrane antigens, IP-10 being predominant, with MCP-1 being significantly reduced in comparison with IP-10, MIP-1 alpha and RANTES. Increased percentages of CD8 cells were induced to produce chemokines in comparison with CD4 cells, indicating a more preferential action on CD8 rather than CD4 cells.

Apoptosis in Porphyromonas gingivalis-specific T-cell lines.

Fluorescence-activated cell sorter analysis and transmission electron microscopy were used to determine the presence of apoptotic cells in Porphyromonas gingivalis-specific T-cell lines established from the peripheral blood of 10 P. gingivalis-infected individuals. P. gingivalis outer membrane antigens were presented to the T cells by autologous Epstein-Barr virus-transformed B cells for 6, 24, 48 and 72 h. Transmission electron microscopy demonstrated the presence of typical apoptotic cells in all cultures. Annexin V-positive cells were present at low concentrations at all 4 four periods. A mean of approximately 2-3% of the CD4 cells and 1-3.5% of the CD8 cells were annexin V-positive, with an increase to around 5.5% positive CD4 cells at 6 h in wells containing P. gingivalis compared with cultures not containing antigen. This difference was not, however, significant at the 0.05 level (P = 0.073). The mean (+/- standard error) CD4:CD8 ratios of the T-cell lines when first established using peripheral blood mononuclear cells as antigen-presenting cells was significantly higher (5.2 +/- 1.1) than when transformed B cells were used as antigen-presenting cell (1.2 +/- 0.5). While this study has shown apoptosis occurring in the T-cell lines, it has not shown definitively that the reversion in the CD4:CD8 ratio in the P. gingivalis-specific T cells following antigen presentation by autologous Epstein-Barr virus-transformed B cells is due to apoptosis of a CD4 population. Alternatively, the reversion in the CD4:CD8 ratio could be due to a selective proliferation of the CD8 population which, in turn, could be relevant to the immunopathology of periodontal disease induced by P. gingivalis.

Antigen-specific T-cell receptor V beta expression in Porphyromonas gingivalis-specific T-cell lines.

FACS analysis was used to determine the expression of 15 T-cell receptor V beta families on CD4 and CD8 cells in Porphyromonas gingivalis specific T-cell lines established from eight P. gingivalis-positive adult periodontitis and seven P. gingivalis-positive healthy or gingivitis subjects. All 15 T-cell receptor V beta families were expressed by the T-cell lines, although a significantly higher proportion of the CD4 cells expressed the 5.2-3 V beta region compared with the other 14 families, including the 5.3 region, suggesting that it is the 5.2 family which is overexpressed. This was also true for the CD8 cells, with the exception of the 3.1 region in adult periodontitis T-cell lines and the 3.1, 13.1/13.3 and 21.3 regions in healthy or gingivitis lines. Between the two clinical groups, a significantly lower percentage of 13.1/13.3-positive CD8 cells was noted in the adult periodontitis lines compared with the healthy or gingivitis lines. There was a significant reduction in DNA synthesis by the lines in the presence of P. gingivalis outer membrane antigens and fixed irradiated lymphoblastoid cell lines compared with cultures containing untreated irradiated lymphoblastoid cell lines and in cultures containing anti-class II major histocompatibility complex antibody in comparison with all other cultures. The results of this study have shown that P. gingivalis preferentially induces the T-cell receptor V beta 5.2 family on CD4 and CD8 cells in P. gingivalis-specific T-cell lines and that activation of T cells by P. gingivalis outer membrane antigens may be by antigen-specific rather than superantigen activity.

Cytokine profiles of lesional and splenic T cells in Porphyromonas gingivalis infection in a murine model.

T cell cytokine profiles in the spleens and Porphyromonas gingivalis-induced lesions of P. gingivalis-immunized mice were examined. BALB/c mice were immunized with P. gingivalis outer membrane (OM) antigens/mouse weekly for 3 weeks followed by challenge with live organisms 2 weeks after the final immunization. Control mice were immunized with PBS. Spleens were excised at 0 and 4 days and lesions at 1, 4, and 7 days after challenge. Splenic and lesional CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10. More than 50% of the T cells in the spleens of immunized mice were IFN-gamma positive at day 0 which was significantly higher than for IL-4 or IL-10, these levels decreasing significantly 4 days after challenge. Less than 6% of the T cells in sham immunized mice were cytokine positive at day 0, although at day 4, there was a significant increase in the percent IL-10 positive CD4 cells and IL-4 and IL-10 positive CD8 cells. There were no differences in the percent IL-4, IFN-gamma, or IL-10 positive T cells in the lesions of immunized mice, but there was a dramatic decrease at day 7 to very low levels in control mice. In conclusion, the results of the present study show a predominant Th1 response in the spleens of BALB/c mice after immunization with P. gingivalis OM antigens, suggesting that a protective immune response to P. gingivalis may involve a strong IFN-gamma response.

Expression of receptor β-chain variable region by T cells in human periodontal disease

T cells play a major part in the immune response in periodontal diseases. In order to determine any selective T-cell receptor (TCR) β-chain variable region (Vβ) usage in the infiltrates of healthy/ gingivitis (H/G) and adult periodontitis (AP), cells were extracted from gingival biopsies, the CD4 and CD8 cells stained with antibodies to eight Vβ regions, and two-colour flow cytometry used to analyse the data. The frequencies of CD4 and CD8 cells expressing each of the TCR-Vβ families varied from 0 to 46% between individuals. A high percentage of CD4 and CD8 cells expressed the Vβ 13 family in several AP biopsies, but, in a number of H/G tissues, a high percentage of T cells expressed up to three families including the Vβ 13 region, these varying from individual to individual. The mean results showed a significantly greater percentage of Vβ 5.2–3-positive CD4 cells ( p = 0.003) and Vβ 5.1- and 5.2–3-positive CD8 cells ( p = 0.003 and 0.025, respectively) isolated from H/G than AP tissues. The percentage of Vβ 3.1-positive CD4 cells extracted from H/G tissues was also higher but not quite significant at the 0.05 level ( p = 0.051). Sections of gingival tissue in biopsies from H/G and AP were stained in situ; there were no significant differences in the mean expression of Vβ 3.1-, 5.1- or 5.2–3-positive cells. A second aim was to determine the effect of Porphyromonas gingivalis on the TCR repertoire. There were no differences in the mean percentage of CD4 or CD8 cells expressing the eight TCR-Vβ regions between the two groups after stimulation in vitro with P. gingivalis outer-membrane antigens. There was, however, a trend towards a decrease in the percentage of positive CD4 and CD8 T cells after culture with the antigen. This was significant for CD4 cells from H/G expressing the Vβ 5.1 and 5.3 TCRs ( p = 0.032 and p = 0.038, respectively). This trend was not evident for Vβ 5.2–3-positive CD4 cells or Vβ 5.1-positive CD8 cells isolated from both H/G and AP nor for Vβ 3.1-positive CD8 cells from AP. The results show that there may be restricted Vβ usage in gingival tissues, particularly in H/G tissues. The Vβ 5 and 3.1 families may be selected for in the gingival tissues and may also be involved in P. gingivalis activation.