CXCL13 expression and follicular dendritic cells in relation to B-cell infiltration in periodontal disease tissues.

BACKGROUND AND OBJECTIVE: B lymphocyte is the dominant infiltrating cell type in periodontitis lesions. CXCL13, produced by follicular dendritic cells, endothelial cells and fibroblasts, is crucial for B-cell trafficking. An association between chronic inflammation and lymphoid organogenesis has been reported in infection and in autoimmune responses, in which T-cell/B-cell follicles with a follicular dendritic cell network are formed. The aim of this study was to examine CXCL13 expression and follicular dendritic cell distribution in relation to B-cell infiltration in chronic inflammatory periodontal lesions. MATERIAL AND METHODS: Fifty-eight gingival tissue biopsies from patients with periodontitis and 25 samples from subjects with gingivitis were analyzed. Gene expression for CXCL13 and for the CD21 long isoform was analyzed using the reverse transcription-polymerase chain reaction. Immunohistochemical analysis was performed using antibodies to CXCL13, CXCR5, follicular dendritic cells, CD3 and CD19 on serial cryostat sections. RESULTS: mRNA for CXCL13 was expressed in both periodontitis and gingivitis tissues. The number of CXCL13+ cells was significantly higher in periodontitis than in gingivitis in connective tissues subjacent to the pocket epithelium and positively correlated with the number of CD19+ cells. CXCL13+ cells were distributed in B-cell-dominant areas both with and without follicular dendritic cells. Although obvious reticular networks of follicular dendritic cells were not found in periodontitis and gingivitis, the accumulation of follicular dendritic cells in B-cell-dominant areas in periodontitis was observed in some patients. CONCLUSION: These findings suggested that CXCL13 and follicular dendritic cells were involved in B-cell recruitment to, and B-cell distribution in, chronic inflammatory periodontal lesions.

Characterization of CD4+ FOXP3+ T-cell clones established from chronic inflammatory lesions.

INTRODUCTION: Our previous study demonstrated that the gene expression of FOXP3, a characteristic marker for CD4(+) CD25(+) regulatory T cells in mice, is upregulated more in periodontitis than in gingivitis at the messenger RNA (mRNA) level. Furthermore, most of the T-cell clones established from periodontitis lesions expressed FOXP3 mRNA. However, role of the FOXP3(+) gingival T cells has not been elucidated. METHODS: The phenotype of FOXP3-expressing cells in periodontitis lesions was determined immunohistochemically. CD4(+) FOXP3(+) gingival T-cell clones were established from three patients with advanced periodontitis by using immunomagnetic beads. Gene expression and phenotype analyses were performed by reverse-transcription polymerase chain reactions and flow cytometry, respectively. The effect of CD4(+) FOXP3(+) T-cell clones on the proliferative response of CD4(+) CD25(-) T cells was examined by [(3)H]thymidine incorporation. RESULTS: FOXP3 expression was found in some CD4(+) T cells and CD25(+) cells but not in CD8(+) T cells by immunohistochemistry. In spite of a substantial expression of the CD25 gene, the expression level of membrane CD25 on the CD4(+) FOXP3(+) gingival T-cell clones was low. While peripheral blood CD4(+) CD25(+) FOXP3(+) cells suppressed the proliferation of CD4(+) CD25(-) T cells, the CD4(+) CD25(low) FOXP3(+) gingival T-cell clones enhanced the proliferation significantly. CONCLUSION: Our study makes it evident that most, if not all, of the FOXP3(+) T cells in periodontitis lesions can be considered to be effector T cells. The effector activity of the gingival T-cell clones could be attributable to the low level of membrane CD25 expression. Further studies are clearly needed to clarify the role of these T cells and their unique characteristics in the pathogenesis of periodontal disease.

Quantitative messenger RNA expression of Toll-like receptors and interferon-alpha1 in gingivitis and periodontitis.

INTRODUCTION: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll-like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, -7, and -9 messenger RNAs (mRNAs) in addition to those of TLR2 and -4, and compared gingivitis and periodontitis. Interferon-alpha1 (IFN-alpha1), which is important for the antiviral response, was also compared. METHODS: Gene expression was analyzed using quantitative real-time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN-alpha, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. RESULTS: The expression levels of TLR2, -4, -7, and -9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and -4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN-alpha1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody-positivity between gingivitis and periodontitis. CONCLUSION: This is the first study to show that a variety of TLRs are up-regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.

Relationship of periodontal infection to serum antibody levels to periodontopathic bacteria and inflammatory markers in periodontitis patients with coronary heart disease.

Several reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.

Balance of inflammatory response in stable gingivitis and progressive periodontitis lesions.

The balance between inflammatory mediators and their counter-regulatory molecules may be crucial for determining the outcome of immune pathology of periodontal diseases. Based on clinical and immunological findings, the immune response in stable gingivitis lesion is supposed to be in balance, whereas the response is skewed towards the predominance of proinflammatory reactivity in progressive periodontitis lesion. However, this hypothesis has not been verified. Therefore, the aim of this study was to compare the gene expression profile of inflammatory mediators including proinflammatory cytokines and other inflammatory molecules, and anti-inflammatory cytokines by using quantitative real-time polymerase chain reaction in gingivitis and periodontitis lesions showing distinct clinical entities. For inflammatory mediators, interleukin (IL)-1beta, interferon (IFN)-gamma and receptor activator of nuclear factor (NF)-kappaB ligand tended to be higher in periodontitis, whereas tumour necrosis factor (TNF)-alpha and IL-12 p40 showed no difference. Heat-shock protein 60 (HSP60) expression was up-regulated significantly in periodontitis. For anti-inflammatory cytokines, transforming growth factor (TGF)-beta1 expression tended to be higher in periodontitis compared with gingivitis, whereas no difference was observed for IL-10 and IL-4. These findings support further our previous finding that autoimmune response to HSP60 may exert in periodontitis lesion, and suggest that perhaps subtle differences in the balance of cytokines may result in different disease expression.

Increased infiltration of CD1d and natural killer T cells in periodontal disease tissues.

BACKGROUND: Natural killer T (NKT) cells are a unique T lymphocyte subset that has been implicated in the regulation of immune responses associated with a broad range of diseases including autoimmunity, infectious diseases, and cancer. In contrast to conventional T cells, NKT cells are reactive to major histocompatibility complex (MHC) class I-like molecule CD1d. Considering the periodontitis having both aspects of infection and autoimmunity in nature, CD1d and reactive NKT cells are of particular importance. OBJECTIVE: The aim of the present study was to examine whether the expression of CD1 isoforms and Valpha24(+) invariant NKT cells is associated with different disease entities, namely gingivitis and periodontitis. MATERIAL AND METHODS: Immunohistochemical analysis was performed on cryostat sections of gingival tissues from 19 patients with periodontitis and eight patients with gingivitis using antibodies to CD1a, b, c, d, Valpha24(+) invariant NKT cells, CD83, CD3 and CD19. RESULTS: Although all four subsets of CD1 molecules were expressed in periodontal lesions, CD1d was most abundant. CD1d expression was more frequent in periodontitis than gingivitis and increased together with increase of invariant NKT cell infiltration. Double immunohistochemical staining showed co-expression of CD1d and CD19 on identical cells and proximate infiltration of CD1d(+) and invariant NKT cells. CONCLUSION: These findings suggest that CD1d-expressing B cells could activate NKT cells by CD1d-restricted manner and this NKT cell activation may play roles in pathogenesis of periodontal diseases.

Gene expression analysis of the CD4+ T-cell clones derived from gingival tissues of periodontitis patients.

The function of T cells infiltrating periodontitis lesions is complex and has not been fully elucidated. Here, we established T-cell clones from the gingival tissues of periodontitis patients and examined their gene expression. A total of 57 and 101 T-cell clones were established by means of immobilized anti-CD3 antibody and IL-2 from gingival tissues and peripheral blood, respectively. The gingival T-cell clones were derived from three patients, and the peripheral blood T-cell clones from two of these patients and a further patient whose gingival T-cell clones were not established. Gingival tissues were also obtained from a further 19 periodontitis patients. The expression of cytokines and molecules related to both regulatory function and tissue destruction were examined by means of reverse-transcription polymerase chain reaction. All the gingival T-cell clones expressed mRNA for TGF-beta1, CTLA-4, and CD25, and all the T-cell clones from peripheral blood expressed IFN-gamma and TGF-beta1 mRNAs. Most but not all the T-cell clones from gingival tissues and peripheral blood expressed mRNA for IFN-gamma and, CD25 and CTLA-4, respectively. The frequency of T-cell clones and gingival tissues expressing FOXP3, a possible master gene for mouse CD4(+)CD25(+) regulatory T cells, was very high (97%, 93%, and 100% for gingival T-cell clones, peripheral blood T-cell clones, and gingival tissues, respectively). Whereas the frequency of IL-4-expressing T-cell clones was lower for gingival T-cell clones (70% vs. 87%), the frequency of the gingival T-cell clones expressing IL-10 and IL-17 was higher than peripheral blood T-cell clones (75% vs. 62% for IL-10, 51% vs. 11% for IL-17). A similar expression profile was observed for gingival T-cell clones compared with gingival tissue samples with the exception of IL-4 expression, where the frequency of positive samples was lower in the gingival tissues (70% vs. 11%). These results suggest that the individual T cells infiltrating gingival lesions can express mRNA for both Th1 and Th2 cytokines as well as regulatory cytokines simultaneously.