Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease

Abstract

Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.

Fig. 1

Forkhead box P3 (FoxP3) expression in CD25-positive cells diminished in periodontal diseased gingival tissue where abundant nuclear factor-kB ligand (RANKL⁺) lymphocytes infiltrate. Healthy gingival tissues (gingival pocket depth < 3 mm; n = 4, one male and three females, ages 25–51 years) and inflamed gingival tissues (gingival pocket depth > 5 mm; n = 4, two males and two females, ages 36–55 years) were analyzed for the expression of FoxP3, CD25 and RANKL on the infiltrating lymphocytes in the tissues. (a) The presence of FoxP3 expression in the human gingival tissues was determined using double-colour confocal microscopy. Fluorescein isothiocyanate (FITC)-conjugated anti-human FoxP3 antibody (green) and Alexa Fluor 647-conjugated anti-human CD25 antibody (red) were reacted with frozen sections of human gingival tissues of healthy (n = 4) and diseased (n = 4) gingival tissues. Representative staining patterns of healthy and diseased gingival tissues are shown. (b) Staining pattern of FoxP3 in the healthy gingival tissues and periodontal diseased lesions with RANKL⁺ lymphocytes was evaluated by staining with FITC-conjugated anti-human FoxP3 antibody (green) and with osteoprotegerin (OPG)-Fc-biotin, followed by TexasRed–avidin (red). The stained tissues were also analysed by the confocal microscope. In the healthy gingival tissue sample, an arrow indicates RANKL-positive cells. (c) The number of FoxP3, CD25 and RANKL-positive cells in the microscopic field (× 1000) were counted. Data from healthy and diseased gingival tissues (n = 4 and n = 4, respectively) are expressed as average cell number ± s.d. per microscopic field (n.d., not detectable). *Significantly higher than the number of FoxP3 or CD25-positive cells in either healthy or diseased tissues by Student's t-test (P < 0·05). (d) The number of FoxP3/CD25 double-positive cells and total CD25 single-positive cells were counted in each microscopic field. The percentage of FoxP3-positive cells in CD25⁺ cells is calculated and shown as an average percentage ± s.d.

Fig. 2

Measurement of soluble nuclear factor-kB ligand (sRANKL), interleukin (IL-10) and IL-1β in gingival tissue homogenates. Gingival tissue homogenates were prepared from inflamed gingival tissues from patients with periodontal disease (gingival pocket depth > 3 mm, average 5·7 ± 1·3 s.d. mm, age 32–58 years, n = 11, eight females and three males) and healthy gingival tissues (gingival pocket depth ≤ 3 mm, age 27–41 years, n = 3, one female and 2 males), following the procedure described in the Materials and Methods. Concentrations of IL-1β, IL-10 and sRANKL in the gingival tissue homogenates were measured using the DuoSet enzyme-linked immunosorbent assay (ELISA) kit. Open or closed symbols indicate the data from healthy or diseased gingival tissue homogenates, respectively. (a) Concentrations of IL-1β and sRANKL and (b) concentrations of IL-10 and sRANKL are shown in scatter diagrams. *Positive correlation between two parameters (n = 14, P < 0·05); **negative correlation between two parameters (n = 14, P < 0·05).

Fig. 3

IL-10 suppressed in vitro soluble nuclear factor – KB ligand (sRANKL) expression by activated peripheral blood mononuclear cells (PBMC). In vitro stimulation of peripheral blood mononuclear cells (PBMC) induced predominant nuclear factor-kB ligand (RANKL) expression in forkhead box P3 (FoxP3)-negative cells, but little or no expression in FoxP3-positive cells. PBMC isolated from a healthy subject were stimulated with Aa Y4 (10⁷/ml) in the presence or absence of anti-human leucocyte antigen D-related (HLA-DR) monoclonal antibody (MoAb) (clone G46-6), control isotype-matched MoAb (PF18), or recombinant interleukin (IL)-10 (50 ng/ml). On day 3, culture supernatant was harvested and subjected to human sRANKL enzyme-linked immunosorbent assay (ELISA). [³H]-thymidine (0·5 µCi) was added to the culture during the last 16 h of a total incubation of 4 days. The data from a healthy subject were expressed as average ± s.d. of (a) [³H]-thymidine incorporation (kcpm = 1000 c.p.m) or (b) secreted sRANKL concentration (pg/ml), respectively. PBMC isolated from another healthy subject and a patient with periodontal disease showed similar expression patterns of sRANKL in response to bacterial stimulation in the presence of IL-10 (not shown). *Significantly lower than Aa stimulation alone (#) by Student's t-test (P < 0·05).

Fig. 4

In vitro stimulation of peripheral blood mononuclear cells (PBMC)-induced predominant nuclear factor-kB ligand (RANKL) expression in forkhead box P3 (FoxP3)-negative cells compared to FoxP3 positive cells. PBMC isolated from healthy subjects were stimulated with formalin-fixed Actinobacillus actinomycetemcomitans (Aa) Y4 (10⁷/ml) in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in the presence or absence of human recombinant interleukin (IL)-10. Three days after incubation, the cells were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-human FoxP3 antibody, Alexa Fluor 647-conjugated anti-human CD25 antibody, Alexa Fluor 647-conjugated anti-human CD4 and osteoprotegerin (OPG)-Fc-biotin/TexasRed-conjugated avidin. The staining pattern for each molecule was analysed using the double-colour confocal microscope. Representative staining patterns of merged images between green and red colours are shown at ×640 magnification. The arrows in (a), (d) and (g) indicate the FoxP3/CD25 double-positive cells. All three samples, including two healthy subjects and one patient with periodontal disease, showed similar expression patterns of CD25, FoxP3 and RANKL after stimulation with Aa in the presence or absence of IL-10. (a–i), PBMC of a healthy subject stimulated for 3 days; (j, k) PBMC of a healthy subject stimulated with Aa for 7 days. The numbers of cells positive for respective molecules were counted in a microscopic field containing a total of 100 PBMC cells (l, FoxP3 and/or CD25; mM, RANKL). *Significantly higher than non-stimulation control by Student's t-test (P < 0·05); **significantly lower than Aa stimulation alone by Student's t-test (P < 0·05).