Differential expression of costimulatory molecules in chronic inflammatory periodontal disease tissue

Abstract

Although B cell activation and subsequent immunoglobulin production are the immunopathological features of chronic inflammatory periodontal disease, in situ expression of costimulatory molecules in humoral immunity has not been investigated. In the present study we examined the expression of CD40, CD40 ligand (CD40L), CD80, CD86, CD28 and cytolytic T lymphocyte-associated antigen-4 (CTLA-4) on lymphocytes immunohistochemically. Cryostat sections were prepared from the gingival tissue samples of 14 patients with moderate to advanced adult periodontitis. In vitro kinetics of the expression of CD40L and CTLA-4 by peripheral blood T cells and that of CD80 and CD86 by peripheral blood B cells were also investigated by flow cytometry. Positive percentage expression of CD40L, CD28 and CTLA-4, and CD40, CD80 and CD86 was calculated for the number of CD3+ and CD19+ cells, respectively. Flow cytometric analysis demonstrated that the expression of CD40L and CTLA-4 on T cells, and CD80 and CD86 on B cells of peripheral blood was up-regulated upon activation. While most T cells and B cells expressed CD28, and CD80 and CD86, respectively, in gingival tissues, the expression of CD40L and CTLA-4 was lower but highly variable between specimens. Furthermore, these two molecules seemed to be expressed reciprocally in the lesion. As both CD40L and CTLA-4 expression are induced transiently by stimulation, variability in the expression of the molecules may reflect immunological activities and participation in the regulation of B cell activation of the lesion.

Fig. 1

Immunoreactivity for costimulatory molecules in the periodontitis lesion. Double staining for CD3/CD19 (a) and single staining for CD28 (b), CTLA-4 (c), CD40L (d), CD80 (e), CD86 (f) and CD40 (g) was performed on sequential sections. Costimulatory molecule immunoreactivity was visualized using alkaline-phosphatase anti-alkaline-phosphatase (APAAP) and Substrate kit III (Vector). For double staining, CD3 was immunolabelled with peroxidase, whereas CD19 was immunlabelled with alkaline phosphatase (×100). PE, Pocket epithelium.

Fig. 3

Expression of each costimulatory molecule. All positive cells for each MoAb in the selected areas were counted. CD28, CTLA-4 and CD40L were expressed as a percentage of the number of CD3⁺ T cells. CD80, CD86 and CD40 were expressed as a percentage of the number of CD19⁺ B cells (n = 16). Data are expressed as mean ± s.d. *CTLA-4 and CD40L expression was significantly lower than CD28 expression (P < 0.01).

Fig. 2

Double staining for CD28/CD80 (a), CD28/CD86 (b), CTLA-4/CD80 (c), CTLA-4/CD86 (d) and CD40L/CD40 (e) in the periodontitis lesion. In CD28/CD80 and CD28/CD86 double staining, CD80 and CD86 were immunolabelled with peroxidase, whereas CD28 was immunolabelled with alkaline phosphatase. In CTLA-4/CD80, CTLA-4/CD86 and CD40L/CD40 double staining, CTLA-4 and CD40L were immunolabelled with peroxidase, whereas CD80, CD86 and CD40 were immunolabelled with alkaline phosphatase (×200). Cognate interaction between receptors and their ligands is seen (arrowheads).

Fig. 4

Frequency of CTLA-4- (•) and CD40L-producing cells (○) for CD3⁺ T cells in periodontitis lesions (n = 16). The plots with the connecting lines represent the individual tissue samples studied.