Mechanism of Ciclosporin-induced gingival hyperplasia An in vitro study of the effect of ciclosporin on human gingival fibroblasts and oral keratinocytes

Abstract

Objectives: Ciclosporin (CsA) is a potent and effective immuno-suppressive drug, but its use is associated with the development of gingival hyperplasia in up to 70% of patients. The mechanism underlying this side effect is unknown and the aim of this in vitro study was to investigate the direct effect of CsA on oral epithelium and connective tissue and to explore whether oral bacterial products modified the response. Methods: 3 human oral and gingival keratinocyte cell lines (OKF6, TR146 and FIBS) and a human gingival fibroblast cell line (HGF) along with reconstructed human gingival and oral epithelial models (RHGE and RHOE respectively, SkinEthic) were used in the study. Cells/tissues were pre-stimulated for 24h with bacterial supernatants (P. gingivalis (Pg), A. actinomycetemcomitans (Aa), F. nucleatum (Fn), and P. intermedia (Pi) at 1/100 or 1/10 respectively and then exposed to a combination of CsA (2000 or 250 ng/ml) and bacterial supernatant for further 24h or 48h. Cell viability, cytokine/ chemokine (IL-1α; IL-6; IL-8) release; cell cycle and proliferation markers (DNA synthesis, cyclin B1, cyclin D1, CCNB1, and CCND1) and apoptosis (Bax, Bcl-2 protein and gene, and Fas-L gene) were assessed using MTT assay, ELISA, FACs analysis, quantitative PCR. Cell cycle analysis, DNA synthesis and expression of cyclins D1 and B1 in cell lines were evaluated simultaneously. Results: CCNB1 and CCND1 were significantly up-regulated in all RHGE cultures exposed to any of the combinations applied compared to controls (CCNB1: CsA+A.a; 1.543± 0.039; CsA; 0.703± 0.032; A.a; 0.669± 0.002; untreated control; 1± 0.047)(CCND1; CsA+A.a; 3.41± 22 versus CsA; 1.046± 0.079; A.a; 2.127± 0.22; and untreated 3 control; 1± 0.106). FACS analysis of monolayer cultures showed an apparent increase in proliferation rate, illustrated by DNA synthesis, of FIBS, OKF6, and HGF consistently with the combination (P.i plus 2000ng/ml CsA) compared to controls. CsA at 2000ng/ml caused a significant increase in cytokine release from both keratinocyte and fibroblast cell lines. Furthermore, bacterial products in combination with CsA had a synergistic effect on IL-6 and IL-8 release from HGF but had a little effect on cytokine release from keratinocytes. Conversely, the combined treatments significantly decreased IL-8 in RHOE. Conclusion: The results suggest that a clinically relevant dose of CsA (2000ng/ml) along with bacterial products stimulate the proliferation of gingival keratinocyte and HGF by activating the cell cycle and DNA replication. Thus, the presence of specific periodontal microorganisms may be important in the development of the condition