Up-regulation of Cyclooxygenase-2 and Prostaglandin E₂ by Human ß-defensin-3

Oral epithelial cells express three antimicrobial peptide human ß-defensins (hBDs) that were previously demonstrated to exert pro-inflammatory effects on immune cells. We hypothesized that these epithelial ß-defensins might also activate inflammation in non-immune cells. Objectives: We wanted to test that hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E₂ (PGE₂) synthesis in human gingival fibroblasts. Methods: Cultured fibroblasts were treated with various concentrations of hBD-1, hBD-2, or hBD-3 for the indicated times. RT-PCR and real-time PCR were conducted to analyze the levels of COX-1 and COX-2 mRNA expression. Whole cell lysates were analyzed for COX-2 protein expression by Western Blot. Cell-free culture supernatants were analyzed for PGE₂ levels by ELISA. The cytotoxicity test was performed by an MTT assay. Results: Ten and forty µg/ml of hBD-3 significantly up-regulated COX-2 mRNA and protein expression in a dose-dependent manner (P<0.01), whereas hBD-1 and hBD-2 did not. COX-1 mRNA was constitutively expressed. The time-course study revealed that hBD-3 significantly up-regulated COX-2 mRNA expression at 6 hours (P<0.05). The PGE₂ levels significantly elevated in cell-free culture supernatants of gingival fibroblasts, treated with 10 and 40 µg/ml of hBD-3 (P<0.01). None of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human ß-defensin-3 plays a role as a pro-inflammatory mediator in controlling arachidonic acid metabolism in a paracrine fashion. This study was supported by the Thailand Research Fund (RMU5080035) and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand.