Differential Expression Patterns of Beta-defensins in Oral and Skin Keratinocytes

Objectives: Human beta-defensins (hBDs) belong to a group of antimicrobial peptides expressed mainly in epithelial cells, including oral and skin keratinocytes. hBD-1 is constitutively expressed in epithelial cells, whereas hBD-2 and -3 are expressed in epithelial cells upon stimulation with pro-inflammatory cytokines. The purpose of the present study was to examine how the hBD expression patterns differ in oral and skin keratinocytes. Methods: Oral keratinocytes derived from gingiva and skin keratinocytes were isolated from 5 healthy individuals. Cells were grown in keratinocyte basal medium (KBM) (modified MCDB 153, Clonetecs). The cells were stimulated with 100ng/ml LPS, 40ng/ml TNF-alpha and 40ng/ml IFN-gamma for 6 and 24hrs. Total RNA was extracted from cultured cells and then reverse-transcribed. A quantitative RT-PCR assay was performed with LightCyclerTM, using the double-stranded DNA dye SYBR Green I (Roche Molecular Biochemicals, Germany) to observe the mRNA levels of hBDs. The transcriptional level of G3PDH, a housekeeping gene, was used as an internal control. Individual experiments were performed in triplicates. In order to detect mRNAs of hBDs in tissue sections, an in situ hybridization method was carried out using digoxigenin-labeled RNA probes for hBDs (Boeringer Mannheim, Germany). Results: The expression levels of hBD-2 and -3 in oral keratinocytes were significantly higher than in skin (p<0.05). Upon stimulation with IFN-gamma, hBD-2 was dramatically upregulated in oral keratinocytes, but only a small effect was seen in skin. By in situ hybridization, signals for hBD-1, -2 and 3 were clearly detected in oral epithelium, but signals for hBD-2 and -3 were faint in skin. Conclusion: The results suggest that hBDs play a more important role in oral epithelium than in skin.