An optimized method for intensive screening of molecules that stimulate beta-defensin 2 or 3 (hBD2 or hBD3) expression in cultured normal human keratinocytes.

Normal human skin controls the intrusion of microorganisms by the production of peptide antibiotics such as defensins. The aim of our study was to develop a culture model of normal human keratinocytes for optimal beta-defensin mRNA detection which allows the screening of molecules able to stimulate hBD2 and hBD3 without inducing pro-inflammatory cytokines. A keratinocyte culture model in 96-well plates, in high calcium medium (1.7 mm) allowed to analyze hBD2 and hBD3 mRNA expression in basal condition and after cell stimulation by products from diverse vegetal extracts. The release of IL-8 and the chemokine MIP-3alpha was also evaluated in cell supernatants by ELISA. Among the 184 extracts tested, 75 showed a stimulatory effect on beta-defensin expression: 40 on hBD2, 26 on hBD3 and nine on both defensins. Fifteen of these substances which also induced the release of pro-inflammatory cytokines were eliminated. Among the other substances, four were selected and were analyzed in a dose-dependent study (n = 4) by real-time quantitative RT-PCR and completed by a measure of MIP-3alpha, IL-8 and IL-1alpha levels. These data underline the important necessity of screening result controls by a quantitative method reproduced at least three times. This new method of intensive screening allowed us to exhibit vegetal extracts that were able to stimulate epidermal beta-defensin expression without inducing an up-secretion of pro-inflammatory cytokines.