Calcium triggers β‐defensin (hBD‐2 and hBD‐3) and chemokine macrophage inflammatory protein‐3α (MIP‐3α/CCL20) expression in monolayers of activated human keratinocytes

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Abstract
The inducible epidermal β-defensins and the chemokine macrophage inflammatory protein-3α (MIP-3α/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of β-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3α by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.09, 0.8 and 1.7 mM) and were stimulated or not with the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α 1–500 ng/ml) or interferon-γ (IFN-γ 1–100 ng/ml). A high calcium concentration (1.7 mM) alone applied in culture medium for 4 days was sufficient to induce hBD-2 and hBD-3 mRNA expression. Whatever interindividual variability in the expression of hBD-2 and hBD-3 mRNA and MIP-3α secretion, the addition of TNF-α for a short duration (16 h), initiated a dose-dependent and coordinated up-regulation of hBD2 mRNA and MIP-3α release in keratinocyte cultures. Unlike hBD-2, hBD-3 mRNA was preferentially stimulated by IFN-γ rather than TNF-α. In our experimental conditions, l-isoleucine, described to stimulate β-defensins in bovine epithelial cells, did not exert any effect either on hBD-2 and hBD-3 transcripts or MIP-3α protein. Taken together, these results confirm the major role of the maturation/differentiation process of normal human keratinocytes in the induction of inducible β-defensins and MIP-3α chemokine, which contribute in vivo to the immunosurveillance of the skin barrier function.