Use of human reconstructed epidermis to analyze the regulation of beta-defensin hBD-1, hBD-2, and hBD-3 expression in response to LPS.
Defensins have been identified as key elements of innate immunity against microbial infections. In the present study, human beta-defensin-2 (hBD-2) mRNA and peptide expression were evaluated by RT-PCR and Western blotting in normal human keratinocytes, in function of their stage of differentiation. In proliferating, non-differentiating keratinocytes generated in serum-free, low-calcium medium, a very low hBD-2 mRNA expression was found. A significantly higher expression was detected in high-calcium cultivated keratinocytes grown either as monolayers or as multilayers under submerged conditions. In an air-liquid interface culture of keratinocytes, allowing epidermis to be reconstructed, hBD-2 mRNA expression level was significantly higher than in the other conditions and displayed inter-individual variability as observed in native epidermis. The peptide was detected only in reconstructed epidermis. These results indicate that hBD-2 gene expression in normal human keratinocytes is dependent upon their stage of differentiation. The level of expression of hBD-1 mRNA was lower and that of hBD-3 was higher than that of hBD-2 in reconstructed epidermis. Exposure of reconstructed epidermis to bacterial lipopolysaccharide (LPS) resulted in an average 4-fold increase in hBD-2 mRNA 18 h after challenge, but not of hBD-1 and hBD-3 gene expression. These results show the selective regulation of hBD-2-encoding gene in an organotypic epidermal model, in response to LPS. They also provide evidence that in vitro reconstructed epidermis represents a useful model for studying regulation of expression of beta-defensins after skin challenge with pathogenic microorganisms in conditions as close as possible to the in vivo situation.
Chadebech
P
P
INSERM U346, affiliée CNRS, Peau Humaine et Immunité, Hôpital Edouard Herriot, Lyon, France.
Goidin
D
D
Jacquet
C
C
Viac
J
J
Schmitt
D
D
Staquet
M J
MJ
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation; D023083:beta-Defensins / Q000096:biosynthesis*
C406407:DEFB1 protein, human; C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D023083:beta-Defensins; D002118:Calcium