Cell-cell Contact Induces Upregulated Expression of Epithelial Antmicrobial Peptides

Objective: Epithelial cells show expression of antimicrobial peptides including beta defensin (hBD) and cathelicidin,CAP18/LL37(LL37). Keratinocyte differentiation induces upregulated expression of these antimicrobial peptides. The present study examined whether cell-cell communication induced expression of hBDs and LL37. Methods: Normal human keratinocyte (NHK, COMBREX,MD) were grown in keratinocyte growth medium (KBM, Switzerland). The cells were cultured in KBM containing 1.8mM Ca2+ at cell density of 25%, 50%, 75% and 100% for 16h. Expression of hBD-1,-2, -3 and -4, and LL37 in NHK cells were observed by RT-PCR and quantitative RT-PCR using TaqMan probes (Applied Biosystem, CA). Data is expressed as the ratio of target mRNA to GAPDH mRNA. Western blot analysis was performed with anti-Cx43 (Sigma-Aldrich) and ant-Cx26 (Zymed lab.) antibodies to evaluate expression level of gap junction proteins. Several experiments were performed, each in triplicate. The data was analyzed using one-way ANOVA. Differences between experimental groups were considered statistically significant at the p<0.05 levels. Results: Western blot analyses confirmed that higher cell density showed higher expression level of Cx43 and 26. By quantitative RT-PCR, expression levels of hBDs and LL37 were highest at cell density of 100%. There was no significant difference in the expression level of hBD-2 between 25% and 50%. There was no significant difference in the expression level of hBD-3 between 75% and 100%. There was no significant difference in the expression level of hBD-4 between 50% and 75%. Conclusion: These results indicate that cell-cell contact may induce upregulated expression of hBDs and LL37.