Effects of Human Neutrophil Defensin-1 on Epithelial Cells

Objectives: Neutrophilic antimicrobial peptides, namely cathelicidins and α-defensins (human neutrophil defensins, HNPs), contribute to the non-oxidative killing of microorganisms. In the present study we tested the effect of HNP-1 on epithelial cell attachment and spread and bacterial adherence on epithelial cells. Methods: Keratinocytes were incubated with 1, 5, 10, 20, 50 μg/ml of HNP-1 for 24 h. Proliferation and cell death of the epithelial cells were determined with proliferation and cytotoxicity kits. Afterwards, keratinocytes were incubated with non-cytotoxic concentrations (1 or 5 μg/ml) of HNP-1 on the fibronectin coated surfaces. Attached and spread cells were counted under a light microscope. Fusobacterium nucleatum ATCC 25586 and Prevotella intermedia ATCC 25611 were used in analyzing the effect of HNP-1 on bacterial adherence to keratinocytes. Keratinocytes, pre-incubated with 1 or 5 μg/ml of HNP-1, were incubated with bacteria. Attachment of bacteria were determined with a standard antibiotic test. All tests were done triplicate and results were statistically analyzed using the t-test. Results: Incubation with 1, 5, and 10 μg/ml of HNP-1 slightly increased the keratinocyte proliferation in a concentration dependent manner. Cellular proliferation decreased in concentrations higher than 10 μg/ml. Concentrations of 10, 20, and 50 μg/ml of HNP-1 increased the cellular death significantly. Concentrations of 1 and 5 μg/ml of HNP-1 increased the cellular attachment, being highest in the concentration of 1 μg/ml. Keratinocytes spread was higher only in the concentration of 1μg/ml. In the concentration of 1 μg/ml, HNP-1 decreased the adherence of F. nucleatum ATCC 25586. In the concentration of 5 μg/ml, HNP-1 increased the adherence of both bacteria, being statistically significant in P. intermedia ATCC 25611. Conclusions: Low doses of HNP-1 play role in the integrity of keratinocytes by increasing their proliferation, attachment, and spread, while higher doses increase the bacterial attachment and keratinocyte death.