Human Neutrophil Defensins and Their Effect on Epithelial Cells

Background: The aims of the present study include: 1) to localize human neutrophil defensin-1 (HNP-1) through HNP-3 in gingiva and in neutrophil extract-treated epithelial cell monolayers; 2) to determine the effects of HNP-1 on the epithelial cell viability, attachment, and spreading; and 3) to analyze the effect of HNP-1 on the bacterial adherence to epithelial cells. Methods: For localization of HNP-1 through HNP-3 in gingival tissue and in preincubated cell monolayers, immunohistochemical and immunocytochemical methods were used. Viability and proliferation of epithelial cells were determined with commercial kits after incubating the keratinocytes with different HNP-1 concentrations (low, 1 to 5 mu g/mL; moderate, 10 mu g/mL; high, 20 to 50 mu g/mL). Attachment and spreading of keratinocytes on fibronectin-coated surfaces in the presence of HNP-1 were evaluated under microscope. Attachment of Fusobacterium nucleatum ATCC25586 and Prevotella intermedia ATCC25611 on keratinocytes preincubated with HNP-1 were determined with a standard antibiotic test. Results: HNP-1 through HNP-3 localized in the junctional epithelium of clinically healthy gingiva and in the pocket epithelium of gingiva with periodontitis. When keratinocyte monolayers were incubated with neutrophil extracts, HNP-1 through HNP-3 were bound to the periphery of the growing cell colonies. In low HNP-1 concentrations, the keratinocyte proliferation enhanced. Moderate and high concentrations of HNP-1 increased the cellular death significantly. HNP-1 increased the attachment and spreading of keratinocytes on fibronectin-coated surfaces and bacterial attachment to keratinocytes in a concentration-dependent manner. Conclusion: HNP-1 plays a role in the integrity of keratinocytes by stimulating their proliferation, attachment, and spreading, whereas higher doses increase the bacterial attachment and keratinocyte death. J Periodontol 2013;84:126-133.