Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes

Epidermal keratinocytes are involved in the skin innate immunity and express toll-like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram-positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear. To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen-associated molecules, and the function of histamine in this process. We investigated the effects of lipopeptide (MALP-2, 1-100 ng mL(-1); ligand for TLR2), peptidoglycan (PGN, 0.02-2 mu g mL(-1); ligand for NOD2) and beta-glucan (1-100 mu g mL(-1); ligand for dectin-1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin-1 as well as human beta-defensin 2 by quantitative real-time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)-1 alpha and IL-8 produced by keratinocytes were measured using enzyme-linked immunosorbent assay. At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP-2 or by beta-glucan; NOD2 was easily induced by all three ligands; and dectin-1 was enhanced by its ligand beta-glucan. These enhanced expressions were further augmented by histamine at 1 mu g mL(-1). While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin-1 was functional, as these pathogen-associated molecules induced the production of IL-1 alpha, IL-8 and defensin, and again, histamine greatly enhanced this production. Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen-associated molecules in a ligand-feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins.