Neutral endopeptidase regulates neurogenic inflammatory responses induced by stimulation of human oral keratinocytes with bacterial lipopolysaccharide and nicotine

Neutral endopeptidase (NEP) is present on various epithelial cells and inactivates numerous physiologically active peptides. Neutral endopeptidase may regulate proinflammatory signals in oral mucosal epithelium. However, the function of NEP in oral mucosal epithelium is unknown. The present study investigated the action of NEP upon proinflammatory signals on human oral keratinocytes and the influence of endothelin-converting enzyme (ECE)-1, an enzyme similar to NEP, on the functions of NEP. Oral keratinocytes were cultured in medium containing inflammatory inducers [lipopolysaccharide (LPS) and nicotine], NEP inhibitors, and ECE-1/NEP inhibitors, either alone or in combination. The concentrations of substance P (SP) and interleukin-1 beta (IL-1 beta) were measured in the supernatant. Additionally, the concentrations of SP and IL-1 beta were measured in the supernatant of cells incubated with LPS or nicotine after transfection with NEP small interfering RNA (siRNA). The concentrations of SP and IL-1 beta were significantly increased in cells incubated with NEP inhibitors and, to a lesser extent, in cells incubated with ECE-1/NEP inhibitors, compared with controls (cells incubated with LPS or nicotine alone). The concentrations of SP and IL-1 beta in cells transfected with NEP siRNA were significantly augmented compared with controls. In conclusion, the present study demonstrated that NEP down-regulated the levels of SP and IL-1 beta produced from human oral keratinocytes, although ECE-1 may be partly related to the down-regulation.