Background and Objective: High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types.Material and Methods: Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [3H]-thymidine incorporation. Cell number was assessed by counting cells in a Bu€rker chamber. Intracellular Ca2+ was monitored by recording Fluo 4-AM fluorescence using a laser scanning confo- cal microscope. Cellular expression of p33 was determined by western blotting.Results: LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50) of 4 lM and a rapid and sustained rise in the intracellular Ca2+ concentration of osteoblasts, sug- gesting that LL-37 forms pores in the cell membrane. p33 (10 lM) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca2+ con- centration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonsti- mulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts.Conclusions: The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteo- blasts from LL-37-induced cell damage in patients suffering from chronic peri- odontitis associated with high levels of LL-37 locally.
