Abstracts

Accumulating evidence suggests that the main humancathelicidin peptide, LL-37, may influence host cell viability andpro-inflammatory signaling when locally overexpressed. LL-37 haspro-inflammatory properties linked to the development of forexample psoriasis, rosacea, SLE and atherosclerosis. However, theimportance of LL-37-induced host cell permeabilization andcytotoxic effects are poorly understood. We hypothesize that theseeffects are implicated in the tissue destruction associated withinflammatory diseases such as chronic periodontitis (CP). CP, thenumber one reason for adult tooth loss globally, is associated withelevated LL-37 levels. Here, tooth detachment is caused bydegradation of the supporting bone. We aimed to investigate effectsof LL-37 on osteoblast cell viability in vitro and the underlyingmechanism in order to explore the detrimental effects of LL-37 andthe possible involvement in tissue destruction associated with CP.In agreement with this, the peptide was found to permeabilize andkill osteoblasts at concentrations relevant for the in vivosituation. Cell permeabilization by LL-37 was associated with LDHrelease, Ca2+-influx, attenuation of cell viability, accumulationof annexin V positive cells and caspase 3 activation, indicative ofapoptosis in MG63 osteoblasts. As LL-37 constitutes a possible drugtarget, we further investigated compounds and endogenous mechanismsthat may serve to inactivate LL-37. We found that the protein p33(gC1qR) may be added extracellularly to rescue osteoblasts fromLL-37-evoked cytotoxicity. Additionally, endogenous p33 appears tobe a mean by which host cells are protected from LL-37-inducedcytotoxicity, as the extent of the toxicity correlates to p33expression levels in various host cell types. Host cell sensitivitytowards LL-37 may moreover be modulated through up- or down-regulation of p33 expression, mediated through transfection with apcDNA3.1 expression vector and siRNA. p33 was found in themitochondria, cytoplasm and in proximity to the cell membrane. Itinactivates LL-37 intracellularly, as cell viability but notcellular LDH release is influenced by p33 expression, andadditionally, LL-37 co-immunoprecipitates with cytoplasmic p33.This indicates that internalization of LL-37 is critical for hostcell cytotoxicity. Furthermore, a comparison of LL-37's effects onvarious cell types indicates that LL-37-evoked cytotoxicity is notdirectly correlated to the degree of cell permeabilization.Theactive form of vitamin D, 1,25D3, is a well-known mediator of LL-37synthesis, although its possible role in dysregulated expression ofLL-37 is yet to be elucidated. Stimulation of LL-37 expressingTHP-1 monocytes with 1,25D3 was shown to attenuate cell viabilityboth of co-cultured periodontal ligament (PDL) cells and the THP-1cells themselves. This effect is reversed by addition ofrecombinant p33 to the cultures, implicating LL-37 as the mediatorof the cytotoxic effect. In skin, RXR protein levels were found tobe critical for LL-37 expression: