Diversity of Porphyromonad gingivalis lipopolysaccharide induced IL-1β, TNF-αand IL-6 levels in HL-60 cells and the associated Toll-like receptors and signaling pathways
Objective To determine the diversity of Porphyromonad gingivalis (Pg) lipopolysaccharide induced IL-1β, TNF-αand IL-6 levels in HL-60 cells and the associated Toll-like receptors and signaling pathways. Methods Pg strain ATCC33277 lipopolysaccharide (Pg-LPS) was prepared using phenol-water method. The levels of IL-1β, TNF-αand IL-6 secreted by HL-60 cells under inducement of Pg-LPS were quantitatively detected using commercial ELISA kits. The blocking test using anti-TLR2 or anti-TLR4 monoclonal antibody (McAb) plus the ELISA were used to determine the types of Pg-LPS binding Toll-like receptors (TLR) on the surface of target cells. The blocking test respectively using the specific inhibitors of JNK, P38MAPK and NF-κB signaling pathways were applied to confirm the associated signaling pathways of Pg-LPS inducing HL-60 cells to secrete IL-1β, TNF-αand IL-6 . A commercial E . coli strain O 1 1 1 : B4 LPS was used as a control in this study . Results When 1μg/ml Pg-LPS acted for 24, 48 and 48 h or 1μg/ml E-LPS acted for 48, 48 and 72 h, the levels of IL-1β, TNF-αand IL-6 secreted by HL-60 cells were remarkably increased ( P 0.01). The maximal TNF-αconcentration induced by Pg-LPS was similar to that induced by E-LPS ( P 0.05), while the maximal concentrations of IL-1βand IL-6 were obviously higher that induced by E-LPS ( P 0.05). The activity of Pg-LPS inducing HL-60 cells to secrete the three cytokines could be blocked with TLR2 McAb alone ( P 0.05). TLR4 McAb showed the effects blocking the three cytokine secretion in HL-60 cells under inducement of E-IJPS ( P 0.05). In HL-60 cells, the signaling pathways responsible for Pg-LPS inducing IL-1β, TNF-αand IL-6 were JNK and NF-κB, all the three pathways, JNK and P38MAPK, respectively (P0.05), while for E-LPS were all the three pathways, P38MAPK and NF-κB, P38MAPK and NF-κB ( P 0.05). Conclusion Pg-LPS shows higher activity inducing HL-60 cells to secrete IL-1β, TNF-αand IL-6 compared to E-LPS. TLR2 is the receptor of Pg-LPS and TLR4 acts as the receptor of E-LPS. The signaling pathways in HL-60 cells to transfer the signals of Pg-LPS inducing the synthesis of cytokines remarkably differ to that of E-LPS. The signaling pathways for Pg-LPS inducing the synthesis of the three different cytokines are also dissimilar.