Objectives: CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. Materials and methods: Following treatment with either interleukin-1β, tumor necrosis factor-α (TNF-α) or γ-interferon (IFN-γ), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-γ, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IκBα was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. Results: IFN-γ stimulated expression of mCD14, whereas -1β and TNF-α did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-γ. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IκBα and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-γ augmented the following activation of MAP kinases and IκBα and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. Conclusions: These results suggest that the augmentation by IFN-γ of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IκBα and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.
- CD14/ toll-like receptor 4/ Myd88
- Human gingival fibroblasts
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