Anti-CD14 antibody reduces LPS responsiveness via TLR4 internalization in human monocytes.

CD14 is an LPS sensing receptor that is primarily expressed in monocytes. CD14 binds and transfers bacterial LPS to the surface TLR4:MD-2 complex to enable its recognition. After recognizing LPS, this complex produces the first intracellular signals via TIRAP and MyD88, after which surface TLR4/LPS complex is rapidly internalized and produces additional signals via TRAM and TRIF. It was recently suggested that CD14 is a key regulator of LPS-induced TLR4 endocytosis and second signaling. In the present study, we showed that surface TLR4 expressions of human primary monocytes and cell line THP-1 were significantly reduced after treatment with anti-CD14 Ab. Among three anti-CD14 Abs with different epitope specificities used in this study, My4, which has an epitope specificity for LPS binding domain of the CD14 molecule, was found to be the most potent at reduction of surface expression of TLR4 as well as CD14. To test the reason for this reduction, we performed an in vitro internalization assay using anti-TLR4 Ab conjugated with toxin. The results of this analysis indicated surface CD14 ligation-mediated TLR4 internalization, and the mechanism of the internalization was found to be partially clathrin-dependent. We next examined NF-κB/AP-1 activation and TNF-α production of THP-1XBlue-CD14 cells in response to LPS challenge with or without My4 pre-treatment. The results revealed that NF-κB/AP-1 activation and TNF-α production of cells treated with My4 were significantly impaired when compared to the control. Our results suggest that membrane CD14 ligation-mediated TLR4 internalization is a novel mechanism for effective down-regulation of surface expression of TLR4 and subsequent reduction of LPS response of human monocytes.