CD14 Is a Co-Receptor for TLR4 in the S100A9-Induced Pro-Inflammatory Response in Monocytes

The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.