Induction of nuclear factor‐κ<scp>B</scp> responses by the <scp>S</scp>100<scp>A</scp>9 protein is Toll‐like receptor‐4‐dependent

Abstract

Summary Interactions between danger‐associated molecular patterns ( DAMP ) and pathogen‐associated molecular patterns ( PAMP ) and pattern recognition receptors such as T oll‐like receptors ( TLR s) are critical for the regulation of the inflammatory process via activation of nuclear factor‐κ B ( NF ‐κ B ) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide ( LPS ) ‐free S 100 A 9 ( DAMP ) protein to activate human and mouse cells compared with lipoprotein‐free LPS ( PAMP ). First, we showed that LPS and S 100 A 9 were able to increase NF ‐κ B activity followed by increased cytokine and nitric oxide ( NO ) secretion both in human THP ‐1 cells and in mouse bone marrow‐derived dendritic cells. Surprisingly, although S 100 A 9 triggered a weaker cytokine response than LPS , we found that S 100 A 9 more potently induced IκBα degradation and hence NF ‐κ B activation. Both the S 100 A 9‐induced response and the LPS ‐induced response were completely absent in TLR 4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S 100 A 9 NF ‐κ B induction were strongly reduced in the presence of specific inhibitors of TLR ‐signalling. Chloroquine reduced S 100 A 9 but not LPS signalling, indicating that S 100 A 9 may need to be internalized to be fully active as a TLR 4 inducer. This was confirmed using A 488‐labelled S 100 A 9 that was internalized in THP ‐1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S 100 A 9 are TLR 4 agonists. Importantly, S 100 A 9 induced stronger NF ‐κ B activation albeit weaker cytokine secretion than LPS , suggesting that S 100 A 9 and LPS activated NF ‐κ B in a qualitatively distinct manner.