This is in line with other previous works failing to find an association between HMGB1 and various cytokines and might reflect the complexity of inflammatory response in the clinical setting. The role of the receptor for advanced glycation end products and its ligands in maintaining and amplifying inflammation has been recently highlighted. RAGE, a member of the immunoglobulin superfamily, interacts with ligands enriched in flamed milieu, includimg lysine-modified adducts and S100/calgranulins, the latter members of a family of Solcitinib proinflammatory cytokines. Blockade of RAGE, employing soluble RAGE, the extracellular ligand-binding domain of RAGE, suppresses injury in hepatic ischemia/reperfusion, as well as the induction of collagen-induced arthritis in mice sensitized to and challenged with bovine type II collagen. In this study, however, the expression levels of Rage mRNA were conversely decreased by administration of LPS/GalN and GLtreatment did not exert any influence on its expression. In contrast, the expression levels of Tlr4 mRNA were significantly increased in LPS/GalN-induced hepatic injury as compared with the control, but GL did not inhibit it. GL binds directly to each of two HMG boxes of HMGB1, as shown by NMR and fluorescence studies. The modest effect of GL on the intranuclear function of HMGB1 is agreement with the absence of cytotoxicity even at high GL concentrations and with the good pharmacological tolerability of GL in rodents and humans. Interestingly, administration of GL does not cause the release of HMGB1 from apoptotic chromatin. The current results suggest that the effective mechanisms of GL are down-stream of initial TLR activation in the injured liver induced by LPS/GalN injection. Inside the cell, HMGB1 binds DNA and regulates transcription, whreas outside the cell, it serves as a cytokine and mediates the late effects of LPS. In the present study,ETH2120 the doubleimmunofluorescence analysis for HMGB1/F4/80 or HMGB1/ CD11c revealed that activated macrophages and dendritic cells expressed HMGB1 in their cytoplasm in the LPS/GalN-induced hepatic injury, respectively. HMGB1 in monocytes and macrophages is extensively acetylated upon activation by LPS, causing localization of the protein to the cytosol. Cytosolic HMGB1 is then concentrated into secretory lysosomes and secreted when the cells received an appropriate second signal. The movement of HMGB1 into the extracellular space has been demonstrated for macrophages stimulated with LPS as well as cells undergoing necrosis or apoptosis. In our previous paper, analyses using TUNEL-method, an oligonucleosomebound DNA ELISA, and microdissection-method showed that the degree of hepatic injury is associated with a substantial number of cells undergoing apoptosis in acute hepatitis induced with a single injection of LPS/GalN. GL-treatment suppressed the apoptosis of liver cells induced by LPS in D-GalN-sensitized mice. The role of HMGB1 in the precise mechanism of apoptotic cell death of hepatocytes in this experimental hepatitis remains to be unknown but is assumed to be implicated in the signal pathways regulating apoptosis. To assess the acetylation of HMGB1 in the inflammatory hepatic specimens, we conducted the immunohistochemical analysis using the antibody to acetylated-lysine. This analysis revealed a few nuclear immunoreactive products in the hepatic cells of control mice.