OBJECTIVETo investigate the influence of high mobility group box-1 protein (HMGB1) on apoptosis of peritoneal macrophages in mice and its receptor mechanism.

METHODSThe peritoneal macrophages were isolated from female BALB/c mice and divided into 4 groups according to different stimuli: i. e, HMGB1 group (with treatment of 10 microg/ml HMGB1 for 24 hours), HMGB1 and anti-receptor advanced glycation end products (RAGE) antibody group (with treatment of 5 microg/ml anti-RAGE antibody for 2 hours followed by HMGB1 stimulation), Recombinant mouse RAGE/Fc chimera (rmRAGE/Fc) and HMGB1 group (10 microg/ml of rmRAGE/Fc and 10 microg/ml HMGB1 were pre-mixed for 2 hours, then the peritoneal macrophages were treated with the mixture), control group (with treatment of phosphate buffer). Expression of RAGE on the surface of macrophages, and the apoptotic rate of the cells were determined by flow cytometry. Laser scanning confocal microscopy was used to observe the apoptosis of the cells.

RESULTSThe percentage of macrophages with positive RAGE expression in HMGB1 group [(54 +/- 12%)] was markedly increased compared to the controls [(13 +/- 5)%, P < 0.01], and fluorescence density of RAGE expression was also significantly different between two groups (126 +/- 10 vs 34 +/- 8, P < 0.01). The occurrence of apoptosis in HMGB1 and rmRAGE/Fc group, as well as in HMGB1 plus anti-RAGE group were much higher than that in control group, and the number of macrophages with apoptosis and necrosis at late stage was obviously increased in HMGB1 group. The apoptic rate in HMGB1 group was (39.5 +/- 2.3)%, which was significantly higher than those in HMGB1 and rmRAGE/Fc group (17.3 +/- 3.6)%, and HMGB1 and anti RAGE group (14.8 +/- 4.8)%, (P < 0.01), which were significantly higher than those in control groups (5.4 +/- 2.3)%, (P < 0.01).

CONCLUSIONRAGE is one of the major receptor to induce apoptosis of macrophages, and the up-regulation of its expression is induced by HMGB1.