| Objectives: (1) To investigate the effects and the potential receptor of the high mobility group box-1 protein (HMGB1) on immune function of peritoneal macrophages in mice. (2) To investigate the differential expression of tumor necrosis factor-α (TNF-alpha), interleukin-10 (IL-10), intercellular adhesion molecule-1 (ICAM-1) in response to HMGB1 in murine peritoneal macrophage. (3) To investigate the effects of HMGB1 on apoptosis of peritoneal macrophages and its receptor mechanisms in mice. (4) To investigate the potential role of caspases 3 and NF-κB signaling in HMGB1- induced peritoneal macrophages apoptosis in mice. Methods : (1) The peritoneal macrophages were isolated from female BALBc mice. (2) After stimulation with different doses of HMGB1 (1, 10, 100, 1000 ng/ml) for 6 hours or with 100 ng/ml HMGB1 for different duration (0, 6, 12, 24, 48, 72 hours), the phagocytosis ability of macrophages was determined by the neutral red dye uptake, its cytotoxicity to L1210 cells was measured with MTT assay, its chemotaxis was assayed with the use of Costar Transwell cell culture chamber inserts, and the expression of I-A~k MHC class II alloantigen on macrophages was determined with flow cytometry. (3) After stimulation with 100 ng/ml HMGB1 for 48h, the receptor for advanced glycation end products (RAGE) expression in macrophages was determined by both flow cytometry and laser scanning Confocal microscopy. After treated with recombinant mouse RAGE/Fc chimera (rmRAGE/Fc)/HMGBl, anti-RAGE antibody/HMGB1, HMGB1 or phosphate buffered saline (PBS), the phagocytosis, cytotoxicity, chemotaxis and expression of I-A~k MHC class II antigen on macrophages were determined. (4) After exposure to different concentration of HMGB1 (1, 10, 100, 1000ng/ml) or PBS for 24 hours, or exposure to 100 ng/ml fordifferent duration (0, 6, 12, 24, 48, 72 hours), the macrophages were denatured in cell culture plates to determine gene expressions of TNF-α, IL-10 and ICAM-1 by Real-Time Quantitative PCR, and cell supernatants were harvested to determine the level of TNF-α, IL-10 and sICAM-1 by ELISA. (5) After exposure to different doses of HMGB1 (10, 100, 1000, 10000ng/ml) or PBS for 24 hours, or exposure to 10 μg/ml for different duration (0, 6, 12, 24, 48 hours), cells were stained with Annexin V-PE and 7-AAD, and the apoptosis of macrophages were determined by flow cytometry and laser scanning Confocal microscopy, respectively. (6) After exposure to 10 μg/ml HMGB1 or PBS for 24 hours, expression of the receptor for advanced glycation end products (RAGE) in macrophages was determined. After treated with recombinant mouse RAGE/Fc chimera (rmRAGE/Fc) + HMGB1, anti-RAGE antibody + HMGB1, HMGB1 or PBS, cells were stained with Annexin V-PE and 7-AAD, then they were examined with flow cytometry and laser scanning Confocal microscopy, respectively. (7) The peritoneal macrophages from female BALBc mice were randomly divided into 3 groups: control groups, HMGB1 groups and caspase 3 inhabitor groups ( 40 μM Z-DEVD-FMK was added before exposure to 10μg/ml HMGB1). Apoptotic cells were stained with Annexin V-PE and 7-AAD and then examined with flow cytometry. Nuclear features of apoptosis were examined by Hoechst33342. Activated caspase-3 was estimated with fluorescence microscopy and flow cytometry after staining with the in situ marker FITC-DEVD-FMK. Nuclear p65 activity was determined using the TransAM Transcription Factor DNA-binding ELISA (NF-κB/p65).Results : (1) The effects of HMGB1 on phagocytic activities, cytotoxicity, chemotaxis and I-Ak antigen expression of macrophages were enhanced in a dose-dependent manner, starting at doses as low as 1ng/ml and with a maximal response at 100 ng/ml (P<0.01). The effects of HMGB1 on macrophages were also time-dependent, with a significant increase in phagocytosis ability, chemotaxis and cytoxicity within 48 h following stimulation with 100 ng/ml HMGB1 (P<0.01). The peak expressions of I-A~k antigen occurred within 12 h (P<0.01). (2) After stimulation with 100 ng/ml HMGB1 for 48h, RAGE expression in macrophages was markedly increased(P<0.01). The phagocytic activities, cytotoxicity, chemotaxis and I-A~k antigen expression of macrophages in HMGB1 groups were significantly higher than those in other groups (PO.01). (3) The protein and gene expressions of TNF-α and ICAM-1/sICAM-1 but not IL-10 were markedly up-regulated. The effects of HMGB1 on TNF-α and ICAM-1/sICAM-1 protein and gene expressions displayed a concentration-dependent manner, starting at dose as low as 1ng/ml and showing a maximal response with 1000 ng/ml (P<0.01). The effects of HMGB1 on TNF-α and ICAM-1 protein and gene expressions were also time-dependent, with a significant increase within 48 h (P<0.01). HMGB1 induced a biphasic TNF response, with the first peak at approximately 6 h and a second peak at 24h. (4) Exposed to HMGB1 for 24 hours resulted in a dose-dependent induction of apoptosis, with increased levels of apoptosis observed at 100ng/ml, 1000ng/ml, 10000ng/ml compared with controls (12.91%±3.89%, 18.76±3.41% and 39.84%±5.98%, respectively, versus 4.49%±1.72%). When macrophages were exposed to HMGB1 at 10 μg/ml for 6, 12, 24, or 48 hours there was a time-dependent increase in apoptosis rate (18.33% ± 4.60%, 25.02 ± 5.27%, 38.3 ± 6.95% and 21.81% ± 7.96%). The percentage of apoptotic cells were markedly increased with a peak value at 24 hours after 10μg/ml HMGB1 stimulation (P<0.01). (5) After stimulation with 10μg/ml HMGB1 for 24 hours, RAGE expression in macrophages was markedly increased (P<0.01). The percentages of apoptosis of macrophages were (39.55 ± 2.31)% in HMGB1 groups, (14.83 ± 4.78)% in HMGB1 + rmRAGE/Fc groups, and (17.29 ± 3.59)% in HMGB1 + anti-RAGE groups, and they were significantly higher than those in control groups (5.37 ± 2.31)% (all P<0.01). (6) The percentage of apoptosis of macrophages was (38.21 ± 4.85)% in HMGB1 groups, (16.47 ± 3.91)% in HMGB1+ caspase 3 inhabitor (Z-DEVE-FMK) groups, and they were significantly higher than those in control groups (4.21 ± 1.64)% (P<0.01). The percentage of apoptotic nuclei staining with Hoechst33342 were higher in HMGB1 groups than that in the other groups. The percentage of caspase 3 positive macrophages were (29.74 ± 4.55)% in HMGB1 groups, and it was significantly higher than those in HMGB1 + Z-DEVE-FMK groups (3.02 ± 1.91)% and those in control groups (7.19± 2.46)% (P<0.01). Thetranscriptional activity of NF-κB/p65 was decreased in HMGB1 groups (P<0.01). Conclusions : (1) These results suggested that HMGB1(1-1000ng/ml) is capable of enhancing immune function of macrophages. (2) HMGB1 is a proinflammatory cytokine and is capable of promoting the release of TNF-α and ICAM-1, but not IL-10 in murine macrophages. (3) HMGB1 (100-10,000 ng/ml) has proapoptotic role on murine peritoneal macrophages. (4) HMGB1 stimulation can result in up-regulation of RAGE expression on surface of peritoneal macrophages. RAGE is the major receptor of HMGB1 in modulating the immune function in murine macrophages. (5) HMGB1 induces apoptosis in murine macrophages via activation of caspase 3 and inhibition of NF-κB. Caspase 3 inhibitor can reduce HMGB1 induced-apoptosis.
|
PDF Full Text Request