The Effect Of High Mobility Group Box-1Protein On Regulated Dendritic Cells In Mice

Objective: High mobility group box1protein (HMGB1) as a late-acting cytokinemediates lethality of sepsis and systemic inflammation. It is of great significance tounderstand the potential mechanism underlying HMGB1in the pathophysiology of sepis,which might help to seek novel therapeutic strategies in clinical settings. With regulatorydendritic cells (DCreg) stimulated with HMGB1in mice in vitro, the present study wasperformed to investigate the effects of HMGB1on splenic DCreg and its protentialregulating mechanism regarding to cell-mediated immunity, thereby preventing thedevelopment of sepsis and subsequent multiple organ dysfunction syndrome.Methods:1. DCregs (CD11clowCD45RBhighDCs) were isolated from the spleens ofmale Balb/c mice. After stimulation with different doses of HMGB1(10,100,1000ng/ml)for12hours or with100ng/ml HMGB1for different duration (2,12,24hours), levels ofinterleukin (IL)-10and IL-12, and expressions of costimulatory molecules including CD40,CD80, CD86, MHC-II were determined to observe the time-dependent and dose-dependentresponses between HMGB1and DCreg. Cytokine protein levels in supernatants weremeasured by ELISA kits. Expressions of costimulatory molecules including CD40, CD80,CD86, and MHC-II were analyzed with flow cytometry.2. CD11c^lowCD45RB^highDCswere stimulated with HMGB1in a concentration of100ng/ml for12hours. After beingstimulated, DCreg were co-cultured with T lymphocytes at1:100for3days, then Tlymphocyte proliferation, polarization, as well as IL-2levels were determined.3. Afterbeing stimulated with HMGB1, the expression of Toll-like receptor4(TLR4) on DCregwas determined with flow cytometry and SYBR Green real-time PCR, respectively. Inaddition, after the treatment with anti-TLR4antibody, the effect of HMGB1on splenic DCreg was investigated to observe changes in levels of interleukin (IL)-10and IL-12, Tlymphocytes proliferation, polarization, as well as release of IL-2.Result:1. Effects of HMGB1on immune function of DCreg in mice:(1) AfterDCreg being stimulated with HMGB1, IL-10, IL-12levels in splenic DCreg wererespectively increased and decreased at12to24hours (P<0.01), and they tended to bestable at12hours. When DCreg were cultured in the presence of10ng/ml,100ng/ml, and1000ng/ml HMGB1for12hours, IL-10, IL-12levels in splenic DCreg were respectivelyenhanced and reduced at12to24hours (P<0.01), and they tended to be stable at100ng/ml.(2) After stimulation with100ng/ml HMGB1for12hours, the costimulatorymolecules including CD40, CD80, CD86, and MHC-II expressions on surface of splenicDCreg were markedly down-regulated compared with control group (P<0.01).2. Effects ofsplenic DCreg treated with HMGB1on immune function of T lymphocytes in mice:DCregs were co-cultured with T lymphocytes at1:100for3days, the proliferativeresponse of splenic T lymphocyte to T cell mitogen were significantly decreased, withmarked reduction in IFN-γ/IL-4ratio and IL-2expressions compared with control group(P<0.01).3. Receptor related mechanism of HMGB1on splenic DCreg in mice:(1) AfterDCreg being stimulated with100ng/ml HMGB1for12hours, TLR4mRNA/proteinexpresions of splenic DCreg were markedly up-regulated (P<0.01).(2) After treatmentwith anti-TLR4antibody, interleukin (IL)-10and IL-12expressions showed no markedchanges compared with the controls, the proliferative response to T cell mitogen andfunctional polarization of T lymphocytes as well as IL-2levels also revealed similarpatterns (P>0.05).Conclusions:1. HMGB1stimulation can result in marked down-regulation of the costimulatorymolecules including CD40, CD80, CD86, and MHC-II expressions on surface of splenicDCreg in mice, while enhance IL-10synthesis as well as release and inhibit IL-12formation. HMGB1appears to be a potential immunostimulatory signal that induces functional differentiation of DCregs.2. Splenic DCreg treated with HMGB1could significantly enhance T lymphocyteproliferative response to T cell mitogen, and induce T lymphocytes differentiation to Th2and inhibit IL-2production.3. HMGB1stimulation can lead to marked up-regulation of TLR4expression ofsplenic DCreg, and TLR4might be a major receptor in mediated maturation anddifferentiation of DCregs.