| Objective: High mobility group box 1 protein (HMGB1) as a late-acting cytokine mediates lethality of sepsis and systemic inflammation. The kinetics of HMGB1 in sepsis suggests its potential therapeutic value in clinical settings. The present study was performed using dendritic cells (DCs) stimulated with HMGB1 in rats in vitro, and to clarify the effects of HMGB1 on splenic DC and its protential regulating mechanism underlying cell-mediated immunity, in turns preventing the development of sepsis and subsequent multiple organ dysfunction syndrome.Methods: DCs isolated from the spleens of male Wistar rats were suspended in RPMI-1640 with 10%FCS at 1×10~5 cells /well on 96-well cell culture plates. In the present experiment, the cells were stimulated with HMGB1 for various length of time or in different concentrations. 1. DCs were denatured in cell culture plates to determine expressions of costimulatory molecules including CD80, CD86, MHC II, and gene expressions of interleukin (IL)-12 as well as timor necrosis factor (TNF)-α. Supernatant was harvested to determine cytokine protein levels. The time-dependent and dose-dependent responses between HMGB1 and costimulatory molecules CD80, CD86 and MHC II expressions were analyzed by flow cytometry. IL-12 and TNF-α mRNA/protein levels were analyzed SYBR Green real-time PCR and ELISA, respectively. 2. The cells were stimulated with HMGB1 in a concentration of 1μg/ml for 48 hours. After being stimulated, DCs were denatured in cell culture plates to determine expression of the receptor for advanced glycation end products (RAGE) with flow cytometry and laser scanning confocal microscopy. The dose-dependent responses between anti-RAGE polyclone antibody and costimulatory molecules including CD80, CD86 andMHC II expressions on the surface of DCs stimulated with HMGB1 were analyzed with flow cytometry. 3. The cells were stimulated with HMGB1 in a concentration of 1μg/ml for 48 hours. After being stimulated, DCs were co-cultured with T lymphocytes at different ratios (1:50, 1:100, 1:150, and 1:200). The cells were collected on days 3 and 5 after culture to determine T lymphocytes proliferation, polarization, gene expressions as well as protein levels of IL-2, interleukin-2 receptor (IL-2R), and the activation of nuclear factor (NF)-κB.Result: 1. Effects of HMGB1 on costimulatory molecules expression on surface of splenic DCs in rats: (1) After stimulation with 1μg/ml HMGB1, the costimulatory molecules CD80, CD86 and MHC II expressions on surface of rat splenic DCs were markedly up-regulated at 24 to 72 hours (P<0.05 or P<0.01), and the expression levels of CD80, CD86 as well as MHC II peaked at 48 hours (P<0.01). When DCs were cultured in the presence of 0.1μg/ml, 1μg/ml, and 10μg/ml HMGB1 for 48 hours, expressions of the costimulatory molecules CD80, CD86 and MHC II were significantly up-regulated (P<0.05 or P<0.01), and values of these three molecules were highest in 1μg/ml HMGB1-treated group (P<0.01). (2) After stimulation with 1μg/ml HMGB1, IL-12, TNF-α protein as well as mRNA expressions in rat splenic DCs were markedly up-regulated at 24 to 72 hours (P<0.05 or P<0.01), and expression levels of IL-12 and TNF-α peaked at 48 hours (P<0.01). When DCs were cultured in the presence of 0.1μg/ml, 1μg/ml, and 10μg/ml HMGB1 for 48 hours, IL-12 and TNF-α expressions were significantly enhanced (P<0.01), and values of both cytokines were highest in 1μg/ml HMGB1-treated group (P<0.01). 2. Effects of HMGB1 on the receptor expression of splenic DCs in rats: after stimulation with 1μg/ml HMGB1 for 48 hours, RAGE expression on surface of rat splenic DC was markedly increased (P<0.01). When DCs were cultured in the presence of 1:50, 1:100 and 1:200 dilution RAGE polyclone antibody for 2 hours, expressions of the costimulatory molecules CD80, CD86 and MHC II after stimulated with HMGB1 in a concentration of 1μg/ml for 48 hours were significantly down-regulated (P<0.01), and values of these molecules were lowest in 1:100 dilution (P<0.01). 3. Effects of splenic DCs treated with HMGB1 on splenic T lymphocyte in rats: (1) splenicT lymphocyte proliferative response to T cell mitogen, concanavalin A (Con A), were significantly increased on days 3 and 5 after cultured in the presence of DC to T lymphocyte ratios in 1:50, 1:100, 1:150 and 1:200 (all P<0.01), with marked production of interferon-γ and reduction in interleukin-4 in supernatants, especially when DC to T lymphocytes ratio was 1:150 and cultured for 3 days (all P<0.01). (2) gene expressions and protein levels of IL-2, IL-2R, the marked activation of NF-κB were demonstrated on 3 days, especially in the presence of DC to T lymphocyte ratios in 1:150 (all P<0.01). Conclusions:1. HMGB1 stimulation can result in marked up-regulation of the costimulatory molecules including CD80, CD86 and MHCII expressions on surface of rat splenic DCs, and IL-12 as well as TNF-α expressions and release. HMGB1 appears to be a potential immunostimulatory signal that induced DC maturation.2. HMGB1 stimulation can lead to marked up-regulation of RAGE expression on surface of rat splenic DCs, and RAGE might be a potential receptor associated with maturation and differentiation of DCs.3. Splenic DCs treated with HMGB1 could significantly enhance T lymphocyte proliferative response to T cell mitogen, and induce T lymphocytes differentiation to Th1.4. Splenic DCs treated with HMGB1 are involved in the regulation of increased IL-2 production and IL-2R expression of splenic T lymphocyte, and NF-κB appears to be the potential signal pathway in cell-mediated immunity.
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