The Effect Of High Mobility Group Box-1 Protein On Dendritic Cells After Thermal Injury In Rats

Objective: The present study was performed (1) to investigate in vivo the changes in high mobility group box 1 protein (HMGB1) and its stimulating effect on the maturation of dendritic cell (DC) after severe thermal injury; (2) to identify if RAGE is the possible receptor on surface of DC to combine HMGB1; (3) to identify in vivo the effect of the HMGB1 stimulated DC on splenic T lymphocyte and its potential mechanisms after severe thermal injury.Methods: A widely used technique for induction of full-thickness scald burns was used in these studies. For rats with burning injury, they were anesthetized, and the dorsal and lateral surfaces of the rats were shaved. Rats were placed on their backs and secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin was immersed in 99°C water for 12 s. Sham-injured rats were subjected to all of the procedures except the temperature of the bath was of room temperature. 40ml/kg Lactated Ringers solution resuscitation was administered i.p delayed for 6 hours after the injury, 4ml at 12, 24, 36 and 48 h after burn injury.One hundred and thirty six male Wistar rats were randomly divided into five groups as follows: normal control group (8 rats), sham burned group (32 rats), burn group (32 rats), burn with Ethryl pyruate (EP) treatment group (32 rats), and burn with anti-RAGE (advanced glycation end-products) antibody treatment group (32 rats), and the last four groups were further divided into four subgroups of 8 rats each, which were sacrificed on post burn days (PBD) 1, 3, 5 and 7 respectively. EP was added to Lactated Ringers solution (EP 28 mM) in EP treatment group. Anti-RAGE antibody 1mg/kg was given via dorsal penile vein at6 h and 24 h respectively after burn injury in anti-RAGE antibody treatment group. Animals of all groups were sacrificed at designated time points, and blood and spleen samples were harvested aseptically to determine organ damage related variables and levels of various cytokines. Spleen was divided into two parts as following: one part was used to detect gene and protein expression levels of HMGB1 and TNF-α, and the other part was used to procure DC by MACS microbeads and T cell by using column of nylon wool. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue, as well as activated NF-κB of T cell was determined by ELISA kits for rats. Gene expression was measured by real-time quantitative PCR taken GAPDH as the internal standard.Result: (1) The significantly elevated expression levels of splenic HMGB1 gene and protein and levels of serum HMGB1 were detected on PBD 1-7, and it was significantly inhibited by treatment of EP, but not by anti-RAGE antibody. (2) DC expressed similar levels of CD80, strongly enhanced levels of CD86 and slightly enhanced levels of MHC class Ⅱ on PBD 1-7 in comparison to DC from sham-injured rats. The capacity of DC to engulf dextran and the levels of IL-12 produced by DC in supernatant decreased markedly after burn injury. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly raise the expression levels of CD80, MHC class Ⅱ of DC and levels of IL-12 production, but not the capacity of DC to swallow the dextran. (3) The markedly increased expression of RAGE on the surface of DC from burned rats was found on PBD 1-7 to compare with sham-injured rats, which was not influenced by treatment of EP, but was blocked by treatment of anti-RAGE antibody. (4) The T cell proliferative activity in response to ConA in burn-injured rats was significantly suppressed on PBD 1-7 as compared with sham-injured rats, and gene or protein expressions of IL-2 and expressions of IL-2Ra of T cell in bum-injured rats were simultaneously suppressed after burn injury to differentextent. EP or anti-RAGE antibody treatment could restore T cell proliferative activity response to Con A, gene or protein expressions of IL-2, and expression of IL-2Rα after burn injury. (5) The levels of IL-4 produced by T cell response to Con A increased markedly after burn injury, whereas, the levels of IFN-γ decreased markedly, indicating that Th cells might develop into Th2 cells. EP or anti-RAGE antibody treatment could significantly inhibite increase of IL-4 and decrease of IFN-γ produced by T cell response to Con A after thermal injury, indicating that EP or anti-RAGE antibody treatment might influence the polarization of T cells in animals subjected to thermal injury and induced Th cells to drift to Th1 cells. (6) The NF-κB activation of splenic T cell was downregulated significantly on PBD 1-7. Treatment with EP or anti-RAGE antibody could completely restored the NF-κB activaty of splenic T cell after burn injury. (7) The serum ALT, AST, Cr, BUN and CK-MB levels were significantly elevated on PBD 1-7, and treatment with EP or anti-RAGE antibody could inhibit these increase to different extent.Conclusion: Serum and splenic HMGB1 were markedly up-regulated for a prolonged period later after severe thermal injury. HMGB1 might play an important role in the development of excessive inflammatory response and subsequent multiple organ dysfunction syndrome. The excessive release of HMGB1 might stimulate splenic DC to mature abnormally (which might mainly induced by binding RAGE on the surface of DC), and further induce suppression of T lymphocyte immune function and drifting to Th2 following burn injury. NF-κB signaling might be involved in the abnormal DC mediating suppression of T lymphocyte associated with excessive release of HMGB1. EP has a therapeutic potential for suppressing HMGB1-induced immune dysfunction and ameliorating multiple organ injury.