Potential Role And Mechanism Of High Mobility Group Box 1 Protein In Macrophage-and Lymphocyte-Mediated Immunity In Mice And Effect Of Escharectomy During Shock Stage On Systemic As Well As Intestinal Immune Function In Thermally Injured Rats

Background: Immunity dysfunction plays a vital role in sepsis. Shift from an initially proinflammatory response to an anti-inflammatory immuno-suppressive state has been identified as sepsis persists, even it can proceed to a state of anergy. Recently, high mobility group box 1 protein (HMGB1), a nuclear and cytosolic protein has been identified as a cytokine mediator of sepsis. Strategies to inhibit HMGB1 activity and release are being investigated. However, whether extracellular HMGB1 participates in regulation of immunological function remains unknown thus far. The aim of this study was to evaluate the roles of HMGB1 in molecular immunity of macrophages and lymphocytes in mice and relationship between production of HMGB1 and immune function in thermally injured rats.Methods: Peritoneal macrophages and splenic lymphocytes harvested from male BALB/c mice were stimulated with HMGB1, then phagocytosis of neutral red and splenic lymphocytes proliferation were assayed, and I-A/-E expression of macrophages, apoptosis and Tcl/Tc2, Thl/Th2 subsets of lymphocytes were assessed using flow cytometry. Secondly, male BALB/c mice were treated with HMGB1 (0.2 ug or 20 ug per mouse, i.p.), and then, phagocytosis of neutral red, splenic and Peyer's Patch (PP) lymphocytes proliferation were assayed, and I-A/-E expression of macrophages, apoptosis, ratio of Tel to Tc2 and Th1 to Th2 of lymphocytes were detected. Levels of interleukin-2 (IL-2) and soluble IL-2R (sIL-2R) of culture supernatant and plasma were assayed by ELISA. Finally, male Wistar ratssubjected to 30% TBSA full-thickness thermal injury were randomly divided into 24 h and 72 h escharectomy groups. Gene expression of HMGBl, interleukin-10 (IL-10), and tumor necrosis factor-a (TNF-alpha) in liver and lung was detected with reverse-transcription PCR, and protein levels of IL-10 and TNF-a in liver and lung tissues were measured by ELISA. HMGBl protein expression in liver and lung was detected with immunohistochemistry. Levels of plasma HMGBl were assayed by Western-blotting. Mucus slgA was quantified with radio-immunoassay (RIA). IL-2 levels in plasma and intestinal tissue were also assayed by ELISA. Moreover, plasma AST and ALT, pulmonary myeloperoxidase (MPO) and intestinal diamine oxidase (DAO) activity were also assayed.Results: (1) HMGBl regulated the phagocytosis of peritoneal macrophages in a time- and dose-dependent manner, but not the expression of I-A/-E. (2) Different culture time and dosage of HMGBl displayed a profound influence on splenic lymphocytes proliferation. (3) Different culture time and dosage of HMGBl did not alter Thl/Th2 and Tcl/Tc2 subsets of splenic lymphocytes, however, the ratio of Thl to Th2 was relatively higher when stimulated with 10 and 100 ng/ml HMGBl for 12 h, and the ratio of Tel to Tc2 was relatively higher when stimulated with 1 ng/ml HMGBl for 12 h or 24 h, but relatively lower when stimulated with 10 ng/ml HMGBl for 12 h. (4) Levels of IL-2 were increased significantly when stimulated with 10 ng/ml HMGBl for 12 h, thus the ratio of IL-2 to sIL-2R was markedly higher. (5) Phagocytosis of peritoneal macrophages was markedly suppressed when challenged with high dose HMGBl for 24 h, while I-A/-E expression was upregulated significantly when challenged with low dose HMGBl for 48 h. (6) Splenocyte proliferation was markedly suppressed when challenged with high dose HMGBl for 24 h, apoptotic death of lymphocytes was increased at 48 h. (7) Significantly less Thl cells weredetected when challenged with HMGBl. Furthermore, low dose HMGBl resulted in an increase in Th2 cells, and high dose HMGBl resulted in a decrease of Tel and Th2, and increase of Tc2 cells at 48 h. (8) Low or high dose HMGBl enhanced lymphocyte proliferation and reduced apoptosis in PP at 24 h. Low dose HMGBl resulted in an enhanced proliferation of PP lymphocytes, while high dose HMGBl resulted in a marked reduction in PP lymphocytes proliferative response and increase in apoptosis at 48 h. (9) HMGBl resulted in Thl and Th2 increase for several times, but lowered...