High Mobility Group Box 1 Protein As An Immune-modulating Factor For Cytokine Gene Expression And Th1/Th2 Polarization In Human T Lymphocytes

Background: Patients with sepsis have impaired host defenses that contribute to the development of multiple organ dysfunction syndrome (MODS) and even death secondary to major insults. Studies indicate that a shift from Th1 to Th2 response contributes to a marked suppression of cell-mediated immunity during sepsis; the mechanism of which remains unknown. The failure of anti-inflammatory agents led investigators to devise new therapeutic strategies for sepsis aims to establish a well-balanced immune response. High-mobility group box 1 protein (HMGB1), widely known as a nuclear structural protein, was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation. The kinetics of HMGBl in sepsis suggests its potential therapeutic value in clinical settings. However, the biological activities of extracellular HMGBl and its effects on adaptive immune response have not been well illustrated. Here we evaluate the roles of HMGBl in molecular immunity of human T lymphocytes in vitro and explore its pathological impression on the immune dysfunction in sepsis.Materials and Methods: Fresh blood was obtained from healthy adult volunteers and PBMC were isolated by Ficoll-Hypaque. The PBMC were suspended in RPMI 1640 with 10% FCS at 2 × 10⁶ cells/ml and cultured with 20 μg of PHA per ml in 5% CO₂ at 37℃. In the first experiment, recombinant human HMGB1 (rhHMGB1) (1-1000 ng/ml) was added to cultures at 24, 48, 60, and 72 hours. Cell viability was assessed by MTT assay after 72-hour incubation. The OD was measured with a spectrophotometer at 560 nm. Four-color flow cytometric(FCM) analysis was used for the detection of CD3, CD8expression and intracellular cytokines IL-4, IFN-γ. Before FCM tests, PBMCs were cultured for 6 hours with 50ng/ml PMA, 1ug/ml ionomycin, and 2μM GolgiStop. For the second experiment, rhHMGBl (10-1000 ng/ml) was added with the PHA and cultures were centrifuged at 12 and 48 hours for cells collecting. Total RNA was extracted from 4 × 10⁶ cells and cDNA was synthesized using an oligo (dT) primer. Polymerase chain reaction (PCR) amplification was performed to detect gene expressions of IL-2, IL-2Ralpha. PCR products were analyzed by electrophoresis. ELISAs for IL-2, sIL-2R protein levels in Cell culture supernatants were performed. Statistical analysis: Analysis of variance (ANOVA) was used to determine differences among groups.Results: (1) Proliferation of T lymphocytes: proliferation was not affected by rhHMGBl in low concentrations (less than 100ng/ml), while continued exposure of T cells to 500-1000 ng/ml rhHMGBl for 48 hours resulted in a degradation of MTT density. (2) CD4 expressions and Thl/Th2 subsets: different stimulating time and dosage of rhHMGBl did not alter CD4 expressions and the number of IFN-γ positive cells (Th1) of CD3⁺ T lymphocytes. rhHMGBl stimulated a dose-dependent and time-dependent increase in Th2 subset and decrease in ratio of Thl to Th2. Polarization of T-helper lymphocyte activity shifting in Th2 direction might lead to immunosuppression in sepsis. (3) Cytokine mRNA production was found to peak at 8 to 12 hours and protein production to peak at 24 to 48 hours. Compared with the untreated cells, coincubated with rhHMGB1 (10-100ng/ml) for 12hours significantly up-regulated both mRNA expressions and protein release of IL-2, IL-2R. At 48 hours, in contrast, gene expression and protein production were relatively lower in cells exposure to 100-1000 ng/ml rhHMGB1.Conclusions: Our results demonstrated that HMGB1 had a dual side influence on immune functions of T lymphocytes. As a cytokine with proinflammatory activities,...