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Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro
Published on: June 1, 2010
The effect of high mobility group box-1 protein on immune function of human T lymphocytes in vitro
Li-feng Huang1, Yong-ming Yao, Hai-dong Meng
1Department of Microbiology and Immunology, Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing 100037, China. c_ff@sina.com
Insights
High mobility group box-1 protein (HMGB1) exhibits a dual effect on human T lymphocyte immune function. While low concentrations are tolerated, higher HMGB1 levels can impair T cell proliferation and alter T helper cell balance.
Area of Science:
- Immunology
- Molecular Biology
Background:
- High mobility group box-1 protein (HMGB1) is a nuclear protein with extracellular functions.
- HMGB1's role in modulating immune responses, particularly T lymphocyte function, requires further elucidation.
Purpose of the Study:
- To investigate the impact of HMGB1 on human T lymphocyte immune function in vitro.
- To explore HMGB1's potential role in cell-mediated immune dysfunction.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with recombinant human HMGB1 (rhHMGB1).
- Cell viability was assessed using MTT assay.
- Flow cytometry (FCM) measured CD3, CD8, and CD4 expression.
- RT-PCR detected gene expression of interleukin-2 (IL-2) and IL-2 receptor alpha (IL-2Rα).
Main Results:
- rhHMGB1 at 500-1000 microg/L for 48 hours decreased T cell viability.
- HMGB1 induced a dose- and time-dependent increase in Th2 subset and decreased the Th1/Th2 ratio.
- Short-term (12 hours) low-dose (10 microg/L) HMGB1 upregulated IL-2 and IL-2Rα mRNA, while long-term (48 hours) high-dose exposure downregulated these genes.
Conclusions:
- HMGB1 exerts a dual influence on T lymphocyte immune functions.
- HMGB1 can modulate T cell proliferation, differentiation, and cytokine gene expression.
Objective:
To investigate the effect of high mobility group box-1 protein (HMGB1) on immune function of human T lymphocytes in vitro and explore its potential role in cell-mediated immune dysfunction.
Methods:
Fresh blood was obtained from healthy adult volunteers and peripheral blood mononuclear cells (PBMCs) were isolated, then rhHMGB1 was added to PBMCs. Cell viability was assessed by thiazolyl blue (MTT) assay. Four-color flow cytometric (FCM) analysis was used for the measurement of CD3, CD8 expression. Reverse transcription-polymerase chain reaction amplification was performed to detect respective gene expression of interleukin-2 (IL-2), IL-2 receptor (IL-2R) alpha.
Results:
(1)Proliferation of T lymphocytes was not affected by rhHMGB1 in low concentrations, while continued exposure of T cells to 500-1000 microg/L rhHMGB1 for 48 hours resulted in a decrease in MTT assay. (2) Different stimulating time and dosages of rhHMGB1 did not alter CD4 expression of CD3+ T lymphocytes. rhHMGB1 stimulation provoked a dose-dependent and time-dependent increase in Th2 subset and lowering in ratio of Th1 to Th2. (3) Compared with the untreated cells, when the cells were co-incubated with rhHMGB1 (10 microg/L) for 12 hours, mRNA expressions of IL-2 and IL-2R were significantly up-regulated. At 48 hours, in contrast, gene expression was relatively lower in T cells after exposure to 100-1000 microg/L rhHMGB1.
Conclusion:
These data demonstrated that HMGB1 had a dual influence on immune functions of T lymphocytes.
