The effect of high mobility group boxª²1 protein on immune function of human T lymphocytes in vitro

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HUANG Liª²fengYAO Yongª²mingMENG Haiª²dongZHAO Xiaoª²dong DONG NingYU YanSHENG Zhiª²yong

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     ¡¾Abstract¡¿Objective   To investigate the effect of high mobility group boxª²1 protein (HMGB1) on immune function of human T lymphocytes in vitro and explore its potential role in cellª²mediated 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. Fourª²color flow cytometric (FCM) analysis was used for the measurement of CD3, CD8 expression. Reverse transcriptionª²polymerase chain reaction amplification was performed to detect respective gene expression of interleukinª²2 (ILª²2), ILª²2 receptor (ILª²2R) alpha. Results   ¢ÙProliferation of T lymphocytes was not affected by rhHMGB1 in low concentrations, while continued exposure of T cells to 5001 000 μg/L rhHMGB1 for 48 hours resulted in a decrease in MTT assay. ¢Ú Different stimulating time and dosages of rhHMGB1 did not alter CD4 expression of CD3£« T lymphocytes. rhHMGB1 stimulation provoked a doseª²dependent and timeª²dependent increase in Th2 subset and lowering in ratio of Th1 to Th2ª±¢Û Compared with the untreated cells, when the cells were coª²incubated with rhHMGB1 (10 μg/L) for 12 hours, mRNA expressions of ILª²2 and ILª²2R were significantly upª²regulated.  At 48 hours,  in contrast, gene expression was relatively lower in T cells after exposure to 1001 000  μg/L rhHMGB1ª±Conclusion   These data demonstrated that HMGB1 had a dual influence on immune functions of T lymphocytes.

     ¡¾Key words¡¿high mobility group boxª²1 protein;immunity;T lymphocytes;proliferation;interleukinª²2

From Department of Microbiology and Immunology, Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing 100037, China.This work was supported by the National Basic Research Program of China (2005CB522602), the National Natural Science Outstanding Youth Foundation of China (30125020), and National Natural Science Foundation of China (30672178).Corresponding author:Prof. YAO Yongª²ming, Email:cff@ sina. com
        High mobility group boxª²1 protein (HMGB1) is a nonhistone, chromatinª²binding protein, which was isolated from calf thymus over 30 years ago¡²1¡³. Recently, HMGB1 has been identified as a proinª²flammatory cytokine that mediates endotoxin lethality, inflammation, and macrophage activation in sepsis¡²24¡³. It has been found in the serum of patients with acute (sepsis) and chronic (rheumatoid arthritis) inflammatory conditions¡²56¡³, thus it may be involved in maladaptive or autoimmune responses. Up to now, the biology of HMGB1 has been extensively studied as a proinª² flammatory cytokine of systemic inflammation, while little is known in regard to its potential contribution to the host cellª²mediated immunity, particularly regarding human T lymphocytes. Clinical data suggest that there is close relationship between the extent of impaired host defense and sepsis as well as multiple organ dysfunction syndrome. Studies have indicated that shift from Th1 to Th2 response was a contriª² butory factor to marked suppression of cellª²mediated immunity in sepsis, though the underlying mechanism has not been fully elucidated¡²7¡³. HMGB1 has recently been identified as a crucial cytokine that mediates the responses to infection,  injury and inflammation. As HMGB1 appears to emerge late after a serious infection, a hope arises that it might be a potential therapeutic target in clinical practice. However, the biological activities of extracellular HMGB1 and its effect on the innate immune response have not been well illustrated. In this study, we investigated the effect of HMGB1 on immunity of human T lymphocytes in vitro and explored its potential role in cellª²mediated immune dysfunction.
1  Materials and Methods
1.1  Experimental design:The study population comprised of 26 healthy volunteers. All of them with no detectable physical or clinical laboratory abnormality were demanded to refrain from taking any drug for one month. Eight volunteers ¡²5 men and 3 women, mean age £¨24±9£© years old, range 1842 years old¡³ participated in the experiment of polarization of the Tª²helper lymphocyte activity; Eight volunteers ¡²4 men and 4 women, mean age £¨25±6£© years old, range 2237 years old¡³ participated in the lymphocyte proliferation assay; Ten volunteers ¡²7 men and 3 women, mean age £¨29±7£© years old, range 2542 years old¡³ were enrolled for the experiment to determine the gene expressions of interleukinª²2 (ILª²2) and ILª²2 receptor (ILª²2R). Written informed consents were signed before the beginning of experiments. The experimental protocol was approved by the Ethical Review Committees of Postgraduate Medical College of PLA, Beijing.
1.2  Peripheral blood T lymphocyte isolation and counting:Thirty milliliters of heparinised blood was diluted in Hanks′ balanced salt solution, and Ficollª²Hypaque (Sigma Chemical Co. , St. Louis, MO) was used for isolation and preparation of peripheral blood lymphocytes. The isolated lymphocytes were washed twice with phosphate buffered saline (PBS), centrifuged (500 g, 4 minutes), resuspended in basal medium (unstimulated culture) RPMIª²1640 (Gibcoª²BRL, Gaithersburg, MD) containing 10% fetal calf serum (Seromed, Berlin, Germany), 1% penicillin/streptomycin (Sigma Chemical Co., St. Louis, MO), and 1% glutamine (ICN, Eschwege, Germany). Then cell populations were counted under a light microscope. Based on the different experimental demand, the peripheral blood mononuclear cells (PBMCs) suspension was diluted with RPMIª²1640 to 2×109 cells/L.
1.3  Assessment of cell viability by methyl thiazolyl tetrazolium (MTT) assay:Cells (2×109  cells/L) were inoculated to 96ª²well plates with 0ª±2 ml per well and incubated for 4 hours at 37 ¡æ in 5% CO2ª±1% phytohemagglutinin (PHA;Sigma Chemical Co., St. Louis, MO) was added to maintain cell proliferation and viability. At 24, 48, 60 hours, recombinant human HMGB1 (rhHMGB1, Sigma Chemical Co. , St. Louis, MO) in different amount or PBS were added to the PBMCs suspension to make final concentration of HMGB1 of 0, 1, 10, 50, 100, 500 and 1 000 μg/L per well, respectively, with 4 wells for each concentration of HMGB1ª±Cells were incubated for 68 hours, and then 100 μl supernatant was procured. MTT (Sigma Chemical Co. , St. Louis, MO) 20 μl was added to each well. After culturing for 4 hours, 100 μl Tritonª²ISOP solution was added. Optical density (OD) value per well at 560 nm wavelength was read in a plate reader 10 minutes later, and mean value was calculated.
1.4  Determination of CD4£«/CD3£« and CD69 expressions on T lymphocytes:Cell suspension was harvested, washed once with PBS, and centrifuged (500 g, 4 minutes), and the cells were resuspended in basal medium 200 μl RPMIª²1640ª±20 μl CD3ª²PerCP (Bectonª²Dickinson, San Jose, CA) and 5 μl CD8ª²APC (Bectonª²Dickinson, San Jose, CA) were added. Cells were stored at room temperature in darkness. Then They were washed once with 2 ml PBS, centrifuged (500 g, 4 minutes), resuspended in 300 μl RPMIª²1640ª±Two samples of supernatant (100 μl) were added in two flow cytometry (FCM) test tubes. Two samples were labeled respectively as the following methods: ¢Ù Negative comparison staining:the method was the same with that of intracellular cytokine staining;¢Ú Cellª²stimulating marker CD69 staining:cells were incubated with 10 μl CD69ª²PE (Bectonª²Dickinson, San Jose, CA) for 20 minutes at 4 ¡æ in the dark and then washed twice with FACS buffer PBS with 1% bovine specific albumin and 0ª±1% sodium azide before FACS acquisition. Before the FCM analysis, PBMCs were cultured for 6 hours with 50 μg/L phorbol 12ª²myristate 13ª²acetate (PMA), 1 mg/L ionomycin, and 2 μmol/L GolgiStop. CD8£«CD69£« T cells expressions were counted by gating on the CD3£« cell population¡²8¡³. (12)×105 cells were analyzed in each experiment using the Cell Quest program (Bectonª²Dickinson, San Jose, CA) after setting the quadrants using isotype controls.
1.5  ILª²2, ILª²2R gene expressions:Cells (2×109 cells/L) were inoculated in 24ª²well plates with 2 ml per well and incubated for 2 hours at 37 ¡æ in 5% CO2ª±Cultures were stimulated with 1% PHA. rhHMGB1/PBS was added in the PBMCs suspensions to make the final concentration of HMGB1 at 0, 10, 100, and 1 000 μg/L per well respecª² tively. There were three parallel wells in each array. Incubated for 12, 48 hours respectively, supernatants and cells were collected.Total cellular RNAs were isolated from cells using Qiagen RNase purification kits (Qiagen, Alameda, CA). The Multiprobe RNase protection assays (RPAs) were performed with 510 μg total RNA according to the manufacturer′s directions (Pharmingen, San Diego, CA). Purity of nucleonic acid was determined as A260/A280 ratio, the following formula calculation was performed to determine total RNA concentration:RNA concentration (g/L)=(£Á260×40×100)/1 000ª±After DNase treatment for RNA specimens, the reverse transcription was performed using the reverse transcriptase and Oligo (dt) 15 Primer in ice bath for cDNA. Reverse transcription system included:1ª±0 μg template £¨RNA£©, 0ª±5  μl rRNasin£¨4×£±£°£· U/L£©, 1ª±0 μl Oligo (dt) 15 Primer (0ª±5 g/L), 4ª±0  μl MgCl2 (25 mmol/L), 2ª±0  μl 10×reverse transcription buffer, 2ª±0  μl dNTP (10 mmol/L), 1ª±5  μl avian myeloblastosis virus (AMV) retroviridase£¨1×10£· U/L£©, 20  μl nonª²RNase liquid. The suspension was mixed well, centrifuged briefly, heated in an aqueous bath for 1 hour at 42 ¡æ, AMV retroviridase was inactivated for 5 minutes at 95 ¡æ£¬ immersed in ice bath for 5 minutes, centrifuged again, cDNA was kept at 20 ¡æ for later use. Reverse transcriptionª²polymerase chain reaction (RTª²PCR) was performed using the following primers:¢ÙILª²2Rα, product size 298 bp¡²9¡³, forward 5′ª² GAA TTT ATC ATT TCG TGG TGG GGC A ª²3′, reverse 5′ª² TCT TCT ACT CTT CCT CTG TCT CCG ª²3′;¢ÚILª²2, product size 457 bp¡²10¡³, forward 5′ª² ATG TAC AGG ATG CAA CTC CTG TCT Tª²3′, reverse 5′ª²GTC AGT GTT GAG ATG ATG ATG CTT TGA Cª²3′;¢Û ⪲actin, product size 383 bp¡²10¡³, forward 5′ª²GTG GGG CGC CCC AGG CAC CA ª²3′, reverse 5′ª² GTC CTT AAT GTC ACG CAC GAT TTC ª²3′. Reverse transcription system for cDNA amplification included: 4  μl cDNA, 0ª±5  μl dNTP (10 mmol/L), 1ª±5  μl MgCl2 (25 mmol/L), 1ª±0 μl upstream primer, 2ª±0  μl 10×reverse transcription buffer, 1ª±0  μl downstream primer, 2ª±0  μl Taq DNA pclymerase (0ª±5 g/L), 25 μl nonª²RNase liquid. Mixed well and centrifuged briefly, covered with 50 μl liquid paraffin, and amplification was performed in a thermal cycler (Perkin Elmer, USA).Denaturating cycle at 97 ¡æ for 5 minutes was first done, and the condition for PCR reactions was shown in Table 1ª±The PCR production was electrophoresed in a 1ª±2% agarose gel containing 0ª±5 g/L ethidium bromide in Tris borate/ethylenediaminetetraacetic acid (EDTA) buffer. Bands were visualized by UVª²transillumination. Pictures were analyzed with LEICA Qª²500IW image analysis software (LEICA Qª²500IW, Germany).
2.3  Changes in T lymphocyte proliferation:With same dosage (11 000 μg/L) but different time of action, no significant difference in T lymphocyte proliferation was noted. However, continued exposure of T cells to 5001 000 μg/L rhHMGB1 resulted in inhibition of their proliferation. No significant difference in T lymphocyte proliferation was observed after being stimulated with different dosage of rhHMGB1 at 12 or 24 hours. After 48 hours, the effect of rhHMGB1 in different dosage on T lymphocyte proliferation showed significant difference (£Ð=0ª±045 3). T lymphocyte reproductive activities with 0, 1 μg/L of rhHMGB1 were stronger than with 500 or 1 000 μg/L (both £Ð<0ª±05), and a highly significant difference was found among 10, 500, and 1 000 μg/L (both £Ð<0ª±01) (Figure 3). Data showed that continued exposure of T cells to high dosages of rhHMGB1 for 48 hours resulted in marked decrease in MTT density.
2.4  CD69 expression:Application of CD3/CD8 gating to examination of Th1/Th2 cells in FCM is a new approach for the more accurate and sensitive detection of Th1/Th2 cells (Figure 4). CD69 expression was an important cellª²stimulating marker. Higher than 85% in CD69/CD3 ratio was demanded for good intracellular cytokine staining. PBMCs were cultured for 6 hours with intracellular cytokine excitomotor (PMA, ionomycin, GolgiStop). CD69£« T cells expression value was consistent with the standard demand (Figure 5£©.
2.5  Changes in CD4£«/CD3£« ratio:The Th1 expression level was markedly lowered when cells were cultured for 72 hours (£Ð<0ª±01), the changes in CD4, Th2 expression levels and ratio of Th1/Th2 were not statistically significant. It was shown that incubation with rhHMGB1 in different dosages ( F=2ª±527 5, £Ð=0ª±010 9 ) and different  time (F=4ª±915 9, £Ð=0ª±010 5) could significantly influence T lymphocytes surface antigen CD4 expression. With the same time (12, 48 hours) but different dosages, no significant difference in CD4 expression was found, but significant differences in CD4 expression at 24 hours were found (F=3ª±214 4, £Ð=0ª±023 9). At same dosage (1, 10, 100, 1 000 μg/L) but different time it was shown that there were significant differences in CD4 expression with dosages of 10 μg/L (F=3ª±355 0, £Ð=0ª±032 9) and 1 000 μg/L (F=3ª±826 7, £Ð=0ª±020 5). It was showed that CD4£«/CD3£« ratio at 48 hours was significantly lower than that at 12 hours (£Ð<0ª±05) when stimulated with 1 000 μg/L rhHMGB1ª± The results suggested that continued exposure of T cells to 5001 000 μg/L rhHMGB1 would result in downª²regulation of CD4 expression, thus, reducing the lymphocyte activities to receive antigen presentation (Figure 6).
2.6  Changes in mRNA expressions of ILª²2 and ILª²2Rα:T cell multiplication was activated with PHA stimulation and the gene expressions reached the peak value at 812 hours. Then, gene expression levels of ILª²2 descended quickly, but high levels of ILª²2Rα gene expressions persisted for 48 hours. At 12 hours, it showed that there was a significant difference in ILª²2 mRNA expressions with different dosages of rhHMGB1 (F=4ª±687 2, £Ð=0ª±007 3). ILª²2 mRNA expressions increased with stimulation of rhHMGB1 in 100 μg/L (£Ð<0ª±01), and this effect was more obvious at 1 000 μg/L (£Ð<0ª±01). There was no marked difference in ILª²2Rα mRNA expressions at different dosages (Figure 7).
       At 48 hours, no significant differences were observed in ILª²2 mRNA expressions with stimulation of rhHMGB1 in different dosages, but a diminishing tendency of ILª²2 mRNA expression was found with increasing concentration of rhHMGB1 (£Ð=0ª±059). There was a significant difference in ILª²2Rα mRNA expressions with different dosages of rhHMGB1 (H=1ª±220, £Ð=0ª±045). The ILª²2Rα mRNA expressions were downª²regulated with 100 μg/L rhHMGB1 stimulation (£Ð<0ª±05), and the downª²regulation was very obvious with 1 000 μg/L rhHMGB1 stimulation (£Ð<0ª±01) (Figures 8 and 9).
    The sequences (from the left to the right) were Marker, primer ⪲actin, 0 μg/L(12 hours), 10 μg/L(12 hours), 100 μg/L(12 hours),1 000 μg/L(12 hours), 0 μg/L(48 hours), 10 μg/L(48 hours), 100 μg/L(48 hours), and 1 000 μg/L(48 hours), respectively  regulatory effect on T cells immune activity.
3  Discussion
      Sepsis is an indocile challenge to human health and economical development world wide. Mortality rate of severe sepsis and septic shock may reach 30%80%¡²11¡³, and it has become the leading cause of death of nonª²cardiac diseases in different countries, mainly because its underlying pathogenetic mechanisms have not been fully elucidated¡²12¡³. The key point in treatment of sepsis is to control inflammatory response in a reasonable degree. The discovery of HMGB1 as a contributory pathogenetic factor in sepsis is of great interest as some investigators found that the inhibition of HMGB1 release is important in controlling inflammatory response in sepsis.
        Our preliminary results had proved that HMGB1 was essential in the process of T lymphocyte adaptive immune responses. The studies in murine models showed that low doses of HMGB1 amplified the cytokine cascade during systemic inflammation. HMGB1, as a cytokine, significantly increased the release of TNFª²α in a doseª²dependent manner. However, with prolonged (2472 hours) and high dosage (1 000 μg/L) of stimulation with HMGB1, the functions of macrophages and T lymphocyte proliferation were inhibited, and apoptosis of T cells intervened (data was not shown). When T cells were treated with HMGB1 in different dosages for different duration, they would transform into different subsets of Th1 and Th2ª±The fact that high dose of HMGB1 can transform Th1 to Th2 subset implies that there is an immune suppression in this event¡²1314¡³. Based on this finding, in order to offer the theoretical evidences to interpret its clinical significance of immunologic competence, we designed to investigate the effects of HMGB1 on human T lymphocyte immune functions.In humans approximately 80% of lymphocytes in peripheral blood are Tª²lymphocytes which mature in the thymus¡²15¡³. Many cellular factors and bioactive substances play a regulatory role in the proliferation of lymphocytes. Cellular DNA synthesis and mitosis are important symbols of cellular proliferation. A costimlatory signal is required for proliferation and differentiation of lymphocytes delivered through Tª²cell and antigen presenting cells (APCs) interaction. ILª²2 is also thought to be able to stimulate Tª²lymphocytes proliferation to secrete immunoglobulins, and to enhance cellular immune responses¡²16¡³. Stimulation of T cells by antigen in absence of coª²stimulatory signal can not cause Tª²cell response. Our experimental results indicated that T lymphocyte cells gradually proliferated after being stimulated by PHA, and it peaked at 24 hours. PHA nearly had no effects on the function of mononuclear cells which were capable to secrete cytokines, and it could not activate the mononuclear cells proliferation. HMGB1 in a low dose appeared to be a stimulatory signal for T lymphocyte proliferation, but continued exposure of T cells to high concentration of rhHMGB1 could result in a marked decrease in MTT density. The results showed that lymphocyte proliferation could be induced by HMGB1 at relative low dose. On the other hand, it was restrained by HMGB1 in high concentration in a timeª²dependent manner, and this was likely to be the direct cause of reduced growth index and decreased number of lymphocytes of the lymphoid organs in vivo¡²17¡³. Excessive release of antiª²inflammatory cytokines is an important mechanism of immunosuppression in sepsis¡²18¡³. Recently, studies have been shown that there is an abnormal polarization of Th1 and Th2 cells in peripheral blood in patients with severe sepsis¡²1920¡³. Because cytokines might play an important role in inducing CD£´£« T ancillary cells differentiation, we speculate that HMGB1 as a late cytokine might influence immune function shift of T lymphocytes from Th1 subset to Th2 subset. Our results showed that Th1/Th2 ratios were significantly decreased after 24 hours of 100 μg/L HMGB1 stimulation, or with 1 000 μg/L HMGB1 stimulation for 12 hours. The results indicate that a prolonged exposure to high dose of HMGB1 can lead to a shift of Th1 to Th2ª±The abnormal polarization of T cells brings about changes in immune state from continual autoimmune responses to immunological depression¡²19¡³. Some clinical observations reported that the levels of ILª²2 and other proinflammatory cytokines began to decrease after 24 hours in septic patients, however, the level of soluble ILª²2R (sILª²2R) increased at the same time in serious trauma, infection and septic patient¡²21¡³. Our data showed that levels of ILª²2 mRNA expression were upª²regulated after 12 hours of 101 000 μg/L HMGB1 stimulation in a doseª²dependent manner. There was a close relation between decreased ILª²2R expressions and increased HMGB1 after 48 hours. As the mediation of APCs such as monocytes, macrophages, and dendritic cells is excluded in vitro, it is our speculation that the mechanism of mediation of ILª²2/ILª²2R mRNA expressions of lymphocytes might be related to the receptors on cell surface. Up to now, receptors which can crosslink with HMGB1 and transduct the immunoregulation signals have not been identified on surfaces of lymphocyte cells. Whether there are other molecules on the lymphocyte surfaces which posses characteristics of the receptors of cytokines to activate the transductions of signals and genetic transcriptions is an unknown subject calling for further investigation.

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