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RELATIONSHIP BETWEEN HIGH-MOBILITY GROUP BOX 1 PROTEIN RELEASE AND T-CELL SUPPRESSION IN RATS AFTER THERMAL INJURY

Zhang, Li-tian; Yao, Yong-ming; Dong, Yue-Qing; Dong, Ning; Yu, Yan; Sheng, Zhi-yong

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doi: 10.1097/SHK.0b013e3181672495
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Abstract

INTRODUCTION

Severe burn injury induces a temporal shift in immune reactivity that can cause septic syndrome or even death. The immune system responds to injury by rapidly producing early and late proinflammatory cytokines, and also suppression of T-cell functions (1, 2). T cell plays an important role in cell-mediated immunity, and it is not only the effector cell but also the modulator cell in immune response. It has long been suggested that the immunosuppression of T cells contributes to diminished host resistance to infection (3, 4). Severe burn injury also alters T cells by inducing an imbalance in T helper (TH) cell functions caused by a phenotypic imbalance in the regulation of TH1 and TH2 immune response. After major injuries, overproduction of TH2 cytokines such as IL-4 and IL-10 is known to inhibit antigen-presenting cells such as macrophages, resulting in a decrease in the resistance to infectious pathogens in host survival (5, 6).

Early cytokines such as IL-1 and TNF-α are important elements of stimuli that might contribute to the initiation or perpetuation of an immune response against pathogens. In contrast, if these cytokines are released chronically and/or in the absence of infection, they can potentially contribute to the activation of self-reactive T cells and take up a role in the development of autoimmunity (7, 8). However, it seems that the response to trauma, burns, hemorrhagic shock, and microbial infection is associated with only a transient proinflammatory period, followed by a more prolonged period of immune suppression (9). Thus, it is postulated that there are some other factors involved in the development of host immune dysfunction after acute insults.

Several lines of evidence have suggested that high-mobility group box 1 protein (HMGB1) plays a role as a late mediator of delayed endotoxin morbidity and lethality (10, 11). High-mobility group box 1 protein plays an important role in the innate immune response by enhancing the production of proinflammatory cytokines such as TNF-α, IL-1 (12), and NO (13). Administration of exogenous HMGB1 to animals causes derangements in intestinal barrier function (13), tissue injury (14-17), and death (10). Moreover, inhibition of the biological activities of HMGB1 protein reduces lethality (10, 18) and protects against organ injury (14-17) in models of experimental sepsis. Ethyl pyruvate (EP) treatment can reduce blood levels of HMGB1 in animals with established endotoxemia or sepsis, and even its administration being initiated 24 h after cecal puncture can significantly increase survival (18).

Because HMGB1 was originally known as a late inflammatory mediator, its effect on T-cell immune function after burn injury is not fully explored hitherto. With this in mind, the present study was performed to investigate the potential role and effect of HMGB1 on T-cell-mediated immunity secondary to severe burns in rats.

MATERIALS AND METHODS

Animals and thermal injury

Wistar rats (weight range, 250-300 g) purchased from the Laboratory Animal Center, Chinese Academy of Medical Sciences, Beijing, China, were housed in separate cages in a 12-h light/12-h dark, temperature- and humidity-controlled room to acclimatize for at least 7 days before experimentation. All animals had free access to water but were fasted overnight before the experiment. Rats were anesthetized with an injection of pentobarbital sodium (60 mg/kg, i.p.), and the hair on the animals' backs was removed with 20% sodium sulfide. A 30% total body surface area full-thickness skin scald was then produced by immersing the back of the animal into boiling water for 12 s. Sham-injured rats were subjected to all of the procedures, except the temperature of the bath was of room temperature. Lactated Ringer solution (40 mL/kg, s.c.) was administered for initial resuscitation 6 h after injury, and, subsequently, 4 mL was administered at 12, 24, 36, and 48 h after injury. All experimental manipulations were undertaken in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals with the approval of the scientific investigation board of the Burn Institute, Postgraduate Medical College, Beijing, China.

Experimental design

Ninety-six rats were randomly divided into three groups as follows: sham burn group (32 rats), burn group (32 rats), and burn with EP (Sigma, St. Louis, Mo) treatment group (32 rats). The three groups were further divided into four subgroups of eight rats each, and they were killed on postburn days (PBDs) 1, 3, 5, and 7, respectively. In addition, eight rats were taken to serve as normal controls (the main parameters determined in the current study were found to be highly consistent with that in sham-injured animals, so the results were not shown.). Ethyl pyruvate was added to lactated Ringer solution (EP, 28 mM) in the EP treatment group. The dosage of EP was determined according to a previous report by Ulloa et al. (18), and they showed that delayed administration of EP in such dosage significantly attenuated the serum levels of HMGB1-induced by endotoxin. Animals of all groups were killed at designated time points, and blood and spleen samples were harvested aseptically to measure levels of various cytokines. Spleen samples were divided into two parts, one of which was snap-frozen and stored in liquid nitrogen until use for detection of mRNA expression of HMGB1, IL-2, and IL-2 receptor (IL-2R) by reverse-transcription polymerase chain reaction (RT-PCR) and nuclear factor of activated T-cell (NFAT) activity with gel mobility shift assay (EMSA). The other portion was prepared for cell culture immediately, and the supernatants were collected later for the measurement of IL-2, IL-4, and interferon (IFN) γ levels by commercially available enzyme-linked immunosorbent assay (ELISA) kits for rats.

HMGB1 Western blot analysis

High-mobility group box 1 protein was analyzed by Western blotting as described by Wang et al. (10). Briefly, serum was filtered through Centricon YM-100 (Millipore, Yonezawa, Japan) to clear the samples from cell debris and macromolecular complexes formed during clotting. Samples were then concentrated 15-fold with Centricon YM-30 (Millipore) and separated on 12% sodium dodecyl sulfate-polyacrylamide gels. Gel electrophoresis was performed at 60 V for 30 min, followed by 100 V for 2 h. Protein was transferred to immunoblot poly (vinylidene difluoride) membrane, and HMGB1 was analyzed by using rabbit polyclonal anti-HMGB1 antibodies and secondary antirabbit horseradish peroxidase (BD PharMingen, San Diego, Calif). The Western blots were then developed by a chemiluminescence reaction kit (Pierce, Rockford, Ill) in a light-lock chamber and were scanned with a silver image scanner (LEICA Q-550IW; Wetzlar, Germany), and the relative band intensity was quantified using National Institutes of Health Image 1.59 software. The levels of HMGB1 were calculated with reference to standard curves plotted with purified recombinant HMGB1 (Sigma).

Splenic T-cell preparation

Spleens were teased in 5 mL RPMI 1640. Cells were dispersed through a 30-μm stainless steel mesh and collected after centrifugation at 300 × g for 10 min, and they were resuspended in 4 mL RPMI 1640. Mononuclear cells were then obtained using Ficoll-Paque density gradient centrifugation and incubated with nylon wool-packed columns for 2 h at 37°C. T cells were obtained by eluting the columns with 30 to 40 mL of RPMI 1640 at a flow rate of one drop per second. Thus, the cells obtained were 90% to 95% CD3+ T cells. T cells with concanavalin A (Con A; Sigma) stimulation were cultured for 18 h at 37°C. Supernatants were collected to determine cytokines, and cells were divided into two portions. One (5× 107/mL) portion was used to extract total RNA, and the other (5 × 105/mL) was used to detect expression levels of IL-2Rα.

T-cell proliferation assay

CD3+ T cells were suspended in RPMI 1640 culture medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin, and placed in 96-well round-bottom plates in sextuplets for proliferation, giving the final cell density of 5 × 106/mL. Cells were treated with or without 5 μg/mL Con A for 68 h at 37°C in 5% CO2/100% humidified air. Thiazolyl blue (MTT; Sigma; 5 mg/mL; 10 μL/well) was added, and incubation was continued for 4 h, and then 100 μg acid isopropanol was added to dissolve the MTT crystals. When all the crystals had been dissolved through repeated blowing with a pipet, the optical density was measured by use of a microplate reader (Spectra MR; DYNEX Technologies, Inc., Chantilly, Va) at a wavelength of 540 nm.

Determination of IL-2Rα

CD3+ T cells (1 × 105) were blocked with 1 μg of Fc blocker (BD PharMingen) for 15 min at 4°C in 100 μL of 5% phosphate-buffered saline (PBS), 0.1% FCS, and then reacted for 20 min in darkness with polyethylene-conjugated CD25 and fluorescein isothiocyanate-conjugated CD3 (BD PharMingen). Cells were then washed twice with PBS 5% FCS, fixed in 10% formaldehyde in PBS (pH 7.2- 7.4), and examined by flow cytometry using a FACScan (BD Biosciences, Mountain View, Calif).

Determination of mRNA expression

Total RNA was extracted from the splenic tissue or T cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer's instruction. First-strand cDNA was synthesized using oligo-dT primer and the AMV reverse transcriptase (Promega Corp., Madison, Wis). The generated cDNA was then added to the reaction mixture with a final concentration of 0.2 μM of specific primers. The PCR mix contained a final concentration of 1× PCR buffer, 2.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate, and 0.7 U/25 μL Taq polymerase (Promega). The gene-specific primers and PCR circles for amplification of the desired cDNA were used as shown in Table 1. The final cycle was then followed by a 10-min soak at 72°C. Glyceraldehyde-3-phosphate dehydrogenase was used as internal controls for standardization of PCR product. DNA was amplified simultaneously.

TABLE 1
TABLE 1:
Primer sequences and PCR circles for amplification

Polymerase chain reaction products and molecular weight markers were subjected to electrophoresis on 2% agarose gels in Tris-acetate-EDTA buffers at 5 V/cm for 1 h and visualized by means of ethidium bromide staining. The number of PCR cycles was selected so that most of the ethidium bromide-stained amplified DNA products were between barely detectable and less than saturation. The gel was then photographed, and the negative was scanned with a densitometer (Pharmacia Corp., Upsala, Sweden). The ratios of HMGB1 (IL-2 or IL-2Rα)/glyceraldehyde-3-phosphate dehydrogenase signals were calculated for each sample. Each experiment included a negative control (sample RNA that had not been subjected to RT). This sample did not yield a PCR product, confirming the absence of extraneous genomic DNA of PCR product contaminating the samples.

Cytokine ELISA measurement

IL-2, soluble IL-2R (sIL-2R), IL-4, and IFN-γ levels in serum and culture supernatant were measured by commercially available ELISA kits for rats. Enzyme-linked immunosorbent assays were performed strictly following the protocols provided by the manufacturer. Enzyme-linked immunosorbent assay kits of IL-2, IL-4, and IFN-γ were purchased from Biosource (Worcester, Mass). Soluble IL-2R ELISA kit was purchased from Market (Kanagawa, Japan).

NFAT activity of splenic CD3+ T cell by EMSA

Reagents

(1) Buffer A: 10 mM (pH 7.8) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.75 mM spermidine, 0.15 mM spermine, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM EGTA, and 0.5 mM phenylmethylsulfonyl fluoride. Just before use, 1 mM NaF, 1 mM NaVO4, 1 mM Na4P2O7, 10 μg/mL aprotinin, 10 μg/mL pepstatin, and 10 μg/mL leupeptin (Calbiochem Corp., La Jolla, Calif) were added. (2) Buffer B: 50 mM Tris-Cl (pH 7.5), 420 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.2 mM dithiothreitol, 10% sucrosum, and 20% glycerine. Just before use, 0.1 mM phenylmethylsulfonyl fluoride (Calbiochem) was added. (3) The oligonucleotide sense probes (SBS Genetech Co., Beijing, China): 5′-GCC CAA AGA GGA AAA ATT TGT TTC ATA CA-3′; 3′-CGG GTT TCT CCT TTT TAA ACA AAG TAT GT-5′ (19).

Extraction of nucleoprotein

Splenic tissue (0.2 g) was ground in liquid nitrogen and then added to a glass Dounce homogenizer with 0.5 mL buffer A to homogenate in ice water. The splenic homogenate was centrifuged for 5 min at 5,000g, 4°C. Supernatant was removed completely, and 0.5 mL buffer A was added, incubated on ice for 30 min, and centrifuged for 5 min at 5,000g, 4°C. Supernatant with cytoplasm protein was then removed completely, and the pellet was resuspended in 0.5 mL buffer B, incubated for 30 min on ice, and centrifuged for 30 min at 14,000g, 4°C. The supernatant with the nucleoprotein was pipetted to a new EP tube and stored in liquid nitrogen.

Electrophoretic mobility shift assay

The protein concentration in the extract was estimated using a LightShift Chemiluminescent EMSA kit (Pierce) with bovine serum albumin as a standard. Binding reactions were loaded onto an 8 × 8 × 0.1-cm 6% polyacrylamide gel in 0.5 × Tris-borate-EDTA buffer and electrophoresed at 100 V at 4°C for 2 h. Biotin-labeled double-stranded DNAs were transferred to positive-charge nylon membrane (Hybond-N; Amersham Pharmacia Biotech, Buckinghamshire, UK) using a capillary method. Biotin-labeled DNA was integrated with streptavidin-horseradish peroxidase conjugate. Finally, chemiluminescence was determined using Light Capture imaging apparatus (AE-6961; ATTO, Tokyo, Japan).

Statistical analysis

Data were expressed as the mean ± SD. Statistical evaluation of the continuous data was performed by one-way ANOVA, followed by Dunnett t test for between-group comparisons. Correlations between variables were tested by Spearman correlation coefficients. These statistical analyses were done using the statistical software SPSS 10.0. The level of significance was considered to be P < 0.05.

RESULTS

HMGB1 gene and protein expression

To examine the kinetic changes in serum and splenic levels of HMGB1, animals were subjected to thermal injury, and blood and spleen were collected at various time points. It was shown that sham burn animals constitutively expressed HMGB1 mRNA in spleen (Fig. 1A). A significant increase in splenic HMGB1 mRNA levels was observed 24 h after thermal insult (2.4-fold of sham burn value; P < 0.01), which remained markedly elevated up to PBD 3 (P < 0.01), whereas EP treatment did not affect HMGB1 mRNA expression during the observation period. Meanwhile, plasma HMGB1 levels of burnt rats rapidly increased (Fig. 1B), peaking on PBD 1 (177.54 ± 68.54 ng/mL) and lasting till PBD 5 (P < 0.05- 0.01), recovering to normal range on PBD 7 (56.35 ± 23.68 ng/mL). Treatment with EP can significantly reduce the elevation of plasma HMGB1 levels (P < 0.05-0.01).

Fig. 1
Fig. 1:
High-mobility group box 1 gene and protein expression. Reverse transcription-PCR analysis was used to determine mRNA expression of splenic HMGB1. A, Levels of HMGB1 mRNA in spleen significantly elevated on PBD 1 and lasted up to PBD 3, whereas EP treatment did not affect HMGB1 mRNA expression during the observation period. Western blot was used to determine serum levels of HMGB1. B, Plasma HMGB1 levels of burnt rats were markedly elevated, peaking on PBD 1, and treatment with EP can reduce an increase in plasma HMGB1 levels. Statistical significance: *P < 0.05, ‡P < 0.01 as burn group versus sham burn group; †P< 0.05, §P< 0.01 as EP group versus burn group.

Splenic T-lymphocyte proliferation

As shown in Figure 2, the proliferative response of splenic CD3+ T cells was significantly suppressed during PBD 7 compared with the with sham burn group (P < 0.05), maximally by 60.4% on the first day. Treatment with EP can significantly improve the suppression of splenic T-lymphocyte proliferative activity at various time points (P < 0.05).

Fig. 2
Fig. 2:
Splenic T-lymphocyte proliferation using MTT assay for T-cell proliferation. T-Cell proliferative activity in response to Con A in burn-injured rats was significantly suppressed PBD 7 as compared with sham-injured rats. Ethyl pyruvate treatment can markedly restore T-cell proliferative activity. Statistical significance: *P < 0.05 as burn group versus sham burn group; †P < 0.05 as EP group versus burn group.

IL-2 production

To evaluate the activity of splenic T cells, we assayed the levels of IL-2 in T-cell culture supernatant or serum and also IL-2 mRNA expression of splenic T cells. As shown in Figure 3A, we found that IL-2 production in culture supernatant was significantly lower among animals subjected to burn injury compared with sham burn injury on PBDs 1 to 5 (P < 0.05). Likewise, in Figure 3B, serum IL-2 levels were significantly lower in animals subjected to thermal injury compared with sham burn injury on PBD 3 (P < 0.05), and the low values were maintained up to PBD 7 (P < 0.05). Ethyl pyruvate administration markedly enhanced IL-2 production in supernatant and serum IL-2 levels (P < 0.05). For Figure 3C, IL-2 mRNA expression in splenic T cells was much lower in the burn group than that in sham burn animals during the observation period (P < 0.05). Treatment with EP, however, can increase expression levels of IL-2 mRNA on PBDs 1 and 3 (P < 0.05).

Fig. 3
Fig. 3:
IL-2 production. Enzyme-linked immunosorbent assay analysis for IL-2 and RT-PCR for IL-2 mRNA were used in the present study. Levels of IL-2 in incubation supernatant (A) and serum (B) and of IL-2 mRNA expression of splenic T cells (C) were significantly lower in animals subjected to burn injury compared with sham burn injury after burns. Ethyl pyruvate treatment can enhance levels of IL-2 in supernatant and serum and expression of IL-2 mRNA after burns (P < 0.05). Statistical significance: *P< 0.05 as burn group versus sham burn group; †P < 0.05 as EP group versus burn group.

IL-2R expression

To test the activation of T cells through self-stimulation, we assayed the IL-2Rα expression on T cells in response to stimulation with Con A, plasma levels of sIL-2R, and gene expression of IL-2R in splenic T cells. As shown in Figure 4A IL-2Rα expression on splenic T cells was significantly lower in the burn group than that in the sham burn group on PBDs 1 and 3 (P < 0.05). Plasma sIL-2R levels were significantly higher in animals subjected to thermal injury compared with sham burn injury during PBDs 1 to 7 (P < 0.05; Fig. 4B). IL-2R mRNA expression of T cells was significantly down-regulated only on PBD 1 in the burn group compared with the sham burn group (0.527 ± 0.237 vs. 0.853± 0.244; P < 0.01), and there were no differences afterward (Fig. 4C). There were no marked changes in the previously mentioned parameters in the EP-treated group in comparison to those without treatment.

Fig. 4
Fig. 4:
IL-2R expression. Immunofluorescence technique was used to measure the expression of IL-2Rα, ELISA for sIL-2R, and RT-PCR analysis for mRNA expression. A, IL-2Rα expression on splenic T cells was significantly lower the in burn group than that in the sham burn group on PBDs 1 and 3 (P < 0.05). B, Plasma levels of sIL-2R were significantly elevated after thermal injury during the 7-day observation period. C, IL-2R mRNA expression of T cells was significantly lower only on PBD 1. There were no marked changes in the previously discussed parameters in the EP-treated group as compared with those without treatment. Statistical significance: *P < 0.05, §P < 0.01 as burn group versus sham burn group.

Polarization of T-lymphocyte cells

Because TH1 cells produce IFN-γ and TH2 cells produce IL-4, we detected the two cytokines produced by T cells by using ELISA to identify polarization of naive T cells. It was shown in Figure 5A IFN-γ levels of the burn group in T-cell supernatants were significantly decreased on PBDs 1 to 7 compared with the sham group (P < 0.05), and in contrast, the IL-4 levels significantly increased on PBDs 1 and 3 (P < 0.01), suggesting that naive T cells might change to TH2 in animals secondary to thermal injury (Fig. 5B). Ethyl pyruvate administration markedly inhibited the decrease in IFN-γ (P < 0.05) and increase in IL-4 (P < 0.01) in supernatants of culture of T cells after injury, implying that treatment with EP can influence the polarization of splenic T cells in animals after thermal injury by inducing naive T cells to shift to TH1 cells.

Fig. 5
Fig. 5:
Polarization of T cells. Enzyme-linked immunosorbent assay was used to determine IFN-γ (A) and IL-4 (B) levels in culture supernatants of T cells. After burn injury, IL-4 levels produced by T-cell response to Con A were markedly increased, whereas IFN-γ levels were markedly lowered. Ethyl pyruvate treatment can significantly revert lowered levels of IFN-γ and elevated levels of IL-4 after thermal injury. Statistical significance: *P < 0.05, ‡P < 0.01, as burn group versus sham burn group; †P < 0.05, §P < 0.01 as EP group versus burn group.

Activation of NFAT in splenic T lymphocytes

To investigate the potential signal pathway with regard to T-cell activation, the activity of NFAT of splenic T cells was detected after burns. As shown in Figure 6, NFAT activity of splenic T cells was markedly down-regulated (P < 0.05) at the early stage (1-3 days) after burns, then gradually restored after the recovery of rat from burn insults. Treatment with EP completely restored the NFAT activity of splenic T cells on PBDs 1 and 3 (P < 0.05).

Fig. 6
Fig. 6:
Electrophoretic mobility shift assay was used to determine NFAT activity. The NFAT activity of splenic T cells was significantly down-regulated at the early stage (1 - 3 days) after burns. Treatment with EP can completely restore the NFAT activity after burn injury. Statistical significance: *P < 0.05 as burn group versus sham burn group; †P < 0.05 as EP group versus burn group.

Correlation analysis

As shown in Table 2, plasma HMGB1 levels were significantly negatively correlated to splenic T-lymphocyte proliferative activity, IL-2 production, and IL-2R expression on T cells in rats after major burns (P < 0.05-0.01).

TABLE 2
TABLE 2:
Correlation analysis between plasma HMGB1 levels and parameters reflecting T-cell mediated immunity in rats after burns

DISCUSSION

Dysfunction of specific immunity is a consequential phenomenon of thermal injury, and it generally is believed that this is attributable to depression of lymphocyte proliferation associated with the loss of function of the TH1 lymphocyte phenotype (19-21). We think that further investigation into the mechanisms underlying the host suppression might hopefully contribute to formulate better therapeutic modalities against injury. Recently, HMGB1 has been shown to act as a late mediator of endotoxic shock, and it exerts a variety of proinflammatory extracellular activities (10, 12). When secreted from LPS-stimulated dendritic cells, HMGB1 contributes to T-cell proliferation, and anti-HMGB1 antibodies are able to prevent this effect and also reduce the number of IFN-γ-secreting T cells in allogeneic mature dendritic cell-T-cell cultures (22). Therefore, the current experiment was planned to examine the hypothesis that released HMGB1 might be a potential factor for inducing suppression of T lymphocytes after severe injury in vivo. We found that the initial significant elevation of splenic or serum HMGB1 level occurred at 24 h after a thermal insult, and it lasted for 3 days in the spleen or 5 days in serum. Meanwhile, the proliferative response of splenic CD3+ T cells was depressed during PBDs 1 to 7, and plasma HMGB1 levels were negatively correlated with spleen T-lymphocyte proliferative activity. Thus, these findings support the view that HMGB1 release might be associated with the suppression of T lymphocytes after severe thermal injury.

To verify our hypothesis, we used the HMGB1 release inhibitor EP (18) to treat burned rats and then to observe the proliferative response of splenic CD3+ T cells to mitogenic stimulation. Ethyl pyruvate, a stable lipophilic pyruvate derivative identified recently by Fink (23) and Sims et al. (24), is an experimental therapeutic that effectively protects animals from I/R-induced tissue injury. Ulloa et al. (18) found that delayed administration of EP with septic mice decreased circulating levels of HMGB1. The molecular basis of EP action is by interfering with signal transduction through the p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB signal transduction pathways (18). Macrophage activation by endotoxin, cytokines, and products of cell injury leads to the nuclear translocation of NF-κB, a transcription factor that enhances the transcription of HMGB1 and other products of the activated macrophage (10) and phosphorylation of p38 MAPK, which stabilizes mRNA of proinflammatory cytokine and increases translation efficiency. Ethyl pyruvate inhibition of NF-κB and p38 MAPK signaling can effectively prevent release of early (TNF-α) and late (HMGB1) inflammatory mediators.

It was shown that an increase in plasma HMGB1 levels from PBD 1 to PBD 5 was markedly inhibited, and the proliferative response of splenic T cells was restored during PBDs 1 to 7 after delayed EP treatment. Therefore, our results suggest that the HMGB1 released might be associated with the development of immune impairment of T lymphocytes after severe injury. It has been demonstrated that EP can also ameliorate intestinal epithelial barrier dysfunction and improve gut function. High-mobility group box 1 protein increased the permeability of Caco-2 enterocytic monolayers and impaired intestinal barrier function (13). From our results, treatment with EP can reduce the level of HMGB1 and might protect against gut impairment, in turn contributing to the prevention of systemic inflammation and immunologic disorder associated with intestinal bacterial/endotoxin translocation after thermal injury (13, 24). In addition, there is accumulating evidence that HMGB1 is a ligand for the receptor for advanced glycation end products (RAGE) (25), and RAGE is expressed on T cells (22, 26). It is likely that the immunomodulatory effect of HMGB1 on T cells might be attributable to its binding with RAGE.

Naive CD3+ T cells are able to develop into two types of effector cells: TH1 cells produce cytokines, including IL-2, IFN-γ, TNF-β, and lymphotoxin, whereas TH2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 (27). Because TH1 cells are principal regulators of cell-mediated immune responses, depressed function of such lymphocyte subset might be deleterious to the ability to resist infection. Indeed, in animal models of injury, therapeutic regimens designed to increase TH1 function have been associated with improved survival after a septic challenge (28, 29). In the present study, splenic naive T cell was found to develop into TH2 in animals subjected to thermal injury and EP treatment, which inhibited the release of HMGB1, can influence the polarization of splenic T cells, and facilitating naive T cells to shift to TH1. Therefore, we postulate that the excessive release of HMGB1 during acute insults might lead to the dysfunction of immune response of splenic T lymphocytes.

Because IL-2 is a major cytokine secreted by activated T lymphocyte, and it is essential to activate T lymphocyte proliferation through autocrine, paracrine, and endocrine patterns, we determined levels of IL-2 in spleen and blood. A significant depression in IL-2 production and mRNA expression was observed in T cells and in plasma from thermal-injured rats compared with those from sham-injured rats. The suppression can be reverted by treatment with EP to inhibit HMGB1 release. In addition, plasma HMGB1 levels were negatively correlated with IL-2 levels in ex vivo culture supernatants. These results indicate that HMGB1 might be associated with inhibition of IL-2 secretion by T cells in the setting of severe burns and further modulate activation of T lymphocytes. Consistently, in previous studies, the levels of IL-2 were found to be low after thermal injury in a murine model, which closely related to mortality (30). In fact, it has been demonstrated that IL-2 is a key regulator of the immune response that is produced by T cells upon antigenic or mitogenic stimulation. By inhibiting IL-2 production, a significant and clinically applicable immunosuppression state can be obtained (31).

IL-2 sensitivity is controlled by IL-2Rα, and the latter is a critical determinant of T-cell proliferation (32). The sIL-2R is a smaller (45 kd) polypeptide than the membrane-bound IL-2R (55 kd) and has a potential role in the modulation of immune responses by inhibiting IL-2 (33). In the present study, expression levels of IL-2Rα on the surface of splenic T cells were significantly decreased in rats after major burns, whereas, the plasma levels of sIL-2R were significantly higher during the observation period, implying that immune suppression of T cells was a sequel of burns. Unexpectedly, EP administration exerted no remarkable effect on both IL-2Rα expression and sIL-2R levels. It was reported that the activity of IL-2Rα specifically depends on the presence of IL-2 (34). IL-2 and IL-2Rα are implicated in an autoregulatory loop that controls cell surface expression of IL-2Rα in T-cell lines (35). Thus, the reason of inconsistency between the changes in IL-2 and IL-2Rα after the use of EP treatment needs to be further explored.

IL-2 is regulated chiefly at the level of transcription of its gene. Activation of the IL-2 promoter during T-cell activation depends on the activation of several obligatory transcription factors, including NFAT, NF-κB, Oct-1, AP-1, Ets, and a CD28 responsive factor (36). These factors collaborate to form a multifactor complex that binds the enhancer region in a stable manner and initiates transcription. Inhibition of one of these factors is sufficient to markedly depress IL-2 production (31). Nuclear factor of activated T cell is the first characterized transcription factor that binds to the IL-2 promoter (37). In the current experiment, NFAT activity was found to be significantly lower in splenic T cells from burn-injured rats compared with that from sham-injured rats on PBDs 1 and 3. Treatment with EP completely prevented the down-regulation of NFAT activity. Our findings are in agreement with previous observation showing that suppression in T-cell IL-2 production from burn-injured rats was accompanied by a decrease in the activation of NFAT and AP-1 (19). Thus, the data presented here suggest that HMGB1 released in the setting of burn injury might be related to the suppression in NFAT complex binding to IL-2 DNA elements, which can potentially suppress IL-2 promoter activity and thereby IL-2 production.

In summary, based on our in vivo study, it is demonstrated for the first time that HMGB1 released after major burns might be associated with the development of immune suppression of splenic T lymphocytes in rats. These findings support the new function for HMGB1 as a putative immunosuppressor cytokine that is complementary to its role in the pathogenesis of septic response in burn injury. Nevertheless, further studies looking at the precise mechanism of its action are merited.

REFERENCES

1. Traber DL: Nitric oxide synthase and tissue injury. Shock 14:243-244, 2000.
2. Ayala A, Chung CS, Grutkoski PS, Song GY: Mechanisms of immune resolution. Crit Care Med 31:S558-S571, 2003.
3. Kerksiek KM, Pamer EG: T cell responses to bacterial infection. Curr Opin Immunol 11:400-405, 1999.
4. Sir O, Fazal N, Choudhry MA, Goris RJ, Gamelli RL, Sayeed MM: Role of neutrophils in burn-induced microvascular injury in the intestine. Shock 14:113-117, 2000.
5. Kelly JL, O'Suilleabhain CB, Soberg CC, Mannick JA, Lederer JA: Severe injury triggers antigen-specific T-helper cell dysfunction. Shock 12:39-45, 1999.
6. Smith JW, Gamelli RL, Jones SB, Shankar R: Immunologic responses to critical injury and sepsis. J Intensive Care Med 21:160-172, 2006.
7. Gallucci S, Matzinger P: Danger signals: SOS to the immune system. Curr Opin Immunol 13:114-119, 2001.
8. Oberbeck R, Dahlweid M, Koch R, van Griensven M, Emmendorfer A, Tscherne H, Pape HC: Dehydroepiandrosterone decreases mortality rate and improves cellular immune function during polymicrobial sepsis. Crit Care Med 29:380-384, 2001.
9. Efron P, Moldawer LL: Sepsis and the dendritic cell. Shock 20:386-401, 2003.
10. Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, Frazier A, Yang H, Ivanova S, Borovikova L, et al.: HMG-1 as a late mediator of endotoxin lethality in mice. Science 285:248-251, 1999.
11. Sunden-Cullberg J, Norrby-Teglund A, Rouhiainen A, Rauvala H, Herman G, Tracey KJ, Lee ML, Andersson J, Tokics L, Treutiger CJ: Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 33:564-573, 2005.
12. Yang H, Tracey KJ: High mobility group box 1 (HMGB1). Crit Care Med 33:S472-S474, 2005.
13. Sappington PL, Yang R, Yang H, Tracey KJ, Delude RL, Fink MP: HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology 123:790-802, 2002.
14. Zhang LT, Yao YM, Lu JQ, Yan XJ, Yu Y, Sheng ZY: Sodium butyrate prevents lethality of severe sepsis in rats. Shock 27:672-677, 2007.
15. Scaffidi P, Misteli T, Bianchi ME: Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191-195, 2002.
16. Tsung A, Sahai R, Tanaka H, Nakao A, Fink MP, Lotze MT, Yang H, Li J, Tracey KJ, Geller DA, et al.: The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med 201:1135-1143, 2005.
17. Zamora R, Grishin A, Wong C, Boyle P, Wang J, Hackam D, Upperman JS, Tracey KJ, Ford HR: High-mobility group box 1 protein is an inflammatory mediator in necrotizing enterocolitis: protective effect of the macrophage deactivator semapimod. Am J Physiol Gastrointest Liver Physiol 289:G643-G652, 2005.
18. Ulloa L, Ochani M, Yang H, Tanovic M, Halperin D, Yang R, Czura CJ, Fink MP, Tracey KJ: Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci U S A 99:12351-12356, 2002.
19. Choudhry MA, Mao H, Haque F, Khan M, Fazal N, Sayeed MM: Role of NFAT and AP-1 in PGE2-mediated T cell suppression in burn injury. Shock 18:212-216, 2002.
20. Patenaude J, D'Elia M, Hamelin C, Garrel D, Bernier J: Burn injury induces a change in T cell homeostasis affecting preferentially CD4+ T cells. J Leukoc Biol 77:141-150, 2005.
21. Phan HH, Cho K, Nelson HA, Shin S, Jeong J, Greenhalgh DG: Downregulation of NF-kappaB activity associated with alteration in proliferative response in the spleen after burn injury. Shock 23:73-79, 2005.
22. Dumitriu IE, Baruah P, Valentinis B, Voll RE, Herrmann M, Nawroth PP, Arnold B, Bianchi ME, Manfredi AA, Rovere-Querini P: Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products. J Immunol 174:7506-7515, 2005.
23. Fink MP: Reactive oxygen species as mediators of organ dysfunction caused by sepsis, acute respiratory distress syndrome, or hemorrhagic shock: potential benefits of resuscitation with Ringer's ethyl pyruvate solution. Curr Opin Clin Nutr Metab Care 5:167-174, 2002.
24. Sims CA, Wattanasirichaigoon S, Menconi MJ, Ajami AM, Fink MP: Ringer's ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats. Crit Care Med 29:1513-1518, 2001.
25. Kokkola R, Andersson A, Mullins G, Ostberg T, Treutiger CJ, Arnold B, Nawroth P, Andersson U, Harris RA, Harris HE: RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages. Scand J Immunol 61:1-9, 2005.
26. Ulloa L, Messmer D: High-mobility group box 1 (HMGB1) protein: friend and foe. Cytokine Growth Factor Rev 17:189-201, 2006.
27. Abbas AK, Murphy KM, Sher A: Functional diversity of helper T lymphocytes. Nature 383:787-793, 1996.
28. Goebel A, Kavanagh E, Lyons A, Saporoschetz IB, Soberg C, Lederer JA, Mannick JA, Rodrick ML: Injury induces deficient interleukin-12 production, but interleukin-12 therapy after injury restores resistance to infection. Ann Surg 231:253-261, 2000.
29. Lyons A, Kelly JL, Rodrick ML, Mannick JA, Lederer JA: Major injury induces increased production of interleukin-10 by cells of the immune system with a negative impact on resistance to infection. Ann Surg 226:450-458, 1997; discussion 458-460.
30. O'Riordain DS, Mendez MV, O'Riordain MG, Molloy RG, Holzheimer RG, Collins K, Saporoschetz I, Mannick JA, Rodrick ML: Molecular mechanisms of decreased interleukin-2 production after thermal injury. Surgery 114:407-414, 1993; discussion 414-415.
31. Abboushi N, El-Hed A, El-Assaad W, Kozhaya L, El-Sabban ME, Bazarbachi A, Badreddine R, Bielawska A, Usta J, Dbaibo GS: Ceramide inhibits IL-2 production by preventing protein kinase C-dependent NF-kappaB activation: possible role in protein kinase Ctheta regulation. J Immunol 173:3193-3200, 2004.
32. Cantrell DA, Smith KA: Transient expression of interleukin 2 receptors. Consequences for T cell growth. J Exp Med 158:1895-1911, 1983.
33. Teodorczyk-Injeyan JA, Sparkes BG, Mills GB, Falk RE, Peters WJ: Increase of serum interleukin 2 receptor level in thermally injured patients. Clin Immunol Immunopathol 51:205-215, 1989.
34. Jankovic DL, Gibert M, Baran D, Ohara J, Paul WE, Theze J: Activation by IL-2, but not IL-4, up-regulates the expression of the p55 subunit of the IL-2 receptor on IL-2- and IL-4-dependent T cell lines. J Immunol 142:3113-3120, 1989.
35. Demaison C, Chastagner P, Moreau JL, Theze J: Ligand-induced autoregulation of IL-2 receptor alpha chain expression in murine T cell lines. Int Immunol 8:1521-1528, 1996.
36. Rothenberg EV, Ward SB: A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc Natl Acad Sci U S A 93:9358-9365, 1996.
37. Kaminuma O, Deckert M, Elly C, Liu YC, Altman A: Vav-Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter. Mol Cell Biol 21:3126-3136, 2001.
Keywords:

High-mobility group box 1 protein; burns; IL-2; IL-2 receptors; splenic T cell

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