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Originally published online as doi:10.1189/jlb.0703314 on November 12, 2004

Published online before print November 12, 2004
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(Journal of Leukocyte Biology. 2005;77:141-150.)
© 2005 by Society for Leukocyte Biology

Burn injury induces a change in T cell homeostasis affecting preferentially CD4+ T cells

Julie Patenaude*, Michele D’Elia*, Claudine Hamelin*, Dominique Garrel{dagger} and Jacques Bernier*,1

* INRS-Institut Armand-Frappier, Pointe-Claire, Quebec, Canada; and
{dagger} Centre hospitalier de l’Université de Montréal (CHUM)-Hôtel-Dieu, Centre des Grands Brûlés, Quebec, Canada

1 Correspondence: INRS-Institut Armand-Frappier, 245 Bld. Hymus, Pointe-Claire, Québec, Canada H9R 1G6. E-mail: jacques.bernier{at}inrs-iaf.uquebec.ca


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ABSTRACT
 
Burn injuries are known to be associated with altered immune functions, resulting in decreased resistance to subsequent infection. In the present study, we determined the in vivo changes in T cell homeostasis following burn injury. Two groups of mice were used: a sham-burn group receiving buprenorphine as an analgesic and a burn group receiving buprenorphine and subjected to burn injury on 20% of the total body surface area. Results showed an important decrease in splenocytes following burn injury. This decrease persisted for 5 days and was followed, at day 10, by a 63% increase in number of cells. In vivo cell proliferation, as determined by the incorporation of 5-bromo-2'-dexoxyuridine, showed a significant increase of cycling splenocytes between days 2 and 10 after burn injury. The percentage of CD4+ and CD8+ T cells in the spleen was altered for 10 days after thermal injury. Analysis of naive (CD62Lhigh CD44low) and effector/memory (CD62Llow CD44high) T cells showed a percent decrease, independent of the expression of CD4 or CD8 molecules. However, early activation markers, such as CD69+, were expressed only on CD4+ T cells after a number of days following injury. Even with an activated phenotype, 10 days post-burn injury, CD4+ naive T cells significantly increased spontaneous apoptosis, detected by using a fluorescent DNA-binding agent 7-amino-actinomycin D. CD8+ T lymphocytes did not express early activation markers and were more resistant to apoptosis. Using purified T cells, we have shown unresponsiveness at day 10. Overall, these results demonstrate that mechanisms of T cell homeostasis were perturbed following burn injury. However, after 10 days, this perturbation persisted only in CD4+ T cells.

Key Words: apoptosis • anergy • macrophages • trauma • mice


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INTRODUCTION
 
Severe injuries such as burns induce a delayed state of immune suppression that predisposes burn and trauma patients to infection and sepsis caused by opportunistic pathogens [1 ]. The extent of immunosuppression appears to be greater after burn injury than following other forms of trauma [2 ]. Effector mechanisms of unspecific and specific host defenses are impaired following thermal trauma (phagocytosis, chemotaxis, lymphocyte proliferation, antibody production) [3 , 4 ]. The hyperproduction of inflammatory cytokines and overstimulation of immune cells during inflammatory response are potential mechanisms involved in immunosuppression [5 , 6 ]. Although injury is often thought of as an inflammatory host response and is thus driven by cells and mediators of the innate immune system, there is considerable experimental and clinical evidence indicating that injury can profoundly suppress adaptive host immune function [7 ]. The immunologic response to burn injury involves a systemic inflammatory response syndrome (SIRS), which is dependent on the extent of burns, followed by a compensatory anti-inflammatory response syndrome, which is linked to the magnitude of SIRS [8 ]. In both syndromes, the interaction between the innate and the adaptive immune systems has been demonstrated to be important [8 ].

The immunological deficit observed in burned patients has been attributed in part to T cell dysfunction. It was demonstrated that T cell unresponsiveness to infection is the consequence of a reduction of T lymphocytes, suppression of cytokine synthesis, and an incorrect activation or an overstimulation of those cells [2 , 4 , 9 ]. Several studies have indicated that production of interleukin (IL)-2 by mitogen-stimulated T cells and the induction of functional membrane IL-2 receptors (IL-2Rs) are profoundly suppressed in burn patients [10 11 12 ]. The dysfunction was associated with T cell signaling defects, including an impairment of Ca2+ signaling and/or defective tyrosine kinase activity [13 ]. Moreover, it has been established that injury dramatically alters the transcriptional activity of activated protein 1 (AP-1) and nuclear factor-{kappa}B in T cells, which are involved in the regulation of many genes, including IL-2 production and T cell activation [14 ]. This abnormal T cell activation can drive activated T cells to an unresponsiveness state (anergy) or death by apoptosis [9 , 15 16 17 18 19 ]. Induction of Fas-mediated responses, release of stress mediators such as tumor necrosis factor {alpha} (TNF-{alpha}), and glucocorticoids may be involved in T cell apoptosis [15 16 17 ]. Particularly, the Fas signaling pathway seems to be involved in burn injury-induced, lethal T cell reactivity against bacterial superantigens [20 ]. The continuous apoptotic cell death, resulting in a residual, anergic T cell population, occurring normally in the control of immune response, can explain the immunosuppression present after burn injury [21 ].

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 [22 23 24 25 26 ]. It is interesting that the significant shift of cytokines toward the Th2 direction has been demonstrated to occur in antigen-driven CD4+T cells and also in CD8+ T cells [27 ]. 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 of the resistance to infectious pathogens in host survival [25 ]. It was also observed that macrophage dysfunctions after burn injury can perturb the T cell functions involved in adaptive immune response [28 ]. The macrophage’s hyperproduction of arachidonic acid metabolites such as prostaglandin E2(PGE2) may suppress certain T lymphocyte functions, such as proliferation and IL-2 production [29 , 30 ]. PGE2 alters macrophage effector functions and depresses the antigen-presenting capacity of monocytes/macrophages following trauma, which may contribute to depressed T cell responsiveness post-trauma. It was also demonstrated in studies by Schwacha and Somers [31 ] that the suppression of splenic T cell proliferation was inducible nitric oxide synthase-dependent and mediated by macrophages in the mixed-cell splenocyte population. Overall, the failure of T lymphocytes to regulate the immune response is considered to be an important immunological consequence of major immunosuppression after thermal injury [32 ].

It was also documented that severe burn injury induces a direct perturbation of T cells. For example, it was established that burn injury primed a naive T cell response, in contrast to a suppressed response from antigen-exposed T cells [33 , 34 ]. Using a T cell receptor (TCR) transgenic CD4+T cell-adoptive transfer approach, it was shown that burn injury did not limit antigen-driven expansion of CD4+ T cells but rather enhanced Th1 reactivity at 1 day following injury, independent of the presence of a specific antigen in host [34 ]. These studies suggest that burn injury can, by itself, affect the T cell populations and homeostasis. Mature T cells are subject to regulation by homeostatic mechanisms that maintain the overall size of the T cell pool at a constant level [35 , 36 ]. As burn injury leads to severe T cell deficiency, spontaneous expansion of the remaining T cells, which can eventually restore the T cell pool to near normal size, can contribute to recovery of normal responses from T cells. At this time, no studies address the question of homeostatic mechanisms involving not only the control of total T cell numbers but also the different susceptibility of naive or memory T cells to burn injury-induced immunosuppression.

The present study addresses T cell homeostasis after burn injury. Using a mouse model of burn injury, splenocyte populations were characterized. T cells expressing CD4+ and CD8+ were subdivided according to their expression of CD62L and CD44 on naive or effector/memory cells, respectively. Naive T cells, which are T cells that have never previously encountered antigen, express high levels of L-selectin CD62LhighCD44low, and the effector/memory T cells down-regulate CD62L expression associated with an increase of CD44. Moreover, we have determined the expression of an early activation marker, namely CD69, and the spontaneous apoptosis of cells at different times following thermal injury. Overall, our results show the loss of T cell homeostasis after burn injury. Naive and effector/memory CD4+T cells are more affected on day 10 following injury.


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MATERIALS AND METHODS
 
Animals
All experiments were performed on 8-week-old male C57Bl/6 mice (Charles River Laboratories, St-Constant, Quebec, Canada). Prior to the initiation of any procedure, the mice were acclimatized for 1 week to our central animal facility with strictly controlled temperature, relative humidity, and a 12-h light/dark cycle. Each cage contained five mice, which were fed standard chow (Richmond standard diet, Lab Diet, Richmond, IN) and water ad libitum. The Institutional Animal Care Committee reviewed and approved all procedures performed in accordance with the Canadian Council on Animal Care guidelines.

Alzet micro-osmotic pumps
Alzet micro-osmotic pumps filled with buprenorphine were used to control pain after burn injury as described previously [37 ]. Briefly, 24 h before the start of experiments, the mice were anesthetized with Isoflurane gas (CDMV, St-Hyacinthe, Quebec, Canada) and shaved, and an incision was made in the upper sternal skin to subcutaneously insert the micro-osmotic pump (Durcet Corp., Cuprentino, Canada). The pump delivered 0.1 ml/kg buprenorphine (Reckitt and Coleman Pharmaceuticals, Richmond, VA) per 12 h, which represents the equivalent of 2.0 µg/12 h buprenorphine for a 20-g mouse. In our previous studies, we have shown that buprenorphine can be used safely and had no detrimental effects on the immune response [36 37 38 ].

Experimental thermal injury
For each day of experiments, animals were randomized into two groups of five mice as follows: buprenorphine-filled pumps with sham injury or buprenorphine-filled pumps with burn injury. The burn-injury model used to induce full-thickness burn was described previously [38 , 39 ]. Briefly, after anesthesia, the animals were placed in a custom-insulated mold exposing 20% of their total body surface area on the dorsal aspect before being immersed for 7 s in room temperature water (22°C) for sham burn or in 90°C water to produce a full-thickness burn. All animals received a 2-ml intraperiteonal (i.p.) injection of 0.9% saline for fluid resuscitation, and a topical antibacterial agent (silver sulfadiazon cream, Smith and Nephew, Lachine, Quebec, Canada) was applied on the burned areas. All mice survived the surgery and the thermal injury. Burnt mice moved, ate, and drank normally throughout the post-burn observation period.

Preparation of spleen cell suspensions
One, 5, and 10 days after the thermal injury, animals were anesthetized with Isoflurane gas (CDMV) and killed by cardiac puncture. Spleens of mice in each group were individually prepared as single-cell suspensions in 3 ml RPMI 1640 (BioMedia, Drummundville, Quebec, Canada), supplemented with 10% heat-inactivated fetal bovine serum (BioMedia), 50 µM 2-mercapthethanol (Sigma-Aldrich, Oakville, Ontario, Canada), and antibiotics [100 U/ml penicillin and 100 µg/ml streptomycin (Biosource, Montreal, Quebec, Canada)]. Splenocyte suspensions were obtained by tearing apart the splenic matrix. The red blood cells were removed by hypertonic lysis (Gey’s solution with NH4Cl). Cells were washed, and cell counting was performed by trypan blue exclusion on a hemocytometer count to determine total cell numbers from organs. Viability of cells was consistently greater than 90%. The cells were then adjusted to desired concentrations in complete RPMI for further studies.

In vivo cell proliferation
In vivo cell proliferation was determined by 5-bromo-2'-dexoxyuridine (BrdU) incorporation as described by Vasseur et al. [40 ]. BrdU is a thymidine analog and is specifically incorporated into DNA during DNA synthesis. Briefly, mice were injected i.p. twice with 100 µl phosphate-buffered saline (PBS) containing 1 mg/ml BrdU (Sigma-Aldrich) 15 h and 1 h before the sacrifice of mice. Thereafter, the spleens were collected for determination of BrdU incorporation. Splenocytes (2.0x106 cells/ml) were fixed overnight in a 70% ethanol solution at 4°C. Cells (105 cells/100 µl) were washed with Hank’s balanced salt solution (HBSS; Sigma-Aldrich) and 1% bovine serum albumin (BSA) and were incubated for 20 min with 3 N HCl solution at room temperature for cellular DNA denaturation. After one wash, the cells were incubated for an additional 5 min in a 0.1-M sodium borate solution at room temperature. The cells were washed twice and were stained with anti-BrdU monoclonal antibody (mAb) fluorescen isothiocyanate (FITC) conjugate for 60 min at room temperature (Roche Molecular Biochemicals, Indianapolis, IN). Cells were washed twice with HBSS and analyzed on a FACScan® (Becton Dickinson, Mississauga, Ontario, Canada). WinMDI software was used for the analysis.

Flow cytometry for splenocyte phenotyping
Cells were analyzed by three-color flow cytometry with a FACScan® (Becton Dickinson) apparatus. Splenocytes (105 cells/100 µl) were incubated for 30 min on ice in the presence of fluorochrome-conjugated anti-mouse mAb: rat anti-CD4 FITC-labeled molecule (eBioscience, San Diego, CA) or rat anti-CD8a FITC-labeled molecule (eBioscience) with anti-CD69 phycoerythrin (PE)-Cy5-conjugated molecule (eBioscience) for activation detection, biotinylated anti-CD62L L-selectine molecule (Cedarlane Laboratories, Hornby, Ontario, Canada) for naive T cells detection, or biotinylated anti-CD44 molecule (eBioscience) for effector/memory T cells detection. Splenocytes were also stained for macrophage markers using a rat anti-F4/80 FITC-labeled molecule (Serotec, Hornby, Ontario, Canada). The cells were then washed twice with cold HBSS, 0.1% BSA, and 0.01% sodium azide (Sigma-Aldrich) and analyzed on a FACScan® (Becton Dickinson). WinMDI software was used for the analysis. Absolute numbers of cells by organ were determined as follows: percent of cells expressing specific marker x total number of cells in organ.

Macrophage and T cell purifications
Splenocytes were depleted of macrophages by two successive series of glass adherence to a fetal calf serum (FCS)-treated petri dish for 1.5 h at 37°C in a humidified atmosphere with 5% CO2. Nonadherent cells were removed from petri dishes with warm PBS, 2% FCS; the resulting adherent cells were scraped with a rubber policeman and represent the macrophage-purified population. Greater than 90% of the adherent cells is positive for F4/80 staining. The nonadherent population (>90% lymphocytes) was essentially free of monocytes (with <1% contamination), as determined by flow cytometry analysis. The lymphocyte population was washed twice with PBS, 2% FCS, adjusted to desired concentrations, and applied to a mouse T cell recovery column kit (Cedarlane Laboratories), according to the manufacturer’s procedure. Mouse T cell recovery columns efficiently removed more than 95% of B cells from the mouse lymphocyte populations. T cell populations were then used for proliferative response assays and flow cytometry analysis.

Flow cytometry apoptosis assessment
T cells were washed before proceeding to apoptosis detection and quantification with three-color flow cytometry by staining with 7-amino-actinomycin D (7AAD; Sigma-Aldrich) and specific mAb. Briefly, T cells (2x105 cells/100 µl) were incubated for 30 min on ice in the presence of fluorochrome-conjugated anti-mouse mAb: rat anti-CD4 FITC-labeled molecule (eBioscience) or rat anti-CD8a FITC-labeled molecule (eBioscience) and biotinylated anti-CD62L L-selectine (Cedarlane) or biotinylated anti-CD44 (eBioscience). Cells were washed and further incubated with streptavidin-PE conjugate (eBioscience) for 30 min at 4°C. As described previously, cells were fixed at 4°C in a 70% ethanol solution overnight, washed three times with PBS, 2% FCS, and incubated in phosphate citrate buffer, pH 7.8, for 30 min at room temperature. Digitonin solution (10 µl of 0.1%) was added and incubated for an additional 30 min at room temperature, followed by the addition of 3 µl RNase solution (1 mg/ml) for a 30-min incubation at 37°C. PBS, 2% FCS washed cells were incubated with 2 µg/ml 7AAD for 30 min at room temperature and analyzed with FACScan® for apoptosis detection in cell cycle (sub-G0).

Lymphocyte-proliferative response assay with macrophages
In a 96-well, flat-bottom, tissue-culture plate (Sarstedt, Montreal, Quebec, Canada), coated with an anti-TCR ß-chain mAb (H57-597; 0.1 µg/ml) [37 , 38 ], 2.5 x 105 isolated T lymphocytes and 2.0 x 104 macrophages from the spleen of sham or burnt mice were added (in triplicate). To determine the potential of IL-2 to restore T cell proliferation, 12.5 U/mL recombinant IL-2 (rIL-2; Endogen Pierce Biotechnologies, Brockville, Ontario, Canada) was added to some wells before incubating the plates at 37°C for 69 h in a humidified atmosphere with 5% CO2. The cultures were pulsed for 3 h with 100 µg/well methylthiazole tetrazolium (MTT; Sigma-Aldrich). Plates were washed once, and 200 µl dimethyl sulfoxide (Fisher Scientific, St-Laurent, Quebec, Canada) per well was added. Absorbance of reduced dye was measured at a wavelength of 570 nm with background subtraction at 630–690 nm as a quantification of cell proliferation.

Statistical analysis
All data are expressed as mean ± SEM. Data were analyzed by a Mann-Whitney U-test and by ANOVA on the Statistica software program. Differences between groups were considered statistically significant when the probability was less than 5%. Each experiment was performed at least in duplicate for statistical purposes.


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RESULTS
 
In vivo, burn injury induces splenocyte depletion associated with an increase in their proliferation
It is well documented that burn injury induces immune cell depletion and impairs function of residual cells [41 ]. In our model, which included burn injury and pain management, we determined the number of splenocytes in this state. As shown in Table 1 , burn injury induced a significant 50% decrease in splenic cells on days 1 and 5 following injury. However, by day 10 post-trauma, the number of cells in this organ was increased significantly by ~40% as compared with the sham group.


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  		Table 1. Burn Injury Induces In vivo Splenocyte Depletion Followed by an Increase of Splenocyte Proliferation

The rapid decrease in spleen cell number after burn injury suggested an absence of splenocyte activation and proliferation. To address this question, we monitored cell proliferation by in vivo BrdU incorporation following burn injury. As shown in Table 1 , BrdU injection at the time of burn injury confirmed the absence of splenocyte proliferation after 24 h. However, BrdU injection at days 4 and 9 showed that splenocyte proliferation was statistically increased for up to 10 days following burn injury. We have also observed an increase of cycling cells in the sham group (day 1 vs. days 5 and 10, P<0.05). When compared with control mice receiving saline by micro-osmotic pumps, we also find the increase of BrdU incorporation, suggesting that buprenorphine mediated the increase of cycling cells in the sham group (data not shown). Overall, burn injury caused rapid splenocyte depletion in the first 24 h followed by an increase in cell proliferation.

Burn injury affects the proportion and absolute numbers of T and B lymphocytes and macrophages differently
We have analyzed whether burn injury causes a change in the percentage or absolute number of T (CD3+) cells for CD4+ and CD8+ T cells or B cells (CD19+) and macrophages (F4/80+) in spleen. As illustrated in Table 2 , no significant changes in percent of CD3+T cells were observed for the first 5 days after thermal injury. However, when compared with the sham group, the percent of splenic CD4+ and CD8+ T cells in the burn group was slightly but significantly increased at day 1 post-injury (P<0.05). At day 10 following burn injury, a decrease in percent of B and T cells for CD4+ and CD8+ T cells was noted, with a concomitant, significant increase by twofold in percent of macrophages (P<0.05). When the change in spleen cell populations was evaluated in terms of absolute numbers, we noted a significant decrease in all populations of ~40% on days 1 and 5 (P<0.05). At day 10, no changes in absolute number of splenic T (CD4+ and CD8+) and B cells were noted between the groups, whereas the number of macrophages were significantly higher in the spleen of burnt mice (P<0.01). Analysis of the ratio between CD4+and CD8+ T cells between the sham or burned group confirms that none of these populations are more affected by the injury (data not shown). Overall, our analysis of CD4+and CD8+T cell populations showed that the depletion observed in first days after injury was not restricted to a specific subpopulation of T lymphocytes in the spleen.


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  		Table 2. Comparative Effect of Burn Injury on Percent and Total Cell Numbers of Splenocytes

Changes in CD4+ and CD8+ T cells can be associated with their own naive cells
Decreases in the number of T cells or elimination of naive or effector/memory cells can be related to increased susceptibility to opportunistic infections, as shown in several pathologies of the immune system [42 ]. Using multiparametric flow cytometry analysis, we identified naive cells (CD4+/CD8+–CD62LhighCD44low) and effector/memory cells (CD4+/CD8+–CD62Llow CD44high; Table 3 ). One day after burn injury, increases in percent of CD4+–CD62Lhigh–CD44low and CD8+–CD62Lhigh–CD44low naive cells in the spleen were noted (P<0.05, Table 3 ). The initial increase in naive CD4+ T cells was followed by a 50% decrease at days 5 and 10 post-burn (P<0.05). Concerning CD8+ T cells, the decrease was only noted at day 5. The decrease of CD62LhighCD44low cannot be related to phenotypic conversion in CD62LlowCD44high (effector/memory) cells, as no significant change in this population was observed at 5 days following burn injury. Also, a decrease of CD8+–CD62Llow CD44high was observed at day 10 (Table 3) .


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  		Table 3. Change in Naive and Effector/Memory T Cell Distribution in the Splenocytes Following Burn Injury

Differential regulation of naive versus effector/memory T cell activation after burn injury
To determine if the increase in naive T cells was associated with their own activation, we determined the percent of cells expressing the CD69, an early activation marker up-regulated on activated T cells. In this experiment, CD69 expression was used for the identification of activated cells, although burn injury causes a perturbation of CD25 expression [19 , 20 , 42 ]. Results show that CD69+ was expressed on 5–20% of T cells for sham and burn groups (data not shown). The ratio of T cells expressing CD69+ from burnt/sham mice in the CD4+–CD62LhighCD44low T cell population showed no significant changes on days 1 (Fig. 1 A )–5 (data not shown). At day 10, a significant increase of the ratio to 2 was observed, indicating an enhancement of activated CD4+T cells in burnt mice as compared with sham mice (Fig. 1B , P<0.05). Concerning effector/memory CD4+T cells (CD4+–CD62LlowCD44high), an increase of 3.5-fold of cells bearing the CD69 molecule was present at day 10 of the post-burn period (Fig. 1B , P<0.05). For CD8+T cells, no changes were noted in CD69 expression in CD8+–CD62LhighCD44low or CD8+–CD62LlowCD44high cell populations between burn and sham groups.



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  		Figure 1. Burn injury activates (CD69+) CD4+ naïve and effector/memory T cells, which express activation marker CD69. Gated T cell subpopulations were analyzed for CD69 expression, an early activation marker. On days 1 (A) and 10 (B), CD4+/CD62Lhigh naive T cells, CD4+/CD44high effector/memory T cells, CD8+ naive, and CD8+ effector/memory T cells were analyzed for their expression of CD69 after burn injury with fluorochrome-labeled anti-CD4/CD8, anti-CD62L, or anti-CD44 and -CD69, as described in Materials and Methods. Results were expressed as activated ratio corresponding to the results obtained for the burned group divided by those of the sham group. *, P < 0.05, buprenorphineburn versus buprenorphine-sham mice.

T cell apoptosis after burn injury persists for 10 days following injury
Our results showed a decrease in splenocyte number in the first 5 days post-burn injury, followed by an increase on day 10. The decrease of immune cell populations post-burn injury can be associated with a release of apoptosis-inducing stress mediators such as TNF-{alpha}, Fas-L, and glucocorticoids [16 , 17 , 20 ]. We therefore investigated the susceptibility of splenic T cells to depletion by apoptosis following burn injury. T cells from sham and burned groups were purified by macrophage and B cell depletion. A 90% T cell purification was obtained from the spleen cells of each group. Purified T lymphocytes, containing CD4+ and CD8+ T cells, were then incubated in normal culture media for 12 h and analyzed for their susceptibility to spontaneous apoptosis. Using flow cytometry assay, we analyzed the presence of apoptotic cells in both populations by determining the sub-G0 population of gated cells. At day 1 following injury, we observed in burned mice a decrease of spontaneous apoptosis in total CD4+T cells (CD4+–CD62LhighCD44low and CD4+–CD62LlowCD44high; Fig. 2 A ). Similar results were obtained for CD8+ T cells but only for CD8+–CD62LlowCD44high. Analysis at 10 days following injury showed that CD4+ T cells were the major cells susceptible to apoptosis (Fig. 2B) . Naive CD4+T cells (CD4+–CD62LhighCD44low) were more susceptible to apoptosis than effector/memory CD4+ T cell population. In contrast, CD8+ T cells from burn injury mice showed a comparable or a resistance (CD8+CD62Lhigh) to apoptosis.



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  		Figure 2. Naive CD4+ T cells are more susceptible to apoptosis 10 days after thermal injury. On days 1 (A) and 10 (B) after thermal injury, T cells of each groups were purified as described in Materials and Methods and incubated in complete RPMI for 12 h at 37ºC. Cells were stained for anti-CD4 FITC or anti-CD8 FITC to discriminate T cell populations and were further distinguished/characterized as naive (CD62Lhigh) or effector/memory (CD44high) cells using streptavidin-PE mAb. Cells were fixed and quantified for apoptosis using flow cytometry with 7AAD as described in Materials and Methods. Results obtained for the sub-G0 population for naive and effector/memory T cells are presented. Data represent the mean obtained for each group of five mice. *, P < 0.05, for buprenorphine-burn versus buprenorphine-sham mice.

T cell activation after burn injury was perturbed independently of macrophage function
Several reports have associated T cell dysfunctions after burn injury with macrophage activation or dysfunction [28 , 30 , 31 ]. To determine the importance of macrophages in T cell dysfunctions after burn injury, we stimulated purified T cells in the presence of purified macrophages obtained from sham or burned groups 10 days following injury. Stimulation of purified T cells from burnt mice in the presence of macrophages isolated from the sham group showed a significant decrease in cell proliferation, demonstrating that normal macrophages cannot restore T cell activation in burnt animals (Fig. 3 A ). Conversely, stimulation of T cells from both groups in the presence of macrophages obtained from burnt mice failed to support normal activation (Fig. 3A) . The response of sham T cells in the presence of anti-TCR plus exogenous IL-2 was greatly increased when sham macrophages were present (Fig. 3B) . However, T cells from burnt mice remained anergic under the latter stimulation conditions. This increase of sham T cell proliferation was completely inhibited in the presence of macrophages from burnt mice (Fig. 3B) . Overall, our results demonstrate that T cells in burnt mice are directly affected or indirectly affected by macrophages.



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  		Figure 3. T cell activation after burn injury was perturbed independently of macrophage functions. Purified T lymphocytes from the buprenorphine-sham group and buprenorphineburn group, as described in Materials and Methods, were incubated with plastic-adherent, isolated macrophages of both groups and stimulated with anti-TCR mAb. Proliferation was determined after 72 h by MTT reduction as described in Materials and Methods (A). The same stimulation condition as in A was used, but cells were incubated in the presence of 12.5 U/mL rIL-2 (B). Data were expressed as mean percentage for each group of mice. Small *, P< 0.05, for buprenorphine-sham versus buprenorphine-burnt mice. Large *, P < 0.05, responses were compared with the response of T cells and macrophages put together isolated from buprenorphine-sham mice. O.D. (Abs), Optical density (absorbance).


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DISCUSSION
 
Burn injury prompts the perturbation of several systems, including the immune system, followed by an increased susceptibility to infections. We and other labs have previously established that severe burn induces an impairment of T cell functions, and the maximal effect occurs at day 10 following burn [38 , 39 , 43 44 45 ]. The spleen cell depletion following burn injury could be explained by an increase in cell migration to the site of the burn or by apoptosis induction. Structural analysis shows that the splenic white pulp was still atrophic with a decrease in B and T lymphocytes 24 h post-injury [46 ], confirming the induction of apoptosis in this organ. Burn injury induced apoptosis in the spleen and the thymus by an increased caspase-3 activation within 3 h post-injury [17 , 47 ]. Apoptosis induction after thermal injury in secondary lymphoid organs was dependent on endogenous glucocorticoid release, as the pretreatment of mice with mifepristone significantly reduced apoptosis [16 , 17 ]. It was also suggested that other factors, such as an endotoxin increase FasL signaling or transforming growth factor-ß (TGF-ß), could be involved [15 , 19 , 20 ]. So, apoptosis of T cells in peripheral immune organs could explain this cell number depletion. Conversely, the migration of CD4+ T cells near the burn wound was also reported and may explain the spleen depletion [48 ]. Lawrence et al. [48 ] showed a fivefold T cell increase in lymph nodes draining the burn wound after dorsal and ventral full-thickness injuries as function of time after burn injury. Thus, changes in the spleen T cell subpopulations of burnt mice could be a result of their apoptosis in the spleen and cell migration to the burn wound area.

Independent of pain management, burn injury initially induced splenocyte depletion, followed by a hyperproliferation as compared with controls. Although spleen cell numbers were decreased, BrdU experiments showed the presence of cycling splenocytes days 5 and 10 after burn injury. Electron microscopy studies also showed the presence of a small number of large, blastic cells and mitotic figures in the spleen early after burn injury, followed by a recovery of T cells at 48 h [46 ]. Recently, Cho et al. [49 ] showed an increase of S-phase entry in the spleen at day 8 following 18% burn injury. These data suggest that burn injury produces a biphasic change in the spleen, characterized by a cell depletion occurring in stress response, followed by an increase in haematopoiesis to restore cell number to normal levels. These last changes can explain the phenotypic changes in spleen after a severe trauma. Considering the phenotypic changes in the spleen, it is interesting to note in our experiments that percent of T and B cells or macrophages remained normal at days 1 and 5. In contrast, on day 10 post-injury, we noted a decrease of the two former populations and a rise of macrophages. However, regarding the absolute number of cells, we demonstrated that these populations decrease in the first 5 days and are normalized or increased at day 10. These patterns of change in lymphocyte populations correlate with the demonstration of apoptosis induced after burn injury [16 , 17 , 20 ]. Moreover, increase of macrophage number is consistent with increase of myelogenous cells in the red pulp of spleen [49 ]. In contrast to our findings, Schwacha et al. [50 ] report at day 7 no change in total splenocyte yields or in percentage of CD3+T cells after thermal injury. Disparity between our results and those of Schwacha et al. [50 ] can be explained by transient normalization of spleen population at day 7 before the cellularity increase. Moreover, others factors, such as sex of mice or antibodies used for phenotyping splenocyte populations, can explain the data difference. Indeed, Schwacha and Somers [31 ] have reported in another study a splenomegaly at 4 and 7 days associated with an increase of monocyte/macrophage percentages and a decrease of lymphocyte percentages.

Our findings demonstrated that T cell homeostasis is perturbed after burn injury. A prompt increase followed by diminution in more long-term naive T cells (CD62LhighCD44low) in either T cell subpopulation occurs after burn injury. In the same conditions, effector/memory CD4+or CD8+ T cells (CD62LlowCD44high) were both decreased at long-term only. Consequently, effector/memory T cells seem to be more resistant than naive T cells to the perturbation induced by the trauma. The question now is to ask if these results make sense in the context of burn injury and T cell homeostasis. Burn injury induces the production of several cytokines such as IL-1, TNF-{alpha}, IL-4, IL-6, TGF-ß, and interferon-{gamma} (IFN-{gamma}) [33 , 34 , 44 , 51 , 52 ]. Among these cytokines, some were identified as having a role in CD4+ or CD8+T cell homeostasis [52 ]. For example, it has been demonstrated that burn injury augmented IFN-{gamma} production by naive CD4+ transgenic T cells [33 ]. IFN-{gamma} increase was also associated with CD8+ T cell homeostasis [53 ]. Moreover, dramatic hormonal changes, including glucocorticoid levels, can influence T cell homeostasis [16 , 54 ]. Furthermore, not only T cell proportions in spleen were altered by burn injury but also their own activation. In our experiments, CD69 expression, an early activation marker, was used for the identification of activated cells, although burn injury causes a perturbation of CD25 expression [10 , 11 , 43 ]. Our analysis showed that only CD4+–CD62LhighCD44low (naive) and CD4+–CD62LlowCD44high (effector/memory) were activated after thermal injury when compared with sham mice. The presence of these activated cells was detected in the spleen at a late time but not immediately following the injury. It has been proposed that the increase of self-antigen or bacterial antigen in circulation after burn injury could potentially influence T cell activation and its homeostasis [19 , 55 , 56 ]. Besides, we also demonstrated at this time a drastic macrophage increase in the spleen, which could possibly facilitate T cell activation. However, other studies have shown a defective antigen presentation on splenic macrophages early after burn injury [28 ]. Overall, burn injury induces an environment that can influence T cell homeostasis. Conversely, we cannot, however, omit the influence of specific and bystander responses of T cells in our model. An increase of CD69 on naive and effector/memory CD4+ T cells favors antigen activation. Indeed, when CD4+ T cells encounter an antigen, they convert from naive CD62LhighCD44low–CD69+ phenotype to activated CD62LlowCD44high–CD69+ phenotype [54 ]. Effector/memory T cells express high levels of ß2 integrins and thus preferentially migrate into inflamed, burn tissues, which can explain their decrease percent in the spleen at a late burn period [48 , 57 , 58 ].

The major change in T cell homeostasis after burn injury occurs 10 days later. This corresponds to the time reported for major perturbations of the immune system [38 , 43 44 45 ]. Analysis of purified T cells showed an increase of spontaneous apoptosis. Analysis of T cell subpopulations showed that cells with high levels of CD44, thus, effector/memory T cells, are more resistant to apoptosis [57 58 59 ]. This resistance can be related to an up-regulation of antiapoptotic gene products, including cellular Fas-associated death domain-like IL-1ß-converting enzyme inhibitory protein, bcl-xl, and bcl-2. Also, it was observed that the CD4+ and CD8+T cell subset expansion can be produced, not only by cell proliferation induction but also by preventing their death [59 ].

We have determined that T cells are directly affected at day 10 following burn injury and that the unresponsiveness of those cells cannot be restored by the addition of rIL-2 in vitro. However, in thermally injured mice, rIL-2 improves immune response [60 ]. Burn induces a depressed T cell activation at several levels, including reduced IL-2 gene transcription by inhibiting c-fos expression [43 ], by decreasing the expression level and activity of IL-2R [43 , 61 , 62 ], or by perturbating the general cell signaling [13 ]. Spleen macrophages also contribute to this anergic state by inhibiting a normal T cells response. Our results are similar with the observations of Schwacha and Somers [31 ], which showed that adherent splenocytes from injured mice suppressed normal and injured lymphocyte proliferation. Macrophages can perturb T cell activation by producing PGE2, which can affect nuclear factor of activated T cells and AP-1 activation in these cells or by the induction of reactive nitrogen intermediates [30 , 31 ].

However, our results diverge from those of Schwacha regarding the capacity of T cells from burn injury to proliferate normally when depleted in adherent cells [30 , 31 ]. Several differences should be considered between the two experiments, including the sex of mice, the time-frame of experiments, and the specificity of mAb used to activate T cells (anti-CD3{varepsilon} vs. anti-TCR-ß). Moreover, we have used purified T cells isolated from spleen of burn injury mice, and Schwacha used nonadherent fraction of spleen. Schwacha also reports a decrease of B cells at day 7 in injury mice, suggesting that the number of T cells can be higher in their preparation of cells from burnt mice [50 ]. Our opinion is that the major point is time-frame, as we have previously reported a normal T cell proliferation from burnt mice at day 7, although a depressed response was noted at day 10 [38 , 39 ].

Overall, burn injury perturbs the T cell homeostasis, possibly by changing hormonal control, creating an environment that can modulate T cell populations and functions. Our results indicated that CD4+ T cells are the most affected cells. The presence of naive and effector/memory CD4+T-activated cells 10 days following thermal injury suggests that these cells are effectively primed, as previously demonstrated in other models [34 ]. Those primed cells could be more susceptible to apoptosis. Thus, multifactorial modulation of T lymphocyte functions following burn injury led to the disruption of the homeostatic controls, which may explain the increase in occurrence of sepsis or infection.

Received July 4, 2003; revised September 20, 2004; accepted October 4, 2004.


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