Major injury disrupts normal immune function and ultimately suppresses the injured host's ability to control opportunistic infections (1, 2). We have shown that suppressed immune function 1 week or more after burn injury is associated with a measurable shift in CD4+ T cell responses toward a T-helper 2 (Th2) phenotype (3). Interestingly, this phenotypic shift in T cells is preceded by an early proinflammatory host response that primes innate and adaptive immune cells for enhanced inflammatory cytokine production (4). The adaptive immune system then undergoes a progressive shift toward an increased Th2-type cytokine production phenotype that is easily detectable 7 days after injury (3, 5). It is believed that this progressive change toward a Th2 phenotype is counterinflammatory and potentially immunosuppressive, resembling the compensatory anti-inflammatory response syndrome noted clinically in seriously injured patients (6-8).
Because the differentiation of naive CD4+ T cells into effector T cell populations with Th1, Th2, or T regulatory (Treg) phenotypes most often requires a T cell receptor (TCR) stimulus, we hypothesized that injury may result in an early T cell activation event that, in turn, drives the differentiation of a unique population of injury-responsive CD4+ T cells. Alternatively, burn injury might activate a small population of memory T cells, natural killer (NK) T cells, or Tregs, which have been shown to play a counterinflammatory function after burn injury (9, 10). Additionally, we were interested in determining whether injury might induce a response resembling that seen after exposure to bacterial superantigens (SAg), potent toxins that activate CD4 and CD8 T cells expressing the appropriate TCR Vβ-chains in an MHC class II-restricted fashion (11, 12). We addressed these possibilities by examining the effects of burn injury on T cell activation and proliferation in the lymph nodes and spleens of mice. We also studied whether burn injury might activate an oligoclonal subpopulation of CD4 or CD8 T cells as judged by TCR Vβ-chain usage.
Our findings indicate that burn injury compared with SAg challenge induces an early proliferation of a restricted set of TCR Vβ-chain-expressing CD4 T cells. We also observed that this injury-induced CD4 T cell response was associated with a significant induction of the T cell activation markers, CD25 and CD152, whereas SAg treatment induced the expression of a somewhat different profile of T cell activation markers (CD25, CD28, and CD69) on CD4 and CD8 T cells in the lymph node and spleen. Thus, this report provides the first evidence to suggest that burn injury initiates an early and specific immune reactivity by CD4 T cells that is qualitatively different than conventional SAg exposure.
MATERIALS AND METHODS
Male C57BL/6J mice, 6 to 7 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME) and were acclimated for at least 1 week in a virus-free animal facility with a diet of standard mouse chow and water ad libitum. All subsequent procedures were carried out in accordance with NIH guidelines and those of the Harvard Medical Area Standing Committee on Animals.
Mouse burn injury model
Mice were subjected to burn or sham injury as described previously (13). Briefly, age- and weight-matched mice, three per group, were randomized to receive sham or burn injury and were anesthetized by intraperitoneal (i.p.) injection of 125 mg/kg ketamine and 6 mg/kg xylazine. The dorsa were shaved and the mice were placed in a plastic mold that exposed 25% of body surface area. The exposed skin was immersed in 90°C water for 9 s. This treatment has been shown to cause an anesthetic full-thickness burn injury with a 5% or lower mortality rate. Sham-injured mice were treated in the same fashion except that they were exposed to isothermic water for 9 s. Sham and burn-injured mice were resuscitated by i.p. injection of 1 mL of sterile, pyrogen-free normal saline solution.
Culture medium (C5) for in vitro studies consisted of RPMI-1640 supplemented with 5% heat-inactivated fetal calf serum, 1 mM glutamine, penicillin/streptomycin/fungizone, 10 mM HEPES buffer, 100 mM nonessential amino acids, and 2.5× 10−5 M 2-mercaptoethanol, all purchased from Gibco-Invitrogen (Grand Island, NY). Peridinin chlorophyll protein-cyanine 5.5 (Cy5)-labeled anti-CD4 and anti-CD8 monoclonal antibodies (Abs) and phycoerythrin (PE)-labeled anti-Ki-67 antigen, anti-CD25, and anti-CD28 anti-CD152 monoclonal Abs were purchased from BD PharMingen (San Diego, CA), as were the mouse TCR Vβ screening panels containing fluorescein isothiocyanate (FITC)-labeled monoclonal Abs specific for Vβ2-, Vβ3-, Vβ4-, Vβ5.1-, and -2-, Vβ6-, Vβ7-, Vβ8.1-, and -2-, Vβ8.3-, Vβ9-, Vβ10-, Vβ11-, Vβ12-, Vβ13-, and Vβ14-chains of the mouse TCR. Staphylococcal enterotoxin B (SEB) was purchased from Sigma Chemical Company (St. Louis, MO).
At varying intervals after sham or burn injury, mice were sacrificed by CO2 asphyxiation and the spleen and lymph nodes (axillary, brachial, and inguinal) were harvested. Cell suspensions in C5 were prepared by mincing tissues through a sterile, stainless-steel wire mesh. Cell suspensions were treated with Tris-ammonium chloride buffer for 3 min to lyse red blood cells and were then washed twice before resuspension in C5 medium.
Lymph node or spleen cells at 1 × 106 per mL were incubated with anti-CD16/CD32 antibody and FC-Block (BD PharMingen), to prevent nonspecific binding to Fc receptors and were then surface stained with Cy5-labeled anti-CD4 or anti-CD8 Abs. The cells were then fixed for 20 min with 100 μL of 2% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, at 4°C. After fixation, the cells were washed once and then resuspended in 100 μL of permeabilization buffer (PBS, 1%bovine serum albumin, 0.1% saponin, and 0.1% sodium azide). Nonspecific binding was blocked by pretreating the fixed and permeabilized cells with 25 μL of a 1 μg/mL solution of normal rat immunoglobulin G (Caltag, Burlingame, CA). For the nuclear staining of cells to detect Ki-67 antigen expression, the cells underwent an additional 30-min permeabilization procedure using a 0.1% NP-40 solution prepared in PBS containing 1% bovine serum albumin and 0.1% sodium azide. The cells were then stained with PE-labeled anti-Ki-67 Ab. Ki-67 expression by CD4+ and CD8+ cells was then determined by flow cytometry on a FACSCalibur instrument (Becton-Dickinson, San Jose, CA) with data analysis by the accompanying CELLQuestPro software program. In other experiments, cell suspensions surface stained with Cy5-labeled anti-CD4 or anti-CD8 Abs were costained with PE-labeled anti-CD25, anti-CD28, or anti-CD152 monoclonal Abs and expression of these surface receptors on CD4+ and CD8+ T cells was again determined by flow cytometry. In experiments to determine T cell receptor usage by Ki-67+ cells, cell suspensions were surface stained with Cy5-labeled anti-CD4 antibody and FITC-labeled antibodies for the various TCR Vβ-chains and then fixed, permeabilized, and stained with PE-labeled anti-Ki-67 Ab as above. Vβ-chain usage by Ki-67+ CD4+ cells was determined by three-color flow cytometry collecting sufficient numbers of events to attain at least 300 individual three-color events.
Comparisons between burn and sham lymph node and splenic CD4+ and CD8+ T cells with respect to expression of Ki-67, costimulatory receptors, and Vβ-chain usage was made by two-tailed t tests and using the PRISM 3.0 software (GraphPad, San Diego, CA) program. P values <0.05 were considered significant.
Burn injury induces an early proliferation of lymph node CD4+ T cells
The Ki-67 antigen is a well-established marker for proliferating cells because it is expressed only in cells that have entered the cell cycle (14, 15). Using this fact, we optimized a flow cytometry procedure that allowed us to examine changes in nuclear Ki-67 expression in CD4 and CD8 T cells after burn injury or SAg treatment. The representative FACS plots shown in Figure 1A illustrate the level of sensitivity we obtained using this approach. In this representative experiment, we found that burn injury caused an increase in Ki-67-expressing CD4 T cells compared with sham-injured or SEB-treated mice in the lymph nodes 12 h after injury. In contrast, we found that SEB given intraperitoneally at a dose of 0.05 mg induced a high level of Ki-67 antigen expression in CD4 T cells 72 h, but not 12 h after SAg exposure. The compiled FACS data examining the effects of burn injury on CD4 and CD8 T cell proliferation responses are shown in Figure 1B. The expression of Ki-67 antigen was significantly increased in lymph node CD4 T cells 12 h after burn injury compared with sham-injured mice. Although the level of Ki-67 antigen expression was not as high as that observed after SAg treatment, this observation does provide evidence that burn injury initiates CD4 T cell proliferation within the peripheral lymph nodes draining the injury site. Interestingly, this injury-induced increase in Ki-67-expressing CD4 T cells after burn injury was not detected at time points after 12 h, which suggests that the injury-induced CD4 T cell proliferation is short-lived. The results of initial experiments also did not show an increase in CD4 T cell proliferation 3 or 6 h after burn injury (data not shown). Interestingly, we did not detect any proliferative response by lymph node CD8 T cells. Moreover, changes in Ki-67 antigen expression in splenic CD4 T cells or CD8 T cells were not observed at any time point postinjury. In marked contrast to what was seen after burn injury, mice exposed to SEB did not display a measurable increase in CD4 or CD8 T cell proliferation at 12 h. Instead, we observed an elevation in Ki-67-antigen expressing CD4+ and CD8+ T cells in the lymph nodes and spleens harvested from mice 72 h after SEB treatment. Of note, the kinetics of SEB-induced T cell reactivity were significantly different from what was observed after burn injury. Specifically, burn injury induced a tightly compartmentalized, very early CD4 T cell response, whereas SEB challenge induced a later, more systemic CD4 and CD8 T cell response.
Burn- and SEB-induced changes in CD4 and CD8 T cell activation and costimulatory receptors
The observation that burn injury induced CD4 T cell proliferation suggested to us that burn injury must provide an early T cell-activating stimulus. To address this possibility, we assessed changes in several major T cell activation markers and costimulatory receptors [CD25, CD28, CD69, and CD152(CTLA-4)] on lymph node and spleen CD4 and CD8 T cells prepared 12 h after sham injury, burn injury, or SEB challenge. As shown in Figure 2, burn injury caused an increase in CD25 and CD152 expression on only lymph node CD4+ T cells, whereas SEB treatment caused a marked elevation of CD25 and CD69 expression on lymph node and spleen CD4 and CD8 T cells. The injury-induced increase in CD25 and CD152 expression on lymph node CD4 T cells was not observed 24 or 72 h after burn injury (data not shown), indicating that it is short-lived or that the activated T cells exited the lymph nodes. These findings are in direct agreement with our findings on injury- versus SAg-induced CD4 and CD8 T cell proliferation responses and they support the concept that the lymph node CD4 T cells, but not CD8 T cells, undergo early activation after burn injury, whereas SAg exposure activates lymph node and splenic CD4+ and CD8+ T cells. Again, these findings indicate that burn injury and SAg responses are qualitatively different. Although they both induce a significant early increase in CD25 expression, burn injury preferentially activates CD152 expression, whereas SEB treatment induces increased CD69 expression.
Burn injury induces the selective proliferation of TCR Vβ4-, Vβ6-, Vβ11-, Vβ12-, and Vβ14-expressing lymph node CD4+ T cells
To determine if a selective population of lymph node CD4+ T cell populations undergo an early proliferative response 12 h after burn injury, we examined by flow cytometry the relative alterations in the percentage of lymph node CD4+/Ki-67+ cells expressing the various TCR Vβ-chains. As shown in Figure 3, the proliferating populations (Ki-67 gated) of CD4+ T cells were found to be expressing Vβ4, Vβ6, Vβ11, Vβ12, and Vβ14 as opposed to the other nine TCR Vβ-chains measured. This selective usage of TCR Vβ-chains displayed by CD4+ T cells after burn injury suggests that injury may specifically activate this subset of Vβ-expressing CD4+ T cells. We next examined if burn injury might cause the in vivo expansion of any of these Vβ TCR-expressing CD4 cells by profiling changes in Vβ TCR usage 3 and 10 days after sham or burn injury. These findings indicated that only the Vβ11 TCR-expressing lymph node CD4+ T cells showed a significant level of selective expansion 3, but not 10, days after burn injury (Fig. 4). We did not detect changes in Vβ TCR usage by CD8+ T cells 3 or 10 days after injury, and splenic CD4+ T cells did not show any detectable change Vβ TCR expression. Thus, we conclude that although there is an early burst of proliferation by an oligoclonal population of lymph node CD4+ T cells 12 h after burn injury, only the Vβ11 TCR-expressing CD4+ T cell population displays a prolonged proliferative response.
Although it is established that serious injury induces a phenotypic change in CD4+ T cell responses toward an enhanced Th2-type response, the direct influence of injury on CD4+ T cell activation and differentiation have not been well defined. In particular, it was not known if burn injury could activate "self" CD4+ or CD8+ T cell responses. Therefore, we initiated these studies to provide an analysis of how injury might influence CD4+ and CD8+ T cell activation in vivo.
Using Ki-67 staining as an approach to measure T cell proliferation, we demonstrate that burn injury supplies a proliferation signal to lymph node CD4+ T cells that is detectable by 12 h after injury. In contrast, CD8+ T cells showed no change in proliferation at this time point. Interestingly, SEB treatment did not elicit a similar early proliferation of CD4+ or CD8+ T cells. Instead, SEB induced a much later (72 h) and more robust proliferation of CD4+ and CD8+ T cells in the lymph nodes and spleen. Several important conclusions can be made based upon these findings. First, burn injury triggers an early, brief proliferation of lymph node CD4+ T cells. Second, CD8+ T cells do not show any detectable sign of increased proliferation at early or late time points after burn injury. Most importantly, however, it appears that the injury effect on T cell activation is much different than a conventional SAg response in that it occurs much earlier, it involves only CD4+ T cells, and it displays markedly different response kinetics.
The rapid response displayed by a selective population of CD4+ T cells in the lymph nodes of burn-injured mice suggests that the responsive subset of CD4+ T cells may be memory T cells. Although this is clearly speculative, these cells may represent a population of CD4+ T cells that respond to endogenous antigens released during the injury response. If this were the case, these cells would have escaped tolerance induction in the thymus. T cells known to have these characteristics include the naturally occurring CD4+ CD25+ regulatory T cell subset (Tregs), which have been shown to react to self antigens and are a subject of ongoing research in our laboratory (16). There is also considerable evidence that Treg cells can be induced in the periphery by several mediators known to be upregulated after injury, interleukin 10 (IL-10) and transforming growth factor β (TGFβ) (17-20). Recent data from our laboratory suggests that burn injury does indeed enhance the Treg activity of CD4+ CD25+ T cells (21).
In addition to measuring T cell proliferation in vivo, we chose to examine the comparative effects of injury versus SEB reactivity on T cell activation by measuring changes in the T cell costimulatory receptors CD25, CD28, CD69, and CD152. The increased expression of these costimulatory receptors on T cells is generally considered to be an indication of T cell activation and would be anticipated to coincide with our T cell proliferation findings. We found that burn, as opposed to sham, injury induced increased surface expression of CD25 and CD152 on lymph node CD4+ T cells 12 h after injury. At this same time point, we observed an early induction of CD25 and CD69 on CD4+ and CD8+ T cells after SEB challenge. Thus, these results reinforce the observation of a restricted proliferative response of CD4+ T cells occurring in the lymph nodes draining the injury site. They also strengthen the observation that the host response to superantigen is fundamentally different than the injury response with respect to the reactive T cells because different costimulatory receptors were modulated by these two immune responses. For example, injury preferentially increased CD152 (CTLA-4) expression on CD4+ T cells, whereas SEB treatment induced a substantial increase in CD69 expression. Because CD152 is known to be an important suppressive/regulatory receptor responsible for controlling and inhibiting T cell responses, it is tempting to speculate that the injury-induced upregulation of CD152 is a component of the regulatory T cell response occurring after severe injury, whereas the lack of upregulated CD152 expression after SEB treatment might indicate a advancement to a fully developed CD4 or CD8 T cell response (22). There is also the possibility that the increase in CD152 expression reflects the preferential activation of an injury-responsive inhibitory CD4+ T cell subset, e.g., Treg cells (23).
Given the observation that burn injury caused an early CD4+ T cell proliferative response, we wished to determine if injury might specifically activate a subset of lymph node CD4+ T cells. Using a panel of FITC-labeled anti-TCR Vβ-chain-specific Abs, along with Ki-67 staining, we show that burn injury induces the selective proliferation of TCR Vβ4-, Vβ6-, Vβ11-, Vβ12-, and Vβ14-expressing lymph node CD4+ T cells 12 h after injury, but only TCR Vβ11+ CD4+ T cells expanded after injury. Interestingly, C57BL/6J mice possess Mtv-8 and Mtv-9 proviral loci, which are known to act as endogenous superantigens and stimulate TCR Vβ11-expressing CD4+ T cells that escape the negative selection deletion process for self-reactive T cells in the thymus (24, 25). We believe these results provide the first evidence to suggest that endogenous antigen(s) may initiate this early T cell response to injury. The source of these antigens may be the injured tissue as suggested by several reports or may be endogenous superantigen-like molecules such as endogenous viral antigens whose expression might be increased after injury (26-29). In fact, it has been shown that mice express increased levels of murine AIDS-related genes after burn injury (30). A similar phenomenon may occur with the endogenous MMTV genes, Mtv-8, Mtv-9, or Mtv-17, in C57BL/6J mice and cause the activation of specifically reactive CD4+ T cells (24). Another source of endogenous antigens could be components of bacteria that are part of the normal flora such as endotoxin or other ubiquitous bacterial antigens. Again, we suspect that the naturally occurring Treg cells might be included in this population of reactive cells, although it has not been conclusively shown that Treg cells use these selective TCR Vβ-chains (16). Our future efforts will focus on determining if the subset of CD4+ T cell activated after injury includes Treg cells.
The results presented here are, to our knowledge, the first reported evidence that severe injury can induce the selective activation and proliferation of a regional subset of CD4+ T cells. These observations underscore the fact that CD4+ T cells are the primary adaptive immune cell type involved in the initiation of the adaptive immune response to severe injury, and they highlight the unique nature of the mammalian injury response. We intend to use these findings as a foundation for the future investigation of factors that promote the development of the counter inflammatory T cell response that occurs after injury. Because the change in adaptive immune function after major injury is associated with a clear-cut increase in susceptibility to sepsis and its potentially lethal complications, an increased understanding of how injury upsets normal immune reactivity and function will suggest strategies to help avert the negative influence of injury on protective immunity (2, 31, 32).
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Adaptive immune system; superantigen; T cell receptor; variable β-chain; costimulatory receptors