Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1102540 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;73:747-755.)
© 2003 by Society for Leukocyte Biology

A role for CD1d-restricted NKT cells in injury-associated T cell suppression

Douglas E. Faunce*, Richard L. Gamelli*, Mashkoor A. Choudhry* and Elizabeth J. Kovacs*,{dagger}

* The Burn and Shock Trauma Institute, Department of Surgery, and
{dagger} Department of Cell Biology, Neurobiology, and Anatomy, Loyola University Medical Center, Maywood, Illinois

Correspondence: Douglas E. Faunce, Ph.D., The Burn and Shock Trauma Institute, Department of Surgery, Loyola University Medical Center, Bldg. 110, Rm. 4221, 2160 So. First Ave., Maywood, IL. E-mail: dfaunce{at}lumc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Natural killer T (NKT) cells are known to modulate T cell responses during autoimmunity, tolerance, and antitumor immunity; however, their potential role in regulating the immune response to injury has not been reported. Using a murine model of burn injury, we investigated whether CD1d-restricted NKT cells played a role in the T cell suppression that occurs early after injury. A functional role for CD1d stimulation of NKT cells in the injury-related immune suppression was demonstrated by experiments in which the suppression of antigen (Ag)-specific delayed-type hypersensitivity and in vitro T cell-proliferative responses were prevented if mice were given anti-CD1d monoclonal antibody (mAb) systemically just before injury. The CD1d-NKT cell-dependent suppression of the T cell response after injury occurred in the absence of quantitative changes in NKT cells themselves or CD1d+ Ag-presenting cells. We observed that elevated production of the immunosuppressive cytokine interleukin (IL)-4 correlated with burn-induced immune dysfunction, and we found that NKT cells but not conventional T cells were the source of IL-4 early after injury. Lastly, we observed that the injury-induced production of NKT cell-derived IL-4 could be blocked by systemic treatment of burn-injured mice with anti-CD1d mAb. Together, our results reveal a novel mechanism involving CD1d stimulation of NKT cells in the onset of T cell suppression that occurs subsequent to injury.

Key Words: natural killer T cells • burns • immune suppression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the early 1950s, it has been well recognized that a major consequence of burn injury is marked immune suppression and the subsequent development of life-threatening, systemic infection or sepsis. Despite our enhanced knowledge of burn and sepsis physiology, the development of advanced antibiotics, and significant advances in the field of immunology, the major cause of death today among patients that sustain serious burn injury remains to be overwhelming infection [1 ]. Over recent years, many investigators have shown that immune suppression after trauma involves a complex, multifaceted cascade of events, including uncontrolled production of multiple proinflammatory cytokines [2 3 4 5 6 7 ], alterations in hematopoiesis and granulopoeisis [8 9 10 ], increased production of T helper cell type 2 (Th2)-like cytokines such as interleukin-4 and -10 (IL-4 and IL-10), as well as transforming growth factor-ß (TGF-ß) [11 12 13 ], all of which are known to suppress various aspects of immune function and decreased production of IL-2 and Th1-associated cytokines including interferon-{gamma} (IFN-{gamma}) and IL-12 [14 , 15 ].

Although a significant amount of research has focused on the roles of conventional T lymphocytes and macrophages in injury-associated immune dysfunction, there is little known about the roles of other nonconventional cell types, such as {gamma}{delta} T cells [16 ] and CD1d-restricted natural killer T (NKT) cells. NKT cells in particular have received considerable attention in recent years as innate lymphocytes that can modulate T cell and antigen (Ag)-presenting cell (APC) functions in autoimmunity, tolerance, and cancer [17 18 19 20 21 22 ]. Additionally, NKT cells are now known to be a major source of the IFN-{gamma}, which is required for early activation of macrophages to perform their bactericidal activity [23 ].

NKT cells are specialized, innate lymphocytes, present in virtually all lymphoid compartments as well as the liver, and are identified by their coexpression of CD3{varepsilon}, T cell receptor (TCR){alpha}ß, and various NK cell markers including NK1.1, Ly49C, AsGM1, and IL-2Rß chain [24 , 25 ]. The majority of murine NKT cells (~85%) expresses an invariant V{alpha}14 J{alpha}18 (formerly J{alpha}281) TCR [26 ] and is restricted by the major histocompatibility complex (MHC) class I-like molecule CD1d, which is expressed by conventional APCs including macrophages, dendritic cells, and marginal zone B cells [26 27 28 29 ]. Like other cells of the innate-immune system, NKT cells can release remarkable quantities of immunomodulatory cytokines within minutes of stimulation of their TCR via CD1d. In particular, NKT cells are known for their ability to rapidly secrete large amounts of IL-4 and IFN-{gamma} after stimulation [30 ], one mechanism by which they are thought to participate in the regulation of the Th cell response. Although the NKT cells’ role in regulating aspects of tolerance, cancer, and autoimmunity is well studied, their potential role in modulation of the immune response to injury has not been investigated.

Using a murine model of burn (dorsal scald) injury, we demonstrate in this report that burn injury results in suppression of Ag-specific immunity including delayed-type hypersensitiviy (DTH) and in vitro T cell-proliferative responses within 24 h after injury. Decreased immunity correlated with decreased Ag-specific IFN-{gamma} production and elevated levels of NKT cell-derived IL-4. Blockade of CD1d-NKT cell activation via systemic administration of anti-CD1d monoclonal antibodies (mAb) prevented the injury-induced suppression of DTH and splenocyte proliferation and prevented the increased production of IL-4. Together, our studies suggest a novel, NKT cell-dependent mechanism that leads to T cell suppression in the early, post-burn period.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight- to 10-week-old male BALB/c mice used in these studies were obtained from Harlan Laboratories (Indianapolis, IN), housed on a 12/12 h light/dark cycle, and provided with food and water ad libitum. All mice were treated humanely and in accordance with guidelines set forth by the Loyola University Institutional Animal Care and Use Committee (Maywood, IL) and the National Institutes of Health (Bethesda, MD).

Burn injury model
All mice were subjected to dorsal scald injury using a method described previously by Walker and Mason [31 ] and modified by Faunce and colleagues [32 ]. Briefly, mice were anesthetized with Nembutal, 10 mg/kg [intraperitoneally (i.p.)], and had their dorsal surfaces shaved with animal clippers. Mice were then placed into a plastic template that exposed 15–20% of their total body surface area, calculated by the method of Spector [33 ]. The mouse and template were immersed into a 100°C water bath for 8 s. Sham control animals were placed in a room temperature water bath. After the water exposure, the mice were dried immediately to prevent further scalding, given 1.0 ml i.p. fluid resuscitation (0.9% normal saline) and were placed under warming lamps until recovery from anesthesia. All procedures were done between the hours of 8 and 10 a.m. to avoid interference of circadian changes in stress-related hormones.

Antibodies
Antibodies used for flow cytometry included CyChromeTM-conjugated anti-TCRß chain mAb, fluorescein isothiocyanate (FITC)-conjugated anti-CD3{varepsilon} mAb, phycoerythrin (PE)-conjugated anti-Ly49C/I mAb (clone 5E6), FITC-conjugated anti-Mac3, and purified anti-CD16/CD32 mAb (FcBlockTM, clone 2.4G2) all from BD PharMingen (San Diego, CA); CyChrome-anti-F4/80 (Caltag, Burlingame, CA); and whole rat immunoglobulin G (IgG; Sigma Chemical Co., St. Louis, MO). Antibodies used in vivo and in cell culture included purified (azide-free, low endotoxin) rat anti-mouse CD1d mAb (clone 1B1) and rat IgG2b (BD PharMingen). Anti-CD1d mAb (and control) used in vivo were given intravenously (i.v.) via the tail vein at a dose of 50 µg/mouse in 100 µl sterile saline. For in vivo experiments, antibodies were given 1 h before injury.

Flow cytometric analyses of NKT cells
Flow cytometric analysis of NKT cells was done as described previously [17 , 18 ]. Briefly, spleens were minced and passed through fine wire mesh, and all debris was removed. Erythrocytes were removed by ammonium chloride lysis, and the remaining cells were resuspended in RPMI 1640 containing 10% fetal calf serum, penicillin-streptomycin, and glutamine. Cell viability was checked by trypan blue exclusion. Splenocytes were then resuspended in staining buffer [phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.1% sodium azide], and nonspecific staining was blocked with anti-CD16/CD32 (FcBlockTM) and whole rat IgG. After blocking, cells were then incubated with CyChromeTM-conjugated anti-TCRß chain, FITC-conjugated anti-CD3{varepsilon}, and PE-conjugated anti-Ly49C/I (clone 5E6), washed twice in staining buffer, and fixed in 4% paraformaldehyde. Flow cytometric determinations were made using a Becton Dickinson FACScalibur flow cytometer and CellQuest Pro software.

DTH and in vivo blockade of CD1d-NKT cell signaling
DTH was induced as described previously [17 ]. Briefly, mice were inoculated subcutaneously (s.c.) at the nape of the neck with 100 µl of an emulsion containing 100 µg ovalbumin (OVA; Sigma Chemical Co.) in complete Freund’s adjuvant (CFA; Sigma Chemical Co.). Seven days later, mice had both ears measured with an engineer’s micrometer (Mitutoyo, Paramus, NJ) and were given an intradermal inoculation of 10 µl PBS containing 200 µg OVA to the left ear pinna and a similar inoculation of human serum albumin (HSA; Sigma Chemical Co.) to the right ear pinna as an Ag-specificity control. Twenty-four hours later, ear measurements were made again, and the change in ear swelling was calculated as an index of DTH. All OVA and HSA solutions used for ear challenge as well as in vitro cultures (described below) were removed of potentially contaminating endotoxin by passage through DetoxiGelTM polymixin-B chromatography columns (Pierce-Endogen, Rockford, IL).

In vitro recall-proliferative response to Ag
Splenocyte suspensions were prepared as described above and plated at 2.0 x 105 cells per well in 96-well plates in RPMI alone or RPMI containing OVA (200 µg/ml; Sigma Chemical Co.) and were cultured at 37°C 5% CO2 for 48 h. After 48 h of culture, 1 µCi 3H-thymidine (3H-TdR; Amersham Pharmacia Biotech, Piscataway, NJ) was added to each well, and the cells were cultured for an additional 16 h. 3H-TdR incorporation was assessed by scintillation counting and used as an index of OVA-specific splenocyte proliferation.

Measurement of Ag-specific production of cytokines
Spleen-cell suspensions were prepared as described above for in vitro-proliferative assays, plated at 2.0 x 105 cells per well in 96-well plates with RPMI alone or RPMI containing OVA (200 µg/ml), and cultured for 24 h. At the end of the culture period, the plates were centrifuged at 1200 rpm for 10 min, and the supernatants were collected and stored at -80°C. The levels of IL-4 and IFN-{gamma} in the supernatants were determined with commercially available enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Woburn, MA), performed according to the manufacturer’s specifications. ELISA plates were read using a SpectraMAX Plus 384 plate reader (Molecular Dynamics, Sunnyvale, CA), and analyses of ELISA data were done with SoftMax ProTM.

Measurement of intracellular cytokines
Splenocytes were collected and cultured overnight ± OVA (200 µg/ml). Brefeldin A (Sigma Chemical Co.) was added for a final concentration of 10 µg/ml for the last 6 h of culture. After culture, the nonadherent cells were collected, washed, and immunostained with CyChrome-anti-TCRß and FITC-anti-Ly49C (eBioScience, SanDiego, CA), then fixed and permeabilized using CytoFixTM and CytoPermTM reagents (BD PharMingen), and then incubated with PE-anti-IL-4 mAb or PE-IgG2b isotype control (BD PharMingen). After final washing and fixation, cells were analyzed by flow cytometry for analysis of surface markers and intracellular cytokine staining.

Statistical analyses
Statistical determinations were made by Student’s t-tests or ANOVA and Neuman-Keuls post-hoc analyses, where appropriate. Statistical significance was determined when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD1d-NKT cell signaling contributes to suppressed T cell immunity after injury
As NKT cells are known to modulate peripheral T cell immunity in humans and in experimental animal models [21 , 22 ], we examined the ability of burn-injured mice to generate an effector T cell response when the CD1d activation of NKT cells in vivo was prevented. As the large majority of murine NKT cells express a TCR that is restricted by CD1d, the use of anti-CD1d-blocking antibodies offered a highly unique and precise method for blocking APC activation of NKT cells. Anti-CD1d mAb given in vivo has been shown by others to prevent the activation of NKT cells [21 ]. In the experiments presented here, anti-CD1d mAb (50 µg/mouse) and control IgG (50 µg/mouse) were given 1 h before injury, and all measurements of immune responsiveness were made 24 h after injury, as our previous reports demonstrated that suppression of T cell immunity in BALB/c male mice appears greatest during the early, post-injury period [34 ].

Compared with sham-injured + control IgG-treated mice, burn-injured + control IgG-treated mice demonstrated a significant (P<0.05) decrease in their ability to generate an OVA-specific DTH response (Fig. 1 ). The DTH response in burn-injured + control IgG-treated mice was only one-fourth the magnitude seen in sham-injured mice. In contrast, if mice were treated with anti-CD1d mAb, an antibody that prevents the activation of NKT cells by CD1d+ APCs [18 , 21 ], and then subjected to burn injury, the burn-induced suppression of OVA-specific DTH was significantly (P<0.05) prevented. Although anti-CD1d mAb treatment did not prevent burn-induced suppression of DTH entirely, burn-injured + anti-CD1d-treated mice displayed DTH responses that were threefold higher than control IgG-treated, burn-injured mice. From this experiment, we conclude that in mice immunized to Ag 1 week before injury, the ability to generate Ag-specific DTH responses was suppressed in a manner that involved signaling by CD1d.



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  		Figure 1. Effects of systemic anti-CD1d mAb treatment on injury-induced suppression of DTH. Seven days after immunization with OVA, mice received control rat IgG or anti-CD1d mAb. One hour later, mice were subjected to sham or burn injury and given an intradermal ear injection of OVA or HSA. Twenty-four hours after ear challenge, DTH was determined. The bar graph represents the change in ear thickness 24 h after ear challenge ± SEM. *, P < 0.05, versus sham + control (Ctrl) IgG; **, P < 0.05, versus burn + Ctrl IgG as determined by ANOVA and Neuman Keuls post-hoc analyses. N = 4. Data are representative of three experiments in which similar results were obtained.

 
Blockade of CD1d activation of NKT cells prevents injury-associated suppression of T cell proliferation in vitro
Although DTH is considered by many to represent a prototypic manifestation of cellular immunity, we also chose to examine the Ag-induced, recall-proliferative response of splenic and lymph node (LN) T cells in vitro. T cell-proliferative responses to OVA were significantly (P<0.05) suppressed in splenocyte cultures obtained from burn-injured, control IgG-treated mice compared with the response of splenocytes from sham-injured, control IgG-treated mice (Fig. 2 ). Like the DTH responses described above, we observed that splenocytes from burn-injured mice displayed only one-fourth the magnitude of proliferation compared with sham controls. In contrast, we observed that the injury-associated suppression was significantly (P<0.05) prevented in splenocyte cultures obtained from burn-injured, anti-CD1d mAb-treated mice. Specifically, we observed that the OVA-specific, proliferative response of splenocytes from burn-injured mice was two- to threefold higher if the mice were pretreated with anti-CD1d-blocking Ab. We also noted that CD1d blockade in sham-injured mice significantly decreased the proliferative recall response to OVA. This finding is in line with reports by others showing the role of NKT cells in amplifying conventional Th1 immunity in vitro. In addition to measuring splenocyte-proliferative responses to Ag, we also measured the Ag-induced, proliferative responses of LN cells. Briefly, we observed that LN cells (pooled axial, brachial, and inguinal) from burn-injured, control IgG-treated mice were significantly suppressed in their ability to proliferate in response to Ag in vitro compared with LN cells from sham-treated mice (data not shown). Like the splenocyte cultures, we observed that the burn-induced suppression of the LN cell-proliferative response was significantly prevented if the mice received anti-CD1d mAb before injury.



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  		Figure 2. Effects of systemic anti-CD1d mAb treatment on injury-induced suppression of in vitro T cell-proliferative response to Ag. Seven days after immunization with OVA, mice were given control rat IgG or anti-CD1d mAb. One hour later, mice were subjected to sham or burn injury. Twenty-four hours later, spleen cells were collected and cultured in the presence or absence of OVA for 48 h, followed by a 16-h pulse with 3H-TdR. The bar graph represents the mean thymidine incorporation for each group expressed as DPM ± SEM. *, P < 0.05, versus sham + control (Ctrl) IgG; **, P < 0.05, versus sham + Ctrl IgG; ***, P < 0.05, versus burn + Ctrl IgG as determined by ANOVA and Neuman Keuls post-hoc analyses. N = 4. Data are representative of three experiments in which similar results were obtained.

 
It has been reported that anti-CD1d mAb treatment does not cause depletion of APCs in vivo [21 ]. However, as some clones of antibodies, when given i.v., can cause depletion of the target cells, and others do not, we questioned whether the effects of anti-CD1d mAb treatment on burn-induced immune suppression might be a result of depletion of APCs in vivo rather than blockade of CD1d-NKT cell signaling. To address this, parallel groups of mice were given anti-CD1d mAb or control IgG, and 24 h later, their spleens were collected and processed to single-cell suspensions. The splenocyte suspensions were then immunostained with antibodies that allowed for the detection of macrophages and dendritic cells (CyChrome-conjugated anti-F4/80 mAb and PE-conjugated anti-Mac3 mAb) and were examined by flow cytometry. We observed that there were no statistically significant differences in the numbers of F4/80 and/or Mac3-positive cells in the spleens of mice that received control IgG or anti-CD1d mAb systemically (Table 1 ). Thus, anti-CD1d mAb did not cause depletion of APCs.


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  		Table 1. Comparison of F4/80 and Mac3-Positive Cells in Splenocyte Suspensions from Anti-CD1d mAb Versus Control IgG-Treated Micea

 
In the experiments described above, we treated mice with anti-CD1d mAb 1 h before injury, followed by various assessments of immunity. Thus, the animals underwent injury and survived the subsequent 24 h with their NKT cell population presumably unable to be stimulated by CD1d+ APCs. To determine whether the T cell suppression could be overcome by blocking CD1d activation of NKT cells after injury, we collected splenocytes from sham or burn-injured mice that were immunized with OVA in CFA (s.c.) 7 days earlier. Splenocyte suspensions were plated in 96-well plates for 1 h in medium containing control IgG or anti-CD1d mAb (10 ng/ml). After 1 h of culture with the respective antibody, OVA (200 µg/ml) was added, and the cells were cultured for 48 h followed by a 16-h pulse with 3H-TdR. Unlike in vivo treatment with anti-CD1d mAb, blockade of CD1d on cells ex vivo did not prevent the injury-associated suppression of Ag-specific T cell proliferation (Fig. 3 ).



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  		Figure 3. Effects of in vitro CD1d blockade on injury-induced suppression of in vitro T cell-proliferative response to Ag. Seven days after immunization with OVA, mice were given sham or burn injury. Twenty-four hours later, spleen cells were collected and cultured in the presence or absence of control IgG or anti-CD1d mAb (10 ng/ml) for 1 h, after which OVA was added to the cultures for 48 h, followed by a 16-h pulse with 3H-TdR. The bar graph represents the mean thymidine incorporation for each group expressed as DPM ± SEM. *, P < 0.05, versus sham + control (Ctrl) IgG; **, P < 0.05, versus sham + anti-CD1d as determined by ANOVA and Neuman Keuls post-hoc analyses. N = 4. Data are representative of two experiments in which similar results were obtained.

 
NKT cell frequency in the spleen after injury
It is known that certain perturbations of the immune response can lead to changes in the frequency and/or function of CD1d-restricted NKT cells throughout the peripheral lymphoid system [17 , 21 ]. Although our previous studies showed no changes in the frequencies of conventional lymphocytes early after burn injury [32 ], others have reported injury-induced alterations in immune function that occur in part through changes in the distribution of immune cell subpopulations [35 ]. As activation of NKT cells contributed to injury-related suppression of immunity, and we did not examine NKT cells specifically in our earlier studies, we next examined whether the frequency or distribution of NKT cells was altered as a consequence of injury.

Mice were subjected to sham or burn injury, and 24 h later, their spleens were collected and processed to single-cell suspensions that were immunostained with a panel of fluorochrome-conjugated antibodies allowing for the identification of NKT cells as described previously [17 ]. The immunostained splenocytes were analyzed by flow cytometry, and NKT cells were identified as cells that coexpressed the CD3{varepsilon}, TCRß chain, and NK marker Ly-49C/I, all at intermediate levels. Although immune function was suppressed (Figs. 1 and 2) , there were no differences found in the frequencies of NKT cells in the spleens of sham versus burn-injured mice (Fig. 4A and 4B ).



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  		Figure 4. Flow cytometric evaluation of NKT cells and CD1d+ APCs in the spleens of sham versus burn-injured mice. Twenty-four hours after sham or burn injury, splenocyte suspensions were prepared and immunostained with anti-CD3{varepsilon}, anti-TCRß, and anti-Ly49C (clone 5E6) or anti-Mac3 and anti-CD1d and were examined by flow cytometry. (A) The dot plots represent cells first gated positively on the CD3{varepsilon}+ population. The percentage of NKT cells (identified as cells that coexpress TCRß and Ly49C) is indicated in the upper-left quadrant of each dot plot. (B) A summary of the groups. (C) A summary of immunostaining of spleen cells for each group that coexpressed Mac3 and CD1d. N = 4. Data are representative of two experiments in which similar results were obtained.

 
As NKT cells are stimulated through their invariant TCR by CD1d molecules expressed on different types of APCs, we also investigated whether injury led to changes in the number of Mac-3+ APCs that expressed CD1d or the relative magnitude of CD1d cell-surface expression. In doing so, we observed no differences in the magnitude of CD1d expression (not shown) or the number of CD1d+ APCs among sham versus burn-injured mice (Fig. 4C) . Thus, CD1d-NKT cell-dependent suppression of T cell immunity after injury is not a result of changes in the number of CD1d+ APCs or overall CD1d expression.

Blockade of CD1d activation of NKT cells prevents injury-induced, Ag-specific production of IL-4
As CD1d activation of NKT cells during the course of injury suppressed Ag-specific T cell responses in the absence of quantitative changes in the NKT cell or CD1d+ APC population, we next investigated whether the suppression correlated with immunomodulatory cytokine production. Briefly, single-cell suspensions of total splenocytes were prepared from mice that received sham or burn injury in conjunction with control rat IgG or anti-CD1d mAb (i.v.) 24 h earlier. After 24 h of culture in the presence or absence of OVA, supernatants were collected and assayed for IL-4 and IFN-{gamma} by ELISA.

We observed that OVA-stimulated splenocytes from burn-injured + control IgG-treated mice produced significantly greater (P<0.05) levels of IL-4 compared with splenocytes prepared from sham-injured + control IgG-treated mice (Fig. 5A ). In contrast, the levels of IL-4 produced by OVA-stimulated splenocytes from burn-injured + anti-CD1d mAb-treated mice were similar to sham-injured mice. We also observed that although OVA-stimulated splenocytes from sham-injured mice produced significant levels of IFN-{gamma}, splenocytes from burn-injured mice did not (Fig. 5B) . Anti-CD1d mAb treatment had no effect on the ability of splenocytes from burn-injured mice to produce IFN-{gamma}. We did note, however, that anti-CD1d mAb treatment reduced the IFN-{gamma} production by splenocytes obtained from sham-injured mice. Therefore, we conclude that during the course of injury, CD1d stimulation of NKT cells leads to the production of IL-4, which correlates with injury-associated suppression of Ag-specific T cell immunity in vitro and in vivo.



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  		Figure 5. Effects of systemic anti-CD1d mAb treatment on injury-induced, Ag-specific cytokine production. Seven days after immunization with OVA, mice were given sham or burn injury. Twenty-four hours later, spleen cells were cultured for 24 h ± OVA (200 µg/ml), after which the supernatants were collected. IL-4 (A) and IFN-{gamma} (B) content in the supernatants was determined by ELISA. The bar graphs represent the mean pg/ml cytokine ± SEM. *, P < 0.05, versus sham + control (Ctrl) IgG; **, P < 0.05, versus burn + Ctrl IgG as determined by ANOVA and Neuman Keuls post-hoc analyses. N = 4. Samples from each individual mouse were measured in duplicate, and data shown are representative of two experiments in which similar results were obtained.

 
NKT cells but not conventional T cells produce IL-4 early after injury
To address the cellular source of the IL-4 that was produced by splenocyte cultures early after injury, splenocytes were collected 24 h after sham or burn injury and control IgG or anti-CD1d mAb treatment and were cultured overnight with or without OVA. After 24 h of culture, the nonadherent fractions were removed and immunostained for TCRß and Ly49C, fixed, permeabilized, and stained for intracellular IL-4 or IFN-{gamma}. We observed that in response to burn injury, NKT cells but not conventional T cells increased their production of IL-4 (Fig. 6 and Table 2 ). Specifically, the number of IL-4-producing NKT cells averaged 15.5% in sham-injured mice and 59% in burn-injured mice (Table 2) . In contrast, only 30% of NKT cells from burn-injured, anti-CD1d-treated mice produced IL-4. We also observed that 58–64% of NKT cells from sham-injured mice produced IFN-{gamma}, and only 24–36% of NKT cells produced IFN-{gamma} after burn. IFN-{gamma} was also produced by conventional T cells from sham-injured mice in response to Ag, and its production was suppressed among T cells from burn-injured mice (data not shown). Treatment of burn-injured mice with anti-CD1d mAb did not restore the ability of NKT cells or conventional T cells to produce IFN-{gamma}. Therefore, we conclude that NKT cells but not conventional T cells serve as the source of IL-4 that is produced within the first 24 h after injury. Additionally, we report that in response to injury, NKT cells acquire a predominantly IL-4-producing phenotype rather than IFN-{gamma}.



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  		Figure 6. Production of IL-4 by T, NKT, and NK cells from sham versus burn-injured mice. Splenocytes were obtained 24 h after sham or burn injury and cultured for 24 h ± OVA. After culture, nonadherent cells were collected and immunostained with CyChrome-anti-TCRß, FITC-anti-Ly49C, and PE-anti-IL-4. IL-4 content among T cells (TCR+ Ly49C–) versus NKT cells (TCRß+ Ly49C+) versus NK cells (TCRß– Ly49C+) was determined by flow cytometry. Data shown are representative of two experiments in which similar results were obtained. N = 3 per group.

 

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  		Table 2. Comparison of IL-4-Positive, Splenic NKT, NK, and T Cells from Mice Given Systemic {alpha}-CD1d Prior to Burn Injurya

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report presents the unique finding that after burn injury, CD1d signaling of NKT cells contributes to the production of immunosuppressive cytokines and the suppression of systemic T cell responses. Using a murine burn-injury model, we demonstrated that in response to injury, NKT cells increased their production of IL-4 and decreased their production of IFN-{gamma}, which correlated with injury-associated suppression of immunity. More importantly, we showed that the production of NKT cell-derived IL-4 and injury-induced immune dysfunction could be prevented if the interaction between APC-expressed CD1d molecules and NKT cells was blocked by a mAb. As anti-CD1d mAb given systemically just before injury prevented T cell suppression, and treatment of cells with anti-CD1d 24 h after injury did not, we conclude that through the course of injury and in the first 24 h thereafter, NKT cells become activated by CD1d and facilitate the suppression of cellular immunity. The fact that suppression of T cell responsiveness could not be overcome by treatment of cells with anti-CD1d mAb in vitro after the injury had already taken place suggested to us that the NKT cell-dependent suppression of T cell immunity occurred very early after injury and that it was not reversible within the timeframe considered here.

It is apparent from clinical studies and experimental animal models that severe injury leads to pronounced defects in immune function, including increased proinflammatory cytokine production, increased Th2-like cytokine production, decreased lymphocyte proliferation and Ag recognition, and altered antibody production [15 , 36 37 38 39 ]. In particular, it has been reported by several investigators that a Th2 response develops among the T lymphocyte population with levels of IL-4 and IL-10 being elevated and levels of IL-2 and IFN-{gamma} being decreased relatively early after injury (1–3 days) [15 , 39 40 41 42 ]. Our current findings are in agreement with the concept of a predominantly Th2-like response early after injury, as we observed elevated production of IL-4 and decreased production of IFN-{gamma} within 24 h after injury. Moreover, our data add to the current understanding by showing that NKT cells are responsible for the IL-4 that is produced early after injury and that they undergo a shift in their cytokine profile from being primarily IFN-{gamma}-producing cells to mainly IL-4 producers. However, a shift in the cytokine response from Th1 to Th2 cannot always be used as a general indicator of immune status after injury, as our laboratory has previously reported immune defects that appear later (7–10 days) after injury in the presence of high levels of IFN-{gamma} [4 ]. Additionally, using a murine model of burn injury combined with acute ethanol exposure, we observed that IL-4 levels were actually suppressed and that administration of IL-4 to achieve levels similar to sham-injured mice actually restored immunity [43 ]. It should be noted, however, that the cytokines reported in those studies were induced by pan stimulation of T cells rather than via Ag-specific pathways. Moreover, in the present study, we observed that early after injury, IFN-{gamma} production by NKT and conventional T cells was suppressed and that it could not be restored by anti-CD1d mAb treatment. This suggests that the restoration of DTH and splenocyte proliferation after blockade of NKT cell activation occurs via non-IFN-{gamma}-dependent mechanisms. A likely candidate that could influence the in vitro-proliferative response as well as the in vivo-DTH response may be IL-2, although we have not yet measured this cytokine in our system.

Investigation of the innate-immune system’s role in injury-associated immune dysfunction has focused largely on macrophages and other APCs as well as granulocytes. In addition to APCs, other innate cells exist that are known to modulate immune function through a variety of mechanisms, including the rapid production of immunoregulatory cytokines. In particular, innate lymphocytes such as CD1d-restricted NKT cells and {gamma}{delta} T cells have recently emerged as cells having powerful, immunoregulatory abilities and are now known to be essential for the regulation of autoimmunity, tolerance, and cancer [21 , 22 , 44 ]. Roles for innate lymphocytes in the immune response to shock, sepsis, and injury are supported by recent reports by others [16 , 45 ]. Dieli and colleagues [45] reported that NKT cell-deficient mice [J{alpha}18-/- (formerly J{alpha}281) on C57BL/6 and BALB/c backgrounds] were resistant to the lipopolysaccharide-induced, systemic Shwartzman reaction, an experimental model of septic shock, and yet another study demonstrated the involvement of NKT cells in mortality following cecal ligation and puncture (CLP)-induced shock [46 ]. Using a murine model of thermal injury combined with CLP, Schwacha and colleagues [16 ] showed that another subset of innate lymphocytes, {gamma}{delta} T cells, contributed significantly to injury and sepsis-related immune dysfunction through the modulation of macrophage effector functions. Related to this, it was recently shown that IFN-{gamma} produced by CD1d-restricted NKT cells is required to activate macrophages to clear bacterial (Pseudomonas) infections [23 ]. Our experiments did not address the role of the NKT cells in survival from bacterial infection superimposed on burn injury. However, based on recent reports that indicate a role for NKT cells in defense against bacterial infections and sepsis, it might be predicted that NKT cells would be required for survival from burn plus infection. Although the role of NKT cells in susceptibility of burn-injured mice to infection is a topic of ongoing investigation by our laboratory, our current findings combined with those of others implicate NKT cells and perhaps other innate lymphocytes as being central to the regulation of the early stages of the immune response to injury. In addition to the roles of innate lymphocytes in peripheral lymphoid organs, future experimentation will also be required to examine the contribution of innate lymphocytes in other organs such as the liver, gut, and lung to injury-induced pathology.

The precise mechanism by which CD1d-NKT cell interactions contribute to injury-associated immune suppression remains to be defined. However, our studies indicate that interaction between NKT cells and CD1d+ APCs during the first 24 h after injury leads to the production of IL-4, a cytokine known to directly suppress the effector functions of T cells, macrophages, and other APCs and promote the differentiation of Th2 responses. The studies reported here illustrate the dependence of injury-associated IL-4 production on CD1d stimulation of NKT cells and support the concept that NKT cells serve as an initial, early source of this cytokine, which may further contribute to an environment that promotes the differentiation of a Th2 response. Whether the IL-4 that is produced after injury serves to directly modulate T cell function, suppress macrophage and other APC effector functions, or both remains to be defined.

Our present studies do not explain why unfractionated splenocytes cultured with OVA led to the production of IL-4 by NKT and not conventional T cells. In such a system, the OVA is presented to T cells by MHC-II expressed on APCs, and the NKT cells are stimulated by CD1d molecules expressed on the APCs. Our findings suggest that after exposure to Ag, APCs become stimulated to up-regulate cell-surface molecules and/or soluble factors that facilitate NKT cell activation differently in sham versus burn (i.e., bias the cytokine production toward IL-4 and perhaps other immunosuppressive cytokines). This idea is supported by other studies in which APCs exposed to OVA in the presence of IL-10 and/or TGF-ß (both of which are produced after burn) increased their expression of CD1d and stimulated NKT cells to promote suppression of T cell immunity (ref. [47 ], and Faunce et al., unpublished observations). Several previous reports have illustrated the central role of macrophages and other APCs in the immune suppression that occurs in the early, post-burn period (1–7 days) [12 , 32 , 48 49 50 ]. Although considerable efforts by our laboratory and others focused on the roles of soluble macrophage/APC-derived mediators in injury-associated suppression of T cell immunity, we expand on those previous findings here by showing that the APC is involved in burn-induced immune suppression through yet another mechanism, CD1d stimulation of NKT cells.

In summary, we present the novel finding that CD1d activation of NKT cells contributes to immune dysfunction that follows severe injury. Traumatic injuries, including burns, remain to be a leading cause of death in the United States, and recovery from such injuries is confounded by substantial immune suppression that renders individuals highly susceptible to bacterial, fungal, and viral infections. Further investigation into the cellular and molecular mechanisms involved in injury-associated immune suppression is needed so that we may begin to develop more effective treatments. The findings presented here suggest that targeting the activation of NKT cells or their interactions with CD1d+ APCs may be potential areas for therapeutic intervention.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from Loyola University Research Funding Committee (to D. E. F.) and National Institutes of Health (AA012034-S1 and AG18859; to E. J. K.). The authors gratefully acknowledge Ms. Jennifer Jarrett, Mr. Timothy Plackett, and Mr. Eric Schilling for their assistance with animal care and tissue collection. We also thank Ms. Patricia Simms in the Loyola University Flow Cytometry Core Facility for her assistance with flow cytometric analyses and interpretation of data.

Received November 11, 2002; revised January 28, 2003; accepted January 29, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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