Hypertonicity rescues T cells from suppression by trauma-induced anti-inflammatory mediators

William H. Loomis, Sachiko Namiki, David B. Hoyt, and Wolfgang G. Junger

Surgical Immunology Research Laboratory, Division of Trauma, Department of Surgery, University of California San Diego, San Diego, California 92103-8236


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Trauma causes the release of anti-inflammatory factors thought to cause infections by inhibiting T cells. We have found that hypertonic saline (HS) enhances functions of normal T cells. Here we studied if HS can rescue T cells from suppression by costimulating interleukin (IL)-2 production. Human peripheral blood mononuclear cells were treated with the immunosuppressive factors IL-4, IL-10, transforming growth factor (TGF)-beta 1, and PGE2 and with serum of trauma patients and stimulated with phytohemagglutinin, and IL-2 production was measured. Costimulation with HS tripled IL-2 production of normal cells. IL-4, IL-10, TGF-beta 1, and PGE2 suppressed IL-2 production with IC50 of 500, 1, 36,000, and 0.01 pg/ml, respectively. Costimulation of suppressed cells with HS restored IL-2 production and increased IC50 values >70-fold. Serum from trauma patients could completely suppress normal cells; however, costimulation with HS restored IL-2 production by up to 80% of the control response. These findings show that HS can restore the function of suppressed T cells, suggesting that HS resuscitation of trauma patients could reduce posttraumatic sepsis.

interleukin-2 expression; immune suppression; p38 mitogen-activated protein kinase; small volume resuscitation; T lymphocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T CELLS PLAY AN important role in the host defense against invading organisms. The activation of T cells requires signals that originate from the T cell receptor (TCR) and from coreceptors like CD28. Signals from the TCR are triggered by its interaction with antigen that is presented by antigen-presenting cells (APC), whereas the CD28 coreceptor is stimulated by its interaction with B7.1/B7.2 molecules on the APC. Under appropriate stimulation conditions, a complex system of intracellular signal pathways initiates a precisely orchestrated sequence of activation steps that include interleukin (IL)-2 transcription and expression and ultimately result in T cell proliferation.

Trauma, hemorrhage, and major injury are known to suppress T cell function (12, 17, 38, 53, 55). This results in immunosuppression that is thought to predispose patients to a high incidence of septic complications and multiple organ failure (11, 12, 53). The cellular mechanisms leading to posttraumatic T cell suppression are not fully understood. However, trauma has been shown to result in altered T cell signaling and gene transcription and to suppress IL-2 mRNA transcription and IL-2 protein expression (10, 12, 18, 46).

These changes in T cell responses have been attributed to anti-inflammatory mediators such as IL-4, IL-10, transforming growth factor-beta 1 (TGF-beta 1), and PGE2. A number of studies have reported elevated levels of these mediators in the circulation of trauma patients (2, 3, 10, 11, 28, 32, 34-36, 39). These anti-inflammatory factors can block T cell function by directly interfering with T cell activation signals or by affecting accessory cell function, thus depriving T cells of necessary costimulatory signals (3, 4, 7, 16, 30, 43, 54, 56). In either case, the failure of T cells to properly respond to cell stimulation increases the risk of infections and sepsis, which remain leading causes of posttraumatic mortality despite the aggressive use of antibiotics.

Hypertonic saline (HS; e.g., 7.5% NaCl) solutions can be used for the resuscitation of trauma patients. At the usual dose of 4 ml/kg body wt, HS resuscitation increases plasma tonicity of trauma patients by up to 40 mM (30). We have previously reported that equivalent levels of hypertonicity markedly enhance IL-2 expression and proliferation of mitogen-stimulated T cells from normal subjects (19, 21, 23). Here we investigated if HS can restore the function of T cells that are suppressed by anti-inflammatory mediators known to cause immunosuppression in trauma patients.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and cell stimulation. Peripheral blood mononuclear cells (PBMC) were isolated from the heparinized blood of healthy human volunteers using Ficoll-Paque density gradient centrifugation (Pharmacia LKB, Piscataway, NJ). Cell viability was >98% as determined with trypan blue dye exclusion. PBMC were brought to a concentration of 1 × 106/ml in RPMI-1640 (Roswell Park Memorial Institute 1640 medium; Irvine Scientific, Santa Ana, CA) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated FCS (Irvine Scientific) and were cultured in 96-well tissue culture plates (200 µl/well) at 37°C in an incubator containing humidified air with 5% CO2. Cells were incubated for 1 h with or without IL-4, IL-10, TGF-beta 1, PGE2, or trauma serum at different doses and were stimulated with 1 µg/ml phytohemagglutinin (PHA; HA 16; Murex Biotech, Dartford, UK); IL-2 release into the supernatants was measured after 20 h. Human recombinant IL-4 and IL-10, TGF-beta 1 from human platelets, and PGE2 were from Calbiochem (La Jolla, CA).

Jurkat T cells (clone E6-1) from American Type Culture Collection (Rockville, MD) were maintained in RPMI-1640 (Irvine Scientific) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin [University of California San Diego (UCSD) core facility, La Jolla, CA], and 10% (vol/vol) heat-inactivated FCS (Irvine Scientific). Cells (105/ml) were pretreated for 1 h with or without 20 µM SB-203580 (Calbiochem) and were stimulated in the presence or absence of HS with a combination of 0.5 µg/ml PHA and 1 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma Chemical, St. Louis, MO). IL-2 production was determined by measuring IL-2 release after 20 h with the ELISA technique described below.

MAPK p38 activation. After the stimulation experiments, Jurkat cells (106 cells/sample) were placed on ice, centrifuged, resuspended in 100 µl SDS sample buffer containing 100 mM dithiothreitol, and lysed by boiling for 5 min. The cell lysates were separated by SDS-PAGE using 8-16% Tris/glycine polyacrylamide gradient gels (Novex, San Diego, CA). Lysed proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA), and the membranes were subjected to immunoblotting. Phosphospecific antibodies recognizing the phosphorylated (on Thr180/Tyr182) and thereby activated form of mitogen-activated protein kinase (MAPK) p38 were obtained from New England Biolabs (Beverly, MA) and were used according to the manufacturer's recommendations. Antibodies recognizing both activated and inactive MAPK p38 were from Santa Cruz Biotechnology (Santa Cruz, CA), and secondary antibody conjugates and the ECL assay kit were from Amersham Pharmacia Biotech (Piscataway, NJ). These reagents were used according to the manufacturer's instructions. In some experiments, MAPK p38 phosphorylation was determined by immunoprecipitation of cell lysates with PY20 anti-phosphotyrosine antibodies (Santa Cruz), followed by SDS-PAGE and immunoblotting with MAPK p38 antibodies (Santa Cruz) as described previously (20).

Trauma patients and serum samples. Serum samples were collected from eight severely injured trauma patients who were admitted to the Trauma Service of the UCSD Medical Center. The injury severity scores of the patients ranged from 41 to 75 points with an average of 55 ± 5 (means ± SE). Blood samples were drawn within 1 wk after admission to the UCSD Trauma Center, and serum samples were stored at -70°C until use. PBMC of healthy subjects were preincubated for 1 h with these serum samples at a final concentration of 5%, or as indicated. Cells were stimulated as above, and IL-2 release was measured.

Hypertonic stimulation. Cells were subjected to HS at 40 mM hypertonicity (unless otherwise indicated) by adding 40 µl/ml of a 1 M solution of NaCl in RPMI. The cell cultures were immediately mixed after the addition of the concentrated NaCl solution to minimize the chances of creating transient increases of osmolarity in excess of the desired levels of hypertonicity. All materials and compounds used in these experiments were sterile and endotoxin free.

IL-2 expression. IL-2 production was measured with an ELISA method using monoclonal mouse anti-human IL-2 as a primary antibody (clone 5355.111), biotinylated goat anti-human IL-2 as secondary antibody (both from R&D Systems, Minneapolis, MN), recombinant human IL-2 as standard (Genzyme Diagnostics, Cambridge, MA), and horseradish peroxidase-conjugated streptavidin (Zymed Laboratories, San Francisco, CA). The ELISA method was performed according to the recommendations provided by R&D Systems. Depending on the cell preparation used, day-to-day variations in the IL-2 response were observed. IL-2 levels of cells that were stimulated under isotonic conditions ranged from 870 ± 17 to 1,360 ± 250 (SD) pg/ml. The IL-2 released by cells that were costimulated with 40 mM HS ranged from 1,390 ± 120 to 2,570 ± 50 (SD) pg/ml. IL-2 production of nonstimulated cells and of cells that were exposed to 40 mM HS was 0.5 ± 2 (SE) pg/ml, with little variations between different cell preparations.

T cell proliferation assay. PBMC were suspended in RPMI supplemented as described above and were plated in flat-bottom 96-well tissue culture plates at a final concentration of 105 cells/well. The cells were stimulated with 0.5 µg/ml PHA, incubated for 3 days at 37°C in an atmosphere consisting of humidified air with 5% CO2, pulsed with 1 µCi/well [methyl-3H]thymidine (NEN Life Science Products, Boston, MA), and incubated for another 18 h. Then the cultures were harvested on glass fiber filter discs using a PHD Cell Harvester (Cambridge Technology, Watertown, MA). The filter discs were transferred to scintillation vials, dried overnight, and covered with 4 ml/vial of scintillation fluid (ScintiVerse BD; Fisher Scientific, Fair Lawn, NJ). Incorporated thymidine was counted with a model 1219 RackBeta liquid scintillation counter (LKB Instruments, Gaithersburg, MD). Data are expressed as counts per minute and represent the average of triplicate measurements.

Statistical analyses. Unless otherwise indicated, data are presented as means ± SD. Sets of data were compared with Student's t-test, using P < 0.05 as the level of significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HS augments IL-2 production and proliferation of T cells by activating MAPK p38. HS at clinically relevant levels rapidly phosphorylated the MAPK p38 of Jurkat T cells (Fig. 1A) and of PBMC (Fig. 1B). We have previously hypothesized that HS could augment T cell function by activating MAPK p38 (19, 20). Here we found that pretreatment of Jurkat T cells with the specific MAPK p38 inhibitor SB-203580 significantly reduced the enhancing effect of HS on IL-2 production (Fig. 2, A and B). This suggests that HS may enhance T cell function directly by costimulating cells via MAPK p38. In support of this conclusion, we found that HS also costimulated IL-2 production of PHA-stimulated human PBMC (Fig. 3A). This effect was dose dependent, and enhancement of IL-2 production was a nearly linear function of the HS level. At hypertonicity levels >60 mM, IL-2 production gradually decreased; HS levels of 100 mM and greater blocked IL-2 production (Fig. 3B). The effect of HS on IL-2 production was closely paralleled by its effect on T cell proliferation of PHA-stimulated PBMC (Fig. 3B). Together these findings suggest that HS augments T cell proliferation by costimulating IL-2 production via MAPK p38.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   Hypertonic saline (HS) activates mitogen-activated protein kinase (MAPK) p38 in human T cells. A: human Jurkat T cells were stimulated with the indicated levels of hypertonicity by adding appropriate volumes of 1 M NaCl. At the time points shown, MAPK p38 activation was determined by immunoblotting with phosphospecific anti-MAPK p38 antibodies (p-MAPK p38). To control for equal protein loading, the membranes were stripped and reprobed with antibodies that recognize phosphorylated and nonphosphorylated MAPK p38 (lanes on bottom; total MAPK p38). B: hypertonic stimulation activated MAPK p38 in human peripheral blood mononuclear cells (PBMC). PBMC were isolated from healthy human volunteers and stimulated with the indicated levels of hypertonicity for 10 min, and MAPK p38 activation was determined by immunoprecipitation with anti-phosphotyrosine proteins and immunoblotting with anti-MAPK p38 as described in MATERIALS AND METHODS. Total cell lysate (TCL) was included as a positive control.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   MAPK p38 inhibitor SB-203580 reduces enhanced interleukin (IL)-2 production in response to hypertonic costimulation. A: Jurkat T cells were pretreated for 1 h with the indicated doses of the MAPK p38 inhibitor SB-203580 in DMSO (black-triangle and ) or with equivalent volumes of DMSO vehicle alone (open circle ) and were stimulated with a combination of phytohemagglutinin (PHA)-phorbol 12-myristate 13-acetate (PMA) in the presence ( and open circle ) or absence (black-triangle) of the 40 mM hypertonicity. Data were compared with Student's t-test (n = 3). *P < 0.005 and **P < 0.001, HS vs. no HS and DMSO control. B: Jurkat cells were pretreated for 1 h with 20 µM SB-203580 (filled bars) or with DMSO vehicle alone (open bars) and were stimulated with a combination of PHA-PMA in the presence of the indicated levels of hypertonicity. After 20 h, IL-2 production was determined by measuring IL-2 released in the supernatants. Data were expressed as means ± SD of triplicate samples, and groups were compared with Student's t-test (n = 3). *P < 0.005 and **P < 0.001. The results are representative of 3 separate experiments.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   HS augments IL-2 production and proliferation of peripheral human T cells. Human PBMC were exposed to increasing levels of hypertonicity by adding increasing volumes of a 1 M NaCl solution. Next, the cells were stimulated with the mitogen PHA, and IL-2 production and T cell proliferation were measured. A: hypertonicity levels between 0 and 80 mM markedly increased IL-2 production of PBMC that were stimulated with 0.5 () or 1 (open circle ) µg/ml PHA. B: at higher HS levels, IL-2 production gradually decreased (open circle ). The pattern of HS-induced enhancement of IL-2 production closely resembles that of T cell proliferation (). Samples were done in triplicate, and data are expressed as means ± SD. Data were compared with isotonic controls (0 mM HS) using Student's t-test (n = 3). **P < 0.001. Figs. 1-9 are representative of at least 3 experiments with cells from different donors.

HS and IL-4 cooperate in costimulating IL-2 production of PHA-stimulated PBMC. IL-4 only partially reduced PHA-stimulated IL-2 production of PBMC. IL-4 at doses between 200 and 700 pg/ml reduced IL-2 production by nearly 50%, whereas higher or lower concentrations of IL-4 had no discernable effects (Fig. 4). Addition of 40 mM HS doubled IL-2 production of untreated cells from 650 ± 9 to 1,500 ± 52 pg/ml. Interestingly, IL-2 production doubled once more in the presence of HS and IL-4, quadrupling the IL-2 response of control cells that were stimulated with PHA alone (2,600 ± 260 vs. 650 ± 9 pg/ml, respectively). Plasma IL-4 levels of trauma patients were reported to reach values of up to 250 pg/ml during the first day after multiple blunt injury and were found to be associated with the severity of injury and outcome (9, 29). These levels of IL-4 suppressed PHA-induced IL-2 production of PBMC by no more than 50%. However, costimulation with HS not only restored the IL-2 response of cells in the presence of such IL-4 levels but it quadrupled IL-2 production.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   HS and IL-4 cooperate in costimulating IL-2 production of HS-treated cells. PBMC were treated with the indicated doses of recombinant human IL-4 for 1 h. Next, the cells were stimulated with 1 µg/ml of PHA in the presence () or absence (open circle ) of 40 mM HS. After incubation for 20 h, IL-2 production was determined by measuring the concentration of IL-2 released in the culture supernatants. Samples were done in triplicate, and data are expressed as means ± SD of the percentage of the full IL-2 response under isotonic conditions. Data were compared with Student's t-test (n = 3). All data points differed significantly (Student's t-test; n = 3; P < 0.001). The results shown are representative of 3 individual experiments with different cell preparations.

HS reduces the suppressive effects of IL-10. Exposure of PBMC to IL-10 markedly reduced IL-2 production in response to PHA stimulation. IL-10 at 1 pg/ml suppressed the IL-2 response by 50%. IL-2 production was completely blocked at IL-10 concentrations of >= 700 pg/ml (Fig. 5). However, when cells were stimulated in the presence of 40 mM HS, the suppressive activity of IL-10 was reduced 1,000-fold, and IL-10 at the highest dose tested was not able to completely suppress IL-2 production. IL-10 plasma levels are elevated during the first day after trauma, reaching plasma levels of ~400 pg/ml (29). These IL-10 levels blocked IL-2 production by >90% under isotonic conditions. However, in the presence of HS, the same IL-10 levels suppressed IL-2 production by a maximum of 30%.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   HS reduces suppression of IL-2 production by IL-10. Human PBMC were pretreated with the indicated doses of IL-10 for 1 h and stimulated with 1 µg/ml PHA under isotonic (open circle ) or hypertonic (40 mM; ) conditions, and IL-2 production was determined. IL-10 at 1 pg/ml suppressed IL-2 production by >50%. In the presence of 40 mM HS, >1,000 times higher IL-10 levels were needed to suppress IL-2 production by 50%. Data were expressed as means ± SD of triplicate samples and were compared with the Student's t-test (n = 3). *P < 0.05 and **P < 0.001. Figure is representative of 3 experiments with different cell preparations.

HS prevents suppression of IL-2 production by TGF-beta 1. Pretreatment of PBMC with increasing levels of TGF-beta 1 gradually suppressed IL-2 production in response to PHA stimulation (Fig. 6). However, TGF-beta 1 inhibited the IL-2 response of PBMC by only 54 ± 7% of the control value, even at TGF-beta 1 doses of >10,000 pg/ml. Hypertonic costimulation of TGF-beta 1-treated cells restored IL-2 production to levels beyond the level of isotonic controls (Fig. 6). Similar to IL-4, TGF-beta 1 doses of 300 pg/ml and greater seemed to act synergistically with HS in augmenting the IL-2 response of PHA-stimulated PBMC. In trauma patients, TGF-beta 1 plasma levels can reach 350 pg/ml (29). This TGF-beta 1 dose reduced the IL-2 production in our experiment to 60% of controls. However, addition of 40 mM HS increased IL-2 production to 200% of isotonic controls.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   HS prevents TGF-beta 1-induced suppression of IL-2 production. PBMC were pretreated with the indicated doses of TGF-beta 1 for 1 h and stimulated with 1 µg/ml PHA under isotonic (open circle ) or hypertonic (40 mM; ) conditions, and IL-2 production was determined. TGF-beta 1 at the highest dose tested (12,500 pg/ml) suppressed IL-2 production by <50% of control values. In the presence of 40 mM HS (), TGF-beta 1 did not suppress IL-2 production but enhanced it to two times the levels of controls. Data are expressed as means ± SD of triplicate samples and were compared with Student's t-test (n = 3). *P < 0.05 and **P < 0.001. Results are representative of 3 separate experiments with different cell preparations.

HS reduces suppression of IL-2 production by PGE2. PGE2 was among the first mediators found to suppress T cell responsiveness in trauma patients (10). PGE2 concentrations as low as 0.01 pg/ml reduced IL-2 production by >50% (Fig. 7). Higher PGE2 levels gradually diminished IL-2 production, with complete suppression at doses >1 µg/ml. HS markedly reduced the suppression of IL-2 production by PGE2. HS restored IL-2 production to isotonic control values or higher at PGE2 levels of up to 10,000 pg/ml. In trauma patients, plasma PGE2 levels of up to 88 ± 20 pg/ml have been reported (33). In our experiment, this PGE2 concentration reduced IL-2 production to 25% of the isotonic control response. Under these conditions, 40 mM HS completely abrogated the suppressive effect of PGE2 and further increased IL-2 production 40% beyond the response of isotonic controls.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   HS reduces PGE2-induced suppression of IL-2 production. PBMC were pretreated for 1 h with PGE2 at the indicated doses and stimulated with 1 µg/ml PHA under isotonic (open circle ) or hypertonic (40 mM; ) conditions, and IL-2 production was determined. Data were expressed as means ± SD of triplicate determinations. Data points were compared with Student's t-test (n = 3), and the results shown are representative of 3 separate experiments. *P < 0.05 and **P < 0.001.

HS restores IL-2 production of cells exposed to serum from trauma patients. The data shown above indicate that costimulation with HS can restore normal IL-2 function of cells that are exposed to anti-inflammatory mediators found in the plasma of trauma patients. HS completely restored the function of cells that were treated with clinically relevant doses of IL-4, TGF-beta 1, and PGE2 and partially recovered the function of cells that were exposed to IL-10 at doses found in the circulation of trauma patients. Thus HS resuscitation could restore T cell function by recovering the ability of T cells to produce IL-2.

However, posttraumatic immunosuppression is thought to be the consequence of multiple anti-inflammatory factors that are released in the circulation of trauma patients (21, 22). Therefore, we examined if HS can restore IL-2 production of cells exposed to serum samples obtained from severely injured trauma patients. At a concentration of 5% (vol/vol), serum from trauma patients reduced IL-2 production to 2.4 ± 1.0% (mean ± SE) of control values (Fig. 8). Serum samples at concentrations of 10-20% (vol/vol) suppressed IL-2 production by >50% (Fig. 9). Addition of 40 mM HS markedly reduced the suppressive effects of the serum samples from patients, increasing IL-2 production from 2.4 to 40.8 ± 10.0% of control values. The degree of restoration of the IL-2 response by HS varied from patient to patient, and the serum concentrations needed for 50% suppression of IL-2 production increased >10 times when 40 mM HS was present (Fig. 9).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   HS restores IL-2 production in the presence of serum from trauma patients. PBMC from healthy volunteers were incubated for 1 h with 5% (vol/vol) of serum samples obtained from 8 severely injured trauma patients. Cells were stimulated with 1 µg/ml PHA in the presence (filled bars) or absence (open bars) of 40 mM HS, and IL-2 production was measured and expressed as a percentage of the response of control cells without serum samples. Different serum samples are identified by the injury severity score (ISS) of their corresponding trauma patients. Samples were done in triplicate, and values are expressed as means ± SD. All data differed significantly from the isotonic controls (cont; Student's t-test, n = 3; P < 0.05).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 9.   Effectiveness of HS in restoring IL-2 production differs between patients. PBMC were pretreated for 1 h with dilutions of serum samples from 3 different trauma patients with ISS of 41 (top), 47 (middle), and 75 (bottom). Cells were stimulated with 1 µg/ml PHA in the presence () or absence (open circle ) of 40 mM HS, and IL-2 production was measured and expressed as a percentage of the response of control cells without serum samples. Data of triplicate determinations were expressed as means ± SD and were compared with Student's t-test (n = 3). *P < 0.05 and **P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Despite aggressive therapeutic and surgical interventions, sepsis and organ failure remain leading causes for the morbidity and mortality among those trauma patients who survive the initial injury. Because of this, much effort has been devoted to finding novel therapeutic approaches to prevent the development of these complications, which are thought to be caused by the patient's own immune response to trauma.

Circulating anti-inflammatory factors like PGE2, TGF-beta 1, IL-4, and IL-10 are thought to cause posttraumatic immunosuppression and sepsis. These mediators impair T cell function either directly by affecting activation signaling of T cells or indirectly by impairing accessory cell functions. PGE2 interacts with the corresponding membrane receptor of T cells, which results in the increase of intracellular cAMP, a second messenger that activates protein kinase A, which can block activation signals triggered by the TCR (45). PGE2 appears to block T cell function by interfering with IL-2 gene expression at multiple stages (1, 6, 37). Exogenous IL-2 can restore T cell proliferation, indicating that PGE2 inhibits T cells by blocking signals that lead to IL-2 expression (37). We found that HS can largely overcome the suppression of T cells by PGE2. Thus HS seems to supplement activation signals that are lost upon exposure of T cells to PGE2.

Trauma has been shown to upregulate the release of PGE2 in the circulation for up to 3 wk, and PGE2 plasma levels of up to 100 pg/ml have been reported (10, 33). Hypertonic costimulation fully recovered IL-2 production of cells that were pretreated for 1 h with these levels of PGE2, indicating that HS resuscitation may be suitable to prevent the suppressive effects of PGE2 after trauma.

IL-10 is another powerful anti-inflammatory mediator that is released in the circulation of individuals who sustain injury from trauma, burns, or sepsis (2, 3, 24, 29, 34). The role of IL-10 as a mediator of sepsis is substantiated by the fact that inactivation of IL-10 increased survival in a mouse model of pneumonia (13). IL-10 directly inhibits the proliferation and IL-2 production of T cells (8, 48). In addition, IL-10 has been shown to induce long-term antigen-specific anergy in CD4+ T cells (14). IL-10 can reach plasma levels of 500 pg/ml in trauma patients and 8,000 pg/ml in baboons after gram-negative sepsis (22, 29). Our results have shown that corresponding IL-10 levels can bring IL-2 production to a complete halt. Costimulation with HS, however, restored IL-2 production, at least in part. IL-10 can inhibit nuclear factor-kappa B activation by monocytes (51). Thus IL-10 may not only affect T cell function directly but also indirectly by interfering with the accessory cell functions of monocytes. It is possible that the combination of these phenomena could lead to an overwhelming suppression of T cell function that cannot be fully compensated by HS.

Elevated TGF-beta 1 levels were found to correlate with posttraumatic immunosuppression (31). TGF-beta 1 exerts multiple inhibitory effects on T cells and accessory cells like monocytes/macrophages (42, 50). In contrast to PGE2 and IL-10, TGF-beta 1 only moderately suppressed IL-2 production in our experiments. We have previously found that TGF-beta 1 fully suppressed T cell proliferation with an IC50 value of 800 pg/ml (21). Although these experiments were done with cells from a different species, the 45-fold difference in the suppressive activity of IL-2 production and proliferation suggests that TGF-beta 1 affects T cell function predominantly at an activation step subsequent to IL-2 expression. This is in agreement with findings of Bright et al. (5) who showed that TGF-beta 1 prevents IL-2-induced cellular signaling in T cells by inhibiting tyrosine phosphorylation and activation of Jak-1 and Stat-5 (5).

Similar to TGF-beta 1, IL-4 had only a limited suppressive effect on IL-2 expression in our experiments. This is perhaps less surprising, since IL-4 is known to inhibit immune responses mostly by affecting macrophage functions (40, 49). For example, IL-4 directly inhibits the secretion of IL-1beta , tumor necrosis factor-alpha , and IL-6 by monocytes (49). When cells were stimulated with HS in the presence of IL-4, a synergistic costimulatory effect on IL-2 production was observed. The reasons for this remarkable costimulation are unclear at this time and should be the focus of future studies. IL-4 plasma levels of up to 250 pg/ml were measured in trauma patients, and IL-4 plasma levels were found to be associated with the severity of injury and the clinical outcome (9). At these levels, IL-4 suppressed IL-2 production in our in vitro assay by no more than one-half. However, in the presence of IL-4, HS increased IL-2 production to levels four times higher than those of control cells. This phenomenon may be important for trauma patients with elevated IL-4 levels, because HS resuscitation could vastly increase T cell responsiveness.

The anti-inflammatory mediators found in the circulation of trauma patients can block T cell activation by inhibiting crucial signaling steps that lead to IL-2 production. Our in vitro data have shown that clinically relevant doses of HS can restore IL-2 production of cells that were exposed to clinically relevant levels of anti-inflammatory mediators (Table 1). We did not attempt to determine the composition of anti-inflammatory mediators in the serum samples from the trauma patients used in this study. However, previous work has shown that the contribution of a large number of different known and unknown anti-inflammatory factors cause immunosuppression in trauma patients (11, 17, 21, 24, 25). The composition of these different factors can vary from patient to patient. Accordingly, we found that the effectiveness of hypertonic costimulation to restore IL-2 responsiveness from the suppression by different serum samples from trauma patients also varied from patient to patient.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   IC50 and plasma levels of anti-inflammatory mediators and the effect of HS

In our in vitro experiments, HS did not fully restore IL-2 production of cells that were exposed for 1 h to trauma serum. It is possible that HS resuscitation of trauma patients could be more potent in restoring T cell function, because HS is administered immediately after trauma. Under these circumstances, HS may be able to more efficiently intervene and prevent the inhibitory effects of the anti-inflammatory mediators released after trauma. In addition, the serum samples in our experiment were obtained from trauma patients who sustained massive injuries, and the potential of HS to restore T cell function seemed to be inversely proportional to the level of injury severity.

We have previously observed that hypertonicity levels similar to those levels found in HS-resuscitated trauma patients can markedly affect leukocyte functions by interfering with activation signal pathways (19, 20, 23). Hypertonic stimulation of neutrophils and T cells activates specific signaling pathways that involve MAPK p38. MAPK p38 is a signaling enzyme that is closely related to high osmolarity glycerol response kinase-1 (HOG-1), a yeast protein known to allow yeast cells to respond to osmotic stimulation and to survive osmotic shock (15, 41, 52). This close relationship between HOG-1 and MAPK p38 suggests that mammalian cells may have retained the ability to detect and respond to changes in extracellular tonicity. However, although yeast cells are faced with wide variations of the extracellular osmolarity in their environment, mammalian leukocytes are comparatively sheltered by the more stable tonicity of blood plasma. Thus the osmosensing system of mammalian leukocytes may have evolved to allow the regulation of mammalian immune cells by relatively subtle changes of plasma tonicity.

We found that hypertonicity activates MAPK p38 in a dose-dependent fashion and concurrently augments IL-2 production and T cell proliferation. This and the fact that the MAPK p38 inhibitor SB-203580 reduced the enhancing effects of HS on IL-2 production (Fig. 2B) provide compelling evidence that MAPK p38 may play a central role in the enhancing effects of HS. However, recent findings have shown that SB-203580 may be less specific than previously thought (27). Thus the data shown in Fig. 2B could be explained by such nonspecific actions of SB-203580, and additional work will be needed to ultimately delineate the potential roles of other signaling enzymes aside from MAPK p38 in the hypertonic enhancement of T cell functions.

MAPK p38 mediates cytokine expression of Th1 effector T cells, and inhibition of MAPK p38 has been shown to block the production of several cytokines, including IL-4, IL-10, IL-2, and interferon-gamma (26, 44, 47). This suggests that HS could augment IL-2 expression by reinforcing activation signaling pathways via MAPK p38. On the other hand, HS-induced MAPK p38 activation could restore the function of suppressed T cells, which are exposed to anti-inflammatory mediators that can block MAPK p38 signaling. Thus, depending on the intracellular mechanisms whereby different trauma-induced anti-inflammatory mediators block T cell activation, HS may be able to recover T cell function at varying degrees.

Yet, although our in vitro findings indicate that HS may enhance T cell functions via MAPK p38, it should be recognized that MAPK p38 could also upregulate the release of anti-inflammatory cytokines like IL-4 and IL-10 (26, 44, 47). This could oppose the enhancing effects of hypertonic costimulation in vivo, and the ultimate response of T cells in a clinical setting is difficult to predict from our in vitro data alone but has to be determined with additional studies.

In summary, our results demonstrate that the suppression of early activation events that lead to IL-2 production can be partially overcome in vitro by hypertonic costimulation of T cells. These findings suggest that the modulation of plasma tonicity, for example, with HS resuscitation, could be useful as a clinical tool to enhance T cell function and prevent sepsis in trauma patients. However, detailed studies are needed to identify the best dosage and timing of HS resuscitation to take full advantage of the potential therapeutic effectiveness of HS as a treatment for sepsis in the intensive care unit. We think that the data presented here may serve as a foundation on which such future studies could be based.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Amnon Altman for helpful suggestions and Crystal Herdon-Remelius and Rachel Lubong for excellent technical support.


    FOOTNOTES

This work was supported in part by National Institute of General Medical Sciences Grants R29 GM-51477 and 1R01GM-60475-01A1 (to W. G. Junger) and Office of Naval Research Grant N00014-00-1-0851 (to W. G. Junger).

Address for reprint requests and other correspondence: W. G. Junger, Surgical Immunology Research Laboratory, Dept. of Surgery, Division of Trauma, Univ. of California San Diego Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8236 (E-mail: wjunger{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 December 2000; accepted in final form 4 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anastassiou, ED, Paliogianni F, Balow JP, Yamada H, and Boumpas DT. Prostaglandin E2 and other cyclic AMP-elevating agents modulate IL-2 and IL-2Ralpha gene expression at multiple levels. J Immunol 148: 2845-2852, 1992[Abstract/Free Full Text].

2.   Ayala, A, Deol ZK, Lehman DL, Herdon CD, and Chaudry IH. Polymicrobial sepsis but not low-dose endotoxin infusion causes decreased splenocyte IL-2/IFN-gamma release while increasing IL-4/IL-10 production. J Surg Res 56: 579-585, 1994[ISI][Medline].

3.   Ayala, A, Lehman DL, Herdon CD, and Chaudry IH. Mechanism of enhanced susceptibility to sepsis following hemorrhage. Interleukin-10 suppression of T-cell response is mediated by eicosanoid-induced interleukin-4 release. Arch Surg 129: 1172-1178, 1994[Abstract].

4.   Ayala, A, Perrin MM, and Chaudry IH. Defective macrophage antigen presentation following haemorrhage is associated with the loss of MHC class II (Ia) antigens. Immunology 70: 33-39, 1990[ISI][Medline].

5.   Bright, JJ, Kerr LD, and Sriram S. TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes. J Immunol 159: 175-183, 1997[Abstract].

6.   Choudhry, MA, Ahmed Z, and Sayeed MM. PGE2-mediated inhibition of T cell p59fyn is independent of cAMP. Am J Physiol Cell Physiol 277: C302-C309, 1999[Abstract/Free Full Text].

7.   De, AK, Kodys K, Puyana JC, Fudem G, Pellegrini J, and Miller-Graziano CL. Only a subset of trauma patients with depressed mitogen responses have true T cell dysfunctions. Clin Immunol Immunopathol 82: 73-82, 1997[ISI][Medline].

8.   De Waal Malefyt, R, Yssel H, and de Vries JE. Direct effects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Specific inhibition of IL-2 production and proliferation. J Immunol 150: 4754-4765, 1993[Abstract/Free Full Text].

9.   DiPiro, JT, Howdieshell TR, Goddard JK, Callaway DB, Hamilton RG, and Mansberger AR, Jr. Association of interleukin-4 plasma levels with traumatic injury and clinical course. Arch Surg 130: 1159-1163, 1995[Abstract].

10.   Faist, E, Mewes A, Baker CC, Strasser T, Alkan SS, Rieber P, and Heberer G. Prostaglandin E2 (PGE2)-dependent suppression of interleukin alpha (IL-2) production in patients with major trauma. J Trauma 27: 837-848, 1987[ISI][Medline].

11.   Faist, E, Schinkel C, and Zimmer S. Update on the mechanisms of immune suppression of injury and immune modulation. World J Surg 20: 454-459, 1996[ISI][Medline].

12.   Faist, E, Schinkel C, Zimmer S, Kremer JP, Von Donnersmarck GH, and Schildberg FW. Inadequate interleukin-2 synthesis and interleukin-2 messenger expression following thermal and mechanical trauma in humans is caused by defective transmembrane signalling. J Trauma 34: 846-854, 1993[ISI][Medline].

13.   Greenberger, MJ, Strieter RM, Kunkel SL, Danforth JM, Goodman RE, and Standiford TJ. Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia. J Immunol 155: 722-729, 1995[Abstract].

14.   Groux, H, Bigler M, de Vries JE, and Roncarolo MG. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells. J Exp Med 184: 19-29, 1996[Abstract].

15.   Han, J, Lee J-D, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808-811, 1994[ISI][Medline].

16.   He, X, and Stuart JM. Prostaglandin E2 selectively inhibits human CD4+ T cells secreting low amounts of both IL-2 and IL-4. J Immunol 163: 6173-6179, 1999[Abstract/Free Full Text].

17.   Hensler, T, Hecker H, Heeg K, Heidecke CD, Bartels H, Barthlen W, Wagner H, Siewert JR, and Holzmann B. Distinct mechanisms of immunosuppression as a consequence of major surgery. Infect Immun 65: 2283-2291, 1997[Abstract].

18.   Horgan, AF, Mendez MV, O'Riordain DS, Holzheimer RG, Mannick JA, and Rodrick ML. Altered gene transcription after burn injury results in depressed T-lymphocyte activation. Ann Surg 220: 342-352, 1994[ISI][Medline].

19.   Junger, WG, Coimbra R, Liu FC, Herdon-Remelius C, Junger W, Junger H, Loomis W, Hoyt DB, and Altman A. Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients? Shock 8: 235-241, 1997[ISI][Medline].

20.   Junger, WG, Hoyt DB, Hamreus M, Liu FC, Herdon-Remelius C, Junger W, and Altman A. Hypertonic saline activates protein tyrosine kinases and mitogen-activated protein kinase p38 in T-cells. J Trauma 42: 437-445, 1997[ISI][Medline].

21.   Junger, WG, Hoyt DB, Liu FC, Loomis WH, and Coimbra R. Immunosuppression after endotoxin shock: the result of multiple anti-inflammatory factors. J Trauma 40: 702-709, 1996[ISI][Medline].

22.   Junger, WG, Hoyt DB, Redl H, Liu FC, Loomis WH, Davies J, and Schlag G. Tumor necrosis factor antibody treatment of septic baboons reduces the production of sustained T-cell suppressive factors. Shock 3: 173-178, 1995[ISI][Medline].

23.   Junger, WG, Liu FC, Loomis WH, and Hoyt DB. Hypertonic saline enhances cellular immune function. Circ Shock 42: 190-196, 1994[ISI][Medline].

24.   Klava, A, Windsor AC, Farmery SM, Woodhouse LF, Reynolds JV, Ramsden CW, Boylston AW, and Guillou PJ. Interleukin-10. A role in the development of postoperative immunosuppression. Arch Surg 132: 425-429, 1997[Abstract].

25.   Koller, M, Clasbrummel B, Kollig E, Hahn MP, and Muhr G. Major injury induces increased production of interleukin-10 in human granulocyte fractions. Langenbecks Arch Surg 383: 460-465, 1998[ISI][Medline].

26.   Koprak, S, Staruch MJ, and Dumont FJ. A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production. Cell Immunol 192: 87-95, 1999[ISI][Medline].

27.   Lali, FV, Hunt AE, Turner SJ, and Foxwell BMJ The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem 275: 7395-7402, 2000[Abstract/Free Full Text].

28.   Mack, VE, McCarter MD, Naama HA, Calvano SE, and Daly JM. Dominance of T-helper 2-type cytokines after severe injury. Arch Surg 131: 1303-1309, 1996[Abstract].

29.   Majetschak, M, Börgermann Waydhas JC, Obertacke U, Nast-Kolb D, and Schade FU. Whole blood tumor necrosis factor-alpha production and its relation to systemic concentrations of interleukin 4, interleukin 10, and transforming growth factor-beta1 in multiply injured blunt trauma victims. Crit Care Med 28: 1847-1853, 2000[ISI][Medline].

30.   Mattox, KL, Maningas PA, Moore EE, Mateer JR, Marx JA, Aprahamian C, Burch JM, and Pepe PE. Prehospital hypertonic saline/dextran infusion for post-traumatic hypotension. The U S A multicenter trial. Ann Surg 213: 482-491, 1991[ISI][Medline].

31.   Meert, KL, Ofenstein JP, Genyea C, Sarnaik AP, and Kaplan J. Elevated transforming growth factor-beta concentration correlates with posttrauma immunosuppression. J Trauma 40: 901-906, 1996[ISI][Medline].

32.   Meert, KL, Ofenstein JP, and Sarnaik AP. Altered T cell cytokine production following mechanical trauma. Ann Clin Lab Sci 28: 283-288, 1998[Abstract].

33.   Menges, T, Engel J, Welters I, Wagner RM, Little S, Ruwoldt R, Wollbrueck M, and Hempelmann G. Changes in blood lymphocyte populations after multiple trauma: association with posttraumatic complications. Crit Care Med 27: 733-740, 1999[ISI][Medline].

34.   Miller-Graziano, CL, De AK, and Kodys K. Altered IL-10 levels in trauma patients' MPhi and T lymphocytes. J Clin Immunol 15: 93-104, 1995[ISI][Medline].

35.   Miller-Graziano, CL, Fink M, Wu JY, Szabo G, and Kodys K. Mechanisms of altered monocyte prostaglandin E2 production in severely injured patients. Arch Surg 123: 293-299, 1988[Abstract].

36.   Miller-Graziano, CL, Szabo G, Griffey K, Mehta B, Kodys K, and Catalano D. Role of elevated monocyte transforming growth factor beta (TGF-beta ) production in posttrauma immunosuppression. J Clin Immunol 11: 95-102, 1991[ISI][Medline].

37.   Minakuchi, R, Wacholtz MC, Davis LS, and Lipsky PE. Delineation of the mechanism of inhibition of human T cell activation by PGE2. J Immunol 145: 2616-2625, 1990[Abstract/Free Full Text].

38.   Napolitano, LM, Koruda MJ, Meyer AA, and Baker CC. The impact of femur fracture with associated soft tissue injury on immune function and intestinal permeability. Shock 5: 202-207, 1996[ISI][Medline].

39.   O'Sullivan, ST, Lederer JA, Horgan AF, Chin DH, Mannick JA, and Rodrick ML. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection. Ann Surg 222: 482-492, 1995[ISI][Medline].

40.   Paludan, SR. Interleukin-4 and interferon-gamma: the quintessence of a mutual antagonistic relationship. Scand J Immunol 48: 459-468, 1998[ISI][Medline].

41.   Posas, F, and Saito H. Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276: 1702-1705, 1997[Abstract/Free Full Text].

42.   Prud'homme, GJ, and Piccirillo CA. The inhibitory effects of transforming growth factor-beta-1 (TGF-beta 1) in autoimmune diseases. J Autoimmun 14: 23-42, 2000[ISI][Medline].

43.   Puyana, JC, Pellegrini JD, De AK, Kodys K, Silva WE, and Miller CL. Both T-helper-1- and T-helper-2-type lymphokines are depressed in posttrauma anergy. J Trauma 44: 1037-1046, 1998[ISI][Medline].

44.   Rincón, M, Enslen H, Raingeaud J, Recht M, Zapton T, Su MS, Penix LA, Davis RJ, and Flavell RA. Interferon-gamma expression by Th1 effector T cells mediated by the p38 MAP kinase signaling pathway. EMBO J 17: 2817-2829, 1998[Abstract/Free Full Text].

45.   Roper, RL, and Phipps RP. Prostaglandin E2 regulation of the immune response. Adv Prostaglandin Thromboxane Leukot Res 22: 101-111, 1994[Medline].

46.   Sayeed, MM. Alterations in cell signaling and related effector functions in T lymphocytes in burn/trauma/septic injuries. Shock 5: 157-166, 1996[ISI][Medline].

47.   Schafer, PH, Wadsworth SA, Wang L, and Siekierka JJ. p38alpha Mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+ CD45RO+ T cells and Th2 effector cells. J Immunol 162: 7110-7119, 1999[Abstract/Free Full Text].

48.   Taga, K, Mostowski H, and Tosato G. Human interleukin-10 can directly inhibit T-cell growth. Blood 81: 2964-2971, 1993[Abstract].

49.   TeVelde, AA, Huijbens RJF, Heije K, De Vries JE, and Figdor CG. Interleukin-4 (IL-4) inhibits secretion of IL-1beta , tumor necrosis factor alpha , and IL-6 by human monocytes. Blood 76: 1392-1397, 1990[Abstract].

50.   Tsunawaki, S, Sporn M, Ding A, and Nathan C. Deactivation of macrophages by transforming growth factor-beta. Nature 334: 260-262, 1988[ISI][Medline].

51.   Wang, P, Wu P, Siegel MI, Egan RW, and Billah MM. Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem 270: 9558-9563, 1995[Abstract/Free Full Text].

52.   Wurgler-Murphy, SM, and Saito H. Two-component signal transducers and MAPK cascades. Trends Biochem Sci 22: 172-176, 1997[ISI][Medline].

53.   Xu, YX, Ayala A, and Chaudry IH. Prolonged immunodepression after trauma and hemorrhagic shock. J Trauma 44: 335-341, 1998[ISI][Medline].

54.   Zedler, S, Bone RC, Baue AE, von Donnersmarck GH, and Faist E. T-cell reactivity and its predictive role in immunosuppression after burns. Crit Care Med 27: 66-72, 1999[ISI][Medline].

55.   Zellweger, R, Ayala A, DeMaso CM, and Chaudry IH. Trauma-hemorrhage causes prolonged depression in cellular immunity. Shock 4: 149-153, 1995[ISI][Medline].

56.   Zhu, XL, Zellweger R, Zhu XH, Ayala A, and Chaudry IH. Cytokine gene expression in splenic macrophages and Kupffer cells following haemorrhage. Cytokine 7: 8-14, 1995[ISI][Medline].


Am J Physiol Cell Physiol 281(3):C840-C848
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society