Surgical Immunology Research Laboratory, Division of Trauma, Department of Surgery, University of California San Diego, San Diego, California 92103-8236
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ABSTRACT |
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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)-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-
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
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INTRODUCTION |
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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-1 (TGF-
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.
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MATERIALS AND METHODS |
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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-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-
1 from human platelets, and PGE2
were from Calbiochem (La Jolla, CA).
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.
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RESULTS |
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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.
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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.
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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%.
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HS prevents suppression of IL-2 production by TGF-1.
Pretreatment of PBMC with increasing levels of TGF-
1
gradually suppressed IL-2 production in response to PHA stimulation (Fig. 6). However, TGF-
1
inhibited the IL-2 response of PBMC by only 54 ± 7% of the
control value, even at TGF-
1 doses of >10,000 pg/ml.
Hypertonic costimulation of TGF-
1-treated cells restored
IL-2 production to levels beyond the level of isotonic controls (Fig.
6). Similar to IL-4, TGF-
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-
1 plasma levels can reach 350 pg/ml
(29). This TGF-
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.
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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.
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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-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.
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DISCUSSION |
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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-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-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-1 levels were found to correlate with
posttraumatic immunosuppression (31). TGF-
1
exerts multiple inhibitory effects on T cells and accessory cells like
monocytes/macrophages (42, 50). In contrast to
PGE2 and IL-10, TGF-
1 only moderately suppressed IL-2 production in our experiments. We have previously found
that TGF-
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-
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-
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-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-1
, tumor necrosis factor-
, 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.
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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- (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.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Amnon Altman for helpful suggestions and Crystal Herdon-Remelius and Rachel Lubong for excellent technical support.
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FOOTNOTES |
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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.
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