Center for Surgical Research, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294
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ABSTRACT |
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The activation of a macrophage
(M)-dependent proinflammatory cascade following thermal injury
plays an important role in the development of immunosuppression and
increased susceptibility to subsequent sepsis in burn patients. In
contrast, although interleukin (IL)-10, an anti-inflammatory cytokine
that can downregulate M
activity, has also been implicated in
postburn immune dysfunction, its role in the regulation of M
function postburn remains unclear. To study this, C57BL/6 female mice
were subjected to a 25% total body surface area third-degree scald
burn, and splenic M
s were isolated 7 days later. Lipopolysaccharide
(LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-
, and
nitric oxide (NO) production were significantly increased in the burn
group compared with shams. Blockade of endogenous IL-10 activity
enhanced IL-6 and TNF-
release, but not NO release, in both groups.
The addition of exogenous IL-10 to the M
cultures dose dependently
suppressed production of these inflammatory mediators in both groups.
The timing of IL-10 addition to the cultures in relation to LPS
stimulation, however, was critical. The suppressive effect of exogenous
IL-10 was attenuated in both groups when the cells were exposed to
IL-10 at 4-6 h after LPS stimulation; however, M
s from injured
mice were significantly better able to maintain inflammatory
mediator-productive capacity. The resistance of M
s from injured mice
to IL-10-mediated suppression correlated with decreased IL-10 receptor
(IL-10R) expression and increased CD11b expression. These findings
suggest that M
s, following thermal injury, display resistance to
suppression by IL-10 due in part to downregulation of IL-10R expression.
interleukin-10 receptor; interleukin-6; tumor necrosis factor-; nitric oxide; immunosuppression
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INTRODUCTION |
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WIDESPREAD
SUPPRESSION of immune responses has been observed following
thermal injury (11, 15, 22, 26, 32, 39), which has been
implicated in the increased susceptibility to subsequent septic
complications (20, 30, 31, 41). Recent findings suggest
that altered expression of macrophage (M) function following thermal
injury is of fundamental importance in the development of immune
dysfunction under such conditions (9, 30, 39). The
activation of a proinflammatory cascade that includes nitric oxide
(NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-
with the
subsequent development of a systemic inflammatory response is important
to the pathogenesis of multiple organ failure, a significant
complication associated with thermal injury as well as other forms of
major trauma (5). Thermal injury is associated with
increased systemic levels of these inflammatory mediators (8,
12), and purified M
populations from thermally injured animals produce elevated levels of these mediators (13, 22, 37,
49). In contrast, other studies suggest that induction of an
anti-inflammatory response is central to the development of postburn
immunosuppression (18, 32, 50). An important mediator in
this anti-inflammatory cascade is the Th-2 cytokine IL-10. IL-10 is a
pleiotrophic cytokine produced by T lymphocytes, monocytes, M
s, B
lymphocytes, and keratinocytes (14, 24). One of the unique
actions of IL-10 is its ability to inhibit the production of
M
-derived proinflammatory cytokines (3, 4, 27, 44). The
observation that M
s are producers of IL-10 suggests that IL-10 may
be an important autocrine regulator of M
proinflammatory activity.
Findings from our laboratory indicate that Ms, following thermal
injury, produce elevated levels of IL-10 as well as proinflammatory mediators (36, 37, 40). It is unclear, however, why M
s, following thermal injury, are capable of producing elevated amounts of
both pro- and anti-inflammatory mediators. This dimorphic response of
M
s in which both the elevated production of anti- and
proinflammatory mediators exists after thermal injury may be related to
a resistance of M
s to the suppressive effect of IL-10. Loss of this
autocrine control of the proinflammatory response following thermal
injury would likely contribute to the expression of a hyperactive M
phenotype, as defined by the increased productive capacity for proinflammatory mediators, such as IL-6, TNF-
, and NO. In this regard, previous studies have implicated M
hyperactivity in postburn immune dysfunction (30, 37, 40). The aim of the present study, therefore, was to determine the role of IL-10 in the regulation of M
hyperactivity following thermal injury.
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MATERIALS AND METHODS |
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Animals. C57BL/6 female mice (18-20 g; 8-10 wk of age; Charles River) were used for all experiments. The mice were allowed to acclimatize to the animal facility for at least 1 wk before experimentation. Animals were randomly assigned into either a thermal injury group or a sham-treatment group. Three to seven mice per group were used for each experimental condition. The experiments in this paper were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. They were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Experimental thermal injury. Mice received a third-degree scald burn as previously described (37, 39). Briefly, the mice were anesthetized by methoxyflurane inhalant, and the dorsal surface was shaved. The animal was placed in a custom-insulated mold that exposed 12.5% of the total body surface area (TBSA) along the dorsum and was immersed in 70°C water for 7 s. The procedure was repeated on the opposite side of the dorsum, yielding a 25% TBSA burn. The mice were then resuscitated with 2 ml of Ringer lactate, administered intraperitoneally, and returned to their cages. The cages were placed on a heating pad (set on medium) for 2 h until the mice were fully awake, at which time they were returned to the animal facility. Sham treatment consisted of resuscitation with Ringer lactate only. Lethality in this thermal injury model was not significant.
Preparation of splenic macrophage cultures.
The mice were killed by methoxyflurane overdose at 7 days after injury,
and the spleens were removed aseptically. We have previously shown that
at 7 days postinjury, maximal immunosuppression and M hyperactivity
occurs (37, 39). Splenocyte suspensions were prepared in
complete media [RPMI 1640 containing 10% heat-inactivated fetal
bovine serum, 5 µg/ml gentimycin, and 100 µg/ml streptomycin and
penicillin (GIBCO BRL, Grand Island, NY)] as described elsewhere (39) at a concentration of 1 × 107
cells/ml. Splenic M
s were purified by adherence. Briefly, 1 × 107 splenocytes were added per well and allowed to adhere
for 2 h. Nonadherent cells were removed by vigorous washing with
warm PBS. More than 90% of the adherent cells displayed typical M
morphology. The adherent cells were cultured in a final volume of 500 µl/well that contained 1 µg/ml of lipopolysaccharide (LPS) and
various concentrations of murine IL-10 (BD Pharmingen, San Diego, CA). In other experiments, splenic M
cultures were treated with 150 ng/ml
of anti-IL-10 (JES5-2A5) or rat IgG1, an isotype control (BD
Pharmingen), along with LPS. On the basis of information provided by
the manufacturer, this concentration of anti-IL-10 represents 2.5 times
the concentration required to neutralize proliferation of the
IL-10-responsive cell line MC/9 by 50%.
Macrophage cytokine production.
Cell-free supernatants were harvested from splenic Ms after various
times in culture and were frozen at
80°C until analysis. Immunoreactive TNF-
, IL-6, and IL-10 present in the supernatants were determined by commercial sandwich ELISA according to the manufacturer's recommendations (OptiEIA, BD Pharmingen).
Macrophage-inducible nitric oxide synthase activity. Inducible nitric oxide synthase (iNOS) activity was determined by measuring the levels of nitrite, a stable degradation product of NO, in cell-free supernatants using the Greiss reaction as previously described (38, 39).
Flow cytometric analysis of macrophage IL-10 receptor and CD11b
expression.
Ms were stained with anti-CD11b fluorescein isothiocyanate (FITC)
and anti-IL-10 receptor (IL-10R) conjugated to phycoerythrin (PE). The
isotype controls were rat IgG2b
conjugated to FITC and IgG1
conjugated to PE. All antibodies were purchased from BD Pharmingen.
Briefly, splenic M
s were unstimulated or cultured with LPS (1 µg/ml) for 2, 4, or 6 h. The cells were scraped up using a
rubber policeman and were washed 1× with PBS/0.1% sodium azide by
centrifugation at 4°C. The cells were incubated with 10 µg Fc Block
(BD Pharmingen) per milliliter of PBS/0.1% sodium azide for 15 min at
4°C in a final volume of 200 µl. The cells were labeled with ~1
µg of each antibody per 1,000,000 cells by adding 1 µg each of
monoclonal anti-CD11b conjugated to FITC and monoclonal anti-IL-10R
conjugated to PE or 1 µg each of respective isotype controls per
tube. Samples were incubated for 30 min on ice in the dark and then
washed 2× with PBS/0.1% sodium azide. Samples were kept on ice in the
dark, and all measurements were analyzed within 30 min after completing
the staining procedure. FITC and PE were analyzed with a Becton
Dickinson FACSort flow cytometer (San Jose, CA) fitted with a 488-nm
argon laser with filter settings for FITC (530-nm-wide band-pass
filter) and PE (575-nm diachronic filter). Appropriate instrument
settings and spectral compensations were made and not changed during
analysis of samples. Stability of the settings and compensations was
regularly checked. A minimum of 10,000 events was assessed using
log-amplified fluorescence signals and linearly amplified side- and
forward-scatter signals. PC-lysis version 1.0 software (Becton
Dickinson) was used to analyze the results.
Systemic IL-10 levels.
Whole blood was obtained by cardiac puncture from mice at 7 days
postinjury or 7 days after the sham procedure and was placed in
Microtainer tubes (Becton Dickinson). The tubes were then
centrifuged at 16,000 g for 15 min at 4°C. Plasma was
transferred to microcentrifuge tubes that were stored at 80°C.
Splenic tissue samples were also obtained from the same animals, snap
frozen in liquid nitrogen, and stored at
80°C until assayed. Before
determination of IL-10 levels, spleen samples were homogenized in
complete protease inhibitor cocktail (Boehringer, Lewes, UK), and
protein levels were determined by Bio-Rad DC protein assay (Bio-Rad,
Hercules, CA). IL-10 plasma and splenic tissue levels were then
determined by commercial sandwich ELISA according to the
manufacturer's recommendations (OptiEIA, BD Pharmingen).
Statistical analysis. Data are expressed as means ± SE of 3-6 animals per group from two independent experiments. Comparisons were analyzed using Student's t-test or the Mann-Whitney's U-test for multiple comparisons. P < 0.05 was considered to be statistically significant for all analyses.
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RESULTS |
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Macrophage IL-10 production.
The ability of splenic Ms to produce IL-10 was assessed by culturing
the cells for 1.5-48 h (Fig.
1). IL-10 levels in the M
supernatants
increased over time in both groups. However, M
s from injured mice
produced significantly (P < 0.05) higher levels of
IL-10 than M
s from sham-treated mice.
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Regulation of macrophage proinflammatory mediator release by IL-10.
The autocrine regulatory effect of IL-10 on M NO, IL-6, and TNF-
was assessed by inhibiting endogenous IL-10 activity with anti-IL-10
(Fig. 2). Splenic M
s from thermally
injured mice produced significantly higher (P < 0.05)
levels of NO, IL-6, and TNF-
, than sham-treated mice in response to
LPS stimulation. Anti-IL-10 did not significantly alter NO production
in either group compared with IgG isotype control (Fig. 2A).
In contrast, an approximate twofold increase in IL-6 production by
M
s from both groups was observed when anti-IL-10 was added to the
culture media compared with IgG isotype control (P < 0.05; Fig. 2B). IL-6 production, however, remained
significantly higher (P < 0.05) in the injury group
compared with sham cells with anti-IL-10 treatment. Anti-IL-10 treatment also significantly increased (P < 0.05)
TNF-
production in both groups to a comparable degree (Fig.
2C).
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Macrophage IL-10R and CD11b expression.
IL-10R expression was determined in Ms at various times following
LPS stimulation by fluorescence-activated cell sorter analysis (Fig.
5A). In the absence of LPS
stimulation, IL-10R expression was approximately twofold higher
(P < 0.05) on M
s from sham mice compared with the
injury group. At 2 h after LPS stimulation, IL-10R expression
increased (P < 0.05) in the injury group to levels
comparable with shams. IL-10R expression 4 h after LPS stimulation
in the sham group was significantly (P < 0.05) higher than that of unstimulated sham cells. In contrast, in the injury group,
IL-10R expression had returned to unstimulated levels. IL-10R
expression in the sham group returned to unstimulated levels by 6 h after LPS stimulation but remained significantly (P < 0.05) higher than the injury group (i.e., similar to unstimulated
cells).
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Systemic IL-10 levels postinjury.
At 7 days postinjury, IL-10 was not detectable in the plasma (Table
1). While significant levels of IL-10
were observed in the spleen of both sham and injured mice, they were
not significantly different.
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DISCUSSION |
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It is well established that suppressed cell-mediated immunity is a
major complication associated with thermal injury (1, 15,
26). While a general loss of immunoresponsiveness postburn has
been implicated in the subsequent increase in susceptibility to sepsis
and infection, recent evidence suggests that activation of a
M-dependent proinflammatory cascade is central to the development of
subsequent deleterious complications such as multiple organ failure
(5). Thus induction of a hyperactive M
phenotype, associated with increased productive capacity for proinflammatory mediators, is important in the development of postburn immune dysfunction (30, 37, 40). In contrast, recent findings
also point to the induction of an anti-inflammatory response that
includes the Th-2 cytokine IL-10 as central to the development of
postburn immune dysfunction (18, 32, 50). With regard to
function of M
s, IL-10 suppresses activation, inhibits their capacity
to release cytokines, and inhibits their ability to serve as accessory cells for T cell activation (24, 25).
Our present findings indicate that thermal injury induces a M
phenotype that is associated with increased productive capacity for
both proinflammatory (NO, IL-6, and TNF-
) and anti-inflammatory (IL-10) mediators. Consistent with the findings of De Waal Malefyt et
al. (4), IL-10 produced by M
s acted in an autocrine
manner to partially attenuate the proinflammatory response under both normal and pathological conditions (i.e., postburn). The suppressive effect of IL-10, however, was more pronounced in the sham group, because in the presence of IL-10, iNOS activity and TNF-
productive capacity remained elevated in the injury group. Moreover, while delayed
exposure of the cells to IL-10 following LPS stimulation attenuated the
suppressive effect of IL-10 in both groups, cells from injured animals
were better able to maintain proinflammatory secretory activity. These
findings indicate that following thermal injury, M
s are partially
resistant to IL-10-mediated suppression. Further analysis of this
response demonstrated that the desensitized response in the injury
group correlated with lower IL-10R expression and elevated CD11b expression.
While IL-10 is primarily thought of as a T cell-derived cytokine, it is
also produced by Ms as well as other cell types (14, 24). Studies suggest that M
-derived IL-10 plays an important role in the autoregulation of M
function (19, 35). Our
findings indicate that endogenous IL-10 differential regulates iNOS
activity and cytokine-productive capacity, since inhibition of IL-10
activity with antibodies upregulated LPS-stimulated IL-6 and TNF-
release, but not NO release. Thus while the hyperactive M
phenotype
following thermal injury is associated with elevated productive
capacity for these inflammatory mediators, their autoregulation by
IL-10 differs. Evidence indicates that the inhibitory effects of IL-10 are primarily at the level of gene expression due to decreased transcription (7, 45). Our findings here are consistent
with this concept, since delayed addition of IL-10 to the culture
media, thus allowing gene induction by LPS, reduced the inhibitory
effect of IL-10 on proinflammatory mediator release in both groups.
IL-10's suppressive effect on M function is via a specific cell
surface receptor. IL-10R activation causes translocation of the nuclear
transcription factor STAT3 to the nucleus, where it binds to the
promoter region of IL-10-responsive genes (6). One such
gene is SOCS-3 (suppressor of cytokine signaling-3) (42). IL-10 rapidly induces SOCS-3 expression and may, in part, explain how
IL-10 inhibits induction of LPS-inducible proinflammatory genes such as
iNOS, TNF-
, and IL-6 in M
s (17). In contrast, O'Farrell et al. (29) suggest that the IL-10 suppression
of proinflammatory genes is independent of STAT3. Our findings point to
the downregulation of IL-10R expression as a potential causative factor
in the resistance of M
s to suppression by IL-10 postinjury. To date,
this is the first study to examine alterations in IL-10R expression
following traumatic injury. The mechanism responsible for decreased
IL-10R expression postinjury is unclear but appears to be independent
of systemic IL-10 levels at 7 days postinjury, since we did not observe
IL-10 in the plasma or increased IL-10 in the spleen. Evidence for the
downregulation of IL-10R expression under pathological conditions comes
from studies by MacDonald et al. (21), in which rheumatoid
synovial dendritic cells were resistant to the immunosuppressive
effects of IL-10 that correlated with decreased IL-10R expression.
Early studies also suggest that synovial monocytes are less susceptible
to the immunosuppressive effects of IL-10 than peripheral blood
monocytes (2, 16). Thus the mechanism by which resistance
to IL-10 is induced in M
s postburn may be similar to that observed
in rheumatoid arthritis, an autoimmune disease also associated with
M
hyperactivity.
Previous studies have shown that prolonged exposure (6-48 h) of
Ms to LPS did not alter IL-10R expression (19, 46). In contrast, our present studies have shown that following LPS
stimulation, there was a rapid but transient upregulation of IL-10R
expression 2-4 h after stimulation, but by 6 h after LPS
stimulation, IL-10R expression had returned to normal in both groups. A
rapid increase in IL-10R expression on M
s following inflammatory
stimuli is consistent with a potent autoregulatory role for this
cytokine, since increased receptor expression would increase the
responsiveness of the cell to IL-10. We observed an approximate twofold
upregulation of IL-10R expression in the sham group at 4 h after
LPS stimulation; however, IL-10R expression in the injury group had
already returned to baseline values by this time. When M
s were
exposed to exogenous IL-10 at this time after LPS stimulation, the
injury group was more resistant to suppression than the sham group. It
is likely that decreased IL-10R expression in the injury group was also associated with decreased STAT3 and SOCS-3 activation, allowing for
elevated proinflammatory gene expression. The notion that differences
in IL-10 responsiveness between the sham and injury groups is due to
altered receptor expression is further supported by the observation
that when IL-10 was added to the cultures at the same time as LPS, the
relative degree of suppression was similar in both groups. While in the
absence of stimulation, IL-10R expression was lower in the injury
group; early after stimulation (2 h), receptor expression was
comparable in both cell populations, thus allowing the exogenously
added IL-10 to suppress both cell populations to a comparable degree.
The differences between IL-10 responsiveness in the sham and injury
groups may be related to factors other than cell surface expression of
IL-10R. For example, IL-10 resistance could be related to an uncoupling
of downstream signal transduction components such as STAT3 and SOCS-3
from the cell surface IL-10R. The ability of IL-10 to inhibit
LPS-induced gene expression is also JAK1 dependent (6).
Activation of STAT3 and JAK1 is mediated by tyrosine phosphorylation; therefore, alterations in tyrosine kinase activity might be causative factors in IL-10 resistance. Additionally, a decreased protein expression of STAT3, JAK1, or SOCS-3, independent of relative IL-10R
cell surface expression, could also contribute to IL-10 resistance
postinjury. Thus even though the present study has shown a strong
inverse correlation between IL-10R expression and IL-10 resistance,
other factors likely contribute to IL-10 resistance postinjury.
Moreover, a component of the LPS-induced activation in the injury group
did not involve IL-10-mediated regulation because NO and TNF-
release remained greater than the sham group in the presence of high
concentrations of IL-10.
CD11b (MAC-1, CR3) is expressed on leukocytes as a
2-intergrin in association with CD18. Compared with
other leukocytes, M
s predominantly express CD11b/CD18 heterodimers
(33). In the present study, M
s from injured mice were
in a higher activation state, based on increased CD11b expression
(10). Increased CD11b expression is also associated with
systemic inflammation and monocyte activation in vivo (34,
43). In our study, the increased CD11b expression in the injury
group inversely correlated with IL-10R expression, suggesting that
"activated" M
s express lower levels of IL-10R. Interestingly,
LPS stimulation did not increase CD11b expression in the injury group,
whereas sham M
CD11b expression was upregulated in a time-dependent
manner. Previous studies have shown that LPS upregulates CD11b
expression on monocytes (10). The reason for the lack of
upregulation of CD11b following LPS stimulation in the injury group may
be due to the fact that CD11b expression was already maximal. The
CD11b/CD18 heterodimer appears to work in concert with CD14 and toll
receptors to elicit optimal LPS responses in M
s (33).
In fact, the CD11/CD18 intergins were originally defined as LPS
receptors (48). Our observation that the increased CD11b
expression correlated with enhanced LPS-stimulated inflammatory
mediator production in the injury group is consistent with this
concept. Moore et al. (23) have also demonstrated that
~50% of the CD14-independent response to particle-bound LPS was
mediated by CD11b/CD18. This finding leads to the speculation that
increased CD11b expression postinjury could perpetuate a hyperactive
M
response to opsonized bacteria. Interestingly, Wooten et al.
(47) have shown that STAT3 activation upregulates CD11b
expression, suggesting a relationship to IL-10. IL-10R ligation leads
to STAT3 activation (6), thus suggesting that IL-10 might be responsible for the increased CD11b expression. Nemoto et al. (28) have shown that resident peritoneal M
s preexposed
to IL-10 (presumably causing STAT3 activation and increased CD11b
expression) displayed elevated iNOS activity in response to LPS
stimulation. Alternatively, the increased response of IL-10-pretreated
cells might also be related to downregulation of IL-10R and loss of autocrine control. While we did not observe elevated levels of IL-10 in
the plasma or spleen at 7 days postinjury, this does not preclude the
possibility that IL-10 levels were elevated earlier postinjury, causing
the observed differences in CD11b expression between the sham and
injury M
s. Yeh et al. (50) have recently shown that
IL-10 plasma levels are markedly increased at 5-20 h postburn,
indicating an early, but transient, systemic rise in IL-10. Moreover,
Sato et al. (35) have shown that IL-10 levels in healing
wounds were markedly elevated 3 h after injury but returned to
normal by 24 h postinjury. It remains to be determined whether an
early increase in systemic IL-10 postburn influences subsequent M
phenotype and responsiveness to this cytokine.
In conclusion, our findings presented here indicate that the
hyperactive proinflammatory M phenotype observed following thermal injury is in part due to downregulation of autocrine and paracrine control of the proinflammatory response by IL-10. Further studies will
be required to more precisely elucidate the causative relationship between IL-10, IL-10R expression, STAT3, and M
hyperactivity following thermal injury.
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ACKNOWLEDGEMENTS |
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We thank Lucretia Vickers for valuable technical assistance.
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FOOTNOTES |
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This work was supported by National Institute of General Medical Sciences Grant GM-58242.
Address for reprint requests and other correspondence: M. G. Schwacha, Univ. of Alabama at Birmingham, Center for Surgical Research, G094 Volker Hall, 1670 Univ. Blvd., Birmingham, AL 35294-0019 (E-mail: Martin.Schwacha{at}ccc.uab.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 26 March 2001; accepted in final form 29 May 2001.
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