Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury

Martin G. Schwacha, Christian P. Schneider, Kirby I. Bland, and Irshad H. Chaudry

Center for Surgical Research, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The activation of a macrophage (Mphi )-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 Mphi activity, has also been implicated in postburn immune dysfunction, its role in the regulation of Mphi 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 Mphi s were isolated 7 days later. Lipopolysaccharide (LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-alpha , 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-alpha release, but not NO release, in both groups. The addition of exogenous IL-10 to the Mphi 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, Mphi s from injured mice were significantly better able to maintain inflammatory mediator-productive capacity. The resistance of Mphi 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 Mphi 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-alpha ; nitric oxide; immunosuppression


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Mphi ) 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)-alpha 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 Mphi 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, Mphi s, B lymphocytes, and keratinocytes (14, 24). One of the unique actions of IL-10 is its ability to inhibit the production of Mphi -derived proinflammatory cytokines (3, 4, 27, 44). The observation that Mphi s are producers of IL-10 suggests that IL-10 may be an important autocrine regulator of Mphi proinflammatory activity.

Findings from our laboratory indicate that Mphi s, following thermal injury, produce elevated levels of IL-10 as well as proinflammatory mediators (36, 37, 40). It is unclear, however, why Mphi s, following thermal injury, are capable of producing elevated amounts of both pro- and anti-inflammatory mediators. This dimorphic response of Mphi s in which both the elevated production of anti- and proinflammatory mediators exists after thermal injury may be related to a resistance of Mphi 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 Mphi phenotype, as defined by the increased productive capacity for proinflammatory mediators, such as IL-6, TNF-alpha , and NO. In this regard, previous studies have implicated Mphi 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 Mphi hyperactivity following thermal injury.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Mphi 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 Mphi 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 Mphi 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 Mphi 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 Mphi s after various times in culture and were frozen at -80°C until analysis. Immunoreactive TNF-alpha , 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. Mphi s were stained with anti-CD11b fluorescein isothiocyanate (FITC) and anti-IL-10 receptor (IL-10R) conjugated to phycoerythrin (PE). The isotype controls were rat IgG2bkappa conjugated to FITC and IgG1kappa conjugated to PE. All antibodies were purchased from BD Pharmingen. Briefly, splenic Mphi 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Macrophage IL-10 production. The ability of splenic Mphi s to produce IL-10 was assessed by culturing the cells for 1.5-48 h (Fig. 1). IL-10 levels in the Mphi supernatants increased over time in both groups. However, Mphi s from injured mice produced significantly (P < 0.05) higher levels of IL-10 than Mphi s from sham-treated mice.


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Fig. 1.   Splenic macrophages (Mphi s) were isolated from sham and burned mice 7 days postinjury and cultured for 1.5-48 h with lipopolysaccharide (LPS; 1 µg/ml). Data are expressed as means ± SE for 3-4 mice per group from 2 independent experiments. *P < 0.05 compared with sham. IL, interleukin.

Regulation of macrophage proinflammatory mediator release by IL-10. The autocrine regulatory effect of IL-10 on Mphi NO, IL-6, and TNF-alpha was assessed by inhibiting endogenous IL-10 activity with anti-IL-10 (Fig. 2). Splenic Mphi s from thermally injured mice produced significantly higher (P < 0.05) levels of NO, IL-6, and TNF-alpha , 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 Mphi 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-alpha production in both groups to a comparable degree (Fig. 2C).


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Fig. 2.   Splenic Mphi nitrite (A), IL-6 (B), and tumor necrosis factor (TNF)-alpha (C) release was determined in the presence and absence of LPS (1 µg/ml), IgG isotype control (150 ng/ml), and anti-IL-10 (150 ng/ml). Mphi cultures were maintained for 48 h for the determination of nitrite and IL-6 and 24 h for the determination of TNF-alpha . Data are expressed as means ± SE for 4-5 mice per group from 2 independent experiments. *P < 0.05 compared with sham; dagger P < 0.05 compared with IgG.

When exogenous IL-10 was added to the culture media, a dose-dependent suppression in the production of NO, IL-6, and TNF-alpha was observed in both groups (Fig. 3). The suppressive effect of IL-10 was maximal at a concentration of 25 U/ml. While NO production was suppressed by IL-10, it remained significantly higher (P < 0.05) in the injury group at all concentrations of IL-10 compared with shams (Fig. 3A). In contrast, IL-6 (Fig. 3B) and TNF-alpha (Fig. 3C) production by Mphi s from injured mice was suppressed by IL-10 to a level that was comparable with the sham group.


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Fig. 3.   Splenic Mphi nitrite (A), IL-6 (B), and TNF-alpha (C) release was determined in response to LPS (1 µg/ml) in the presence of various concentrations of exogenous IL-10. Mphi cultures were maintained for 48 h for the determination of nitrite and IL-6 and for 24 h for the determination of TNF-alpha . Data are expressed as means ± SE for 4-6 mice per group from 2 independent experiments. *P < 0.05 compared with sham; dagger P < 0.05 compared with 0 U/ml IL-10.

The timing of the exposure of splenic Mphi s to exogenous IL-10 following LPS stimulation markedly altered its suppressive effect (Fig. 4). IL-10-mediated suppression of NO production was attenuated when the cells were treated with IL-10 at 4 or 6 h after LPS stimulation (Fig. 4A). In the injury group, IL-10 did not significantly suppress NO production when it was added at 4 or 6 h after LPS stimulation (Fig. 4A). In the sham group, IL-10 significantly suppressed (P < 0.05) NO release when it was added 4 h, but not 6 h, after LPS stimulation. The suppressive effect of exogenous IL-10 on IL-6 production was also significantly (P < 0.05) attenuated when the cells were exposed to IL-10 at 4-6 h after LPS stimulation. However, unlike NO, IL-6 release was still significantly (P < 0.05) suppressed when IL-10 was added to the culture media at 6 h after LPS stimulation (Fig. 4B). In addition, IL-6 levels were significantly higher (P < 0.05) in the injury group compared with shams when IL-10 was added 2-6 h after LPS stimulation. Delaying the addition of IL-10 to the culture media following LPS also attenuated the suppression of TNF-alpha production (Fig. 4C). IL-10 added 6 h after LPS significantly (P < 0.05) suppressed TNF-alpha production in the sham group but not in the injury group.


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Fig. 4.   LPS (1 µg/ml) stimulated nitrite (A), IL-6 (B), and TNF-alpha (C) release by splenic Mphi . Exogenous IL-10 (25 U/ml) was added to the Mphi cultures 0, 2, 4, or 6 h after LPS stimulation. Mphi cultures were maintained for 48 h for the determination of nitrite and IL-6 for 24 h for the determination of TNF-alpha . Data are expressed as means ± SE for 4-5 mice per group from 2 independent experiments. *P < 0.05 compared with sham; dagger P < 0.05 compared with LPS only.

Macrophage IL-10R and CD11b expression. IL-10R expression was determined in Mphi s 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 Mphi 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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Fig. 5.   IL-10 receptor (IL-10R) expression (A) and CD11b expression (B) on splenic Mphi . Mphi cultures were either unstimulated or stimulated with LPS (1 µg/ml) for 2, 4, and 6 h. IL-10R expression of CD11b+ cells was determined by dual color fluorescence-activated cell sorter (FACS) analysis as described in MATERIALS AND METHODS. CD11b expression was determined by single-color FACS analysis. Data are expressed as means ± SE of the mean fluorescent intensity (MeanX) of 4 separate analyses. *P < 0.05 compared with sham; dagger P < 0.05 compared with unstimulated cells (no stim).

CD11b expression, a marker of Mphi activation, was higher in the injury group compared with shams in the absence of LPS stimulation (Fig. 5B). Statistical significance was not reached, however, due to high variability between samples. After LPS stimulation, CD11b expression significantly (P < 0.05) increased in shams, whereas no change was observed in the injury group.

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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Table 1.   Systemic interleukin-10 levels at 7 days postinjury


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Mphi -dependent proinflammatory cascade is central to the development of subsequent deleterious complications such as multiple organ failure (5). Thus induction of a hyperactive Mphi 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 Mphi 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 Mphi phenotype that is associated with increased productive capacity for both proinflammatory (NO, IL-6, and TNF-alpha ) and anti-inflammatory (IL-10) mediators. Consistent with the findings of De Waal Malefyt et al. (4), IL-10 produced by Mphi 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-alpha 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, Mphi 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 Mphi s as well as other cell types (14, 24). Studies suggest that Mphi -derived IL-10 plays an important role in the autoregulation of Mphi 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-alpha release, but not NO release. Thus while the hyperactive Mphi 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 Mphi 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-alpha , and IL-6 in Mphi 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 Mphi 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 Mphi s postburn may be similar to that observed in rheumatoid arthritis, an autoimmune disease also associated with Mphi hyperactivity.

Previous studies have shown that prolonged exposure (6-48 h) of Mphi s 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 Mphi 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 Mphi 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-alpha 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 beta 2-intergrin in association with CD18. Compared with other leukocytes, Mphi s predominantly express CD11b/CD18 heterodimers (33). In the present study, Mphi 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" Mphi s express lower levels of IL-10R. Interestingly, LPS stimulation did not increase CD11b expression in the injury group, whereas sham Mphi 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 Mphi 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 Mphi 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 Mphi 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 Mphi 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 Mphi phenotype and responsiveness to this cytokine.

In conclusion, our findings presented here indicate that the hyperactive proinflammatory Mphi 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 Mphi hyperactivity following thermal injury.


    ACKNOWLEDGEMENTS

We thank Lucretia Vickers for valuable technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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