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The Journal of Immunology, 2001, 167: 3919-3927.
Copyright © 2001 by The American Association of Immunologists

Critical Role of Kupffer Cell-Derived IL-10 for Host Defense in Septic Peritonitis1

Klaus Emmanuilidis*, Heike Weighardt*, Stefan Maier*, Klaus Gerauer*, Tanja Fleischmann*, Xin X. Zheng{dagger}, Wayne W. Hancock{ddagger}, Bernhard Holzmann2,3,* and Claus-Dieter Heidecke3,*

* Department of Surgery, Klinikum rechts der Isar, Technische Universität, München, Germany; and {dagger} Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, and {ddagger} Department of Pathology, Harvard Medical School, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intra-abdominal infection in patients following major visceral surgery is associated with high mortality. Using a macrophage depletion technique, we demonstrate that in murine septic peritonitis, Kupffer cells are a major source of systemic IL-10 levels. Kupffer cell-depleted mice were highly susceptible to the lethal effects of septic peritonitis and exhibited an increased bacterial load. Kupffer cell-depleted mice were protected by the administration of an IL-10-Fc fusion protein. Loss of Kupffer cell-derived IL-10 was associated with a weak increase in serum IL-12 levels, whereas TNF, IL-1{alpha}, and IL-18 levels were not significantly elevated, suggesting that the loss of Kupffer cell-derived IL-10 did not result in a toxic cytokine release syndrome. Instead, loss of Kupffer cell-derived IL-10 was associated with a reduced splenocyte production of IFN-{gamma} that is required for immune protection in murine septic peritonitis. Therefore, the results suggest that the protective function of IL-10 in septic peritonitis may not be restricted to the anti-inflammatory activities of IL-10.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-10 was described as a cytokine that exhibits potent anti-inflammatory activities (1, 2, 3). Thus, exposure of mononuclear phagocytes or dendritic cells to IL-10 inhibits the synthesis of proinflammatory cytokines, the release of reactive oxygen and nitrogen intermediates, as well as the Ag-presenting capacity of these cells. IL-10 may also suppress proliferative and cytotoxic T cell responses and cytokine production by Th1 cells. Treatment with IL-10 protects mice against endotoxic shock by preventing excessive production of proinflammatory cytokines (4, 5, 6, 7, 8, 9). Investigations in models of rheumatoid arthritis, thyroiditis, autoimmune diabetes, and collagen-induced arthritis further support the notion that IL-10 limits autodestructive immune responses (10, 11, 12, 13). Furthermore, IL-10 knockout mice exhibit polarized Th1 immune responses and develop chronic enterocolitis that is related to a persistent stimulation with intestinal microorganisms (14).

In addition, IL-10 was reported to have several proinflammatory properties. Thus, IL-10 enhances TNF production of peripheral monocytes, if the cells are maintained in whole blood or if cell adherence is prevented (15). IL-10 also promotes NK cell proliferation, cytotoxicity, or secretion of IFN-{gamma}, GM-CSF, and TNF when combined with IL-2, IL-18, or IL-12 (16, 17, 18). Moreover, IL-10 costimulates the proliferation of thymocytes, mast cell progenitors, and B cells, and increases expression of MHC class II proteins and Ab secretion by B cells (19, 20, 21, 22). Mouse models of allotransplantation have provided evidence that IL-10 may also exhibit potent proinflammatory activities during immune responses in vivo. Thus, viral IL-10 or mutant forms of IL-10, which lack proinflammatory functions, significantly delayed the rejection of cardiac allografts, whereas wild-type IL-10 did not (23). Similarly, treatment of mice with an IL-10-Fc fusion protein accelerated the destruction of pancreatic islet cell allografts and augmented the granzyme B expression in local draining lymph nodes (9).

The concept that the pathogenesis of sepsis results from an unopposed inflammatory reaction has been challenged by the failure of recent clinical trials to show beneficial effects of anti-inflammatory treatment strategies (24, 25, 26, 27, 28). Instead, evidence is accumulating to indicate that immunosuppression may represent an important pathogenic process in sepsis. Mononuclear phagocytes of sepsis patients show a severely decreased ability to produce cytokines such as IL-12, IL-1{beta}, IL-6, and TNF, as well as a reduced expression of MHC class II molecules (29, 30, 31, 32). Impaired monocyte IL-12 secretion or MHC class II expression was found to even precede infection and to be associated with an increased susceptibility of surgical patients to severe postoperative sepsis (33, 34). Moreover, T cells of patients with lethal sepsis displayed a reduced secretion of IL-2 and TNF and a diminished proliferative response (35). These immune defects in sepsis patients appear to be of considerable significance, because their severity and persistence were found to correlate with sepsis mortality. However, it should be noted that although immunosuppression could result from an excessive production of anti-inflammatory mediators such as IL-10, we rather observed that IL-10 production was diminished in monocytes and unaltered in T cells of sepsis patients (32, 35).

Although mononuclear phagocytes are considered to be important for the host defense in polymicrobial sepsis, little is known about the specific role of distinct macrophage subsets for the cytokine response to a septic challenge. Using a macrophage depletion technique, we identify Kupffer cells as the major source of systemic IL-10 during septic peritonitis. Macrophage depletion resulted in an increased mortality of septic peritonitis that was completely prevented by administration of an IL-10-Fc fusion protein. However, loss of Kupffer cell-derived IL-10 did not result in an unopposed inflammatory reaction to the septic challenge, but was associated with a reduced splenocyte IFN-{gamma} production. Therefore, it appears that the protective effects of IL-10 may not be mediated by its anti-inflammatory activities.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Colon ascendens stent peritonitis (CASP)4 model of polymicrobial sepsis and treatment protocols

Female C57BL/6 mice were used at 8–12 wk of age. The technique used for induction of CASP was performed as previously described (36). Briefly, the colon ascendens was exteriorized and a 7/0 ethilon thread (Ethicon, Nordersted, Germany) was stitched through the antimesenteric portion of the colon ascendens ~10 mm distal of the ileocecal valve. A 16-gauge venous catheter was punctured antimesenterically through the colonic wall into the intestinal lumen, directly proximal of the pretied knot, and fixed. To ensure proper intraluminal position of the stent, stool was milked from the cecum into the colon ascendens until a small drop appeared. Fluid resuscitation of the animals was performed by flushing 0.5 ml of sterile saline into the peritoneal cavity before closure of the abdominal wall.

Liposome-encapsulated clodronate was prepared as described (37). To deplete specific macrophage populations, 40 µl of the clodronate-liposome suspension was diluted with 160 µl of PBS and injected i.v. 24 h before performing the CASP procedure. Control animals received 200 µl of PBS.

In some experiments, clodronate liposome-treated mice were given a single i.v. injection of 0.5 or 2 µg of IL-10-Fc in 200 µl of PBS 1 h before CASP. Control animals received the same volume of PBS. The IL-10-Fc chimeric protein was constructed by the fusion of murine IL-10 with the hinge, CH2, and CH3 regions of mouse IgG2a. In addition, amino acid mutations were introduced in the complement and Fc{gamma}R 1 binding sites to prevent Ab-dependent cell-mediated cytotoxicity and complement-directed cytolysis by the IL-10-Fc fusion protein (9).

Bacterial load of peripheral organs

Lungs, livers, and spleens were removed 12 h after CASP surgery and homogenized in 10 ml of sterile PBS. Serial dilutions of organ homogenates in PBS were plated onto blood agar plates (BD Biosciences, Heidelberg, Germany). CFU were counted after incubation at 37°C for 24 h and calculated as CFU per whole organ.

Immunohistochemistry

Cryostat sections were fixed in acetone for 10 min, dried, and stored at -80°C. Endogenous peroxidase activity was blocked by preincubation of the sections with 0.1% H2O2. The sections were incubated for 1 h with mAbs F4/80, MOMA-1 (both obtained from Serotec, Raleigh, NC), or ER-TR9 (Dianova, Hamburg, Germany), and subsequently with peroxidase-conjugated mouse anti-rat IgG Ab (Dianova) for 1 h. The sections were stained for 10 min in 50 mM acetate buffer containing 0.01% H2O2 and 5 mg/ml 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO), which was predissolved in N,N'-dimethylformamide. Nuclear counterstaining was performed with Mayer’s hematoxylin.

Immunostaining of IL-10 was performed as described previously (13). Briefly, cryostat sections were incubated overnight with 5 µg/ml rabbit Ab against murine IL-10 (PeproTech, Rocky Hill, NJ). Ab reactivity was detected by a peroxidase-labeled polymer using the anti-rabbit Envision detection system (DAKO, Carpenteria, CA).

Flow cytometry analysis

Peritoneal lavage fluid or lungs were removed 24 h after injection of clodronate liposomes or PBS. Lungs were extensively perfused with PBS, minced, and incubated in RPMI 1640 medium containing 670 U/ml collagenase IV (Sigma) for 45 min at 37°C. Single cell suspensions were obtained by filtration through a nylon mesh of 70 µm diameter. Treatment with collagenase IV resulted in complete digestion of lung tissue without any visible cell clumps left after filtration. Fluorochrome- or biotin-labeled rat mAbs against murine CD45 (30F11.1), Mac-1 (M1/70), and Mac-3 (M3/84) were purchased from BD PharMingen (San Diego, CA). Abs were incubated for 30 min at 4°C in PBS containing 1% BSA. Reactivity of biotin-labeled Abs was detected using streptavidin conjugated with FITC (Dianova) or PE (BD Biosciences). In each experiment, the appropriate isotype-matched controls were included. After washing with PBS, cells were analyzed on an EPICS XL cytometer (Coulter, Hialeah, FL).

Cytokine protein measurement

Serum samples or peritoneal lavage fluid were collected at various time intervals after CASP surgery. Splenocyte supernatants were collected 16 h after in vitro culture of 4 x 106 cells in 200 µl of RPMI 1640 medium containing 10% FCS. Cytokine levels were determined by specific ELISA according to the manufacturer’s protocols. Except for IL-10 (Endogen, Woburn, MA), all ELISA kits were purchased from R&D Systems (Minneapolis, MN). The levels of sensitivity were 12 pg/ml for IL-10, 4 pg/ml for IL-12, 8 pg/ml for IL-18, 5 pg/ml for TNF, 2 pg/ml for IFN-{gamma}, and 2 pg/ml for IL-1{alpha}.

RNase protection assay

Organs were snap frozen in liquid nitrogen at -80°C immediately after removal. Total RNA was extracted using the method of Chomczynski and Sacchi (38). RNase protection assays were performed using the Riboquant kit (BD PharMingen) in accordance with the manufacturer’s protocol, as described previously (39). IL-10 mRNA expression was analyzed using a phosphor imaging system and ImageQuaNT v4.2a software (Amersham Pharmacia Biotech, Piscataway, NJ). For each sample, the intensities of IL-10-specific signals were normalized to the corresponding values of the housekeeping gene GAPDH.

Statistical analysis

Statistical analysis of the data was performed using the Mann-Whitney U test or Student’s t test where appropriate. Survival data were analyzed using the log-rank test. All data are presented as mean ± SEM. The level of statistical significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kupffer cells determine systemic IL-10 levels during septic peritonitis

Mononuclear phagocytes are considered to play a central role for the regulation of the cytokine response to a septic challenge. In the present study, we attempted to examine the role of specific macrophage populations for the cytokine response during septic peritonitis. Previous work has shown that macrophages can be depleted based on the liposome-mediated intracellular delivery of clodronate (37, 40). It is also well established that different macrophage populations can be selectively eliminated depending on the route of clodronate-liposome injection. Thus, several studies demonstrated that i.v. administration of clodronate liposomes efficiently depletes Kupffer cells and some splenic macrophage subsets, but not macrophages in the lung, lymph nodes, peritoneal cavity, or thymus (37, 41, 42). Consistent with these reports, we found that the i.v. injection of clodronate liposomes resulted in the rapid (within 24 h) and complete depletion of Kupffer cells as well as splenic metallophilic and marginal sinus macrophages (Fig. 1Go, A and B). In contrast, the numbers of splenic red pulp macrophages were not reduced by this treatment (Fig. 1GoB). Repopulation of the hepatic and splenic macrophage compartments did not occur for at least 72 h after injection of clodronate liposomes (Fig. 1GoA and data not shown). As a control for the selective depletion of macrophage compartments, we could confirm previous findings (37, 41, 42) showing that i.v. injection of clodronate liposomes does not affect the numbers of peritoneal or lung resident macrophages (Table IGo).



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  		FIGURE 1. Depletion of Kupffer cells and splenic macrophages by clodronate liposomes. Mice were injected i.v. with clodronate liposomes or PBS, and organs were removed at various time points thereafter. A, Frozen sections of liver tissues were stained with mAb F4/80 to detect Kupffer cells. B, Frozen sections of spleens removed 24 h after clodronate-liposome injection were incubated with mAbs F4/80, MOMA-1, or ER-TR9 to label red pulp, marginal sinus, or metallophilic macrophages, respectively. Staining of liver or spleen sections was not detectable with isotype-matched negative control Ig (not shown). Original magnifications were x400 (A) and x200 (B).

 

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  		Table I. Effects of clodronate-liposome treatment on peritoneal and lung macrophages1

 
To identify the contribution of different macrophage subsets to the cytokine response during sepsis, mice were injected i.v. with clodronate liposomes, and 24 h later septic peritonitis was induced using the CASP model (36). The results in Fig. 2Go demonstrate that in control mice, serum IL-10 levels were strongly increased as early as 3 h after CASP and remained at this high level for at least 12 h. In contrast, injection of clodronate liposomes 24 h before CASP strongly reduced serum IL-10 levels (Fig. 2Go). IL-10 levels were substantially lower in these mice than in controls at all time points analyzed (Fig. 2Go). However, it should be noted that at all time points tested during septic peritonitis, residual IL-10 levels in clodronate liposome-treated mice were at least 25-fold greater than IL-10 levels in nonseptic control mice (Fig. 2Go).



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  		FIGURE 2. Effects of clodronate-liposome treatment on serum IL-10 levels during septic peritonitis. Mice were injected i.v. with clodronate liposomes (filled bars) or solvent control (hatched bars) 24 h before CASP. Serum samples were obtained before CASP (0 h) or 3, 6, and 12 h thereafter. IL-10 concentrations were determined by specific ELISA. The results are derived from three to five mice per group. *, p < 0.01; #, p < 0.001.

 
Because the i.v. injection of clodronate liposomes was observed to deplete macrophages in liver and in spleen, but not in other compartments, we attempted to determine the relative contribution of these two subsets to systemic IL-10 levels more precisely. In a first set of experiments, mice were splenectomized before CASP and serum IL-10 was measured. The results in Fig. 3Go clearly show that serum IL-10 levels were not affected by splenectomy, suggesting that splenic macrophages do not contribute significantly to systemic IL-10 during septic peritonitis. In additional experiments, IL-10 mRNA levels were analyzed in various organs 6 h after CASP. The results presented in Fig. 4Go revealed no significant differences of IL-10 induction in lungs and spleens of both control and clodronate liposome-treated mice. In contrast, IL-10 mRNA levels were up-regulated 6 h after CASP in livers of septic control, but not clodronate liposome-treated mice (Fig. 4Go). To further examine IL-10 production by Kupffer cells, IL-10 protein was localized immunohistochemically in liver sections of mice 9 h after CASP. The results depicted in Fig. 5Go show that IL-10 was readily detectable in livers of control, but not clodronate liposome-treated mice. Furthermore, the IL-10-staining pattern in livers of septic control mice was consistent with its production by Kupffer cells (Fig. 5Go, middle panel). Considered together, these experiments indicate that during septic peritonitis, Kupffer cells are a major source of systemic IL-10.



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  		FIGURE 3. Effects of splenectomy on serum IL-10 levels during septic peritonitis. Mice were either splenectomized or left untreated, and the CASP procedure was performed. Serum was obtained 6 h later, and IL-10 concentrations were determined by specific ELISA. The results are derived from four mice per group.

 


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  		FIGURE 4. Induction of IL-10 mRNA during septic peritonitis. Mice were injected with clodronate liposomes (filled bars) or with PBS (hatched bars), and the CASP procedure was performed 24 h later. Lungs, livers, and spleens were removed for RNA preparation 6 h after CASP. Amounts of IL-10 mRNA in organs were determined by RNase protection assay. Results are given as relative intensities of IL-10 signals normalized by the housekeeping gene GAPDH. In each group, at least three independent mice were analyzed. Differences in splenic IL-10 levels were not statistically significant. *, p = 0.05 (clodronate liposome-treated vs control mice).

 


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  		FIGURE 5. Immunohistochemical localization of IL-10 in livers of septic mice. Livers were removed from untreated mice or from mice 9 h after CASP, and frozen sections were stained with an IL-10-specific Ab. Representative sections from mice without CASP (A), with control PBS treatment and CASP (B), or with clodronate-liposome treatment and CASP (C) are shown. Original magnifications were x200.

 
Increased susceptibility to septic peritonitis following Kupffer cell depletion and effects on serum cytokine levels

To determine the consequences of macrophage depletion and the reduction of serum IL-10 for host defense, the survival of septic peritonitis and the bacterial load of various organs were analyzed. The results in Fig. 6GoA clearly demonstrate that mice injected with clodronate liposomes 24 h before CASP were highly susceptible to septic peritonitis, showing a significantly reduced survival when compared with controls. Consistent with the loss of Kupffer cells, clodronate liposome-injected mice had significantly more bacterial counts in liver than control mice (Fig. 6GoB). However, the number of bacterial colonies in spleen and lung was not significantly different between both groups (Fig. 6GoB).



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  		FIGURE 6. Impaired immune defense against septic peritonitis following clodronate-liposome treatment. A, Mice were injected i.v. with clodronate liposomes (filled triangles, n = 24) or solvent control (open circles, n = 24) 24 h before CASP. Data were pooled from four independent experiments, each showing reduced survival of clodronate liposome-treated mice as compared with controls. Cumulative survival was monitored over a period of 10 days. B, Livers, spleens, and lungs were obtained 20 h after CASP from mice pretreated with clodronate liposomes (filled bars) or solvent control (hatched bars). Organ homogenates were prepared, and bacterial numbers were determined by plating serial dilutions of each homogenate on blood agar. The results are derived from six to eight mice per group. #, p < 0.01.

 
It is conceivable that the reduction of systemic IL-10 in Kupffer cell-depleted mice could result in an unopposed proinflammatory response to the septic challenge, leading to a toxic cytokine release syndrome. To examine this possibility, serum levels of various proinflammatory cytokines were determined in clodronate liposome-treated mice during septic peritonitis. The results in Fig. 7Go show that concomitant with the loss of IL-10, the serum levels of IL-12 were significantly augmented at 6 and 12 h, but not 3 h, after CASP. However, it should be noted that the increase in serum IL-12 was weak as compared with the substantial loss of IL-10. Thus, IL-12 levels were only increased by 1.7- and 2.2-fold at 6 and 12 h after CASP as compared with a 5-fold reduction of IL-10 (Figs. 2Go and 7Go). The serum levels of IL-18 and TNF did not show statistically significant differences between Kupffer cell-depleted mice and controls at any time point investigated (Fig. 7Go). Moreover, IL-1{alpha} levels were also not elevated in clodronate liposome-treated mice, but were even slightly reduced at 12 h after CASP (Fig. 7Go). Together, the results suggest that the substantial loss of systemic IL-10 in Kupffer cell-depleted mice does not lead to a toxic cytokine release during septic peritonitis.



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  		FIGURE 7. Effects of clodronate-liposome treatment on proinflammatory cytokine levels during septic peritonitis. Mice were injected i.v. with clodronate liposomes (filled bars) or solvent control (hatched bars) 24 h before CASP. Serum samples were obtained before CASP (0 h) or 3, 6, and 12 h thereafter. Cytokine concentrations were determined by specific ELISAs. The results are derived from three to five mice per group. *, p < 0.05; #, p < 0.01.

 
Depletion of Kupffer cell-derived IL-10 is associated with reduced splenocyte IFN-{gamma} production

Survival of septic peritonitis in the CASP model was previously shown to depend on IFN-{gamma} (36). Because IL-10 also exhibits proinflammatory activities such as stimulation of IFN-{gamma} production by NK cells (16, 17, 18), we investigated whether treatment with clodronate liposomes might affect IFN-{gamma} production during septic peritonitis. However, IFN-{gamma} was not detectable in serum samples after CASP (data not shown). Therefore, splenocytes were isolated 6 h after CASP, and in vitro IFN-{gamma} production was determined without additional stimulation, because we argued that these cells would have received IFN-{gamma}-inducing stimuli in vivo. The results in Fig. 8Go demonstrate that IFN-{gamma} production was low in nonseptic mice and did not differ between clodronate liposome-injected and control mice. Septic peritonitis induced a small, but significant increase of splenocyte IFN-{gamma} production in control mice, but not in macrophage-depleted mice (Fig. 8Go). Moreover, splenocyte IFN-{gamma} secretion was significantly greater in septic control mice than in septic mice pretreated with clodronate liposomes (Fig. 8Go).



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  		FIGURE 8. Sepsis-induced IFN-{gamma} production in spleen is impaired in clodronate liposome-treated mice. Mice were injected i.v. with clodronate liposomes (filled bars) or PBS (hatched bars), and the CASP procedure was performed 24 h later. Splenocytes were isolated 6 h after CASP and cultured in vitro for 16 h without additional stimulation. Culture supernatants were analyzed for IFN-{gamma} content by ELISA. Results are derived from 12–18 mice per group. #, p < 0.005 (compared with untreated PBS controls), and *, p < 0.05 (compared with septic PBS controls).

 
Protective role of Kupffer cell-derived IL-10 for survival of septic peritonitis

IL-10 was previously reported to exert a protective role in septic peritonitis (43, 44). Therefore, we addressed the question as to whether the increased susceptibility of clodronate liposome-treated mice to septic peritonitis may result from the lack of Kupffer cell-derived IL-10. Because the results in Fig. 2Go show that systemic IL-10 levels rise very rapidly following CASP, with plateau levels being reached after 3 h, we argued that potential protective effects of IL-10 might depend on its presence during the early phase of septic peritonitis. Therefore, IL-10 reconstitution experiments were designed to allow for the substitution of early IL-10 levels. Moreover, we have chosen to administer a noncytolytic IL-10-Fc fusion protein rather than rIL-10 because it exhibits an extended t1/2 (30 min for IL-10 vs 31 h for IL-10-Fc) (6, 9). This was expected to ensure the continuous presence of IL-10 activity during the course of the experiment. Although the fusion protein may cross-link IL-10R, our previous work has shown that on a molar basis the IL-10-Fc fusion protein is as effective as rIL-10 (9). Clodronate liposome-treated mice were injected with different amounts of an IL-10-Fc fusion protein or with solvent control, and 1 h later septic peritonitis was induced by the CASP procedure. The results in Fig. 9GoA clearly demonstrate that the administration of the IL-10-Fc fusion protein improved survival in a dose-dependent manner. Significant protection was observed when mice received a single dose of 2 µg IL-10-Fc (Fig. 9GoA). Notably, the survival rate of clodronate liposome-injected mice treated with the IL-10-Fc protein was comparable with that of untreated control mice (Figs. 5GoA and 8A). Moreover, the bacterial load in liver, lung, and spleen was not altered by the administration of the IL-10-Fc fusion protein (Fig. 9GoB), suggesting that the increased numbers of viable bacteria in livers of clodronate liposome-treated mice following CASP (Fig. 5GoB) result from the loss of Kupffer cells’ phagocytic and antimicrobial activity, but not from partial IL-10 deficiency. Nonetheless, reconstitution of IL-10 activity was sufficient to restore resistance against septic peritonitis.



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  		FIGURE 9. Treatment with IL-10-Fc restores resistance to septic peritonitis in mice injected with clodronate liposomes. Mice were injected i.v. with clodronate liposomes, and 24 h later the CASP procedure was performed. One hour before CASP mice were treated i.p. with 0.5 µg IL-10-Fc (open circles, n = 12), 2 µg IL-10-Fc (open squares, n = 6), or solvent control (filled triangles, n = 11). Data were pooled from three independent experiments, each showing an improved survival of IL-10-Fc-treated mice as compared with controls. Cumulative survival was monitored for 10 days. The p value indicates the statistical comparisons between the treatment group receiving 2 µg IL-10-Fc and controls.

 
Treatment of mice with clodronate liposomes did not result in a marked overproduction of inflammatory cytokines, suggesting that the contribution of Kupffer cell-derived IL-10 to the total IL-10 pool is mostly dispensable for controlling serum levels of proinflammatory cytokines during septic peritonitis (Fig. 7Go). Thus, if the amount of IL-10-Fc protein administered to clodronate liposome-treated mice was just enough to compensate for this partial loss, additional anti-inflammatory effects should not be observed. However, if the dose of IL-10-Fc applied would exceed by far the levels endogenously produced in septic mice, exogenous IL-10-Fc could suppress the production of inflammatory cytokines; therefore, the effects of this treatment could differ from the activities of endogenous IL-10. To address this concern, clodronate liposome-treated mice received 2 µg IL-10-Fc 1 h before CASP, and cytokine levels were determined in serum and peritoneal lavage fluid 12 h after CASP. The results depicted in Fig. 10GoA indicate that the treatment with the IL-10-Fc fusion protein did not significantly alter sepsis-elicited serum levels of TNF, IL-1{alpha}, IL-12, and IL-18. Moreover, the amounts of TNF and IL-1{alpha} present in the peritoneal cavities of septic mice were also not significantly affected by the injection of IL-10-Fc (Fig. 10GoB). IL-12 and IL-18 levels were below the limits of detection in peritoneal lavage fluids (data not shown). Considered together, the results indicate that the effects of macrophage depletion on the survival of septic peritonitis can be completely reversed by the administration of the IL-10-Fc fusion protein, and suggest an essential protective role for Kupffer cell-derived IL-10.



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  		FIGURE 10. Effects of IL-10-Fc treatment on sepsis-induced cytokine production. Mice were injected i.v. with clodronate liposomes, and 24 h later the CASP procedure was performed. IL-10-Fc fusion protein (2 µg) was administered i.p. 1 h before CASP. Serum samples (A) or peritoneal lavage fluid (B) were obtained 12 h after CASP. Cytokine concentrations were determined by specific ELISA. The results are derived from at least six mice per group. There were no statistically significant differences in cytokine levels between the two groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study identify Kupffer cells as a major source of systemic IL-10 levels during septic peritonitis. The effects of macrophage depletion by i.v. injection of clodronate liposomes on IL-10 protein and mRNA levels in various compartments, the analysis of IL-10 production in splenectomized mice, and the direct immunohistochemical localization of IL-10 in Kupffer cells during sepsis support this conclusion. Our observations are consistent with previous work showing that Kupffer cells secrete IL-10 in response to a LPS challenge (41, 45), although the contribution of Kupffer cells to systemic cytokine levels was not addressed in these studies. Moreover, Kupffer cells were reported to produce IL-10 and IL-6, but not IL-12 or IL-18, when exposed to Schistosoma mansoni Ag (46). Following infection with S. mansoni, Kupffer cells also secreted IL-4 and IL-13, thereby contributing to the Th2 differentiation of hepatic T cells (46). Together with the results of the present report, these findings suggest that Kupffer cell cytokine production is of critical importance for regulating immune responses to infection.

Our results show that the deleterious effects of Kupffer cell depletion on the survival of septic peritonitis were completely prevented by the administration of an IL-10-Fc fusion protein, suggesting that the production of IL-10 is an essential function of Kupffer cells in septic peritonitis. The IL-10-Fc fusion protein was injected before the induction of septic peritonitis, because production of IL-10 in response to a septic challenge was observed to be extremely rapid (Fig. 2Go) (44), and we argued that this fast kinetics may be important for IL-10 function. However, the data available indicate that depending on the time point of intervention, modulation of IL-10 levels in experimental animals may have either beneficial or harmful effects on host resistance against a septic challenge. Although partial or complete depletion of IL-10 before infection results in an increased mortality of septic peritonitis (Fig. 6GoA) (44), neutralization of IL-10 several hours after the onset of infection appears to exert beneficial effects (47, 48). This apparent discrepancy might be resolved by assuming that IL-10 exhibits a dual role in septic peritonitis. Although rapid production of IL-10 during the early phase of sepsis may be required for efficient host defense, sustained high levels of IL-10 may be detrimental during the late phase of sepsis possibly by promoting immunosuppression and/or a Th1/Th2 dysbalance (47). Interestingly, administration of exogenous IL-10 to normal mice that do not exhibit any overt IL-10 deficiency does not affect sepsis-associated mortality or morbidity, suggesting that saturating amounts of IL-10 are produced endogenously (49).

The protective effect of IL-10 in septic peritonitis could be explained by its anti-inflammatory activities (1, 2, 3). Impaired production of IL-10 during sepsis could lead to an unopposed inflammatory response similar to the cytokine release syndrome induced by bacterial toxins such as LPS. Excessive cytokine production and toxicity following LPS administration are known to be mitigated by IL-10 (4, 5, 6, 7, 8). However, our results demonstrate that despite the strong reduction of systemic IL-10, serum levels of TNF, IL-1{alpha}, and IL-18 were not significantly altered, and the increase in IL-12 was only weak. Although these data are consistent with studies showing that Kupffer cell blockade by gadolinium chloride does not affect systemic TNF levels following LPS administration or induction of septic peritonitis (50, 51), the question arises as to why proinflammatory cytokines do not overshoot, despite the substantial loss of IL-10. Several explanations can be provided for this observation that are not mutually exclusive. First, other cytokines such as TGF-{beta} may contribute to anti-inflammation in septic peritonitis. Second, residual IL-10 levels in clodronate liposome-treated mice may be sufficient to mediate anti-inflammatory activities. Consistent with this notion, the results in Fig. 2Go clearly show that residual IL-10 levels in clodronate liposome-treated mice are still highly elevated when compared with basal IL-10 levels in nonseptic mice. Third, an unopposed increase of inflammatory cytokines in serum may not be observed, because macrophage populations that are depleted by clodronate liposomes may contribute to proinflammatory cytokine levels. Thus, exaggerated cytokine production by other cell populations may only compensate for this loss, but may not result in high systemic amounts. Production of proinflammatory cytokines by Kupffer cells has been shown in several previous reports (52, 53). Fourth, overproduction of inflammatory cytokines may be compartmentalized to individual organs and may not be strong enough to contribute to systemic cytokine levels. Nonetheless, this local overproduction of inflammatory cytokines could lead to an enhanced organ injury. Consistent with this possibility, we have observed significantly increased neutrophil numbers in bronchoalveolar lavage from clodronate liposome-treated mice during septic peritonitis as compared with controls (data not shown). Therefore, it is conceivable that treatment with IL-10-Fc may protect from compartmentalized hyperinflammation and organ injury without altering systemic cytokine levels.

Along with its anti-inflammatory functions, IL-10 was also found to exhibit various proinflammatory activities. It was reported that IL-10 promotes NK cell proliferation, cytotoxicity, and production of IFN-{gamma}, GM-CSF, and TNF when combined with IL-2, IL-12, or IL-18 (16, 17, 18). Several lines of evidence suggest that the immunostimulatory activities of IL-10 may also affect the host defense against infection. Using recombinant vaccinia viruses, it was found that infection of SCID mice with a virus expressing IL-10 resulted in greater NK cell activity and lower virus replication than infection with control virus (54). In addition, it was demonstrated that administration of IL-10 increases the serum levels of IFN-{gamma}, IL-12, interferon-inducible protein-10, and monokine induced by IFN-{gamma}, and activates CTLs and NK cells during human endotoxemia (55). Interestingly, the results of the present study also reveal that the reduction of systemic IL-10 levels in clodronate liposome-treated mice is associated with an impaired production of IFN-{gamma} by splenocytes from septic mice. Although being statistically significant, the differences in IFN-{gamma} release by splenocytes were small on a quantitative basis. However, when interpreting the absolute levels of IFN-{gamma} release, it should be considered that splenocytes were isolated from septic mice, but did not receive additional stimuli during the subsequent in vitro culture. Thus, efficient IFN-{gamma} secretion might have only occurred during part of the in vitro culture period. In addition, total splenocytes were used for these experiments, whereas IFN-{gamma} production may be restricted to distinct subpopulations such as NK cells or dendritic cells. Finally, survival of mice in the CASP model is highly dependent on IFN-{gamma} (36), although the amounts of IFN-{gamma} released in this model are not sufficient to be detected in the serum. Therefore, it is conceivable that even small amounts of IFN-{gamma} may be biologically significant in the CASP model, and it is tempting to speculate that the protective effects of IL-10 in septic peritonitis may at least in part be linked to its proinflammatory activities.

In accordance with our results, Ab-mediated neutralization of IL-10 was previously found to result in an increased mortality of septic peritonitis in the cecal ligation and puncture model (43, 44). In these studies, IL-10-depleted mice also exhibited elevated levels of TNF in the peritoneal cavity and in serum, which appears to differ from our observation of unaltered serum TNF in clodronate liposome-treated mice. However, it should be noted that Ab treatment most likely results in a nearly complete neutralization of IL-10, whereas depletion of Kupffer cells only leads to a partial, although substantial reduction. Importantly, van der Poll and coworkers (44) have also demonstrated that the administration of anti-TNF Abs did not prevent the increased mortality of anti-IL-10-treated mice. These observations are consistent with our results and indicate that lethality of septic peritonitis in IL-10-depleted mice is not caused by a toxic cytokine release syndrome. Although the underlying mechanisms will have to be resolved in future studies, it is conceivable that the proinflammatory activities of IL-10 may have contributed to the protective effects in both models of septic peritonitis.

Several previous reports using gene-deficient mice have supported the concept that the anti-inflammatory activities of IL-10 are important for the control of immune responses to microbial pathogens or intestinal bacteria. For example, IL-10 knockout mice develop chronic enterocolitis that is associated with uncontrolled cytokine production by activated macrophages and Th1-like T cells (14, 56, 57). The severity of enterocolitis is reduced in mice maintained under specific pathogen-free conditions as compared with conventional housing and is ameliorated by Ab-mediated neutralization of IFN-{gamma} (14, 56). IL-10-deficient mice were also found to be highly susceptible to infection with Toxoplasma gondii (58). Early mortality in this model was prevented through treatment with IL-10 and was delayed by Abs against IL-12 or IFN-{gamma} (58).

Considered together, the results of the present report and previous studies are consistent with the concept that depending on the type of infection and the type of immune response triggered, either proinflammatory or immunosuppressive activities of IL-10 may be crucial for the generation of protective immune responses.


   Footnotes
 
1 This work was supported by Grant Si 208/5-4 from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Bernhard Holzmann, Department of Surgery, Klinikum rechts der Isar, Technische Universität, Ismaninger Strasse 22, 81675 München, Germany. E-mail address: holzmann{at}nt1.chir.med.tu-muenchen.de Back

3 B.H. and C.-D.H. contributed equally to this work. Back

4 Abbreviation used in this paper: CASP, colon ascendens stent peritonitis. Back

Received for publication November 7, 2000. Accepted for publication July 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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