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Originally published online as doi:10.1189/jlb.0405179 on January 13, 2006

Published online before print January 13, 2006
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(Journal of Leukocyte Biology. 2006;79:809-817.)
© 2006 by Society for Leukocyte Biology

The Kupffer cell protects against acute lung injury in a rat peritonitis model: role of IL-10

Hiroshi Kono*,1, Hideki Fujii*, Yu Hirai*, Masato Tsuchiya*, Hidetake Amemiya*, Masami Asakawa*, Akira Maki*, Masanori Matsuda* and Masayuki Yamamoto{dagger}

* First Department of Surgery, University of Yamanashi, Japan; and
{dagger} Department of Surgery, Shinko Byoin Hospital, Hyogo, Japan

1Correspondence: First Department of Surgery, University of Yamanashi, 1110 Shimokato, Tamaho, Nakakoma, Yamanashi 409-3898, Japan. E-mail: hkouno{at}res.yamanashi-med.ac.jp


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ABSTRACT
 
The possibility that Kupffer cells (KCs) play key beneficial and deleterious roles in multiple organ injury in sepsis has been discussed. The role of KCs in lung injury in a rat peritonitis model was investigated. Specifically, the involvement of interleukin (IL)-10, which has anti-inflammatory effects, was examined. Rats were given saline or gadolinium chloride (GdCl3), a KC toxicant, 24 h before cecal ligation and puncture (CLP). Survival was assessed for 7 days after CLP. The liver, lung, and serum were harvested, and the expression of cytokines was assessed. Macrophages were isolated from each organ after CLP, and the mRNA expression of inflammatory mediators was assessed. GdCl3 treatment increased lung injury and mortality. Plasma endotoxin levels were significantly greater, whereas serum IL-10 levels were lower in the GdCl3 than in the control group after CLP. IL-10 levels were significantly greater in the aorta than the hepatic vein. The mRNA expression of IL-10 was less in KCs from the GdCl3 than the control group. In the liver, the expression of IL-10 increased rapidly and continuously, up to 9 h in the control group, but values were significantly lower in the GdCl3 group. Rabbit anti-rat IL-10 antibodies were injected just after CLP to investigate the effects of immunoneutralization of endogenously produced IL-10. In the antibody-treated group, lung injury and mortality increased compared with animals treated with rabbit immunoglobulin G. Taken together, these results indicate that KCs play a protective role in lung injury in sepsis by production of IL-10.

Key Words: acute respiratory distress syndrome • inflammatory cytokine • lung macrophage and endotoxemia


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INTRODUCTION
 
Under conditions of sepsis, lung dysfunction is the first step in the development of multiple organ failure (MOF) [1 ]. Although the exact sequence of events leading to acute respiratory distress syndrome (ARDS) remains unknown, experimental and clinical studies have suggested that the migration of polymorphonuclear granulocytes into the lung tissue plays a key role in the cascade of events leading to ARDS [2 ]. To clarify the role of Kupffer cells (KCs) during endotoxemia, we previously used a lipopolysaccharide (LPS) infusion model in which one lethal dose of endotoxin was injected into the peripheral circulation. In this model, serum endotoxin levels are increased rapidly to high levels after the injection and then disappear rapidly from peripheral blood. The high level is sufficient to cause direct organ damage. However, in those experiments, lung as well as liver injuries were prevented by a KC toxicant, gadolinium chloride (GdCl3), suggesting that KCs are involved in acute lung injury as a result of endotoxemia [3 ].

Previously, it was reported that GdCl3 prevented the occurrence of hepatocellular dysfunction and the up-regulation of interleukin (IL)-1ß and IL-6 in a mouse septic peritonitis model [4 ]. Conversely, it was recently reported that inhibition of KCs increased mortality in the mouse septic peritonitis model [5 ]. Thus, the role of KCs in inflammation is not fully understood and is controversial, and their role may differ in various pathophysiological conditions during inflammation.

In the present study, the role of KCs was investigated in a rat septic peritonitis model, cecal ligation and puncture (CLP). A characteristic of this model is persistent release of bacteria from the abdominal cavity to the liver through the portal circulation. This model has been shown to induce a polymicrobial sepsis that closely mimics human sepsis [6 , 7 ]. Initial and direct modulations in the liver caused by endotoxin and other substances produced in the cecum may contribute to lung injury.

KCs are the major source of IL-10, a cytokine that has potent anti-inflammatory activities [8 ]. Exposure of mononuclear phagocytes or dendritic cells to IL-10 inhibits the synthesis of proinflammatory cytokines and the release of reactive oxygen and nitrogen intermediates, as well as the antigen-presenting capacity of these cells. Furthermore, IL-10 protects mice against endotoxin shock by preventing excessive production of proinflammatory cytokines [6 ]. Thus, IL-10 can turn off the signaling of the inflammatory cytokine cascade and inhibit development of multiple organ injury such as ARDS. Accordingly, the specific purpose of this study was to investigate the role of KCs and IL-10 in mortality and acute lung injury in a rat peritonitis model.


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MATERIALS AND METHODS
 
Animals and treatments
Male Sprague-Dawley rats (200–250 g body weight, Japan SLC Inc., Shizuoka) were used in these experiments. The experimental protocol followed the Institutional and the National Research Council’s criteria for the care and use of laboratory animals in research. All animals received humane care in compliance with institutional guidelines. To eliminate KCs, GdCl3 (10 mg/kg, Sigma Chemical Co., St. Louis, MO) was administered to rats via the tail vein. A saline vehicle (1 ml/kg) was administered as a control. After 24 h, septic peritonitis was induced by CLP as described previously [9 ]. Briefly, rats were anesthetized with 50 mg/kg intraperitoneally administered sodium pentobarbital (Dainippon Pharmaceutical Co., Osaka, Japan), and the cecum was ligated below the ileocecal junction; intestinal continuity was maintained. The cecum was punctured twice with an 18-gauge needle, and a small amount of cecal contents was expressed through the punctures. The incision was closed, and 10 ml sterile saline was administered subcutaneously for fluid resuscitation. After surgery, rats were placed on a heating pad until they recovered from anesthesia. Rats were given food and water ad libitum throughout the study.

Animals were divided into four groups: Group I, saline + sham operation; Group II, GdCl3 + sham operation; Group III, saline + CLP; and Group IV, GdCl3 + CLP. After the CLP operation, survival was assessed for the following 7 days (n=14 in each group).

Treatment with rabbit anti-rat IL-10 antibodies
The effects of neutralization of IL-10 were investigated by administering rabbit polyclonal anti-rat IL-10 neutralizing antibodies (anti-IL-10 antibodies; 500 µg/kg, Biosource International, Camarillo, CA) immediately after CLP (n=6 in each group) or sham operation (n=4 in each group). For purposes of immunoneutralization of endogenously produced IL-10, ~5 µg/mL antibody was used to neutralize 5 ng/mL rat IL-10. Rabbit isotype immunoglobulin G (IgG; Santa Cruz Biotechnology, CA) served as control IgG. After the operation, survival was assessed for the following 7 days. In another set of experiments, animals were killed 9 h after CLP, and serum and tissues were collected (n=4 in each group). Furthermore, lung injury was assessed by measuring the wet/dry weight ratio and lung microvascular permeability (n=4 in each group).

Sample collections and measurement of cytokine and endotoxin levels
Tissues from the liver and lung and blood samples from the aorta were collected 0, 60 min, 90 min, 6 h, 9 h, and 12 h after CLP or the sham operation, as animals started to die 12 h after CLP. Blood samples were collected from the aorta at each time-point after CLP for measurement of serum IL-6 and IL-10 (n=6) and were centrifuged at 1200 g for 10 min at 4°C. Tissues and serum samples were stored at –80°C until assays. Serum cytokine levels were determined using an enzyme-linked immunosorbent assay kit (Cosmo Bio Co., Tokyo, Japan).

In another set of experiments, blood samples were collected from the portal and hepatic vein 9 h after CLP (n=6) to investigate the role of the KC in production of IL-10 and removal of endotoxin.

Blood samples were collected in pyrogen-free heparinized syringes (n=6) and centrifuged at 1200 rpm for 10 min. Plasma was stored at –20°C in pyrogen-free glass tubes until assay using a limulus amebocyte lysate test kit (Kinetic-QCL, BioWhittaker, Walkersville, MD) [10 ].

Wet/dry lung weight ratio
Nine hours after CLP, blood was collected from the abdominal aorta, and subsequently, the lungs were removed (n=6 in each group). The lungs were placed in a tared plastic petri dish for weighing. They were then dried in a vacuum lyophilizer at –50°C and atmospheric pressure of 10 mmHg for 72 h (Refrigeration for Science, Island Park, NY). This process removed virtually all gravimetrically detectable water. The dry lung weight was determined, and wet/dry weight ratios were calculated to assess pulmonary edema [11 ].

Lung microvascular permeability
Pulmonary microvascular permeability was quantified by measuring the concentration of Evans blue dye (EBD) [12 ] within the lung (n=6 in each group). Nine hours after CLP, animals were killed. Animals received 30 mg/kg dye intravenously (i.v.) 30 min prior to termination of the experiment [13 ]. The lungs were perfused with phosphate-buffered saline (PBS) and then removed and dried as described previously. The dry lung weight was determined. The concentration of EBD was determined from a standard curve of EBD-formamide solutions and expressed as milligrams of EBD per gram of dry lung weight.

Isolation of tissue macrophages
It was reported that serum endotoxin levels rose 1 h after CLP [14 ]. Therefore, tissue macrophages were isolated 2 h after CLP to investigate the mRNA expression of inflammatory mediators. KCs were isolated from rats by collagenase digestion and differential centrifugation using Nycodenz (Nycomed Pharma AS, Oslo, Norway), as described in our previous work with some modifications [15 ]. In a previous study, small KCs, which are ED1-positive and ED2-negative cells, were isolated from rats treated with GdCl3 (Hiroshi Kono et al., unpublished data). The alveolar macrophages were collected, and lavage of the lungs was repeated 20 times, until only occasional cells were present in the lavage fluid. Thereafter, the lung tissue was sliced into small pieces. The interstitial lung cells were isolated according to the method of Holt et al. [16 ], as modified by Sjöstrand et al [17 ]. For isolation of splenic macrophages, single-cell suspensions were recovered from the spleen by disaggregating them on a wire mesh and sieving through 1 mm gauze wire mesh to remove large pieces of tissue [18 ]. For isolation of the peritoneal macrophages, contents of the peritoneum were lavaged by injection of 50 ml PBS. Peripheral blood was also collected from the aorta, and peripheral mononuclear cells were isolated by differential centrifugation using Nycodenz (NycoPrep 1.077A, Nycomed Pharma AS) [19 ]. Isolated cells were seeded onto the culture dishes and incubated for 1 h in Dulbecco’s modified Eagle’s medium (Gibco Laboratories Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum and antibiotics. After incubation, adhesive cells were collected using a scraper and then were washed three times with PBS for mRNA extraction.

Measurement of the mRNA expression of IL-6 and IL-10 by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
The mRNA expression of IL-6 and IL-10 was assessed by semiquantitative RT-PCR (n=6 in each group). Total RNA was isolated using an RNA purification kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and was used for the PCR assay to detect mRNA expression. RT of total RNA (2 µg) was performed in a final volume of 100 µl containing 1x TaqMan RT buffer, 5.5 mM MgCl2, 500 µM/L each deoxy-unspecified nucleoside 5'-triphsophate, 2.5 µM random hexamers, 0.4 U/µl RNase inhibitor, and 1.25 U/µl multiscribe RT. PCR primers for IL-6 and IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contained the following sequences [20 , 21 ]: IL-6 sense (5'-TTGCCGAGTAGACCTCATAGTGACC-3'), antisense (5'-CAAGAGACTTCCAGCCAGTTGC-3'); IL-10 sense (5'-GTGAAGACTTTCTTTCAAACAAAG-3'), antisense (5'-CTGCTCCACTGCCTTGCTCTTATT-3'); GAPDH sense (5'-TGAAGGTCGGAGTCAACGGATTTGGT-3'), antisense (5'-CATGTGGGCCATGAGGTCCACCAC-3').

The size of amplified PCR products was 614 base pairs (bp) for IL-6, 274 bp for IL-10, and 983 bp for GAPDH.

Aliquots (5 µL) of synthesized cDNA were added to 45 µL PCR mix containing 5 µL 10x PCR buffer, 1 µL each deoxynucleotide (1 mmol/L each), 0.5 µL sense and antisense primers (0.15 mmol/L), and 0.25 µL DNA polymerase (Gene Amp PCR kit, Perkin Elmer Cetus, Norwalk, CT). The reaction mixture was covered with a wax gem (Perkin Elmer Cetus), and amplification was initiated by 1 min denaturation at 94°C for 1 cycle, followed by multiple (20–35) cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min using a GeneAmp PCR system 9800 DNA thermal cycler (Perkin Elmer Cetus). After the last cycle of amplification, samples were incubated for 7 min at 72°C.

The amplified PCR products were subjected to electrophoresis at 100 V through a 2% agarose gel (Gibco Laboratories Life Technologies) for ~30 min. The agarose gels were stained with 0.5 mg/mL ethidium bromide Tris-borate-ethylene diaminetetraacetic acid buffer (ICN, Costa Mesa, CA) and photographed with Type 55 Polaroid positive/negative film. Densitometric analysis of the captured image was performed on a Macintosh computer using NIH Image 1.54 Analysis software. The area under the curve was normalized for GAPDH content.

Evaluation of pathology and distribution of macrophages by immunohistochemistry
To investigate the effect of GdCl3 and CLP on the distribution of tissue macrophages, immunohistochemistry was performed on liver and lung tissue using the antimacrophage antibodies, ED1 (CD68, a standard macrophage marker, Serotec, Oxford, UK) [22 , 23 ] and ED2 ({alpha}Dß2 integrin, a KC marker, Serotec) [22 , 23 ]. Tissue samples were collected from rats killed 9 h after CLP or sham operation, fixed in formalin, embedded in paraffin, and serially sectioned (5 µm-thick). Some sections were stained with hematoxylin-eosin to assess inflammation and necrosis. Others were used for immunohistochemistry using a labeled streptavidin biotin kit (Dako, Carpinteria, CA). Immunohistochemistry was performed as described by Haratake et al. [24 ] with some modifications. The number of ED1- and ED2-positive cells was counted in five different high-power fields, and the number of cells per 400 hepatocytes in the liver or 0.25 mm2 in the lung was assessed (n=6 in each group). Polymorphonuclear neutrophils were identified by morphology and counted in five low-power fields, and the number of cells per 400 hepatocytes in the liver or 0.25 mm2 in the lung was assessed.

Statistical analysis
Data are expressed as mean ± SEM. ANOVA with Bonferroni’s post-hoc test or the Student’s t-test was used for the determination of significance as appropriate. A P value less than 0.05 was selected before the study as the level of significance.


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RESULTS
 
Effect of CLP, GdCl3, and anti-rat IL-10 antibodies on mortality
In the sham operation group, all animals survived 7 days after surgery, and GdCl3 treatment did not affect mortality (data not shown). Mortality was ~85%, 7 days after CLP in the control group (Fig. 1 ). Furthermore, all animals died within 20 h after CLP in the GdCl3 group.


Figure 1
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  		Figure 1. Effect of GdCl3 and CLP on mortality, which after CLP, was determined as described in Materials and Methods (n=14). Control, CLP treatment with a saline vehicle; GdCl3, treatment with GdCl3. *, P < 0.05, compared with the control group by Fisher’s exact test.

After the sham operation, all animals survived in the anti-IL-10 antibody and IgG groups (Table 1 ). In animals treated with control IgG, mortality was 50%, 24 h after CLP, and 83%, 7 days after CLP. In animals treated with anti-IL-10 antibodies, mortality was 100%, 24 h after CLP.


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  		Table 1. Effects of Neutralizing Anti-Rat IL-10 Antibodies on Survival Rate after CLP

Effect of GdCl3 and CLP on pathological changes of the liver and lung
The sham operation and GdCl3 itself did not cause pathological changes in the liver and lung (data not shown). Pathological changes were not observed in the liver 9 h after CLP (Fig. 2A and B ). Conversely, CLP caused acute lung injury in the control group (Fig. 2C ). These changes were more severe in the GdCl3 group (Fig. 2D) .


Figure 2
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  		Figure 2. Effect of CLP, GdCl3, and anti-rat IL-10 antibodies on pathology of the liver and lung. (A) Liver from a saline-treated rat 9 h after CLP; (B) liver from a GdCl3-treated rat 9 h after CLP; (C) lung from a saline-treated rat 9 h after CLP; (D) lung from a GdCl3-treated rat 9 h after CLP; (E) lung from a rat treated with rabbit IgG; and (F) lung from a rat treated with rabbit anti-IL-10 antibodies. Control, Treatment with a saline vehicle; GdCl3, treatment of GdCl3; IgG, treatment with control IgG; and IL-10 Abs, treatment with rabbit anti-rat IL-10 antibodies. Original magnification, x200. Representative photomicrographs.

In the sham operation groups, lung injury was not observed in any of the animals. After CLP, acute lung injury was observed in animals treated with control IgG (Fig. 2E) . The pathology was more severe in animals treated with anti-IL-10 antibodies (Fig. 2F) .

Immunohistochemical analysis of ED1 and ED2 in the liver and lung
Immunohistochemistry was performed to evaluate the effect of GdCl3 and CLP on the distribution of macrophages in the liver and lung. ED1-positive cells were detected in both organs (Fig. 3 ). However, ED2-positive cells, which are recognized as KCs, were detected only in the liver (Table 2 ) and were located mainly around the periportal area. Furthermore, no ED2-positive cells were detected in the liver after GdCl3 treatment (Groups II and IV). The number of ED2-positive cells did not change after CLP (Groups III and IV). In contrast, the number of ED1-positive cells and neutrophils increased significantly in both organs, 9 h after CLP (Group III), and the number of positive cells was significantly greater in the GdCl3 group than in the control group (Group IV).


Figure 3
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  		Figure 3. Immunohistochemical staining for ED1 in the liver and lung. Immunohistochemical staining for ED1 was performed as described in Materials and Methods. (A) ED1 staining of liver from a saline-treated rat, 9 h after CLP; (B) ED1 staining of liver from a GdCl3-treated rat 9 h after CLP; (C) ED1 staining of lung from a saline-treated rat 9 h after CLP; and (D) ED1 staining of lung from a GdCl3-treated rat 9 h after CLP. Control, Treatment with a saline vehicle; GdCl3, treatment with GdCl3. Original magnification, x400. Representative photomicrographs.


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  		Table 2. Number of ED1- and ED2-Positive Cells and Neutrophils in the Liver and Lung

Effect of GdCl3 and anti-rat IL-10 antibodies on lung edema and lung microvascular permeability after CLP
In the control group, the wet/dry lung weight ratio was not significantly different 9 h after CLP compared with normal rats (Fig. 4A ). Conversely, this ratio increased significantly ~1.2-fold in the GdCl3 group. It is important that this ratio was also increased significantly by treatment of anti-IL-10 antibodies compared with control IgG. Furthermore, lung microvascular permeability assessed by EBD significantly increased approximately threefold, 9 h after CLP in the control group compared with normal rats (Fig. 4B) . GdCl3 treatment significantly increased the permeability approximately twofold compared with the control group after CLP. The permeability was increased significantly by treatment with anti-IL-10 antibodies compared with control IgG. Thus, inhibition of the KC by GdCl3 probably increased respiratory disturbances caused by lung injury in sepsis.


Figure 4
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  		Figure 4. Effect of CLP, GdCl3, and anti-rat IL-10 antibodies on lung edema and lung microvascular permeability. Lung tissues were collected 9 h after CLP. The wet/dry lung weight ratio was determined as described in Materials and Methods (A). In a different set of experiments, lung microvascular permeability was measured using EBD as described in Materials and Methods (B). Normal, Normal rats; VEH, treatment with a saline vehicle; GdCl3, treatment with GdCl3; IgG, treatment with control IgG; and Abs, treatment with rabbit anti-rat IL-10 antibodies. Data represent mean ± SEM. *, P < 0.05, compared with normal rats; #, P < 0.05, compared with rats in the GdCl3 group; ##, P < 0.05, compared with rats in the IgG group by ANOVA with Bonferroni’s post-hoc test.

Plasma endotoxin levels in the portal and hepatic vein
Endotoxin was not detectable in plasma from the portal and hepatic vein in the sham operation group (data not shown). In contrast, 9 h after CLP, values were significantly greater in plasma from the portal vein than the hepatic vein in the control group (Fig. 5 ). Furthermore, values were significantly greater in plasma from the portal and hepatic vein in the GdCl3 group compared with the control group.


Figure 5
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  		Figure 5. Plasma endotoxin levels after CLP. Plasma endotoxin levels were determined as described in Materials and Methods. Control, Treatment with a saline vehicle; GdCl3, treatment with GdCl3; P, plasma in the portal vein; and H, plasma in the hepatic vein. Data represent mean ± SEM (n=6 in each group). *, P < 0.05, compared with values in the portal vein in the control group; #, P < 0.05, compared with values in the hepatic vein in the control group by Student’s t-test.

Effect of GdCl3 and CLP on the mRNA expression of cytokines in isolated KCs and tissue macrophages
The mRNA expression of inflammatory mediators was barely detectable in isolated macrophages from the sham operation group (data not shown). After CLP, in the control group, the mRNA expression of IL-6 was detected in macrophages isolated from each organ, and this expression was greatest in peripheral mononuclear cells (Fig. 6 ). The expression was reduced significantly in KCs isolated from the GdCl3 group compared with the control group. After CLP, in the control group, the mRNA expression of IL-10 also increased significantly in KCs and tissue macrophages, except alveolar macrophages. In the GdCl3 group, however, the expression of IL-10 decreased in KCs, but the expression increased in the interstitial pulmonary macrophages. The exact mechanisms underlying these results are not yet understood, and further investigation is needed to clarify this issue.


Figure 6
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  		Figure 6. Effect of GdCl3 and CLP on the mRNA expression of IL-6 and IL-10 in isolated tissue macrophages. The mRNA expression of IL-6 and IL-10 was determined as described in Materials and Methods. PE, Peritoneal macrophages; AV, alveolar macrophages; IT, interstitial pulmonary macrophages; SP, splenic macrophages; PH, peripheral macrophages; CON, treatment with a saline vehicle; and GD, treatment with GdCl3. Data represent mean ± SEM (n=6 in each group). *, P < 0.05, compared with interstitial pulmonary macrophages in the control group; #, P < 0.05, compared with KCs in the control group by ANOVA with Bonferroni’s post-hoc test.

Effect of GdCl3 and CLP on the mRNA expression of cytokines in the liver and lung
The mRNA expression of the cytokines studied was not detected in the sham operation groups (data not shown). After CLP, the expression of IL-6 was greater in the lung than in the liver (Fig. 7 ), where the mRNA expression of IL-6 increased rapidly, with a peak at 6 h, and was significantly greater in the control group than the GdCl3 group until 6 h. In the lung, this expression increased with a peak at 6 h after CLP in the control group. Furthermore, it was significantly greater in the GdCl3 group than the control group, 6 h after CLP. In the liver, the expression of IL-10 increased rapidly and remained high until 9 h. This expression was significantly greater in the control group than the GdCl3 group, except at 6 h after CLP. In the lung, the expression of IL-10 was significantly greater in the GdCl3 group than the control group 9 h after CLP.


Figure 7
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  		Figure 7. Effect of GdCl3 and CLP on the mRNA expression of IL-6 and IL-10 in the liver and lung. The mRNA expression of IL-6 and IL-10 in each organ was determined as described in Materials and Methods. (A) mRNA expression in the liver; (B) mRNA expression in the lung. CON, Treatment with a saline vehicle; GD, treatment with the GdCl3 group. Data represent mean ± SEM (n=6 in each group). *, P < 0.01, compared with the control group by Student’s t-test.

Serum IL-10 levels in the portal and hepatic vein
Serum IL-10 levels were minimal in the sham operation group (data not shown). IL-10 levels in the portal and hepatic vein were markedly greater in the control group than the GdCl3 groups (Fig. 8 ). Furthermore, these levels were significantly greater in the hepatic vein compared with the portal vein in the control group. Conversely, there were no significant differences within the GdCl3 group.


Figure 8
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  		Figure 8. Serum IL-10 levels in the portal vein and hepatic vein after CLP. Serum was collected 9 h after CLP via the portal and hepatic vein, and serum IL-10 levels were determined as described in Materials and Methods. Control, Treatment with a saline vehicle; GdCl3, treatment with GdCl3; P, serum in the portal vein; and H, serum in the hepatic vein. Data represent mean ± SEM (n=6 in each group). *, P < 0.05, compared with values in the portal vein in the control group; #, P < 0.05, compared with values in the hepatic vein in the control group by Student’s t-test.

Serum IL-6 and IL-10 levels after CLP
Serum IL-6 levels were minimal until 90 min after CLP in the control and GdCl3 groups. Levels then gradually increased for up to 12 h in the control group and peaked at 9 h in the GdCl3 group. There were no significant differences between the two groups, except at 12 h (Fig. 9 ). IL-10 expression was not detected until 90 min after CLP in the GdCl3 group. Levels gradually increased for up to 12 h in both groups, although they were markedly greater in the control group than the GdCl3 group.


Figure 9
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  		Figure 9. Serum IL-6 and IL-10 levels and IL-6/IL-10 ratio after CLP. Serum was collected at each time-point after CLP via the aorta, and serum IL-6 and IL-10 levels and the IL-6/IL-10 ratio were determined as described in Materials and Methods. Data represent mean ± SEM (n=6 in each group). CON, Treatment with a saline vehicle; GD, treatment with the GdCl3group. *, P < 0.05, compared with values in the control group by Student’s t-test.


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DISCUSSION
 
Functional differences between KCs and lung macrophages during inflammation
Functional differences between KCs and macrophages in the lung have been reported. Although only a few studies have compared the liver and pulmonary macrophages directly, these reports clearly indicate that significant heterogeneity exists between these two populations [25 ]. KCs, the most abundant mononuclear phagocytes in the body, are predominantly localized in the lumen of hepatic sinusoids and are anchored to the endothelium by long cytoplasmic processes [26 ]. Like KCs, macrophages in the lung also play an important role in nonspecific host defense, as well as in specific immune responses. In a preliminary study, KCs showed extensive phagocytosis and produced IL-10, whereas the pulmonary macrophages took up few latex beads and produced significantly greater quantities of the proinflammatory cytokine tumor necrosis factor {alpha} (TNF-{alpha}) and chemokine monocyte chemoattractant protein-2 (H. Kono et al., unpublished data), consistent with previous reports [27 ].

At least two different subpopulations of macrophages, the alveolar macrophages and the interstitial macrophages, are localized in distinct anatomical compartments in the lung, including the air spaces and lung connective tissue, respectively. Interstitial pulmonary macrophages are quite prominent in the lung, constituting ~40% of the total macrophages in lung tissue [28 ]. Morphometric studies show that the number of macrophages within the interstitium of the lung approximates or exceeds the number of alveolar macrophages [29 ]. Furthermore, as interstitial pulmonary macrophages are in direct contact with matrix and other pulmonary connective tissue components, the release of mediators or enzymes by these cells may have greater biological and/or pathological effects than those released by the alveolar macrophages. Indeed, the mRNA expression of IL-10 was greater in the interstitial pulmonary macrophages than the alveolar macrophages after LPS stimulation in vivo (Kono et al., unpublished data). Furthermore, in this study, the mRNA expression of IL-10 was detected in the interstitial pulmonary macrophages but not in the alveolar macrophages in the control group after CLP (Fig. 6) .

Significance of inflammatory mediators in septic peritonitis and endotoxemia
TNF-{alpha} was expressed markedly in the liver and lung in a LPS infusion model, as reported previously, and the mRNA expression was significantly greater in the control group than in the GdCl3 group [3 ]. It is important that GdCl3 inhibited this increase significantly and improved mortality in that study. Conversely, the expression of TNF-{alpha} was minimal in the liver and lung in the present study (data not shown). After an i.v. bolus injection of 10 mg/kg endotoxin, endotoxin levels in the aorta increased rapidly and then gradually decreased for up to 9 h (Kono et al., unpublished data). Thus, when high levels of endotoxin are present in serum, KCs produce a significant amount of TNF-{alpha}, which is the most potent and critical factor in pathophysiology. Conversely, in the septic peritonitis model, IL-10 produced by KCs rather than TNF-{alpha} was a critical factor (Figs. 2 , 4 , and 9 and Table 1 ). This difference may result from the total amount of endotoxin and the kinetics after injection. Thus, the role of KCs varies in inflammatory conditions. Taken together, these data indicate that the function of the pulmonary macrophages was clearly affected by KCs in endotoxemia; however, details of the regulatory mechanisms and the key molecule bridging the liver and lung are still unclear. Further investigation is needed to clarify these important issues.

IL-6 is produced by monocytes/macrophages, endothelial cells, fibroblasts, and smooth muscle cells in response to stimulation by endotoxin, IL-1ß, and TNF-{alpha} [30 ]. Elevated levels of IL-6 have been described in a number of severe conditions, such as burns, major surgery, and sepsis [31 ]. IL-6 levels increase continuously in patients with septic shock or MOF [32 ]. The significance of IL-6 in the acute-phase response is confirmed by the observation that IL-6 stimulates the synthesis of acute-phase proteins, including C-reactive protein from hepatocytes [33 ]. Thus, the circulating IL-6 level is widely recognized as a useful predictor of the severity of ARDS in the clinic [34 35 36 ]. Therefore, serum IL-6 levels were assessed in the present study. It is surprising that there were no significant differences between the two groups at each time-point until 9 h after CLP (Fig. 9) . As the mRNA expression of IL-6 was greatest in peripheral mononuclear cells (Fig. 6) , and there were no significant differences in serum IL-6 levels between the control and GdCl3 group, except 12 h after CLP, serum IL-6 levels may reflect its production from peripheral mononuclear cells (Fig. 9) . Alternatively, in the liver, the mRNA expression of IL-6 increased rapidly, with a peak at 6 h, and was significantly greater in the control group than the GdCl3 group until 6 h. In the lung, the expression was significantly greater in the GdCl3 group than the control group 6 h after CLP (Fig. 7) . Thus, in addition to the pathophysiological roles of systemic levels of IL-6, there is evidence that IL-6 may have important local, biological effects.

Anti-inflammatory cytokine IL-10 derived from the KC is a key factor in acute lung injury in septic peritonitis
Elimination of KCs by clodronate liposomes increased mortality in a mouse septic peritonitis model [5 ]. In that study, serum IL-10 levels were decreased by elimination of KCs, consistent with the present study. Furthermore, the mRNA expression of IL-10 in the liver and serum IL-10 levels decreased in rats treated with GdCl3 (Figs. 7 and 9) . These results indicate that KCs are a predominant source of systemic IL-10 levels. In the present study, lung injury and mortality increased when KCs were eliminated by GdCl3 (Figs. 1 , 2 , and 4) . As immunoneutralization of endogenously produced IL-10 also increased lung injury and mortality in the present study (Figs. 2 and 4 and Table 1 ), IL-10 derived from the KC plays a pivotal role in acute lung injury and host survival in sepsis. Plasma IL-10 levels increased in endotoxemia and inhibited the release of proinflammatory cytokines, such as IL-6 and TNF-{alpha}, from macrophages, preventing subsequent tissue injury [37 ]. It was reported that IL-10 also stimulates production of IL-1 receptor antagonist (IL-1ra) and release of the soluble p75 receptor, thereby neutralizing the proinflammatory action of these cytokines [33 ]. Indeed, the mRNA expression of IL-1ra in the liver and the lung was greater in the control group than the GdCl3 group in the present study (data not shown). Furthermore, its serum levels were greater in the control group than the GdCl3 group, although the difference was not significant. Thus, in addition to direct anti-inflammatory effects of IL-10, an indirect mechanism may also be involved. Further investigation is needed to clarify this important issue. It is important that IL-10 inhibits production of proinflammatory mediators involved in ARDS by alveolar macrophages [38 ]. Patients with ARDS had lower circulating IL-10 levels than those who were supposedly at risk but had not developed the disease [39 ]. In another study, deficient IL-10 responses contributed to the septic deaths of burnt patients [40 ]. Taken together, these results indicate that the proinflammatory versus the anti-inflammatory imbalance in ARDS may be reflected by the ratio of serum IL-10 and IL-6 levels, which may be a good indicator of risk of ARDS in septic conditions (Fig. 9) .


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ACKNOWLEDGEMENTS
 
This work is supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.

Received April 5, 2005; revised November 11, 2005; accepted November 28, 2005.


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