Neutralization of IL-10 Exacerbates Cycloheximide-Induced Hepatocellular Apoptosis and Necrosis

Abstract

Cycloheximide (CHX)-induced liver injury in rats has been characterized by hepatocellular apoptosis and necrosis. We previously reported that Kupffer cell inactivation causes a reduction of IL-10 production, resulting in the exacerbation of CHX-induced liver injury. In this study, we directly evaluate the role of IL-10 in liver injury by a pretreatment with anti-IL-10 neutralizing antibody (IL-10Ab). Rats were given goat IgG or IL-10Ab before being treated with CHX (CHX group or IL-10Ab/CHX group). In the CHX group, the CHX treatment markedly induced hepatic mRNA and serum protein levels of IL-10. The up-regulation of IL-10 was significantly suppressed in the IL-10Ab/CHX group. Blocking IL-10 in the IL-10Ab/ CHX group led to greater increases in hepatic mRNA and serum levels of proinflammatory cytokines, such as TNF-α and IL-6. The IL-10Ab/CHX group developed more severe hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes compared with the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in the livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results demonstrate that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines, such as a TNF signaling cascade, and in attenuating the CHX-induced liver injury.

Introduction

Hepatocellular apoptosis has been implicated in a number of liver diseases, such as alcoholic or drug-induced liver injury, viral and autoimmune hepatitis, and cholestatic diseases (Neuman 2001). In toxic liver injuries, the mechanistic distinction between hepatocellular apoptosis and necrosis is ill defined because both processes share the same initiation mechanisms, such as the disruption of calcium and mitochondrial homeostasis, accumulation of reactive oxygen species, termination of protein synthesis, or membrane damage (Eichhorst 2005). Previous experimental data also indicate that hepatocellular apoptosis in pathophysiologic conditions promotes neutrophil transmigration and necrosis in livers (Jaeschke et al. 1998; Lawson et al. 1998; Canbay et al. 2003). Under such conditions, the proinflammatory and anti-inflammatory cytokine balance is crucial for the progression of liver injuries. The equilibrium of these cytokines is mainly regulated by Kupffer cells, which are the resident macrophages and the primary source of the cytokines in the liver (Dahle et al. 2004).

A protein synthesis inhibitor, cycloheximide (CHX) induces not only hepatocellular apoptosis but also necrosis of the hepatocytes with slight neutrophil infiltration in rats (Alessenko et al. 1997; Ito et al. 2006; Kumagai et al. 2006). Significant increases in the hepatic mRNA levels of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and an anti-inflammatory cytokine, IL-10, occurred either earlier or simultaneously with the apoptotic induction by CHX (Ito et al. 2006). On the other hand, in the CHX-treated rats, Kupffer cell inactivation by gadolinium chloride (GdCl₃) caused a decrease in the mRNA levels of the cytokines, in particular a loss of ability to produce IL-10, resulting in the aggravation of the necroinflammatory change compared with those not treated with GdCl₃ (Kumagai et al. 2007). One of the important factors in the deterioration of the necrotic change was related to the activation of TNF-signaling as suggested by the up-regulation of hepatic mRNA levels of chemokines and adhesion molecules such as chemokine (C-C motif) ligand (Ccl) 20, oxidized low-density lipoprotein (lectin-like) receptor 1 (LOX-1), and E-selectin in the microarray analysis.

The role of IL-10 in regulating hepatic injuries under pathological conditions has been investigated in some animal models. The data from a genetic knockout model of IL-10 or from animals treated with an anti-IL-10 neutralizing antibody have shown that IL-10 has the capacity to inhibit the inflammatory process or reduce the mortality against several hepatotoxic causes, such as acetaminophen (Bourdi et al. 2002), concanavalin A (Louis, Le Moine, Peny, Quertinmont, et al. 1997; Di Marco et al. 1999), lipopolysaccharide (LPS) (Berg et al. 1995), coadministration of LPS, and galactosamine (Louis, Le Moine, Peny, Gulbis, et al. 1997). More recently, the blocking of endogenous IL-10 by genetic knockout or by immunoneutralization has been shown to enhance hepatocellular apoptosis in LPS-treated mice (Zhong et al. 2006) and in a rat model of hepatic ischemia and reperfusion (Dinant et al. 2007).

The purpose of this study was to investigate the role of IL-10 in the CHX-induced liver injury by direct inhibition using an anti-IL-10 neutralizing antibody. We evaluated the effect of IL-10 inhibition on the expression of multiple cytokines and hepatic cell death in CHX-treated rats. We also examined whether the gene expression changes of Cc120, LOX-1, and E-selectin are responsible for the hepatic injury in this model.

Materials and Methods

Materials and Animal Treatment

Six-week-old male F344/DuCrlCrlj rats were purchased from Charles River Laboratories (Yokohama, Japan). They were maintained for two weeks under controlled conditions (24 ± 2°C temperature, 40% to 70% relative humidity, and a 12:12 light-dark cycle), with free access to a normal laboratory diet and water. A total of fifty animals were used at eight weeks of age, and rats were randomly divided into the following experimental groups (ten rats in each group):

Control group: This group of rats was treated intravenously with 50 μg/rat of normal goat IgG (R&D Systems, Inc., Minneapolis, MN, USA) and was treated intravenously with saline 30 min after goat IgG administration.

CHX group: Groups of rats were treated intravenously with 50 μg/rat of normal goat IgG and were treated intravenously with 3 or 6 mg/kg of CHX 30 min after the goat IgG administration.

IL-10Ab/CHX group: Groups of rats were treated intravenously with 50 μg/rat of antirat polyclonal IL-10 antibody (IL-10Ab, R&D Systems) and were treated intravenously with 3 or 6 mg/kg of CHX at 30 min after the IL-10Ab administration.

Five animals from each group were sacrificed at 2 or 6 h after saline or CHX treatment. Blood was drawn from the abdominal aorta of each animal under anesthesia. The left lateral lobes of the livers were removed and fixed in 10%(v/v) neutral buffered formalin for histopathological evaluation, or stored at −80°C for other examination until use. The experimental protocol was approved by the Ethics Review Committee for Animal Experimental of Daiichi Sankyo Co., Ltd.

Analysis of Liver Enzymes in Serum

Serum samples from the animals autopsied 6 h after the treatment were prepared by the routine procedure. The serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using an automated clinical analyzer TBA-200FR (Toshiba Medical Systems Co., Ltd., Tokyo, Japan).

Histopathology

The fixed samples were dehydrated through graded alcohols and embedded in paraffin. Serial sections of 2 μm thickness were stained with hematoxylin and eosin for routine pathological examination.

Assessment of Hepatocellular Apoptosis

TUNEL Assay:

Each of the liver sections from the animals autopsied 2 h after the treatment was used for the TUNEL assay. The TUNEL assay was performed using an ApopTag^® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., Temecula, CA, USA) according to the manufacturer’s instructions. The reaction products were visualized with 3,3’-diaminobenzidine tetrahydrochloride. The sections were counterstained with hematoxylin. A quantitative analysis for the TUNEL assay was performed to evaluate the percentage of TUNEL-positive nuclei in the hepatocellular nuclei in a total area of approximately 4 mm² (53 microscopic fields, 0.075 mm² per field) per rat. Briefly, the number of hepatocellular nuclei was counted using an image-processing apparatus (IPAP-WIN, Sumika Technoservice Corporation, Hyogo, Japan). Next, the TUNEL-positive nuclei were manually counted in the same pictures. Each quantitative result of TUNEL-positive nuclei was normalized to each quantitative result of the hepatocellular nuclei.

Caspase Assays:

The caspase 3/7, 8, and 9 activities in liver tissues were measured using Caspase-Glo^® assay kits (Promega, Madison, WI, USA) based on the method previously reported (Liu et al. 2004). Briefly, liver tissues were homogenized in 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl₂, 1 mM EGTA, 1 mM Pefablock, 1 μg/ml each pepstatin, leupeptin, and aprotinin. After centrifugation at 13,000 rpm at 4°C for 15 min, the protein concentrations in each supernatant were determined using a bicinchoninic acid protein reagent kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Then 10 μg/ml of diluted sample lysates were incubated with an equal volume of Caspase-Glo^® 3/7, 8, or 9 assay reagents in a white-walled 96-well plate at RT for 1 h. Finally, the luminescence of each sample was measured using a plate-reading luminometer.

Quantification of Hepatic Neutrophils

The liver sections from the animals autopsied at 6 h after the treatment were stained with a myeloperoxidase (MPO) rabbit polyclonal antibody (Dako Japan Inc., Kyoto, Japan) for identifying neutrophils in the liver. The sections were incubated with 0.1% of trypsin solution at 37°C for 10 min. After the pretreatment, endogenous peroxidase activity was quenched by incubation in 3% v/v H₂O₂ at room temperature for 15 min. Next, the sections were incubated with a Dako nonspecific staining blocking reagent (Dako Japan Inc.) at room temperature for 5 min. The sections were then incubated with the MPO antibody, which was prediluted by the manufacturer, followed by incubation with Envision⁺ reagent for mice (Dako Japan Inc.). The reaction products were visualized with a 3,3’-diamino-benzidine tetrahydrochloride substrate. Each section was counterstained with hematoxylin. The number of MPO positive cells was quantitated by manually counting in 100 random microscopic fields (0.075 mm² per field).

Real-Time Quantitative RT-PCR Analysis

Liver sampleswere homogenized with the RLT buffer supplied in the RNeasy^®Mini Kit (QIAGEN, Valencia, CA, USA), and the total RNA was isolated according to the manufacturer’s instructions. The total RNA of 5 μg was treated with 5 U of DNaseI (Takara Bio Inc., Shiga, Japan) in the manufacturer’s buffer containing 40 U RNase inhibitor (TOYOBO Co., Ltd., Osaka, Japan) in a final volume of 50 μL, and subjected to phenol-chloroform purification. DNaseI-treated total RNA 2 μg was reverse-transcribed in the manufacturer’s buffer supplement with 10 mM dithiothreitol, 0.5 mM dNTPs, 200 U of Superscript^®II (Invitrogen Corporation, Carlsbad, CA, USA), and 40 U RNase inhibitor in a final volume of 40 μL. The gene sequences of Cc120 (NM_019233) and LOX-1 (NM_133306) were downloaded from the GenBank. Primer and probe sequences were as follows:

Cc120, forward: 5’-CAAGAAGATGTAAAAACGGATGCTT-3’;

reverse: 5’-CCCCAGCTGTGATCATTTCC-3’;

probe: FAM-TCTGGGATGGAATTGGACACAGCCC-TAMRA.

LOX-1, forward: 5’-TCCCTTAAATTGCATACTTTGTAGACA-3’;

reverse: 5’-TTTGGTCATGTATGTTTCTAAGTCATAGC-3’;

probe: FAM-CTCTCCTTGACCCTGCCATGCCA-TAMRA.

We referred to the previous reports for the sequences of the following primers and probes: TNF-α, IL-6, and IL-10 (Kumagai et al. 2007), and E-selectin (Berti et al. 2002), respectively. Real-time quantitative RT-PCR primers and probes were purchased from Nippon EGT Co., Ltd. (Toyama, Japan). Quantitative RT-PCR was performed using qPCR^™ Mastermix Plus (Eurogentec, Philadelphia, PA, USA), and the mRNA content was quantified with a GeneAmp^® 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. For an internal control, a Rodent GAPDH Control Kit (Applied Biosystems) was used. The relative mRNA levels of the selected genes compared to that of GAPDH gene were calculated individually for all animals.

Cytokine Assay

A panel of cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) were assayed in sera with a Bio-Plex Protein Array System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The sera were assayed using a Bio-Plex rat 9-plexed cytokine assay kit combined with a Bio-Plex Cytokine Reagent Kit and a Diluent Kit for serum (Bio-Rad Laboratories Inc.). Beads coated with capture antibodies (5,000 beads per cytokine) were incubated with premixed standards or sera (50 μL) in 96-well filter plates. The plates were shaken at 1,000 rpm for 30 s and then incubated at room temperature at 300 rpm for 30 min. After incubation, premixed detection antibodies (1 μg/ml) were added and the plates were shaken and incubated as before. After washing, streptavidin-phycoerythrin (2 μg/ml) was added to the wells, and the plates were incubated at room temperature for 10 min with shaking. After washing, the beads were resuspended in 125 μL of Bio-Plex cytokine assay buffer and read using the Bio-Plex array reader. The data were analyzed with Bio-Plex Manager software version 2.0.

Statistical Analysis

Comparisons between the CHX group and the IL-10Ab/ CHX group at the same dose of CHX were analyzed by an F-test to evaluate the homogeneity of variance. If the variance was homogeneous, a Student’s t-test was applied. The data were expressed as the mean ± SD and were considered significant at the level of a p value < .01 or < .05.

Results

Effect of IL-10 Neutralization on mRNA Levels of Cytokines in CHX-Treated Rats

To determine whether the inhibition of IL-10 activity alters the mRNA levels of TNF-α, IL-6, or IL-10, the hepatic mRNA contents were determined by quantitative RT-PCR analysis.

At 2 h after the CHX treatment, the IL-10 mRNA levels in the CHX groups were 14.7-fold and 57.8-fold at the dose of 3 and 6 mg/kg of CHX higher than in the control group, respectively. The IL-10Ab treatment caused significant decreases in the IL-10 mRNA levels to 25.5% and 6.7% in the 3 mg/kg and 6 mg/kg of the CHX treatment compared with the corresponding CHX group (p < .01, Figure 1A). On the other hand, TNF-α and IL-6 mRNA contents in the IL-10Ab/CHX groups were significantly higher than those of the CHX group (p < .01, Figure 1A).

At 6 h after the CHX treatment, the IL-10 mRNA contents in the IL-10Ab/CHX groups showed no differences from those in the CHX groups. As well, the mRNA levels of TNF-α in the IL-10Ab/CHX groups were significantly different from the CHX groups (p < .01), although these increased degrees were reduced through the CHX and IL-10Ab/CHX groups relative to the levels at 2 h postdose (Figure 1B). Moreover, the IL-6 mRNA levels in the IL-10Ab/CHX groups were higher than in the CHX groups (p < .01). The marked induction of IL-6 mRNA was observed in the 6 mg/kg CHX group and both IL-10Ab/CHX groups, compared with those at 2 h after the treatment (Figure 1B).

Effect of IL-10 Neutralization on Serum Cytokines in CHX-Treated Rats

The serum cytokine levels of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α were measured to evaluate the effect of IL-10 inhibition on the production of multiple cytokines in CHX-treated rats. Serum IL-1α, IL-4, or GM-CSF were not detected in any of the groups at any time points. There were no changes in serum IL-2 and IFN-γ among the groups at any time points.

At 2 h after the CHX treatment, the serum IL-10 in the CHX groups was increased twofold over that of the control group. In contrast, the neutralization of IL-10 completely prevented an increase in the serum IL-10 in the IL-10Ab/CHX groups (Figure 2A). Serum TNF-α in the IL-10Ab/CHX groups was increased more than fivefold to tenfold, as compared with the control group. In addition, a significant difference was observed between the serum TNF-α levels in the CHX and IL-10Ab/CHX groups (p < .01, Figure 2A). Similarly, serum IL-1β and IL-6 levels were found to be comparable between the control and CHX groups, but were significantly elevated in the IL-10Ab/CHX groups (p < .01 relative to the CHX group, Figure 2A).

At 6 h after the treatment, the CHX treatment resulted in an increase in the serum IL-6, and reached a tenfold increase over the control group in the CHX groups. Moreover, the marked elevation of IL-6 levels was observed in the IL-10Ab/CHX groups (sixty- to ninety-fold increase over the control) and was significantly higher than those of the CHX groups (p < .01, Figure 2B). The serum TNF-α and IL-1β also remained significantly increased in the IL-10Ab/CHX groups compared with the CHX groups (Figure 2B). On the other hand, decreased levels of serum IL-10 were observed in the IL-10Ab/CHX groups (Figure 2B).

Effect of IL-10 Neutralization on CHX-Induced Hepatocellular Apoptosis

Quantitative Analysis of TUNEL Assay:

Previous studies demonstrated that CHX-induced hepatocellular apoptosis reached a peak at 2 h after the CHX treatment (Ito et al. 2006; Kumagai et al. 2007). Therefore, we examined the effect of the neutralization of IL-10 on the induced level of hepatocellular apoptosis in CHX-treated livers at this time point.

The basal value of TUNEL-positive nuclei was 0.50% ± 0.10% of the hepatocytes in the control group. The number of TUNEL-positive nuclei was markedly increased in both the CHX and IL-10Ab/CHX groups compared with the control group (Figure 3). As shown in Figure 3, the pretreatment with IL-10Ab exacerbated the extent of apoptotic induction by the CHX treatment in both 3 mg/kg (TUNEL-positive nuclei of 8.39% ± 0.53% and 4.65% ± 0.26% for the IL-10Ab/CHX and CHX groups, respectively, p < .01) and 6 mg/kg (TUNEL-positive nuclei of 10.59% ± 0.53% and 5.90% ± 0.53% for the IL-10Ab/CHX and CHX groups, respectively, p < .01).

Enhanced Activation of Initiator and Effector Caspases by IL-10 Inhibition:

We measured the activated levels of initiator caspases 8 and 9 and effector caspase 3 (and caspase 7) in CHX-treated rat livers at 2 h after the CHX treatment. As shown in Figure 4, the CHX administration increased caspase 3 activities in both the CHX and IL10Ab/CHX groups. Moreover, caspase 3 activities in the IL-10Ab/CHX groups were much higher than those in the CHX groups (2.8-fold increase in 3 mg/kg of the CHX treatment, 2.2-fold increase in 6 mg/kg of the CHX treatment, respectively, p < .01). Pretreatment of IL-10Ab significantly increased caspases 8 and 9 activities by 1.3- to 1.8-fold in IL-10Ab/CHX groups compared with the CHX groups (p < .01). From these results, we concluded that IL-10Ab pretreatment enhanced CHX-induced hepatic apoptosis.

Effect of IL-10 Neutralization on Development of the Chx-Induced Necrotic Changes in Hepatocytes

Analysis of Liver Enzymes in Serum:

We showed that the CHX treatment also induced necrotic changes in hepatocytes in addition to hepatocellular apoptosis in previous reports (Ito et al. 2006; Kumagai et al. 2007). Thus, the impact of blocking endogenous IL-10 on hepatic necroinflammatory changes was evaluated at the same time point.

The results of the serum ALT and AST activities are presented in Figure 5. At 6 h post-CHX treatment, the neutralization of IL-10 by the IL-10Ab resulted in significant increases of ALT and AST activities at the dose of 3 and 6 mg/kg of CHX in the IL-10Ab/CHX groups compared with the CHX group (p < .01).

Histopathology:

In the control group, no apoptotic or necrotic changes were observed in the livers at any time points. In the CHX groups, small necrotic foci of the hepatocytes with slight neutrophil infiltrations were observed 6 h after the CHX treatment (Figure 6A). In the IL-10Ab/CHX groups, hepatocellular necrotic lesions were observed more frequently at 6 h after the CHX treatment (Figure 6B). Neutrophils in the sinusoids also increased in the IL-10Ab/CHX groups, compared with the CHX groups at the same time point.

IL-10 Inhibition Increases Infiltration of Neutrophils in the Liver:

To assess the influence of IL-10 inhibition on the infiltration of neutrophils in the liver, the number of MPO-positive cells was measured in the sections from the animals necropsied 6 h after the treatment. The IL-10Ab treatment caused a significant increase in the number of neutrophils in the IL-10Ab/CHX groups compared with the CHX groups (2.3-fold increase in 3 mg/kg of the CHX treatment, p < .01; 1.5-fold increase in 6 mg/kg of the CHX treatment, p < .01; Figure 7).

IL-10 Neutralization Up-Regulates the mRNA Levels of Cc120, LOX-1, and E-selectin in CHX-Treated Rat Livers

The hepatic mRNA contents of Cc120, LOX-1, and E-selectin were determined by quantitative RT-PCR analysis. Thesedata are summarizedin Figure 8. There was no detectable orvery low gene expression of Cc120, LOX-1, or E-selectin in any of the control groups. The expression of Cc120 and LOX-1 in the IL-10Ab/ CHX groups was greatly up-regulated compared with the CHX groups at 2 h and 6 h after the CHX injection (p < .01). In the mRNA contents of E-selectin, a significant increase was only observed at 2 h following the treatment in the IL-10Ab/CHX groups, as compared with each CHX group (p < .01).

Discussion

The objective of this study was to investigate the role of IL-10 in the CHX-induced liver injury by IL-10 inhibition using the neutralizing antibody. The marked induction of IL-10 in the CHX group was suppressed in the IL-10Ab/CHX group. IL-10 neutralization led to greater increases in proinflammatory cytokines such as TNF-α and IL-6. The hepatocellular apoptosis, neutrophil transmigration, and necrotic change of hepatocytes in IL-10Ab/CHX group were more severe than those in the CHX group. The caspase activities and mRNA levels of Cc120, LOX-1, and E-selectin in livers were significantly higher in the IL-10Ab/CHX group than the CHX group. These results suggest that IL-10 plays an important role in counteracting the effect of proinflammatory cytokines on apoptotic and necrotic changes by CHX.

IL-10 is suggested to protect the liver directly by down-regulating apoptotic cytokine secretion and indirectly by counteracting the proapoptotic action of proinflammatory cytokines (Nagaki et al. 1999). In fact, in this study the pretreatment of IL-10Ab resulted not only in the significant induction of TNF-α and IL-6 mRNAs, but also in the marked increase in TNF-α, IL-1β, and IL-6 proteins in the serum. Zhong et al. (2006) have reported that in LPS-treated IL-10 KO mice, the deficiency of IL-10 production increases not only proinflammatory cytokine activities but the expression of those receptors as well, resulting in the further induction of hepatocellular apoptosis. On the other hand, in the CHX group, no or slight increase in the serum TNF-α and IL-1β was observed, as compared to the high serum level of IL-10. One of the reasons that IL-10 directly inhibits the secretion of proinflammatory cytokines is that it promotes degradation of mRNA for these cytokines (Rai et al. 1997; Opal and DePalo 2000). Our findings regarding the hepatic mRNA and plasma levels of proinflammatory cytokines in the CHX groups agree with this suggestion.

In this study, the treatment of IL-10Ab resulted in the clear inhibition of the up-regulation of IL-10 mRNA. The expression of IL-10 mRNA in macrophages can be controlled by IL-10 itself in an autocrine fashion. A negative autoregulatory effect of IL-10 on its gene expression has been previously reported in LPS-treated macrophages (de Waal Malefyt et al. 1991) and Kupffer cells (Knolle et al. 1998). These studies also reported that the treatment of IL-10Ab in the LPS-activated cells resulted in a higher induction of IL-10 mRNA by negative feedback inhibition. However, the opposed feedback role for autocrine IL-10 has been described in other groups (Corinti et al. 2001; Staples et al. 2007). Recently, Staples et al. (2007) demonstrated that the treatment of primary macrophages with IL-10 can induce the transcription of IL-10. They also suggested that IL-10 induction in IL-10-treated macrophages was mediated by the activation of the transcription factor Stat3, while that in LPS-treated macrophages was via a different signaling mechanism. Based on the above, a positive feedback loop may be involved in the increased IL-10 gene expression in CHX-treated rat liver.

The caspase-3, -8 and -9 activities in the liver were found to rise in the CHX and IL-10Ab/CHX groups at 2 h after the treatment. We also showed that the IL-10 blockage significantly increased these caspase activities compared with the CHX group. In the liver, two major pathways lead to the activation of initiator caspases 8 and 9 (Cryns and Yuan 1998). One of these, the “extrinsic” pathway, utilizes death receptors such as Fas, TNF receptor 1 (TNFR1), and TNF-related apoptosis inducing ligand receptors (TRAIL-Rs) to initiate cleavage of procaspase 8 to active caspase 8 through interaction with their corresponding ligands (Fas ligand, TNF-α, and TRAIL, respectively) (Malhi, Gores, and Lemasters 2006). Caspase 9 is activated by the “intrinsic” pathway through the release of cytochrome c from mitochondria. In TNFR1-mediated hepatocellular apoptosis, the mitochondrial death pathway is essential for the execution of an apoptotic program (Yoon and Gores 2002). In contrast, active caspase 9 plays an amplifier role in Fas-mediated apoptotic signaling because Fas can presumably lead to hepatocellular apoptosis through both extrinsic and intrinsic pathways (Scaffidi et al. 1998; Imao et al. 2006). Thus, we suggest that the excessive production of TNF-α in the IL-10Ab/CHX groups was involved in the higher activities of caspases 8 and 9 through TNFR1, which resulted in the elevated activation of effector caspase 3 and in the aggravation of CHX-induced hepatocellular apoptosis.

As we have shown in previous studies (Ito et al. 2006), the CHX treatment induced spotty hepatocellular necrosis in rat livers at 6 h after the treatment. In this study, the CHX-induced necroinflammatory changes were aggravated by the direct inhibition of IL-10 activity. This finding agrees with our hypothesis in the previous report that the Kupffer cell inactivation resulting from GdCl₃ treatment causes a loss of the capacity of IL-10 production, leading to exacerbation of the CHX-induced necrotic changes (Kumagai et al. 2007). A microarray analysis in rat livers exposed to GdCl₃ and CHX previously indicated that the specific up-regulation of hepatic Cc120, LOX-1, and E-selectin might be a precipitating factor of necroinflammatory change. In the present study, the enhanced expression of these genes was also observed in the IL-10Ab/CHX groups. It has been reported that proinflammatory cytokines regulate the transcription of Cc120 (Hromas et al. 1997; Sugita et al. 2002), LOX-1 (Honjo et al. 2003), and E-selectin (Wolf et al. 2001). Shimizu et al. (2001) reported that Cc120 could be produced by macrophages located near the necrotic foci in the liver. Cc120 is suggested to be involved in the migration of activated neutrophils (Yamashiro et al. 2000). Moreover, E-selectin expressed on endothelial cells mediates the rolling of leukocytes along the vascular surface (Tedder et al. 1995). LOX-1 is also expressed on endothelial cells. It has been suggested that one of the functions of LOX-1 is to support neutrophil-endothelial interaction under inflammatory conditions (Honjo et al. 2003). Activated neutrophils promote hepatic cell damage by secreting toxic proteases and reactive oxygen species. Thus, our finding of significantly increased neutrophils in the IL-10Ab/CHX groups suggests that endogenous IL-10 can suppress a necroinflammatory change by counteracting proinflammatory cytokines.

The present study provides additional support for the hypothesis that endogenous IL-10 attenuates apoptotic and necrotic changes of hepatocytes by CHX through decreasing the level of proinflammatory cytokines. Attenuation of hepatic injury by CHX was associated with the inhibition of caspase activities and neutrophil transmigration. Taken together, these results highlight the importance of a balance between proinflammatory and anti-inflammatory cytokine in toxic liver injuries.