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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2004 Dec;85(6):335–343. doi: 10.1111/j.0959-9673.2004.00401.x

Induction of macrophage scavenger receptor MARCO in nonalcoholic steatohepatitis indicates possible involvement of endotoxin in its pathogenic process

Mika Yoshimatsu *, Yasuhiro Terasaki *, Naomi Sakashita *, Emi Kiyota *, Hiroo Sato *, Luc J W van der Laan †, Motohiro Takeya *
PMCID: PMC2517536  PMID: 15566430

Abstract

Nonalcoholic steatohepatitis (NASH) is one of the life-threatening hepatic diseases; however, its pathogenesis is still unknown. To evaluate the causative role of hyperlipidaemia and high-fat diet, we compared C57BL/6 mice with inherited hyperlipidaemic model mice (LDLR–/–mice and ApoE–/– mice) fed a normal or a high-fat diet. LDLR–/– and ApoE–/– mice fed the normal diet showed significantly higher serum cholesterol level than that of C57BL/6 mice fed the high-fat diet. These mice, however, have shown neither significant elevation of serum alanine transaminase (ALT) level nor histopathologic features of steatohepatitis. High-fat diet groups of all three strains showed histopathological characteristics of steatohepatitis with elevated serum ALT levels and high expression of macrophage scavenger receptor MARCO mRNA in the liver. Semiquantitative endotoxin analysis showed an elevated serum endotoxin level in the portal vein but not in the vena cava in ApoE–/– mice fed the high-fat diet. These results indicate that long-term feeding of a high-fat diet induces NASH, whereas hyperlipidaemia alone is not enough to induce NASH. Liver-restricted induction of MARCO in mice with high-fat diet and portal endotoxaemia in ApoE–/– mice fed the high-fat diet suggest the possible involvement of endotoxin in the pathogenesis of NASH.

Keywords: endotoxin, high-fat diet, hyperlipidaemia, MARCO, nonalcoholic steatohepatitis

Introduction

Nonalcoholic steatohepatitis (NASH), which was first documented in 1980 (Ludwig et al. 1980), is a common liver disease that resembles alcoholic liver disease but occurs in persons who drink little or no alcohol. The histological features of NASH are indistinguishable from those of alcohol-related steatohepatitis and include the presence of steatosis, as well as infiltration of inflammatory cells into the lobular and/or portal area, with or without fibrosis (Ludwig et al. 1980; Brunt 2001). Some patients with NASH progress to liver cirrhosis and liver-related death (Harrison et al. 2003; Hui et al. 2003). The main features of metabolic syndromes such as obesity, diabetes, insulin resistance and hyperlipidaemia have been reported as risk factors of NASH (García–Monzón et al. 2000; Dixon et al. 2001; Chitturi et al. 2002). Intimate involvement of diabetes mellitus (Paradis et al. 2001) and obesity (Santamaría et al. 2003) in NASH pathogenesis have been well documented; however, there are few reports about the relationship between hyperlipidaemia and NASH, despite the high morbidity of hyperlipidaemia in NASH patients. To evaluate the causative role of hyperlipidaemia and high-fat diet in NASH, we compared wild-type mice (C57BL/6) with inherited hyperlipidaemic model mice such as low-density lipoprotein (LDL) receptor-deficient (LDLR–/–) mice and apoprotein E-deficient (ApoE–/–) mice fed a normal and a high-fat diet.

Considerable experimental evidence suggests that gut-derived endotoxin play a key role in the pathogenesis of alcohol-related steatohepatitis (Bjarnason et al. 1984; Nanji et al. 1994; Adachi et al. 1995; Enomoto et al. 1998; Uesugi et al. 2002). A previous study found that patients with NASH have some degree of intestinal bacterial overgrowth (Wigg et al. 2001). In animal experiments, administration of probiotics has suppressed lipid deposition within hepatocytes (Li et al. 2003). These data implicate the involvement of gut-derived endotoxin in the pathogenesis of NASH, as has been suggested for alcoholic steatohepatitis. To evaluate the involvement of endotoxin in NASH, we have examined a receptor – macrophage receptor with a collagenous structure (MARCO) which is known to be induced by endotoxin (Elomaa et al. 1995; van der Laan et al. 1997, 1999; Sankala et al. 2002; Takahashi et al. 2002). Serum endotoxin levels in the portal vein and vena cava of the mice were also evaluated.

Materials and methods

Mice and diets

Female C57BL/6 mice, LDLR–/– mice and ApoE–/– mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The LDLR–/– and ApoE–/– mice are maintained on the C57BL/6 background strain. Mice were given access to food and water ad libitum in the Animal Resource Facility at Kumamoto University under specific pathogen-free conditions. All animal procedures were approved by the Animal Research Committee at Kumamoto University, and all procedures conformed to the Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources.

Two diets, a normal diet and a high-fat diet, were used in the experiment. The normal diet was a regular diet for mouse containing 0.1% cholesterol (CE-2 from CLEA, Tokyo, Japan), and the high-fat diet contained 1.25% cholesterol, 50% sucrose, 20% casein, 1% corn oil, 0.5% cholic acid, 0.3% dl-methionine, 15% cocoa butter, 5% AIN mineral mixture, 4.95% cellulose, 1% AIN vitamin mixture and 1% choline chloride (CLEA, Tokyo, Japan).

Feeding of the high-fat diet to C57BL/6, LDLR–/– and ApoE–/– mice was started when the mice were between 8 and 10 weeks old and continued for 12 weeks. At 2, 4, 8 or 12 weeks after feeding with the high-fat diet, mice were killed with ether anaesthesia, and assays were performed as follows. Age-matched mice fed a normal diet were used as controls. Blood was sampled from the inferior vena cava and the portal vein in all mice. Then, the liver, spleen, lymph nodes, thymus, lung, stomach, small intestine, large intestine, kidney, heart and brain were removed. For analysis of lipopolysaccharide (LPS)-induced MARCO expression, 100 µg of LPS (O111; Sigma-Aldrich Co., St. Louis, MO, USA) was injected into the tail vein of C57BL/6 mice and were killed with ether anaesthesia after 24 h. Serum samples were stored at −80 °C until total cholesterol and alanine transaminase (ALT) levels were analysed by standard enzymatic procedures (Richmond 1973; Allain et al. 1974; Bergmeyer et al. 1978; Bergmeyer 1988).

Histopathology

Liver tissues were fixed with 10% neutral-buffered formaldehyde and were embedded in paraffin. Tissue specimens were cut into 3-µm-thick sections and were stained with haematoxylin and eosin for pathological evaluation and with Elastica Masson–Goldner methods for the detection of fibrosis. For lipid staining, liver tissues were fixed in 2% periodate–lysine–paraformaldehyde solution at 4 °C for 4 h, washed for 4 h with phosphate-buffered saline containing 10, 15 or 20% sucrose, respectively, embedded in OCT compound (Miles, Elkhart, IN, USA) and frozen in liquid nitrogen. Tissue sections were cut with a cryostat (Microm, Waldorf, Germany) into 6-µm-thick sections and were stained with Oil red O.

Immunohistochemistry

For immunohistochemistry, the following antibodies were used: F4/80 (Serotec, Oxford, UK), a monoclonal antibody against murine macrophages (Hume & Gordon 1983); GR-1 (LY-6G) (Southern Biotechnology Associates, Birmingham, AL, USA), a monoclonal antibody against murine neutrophils (Dubois et al. 1993); and ED31, a monoclonal antibody against the murine MARCO (van der Laan et al. 1997, 1999).

After inhibition of endogenous peroxidase activity by methanol and H2O2, 6-µm-thick frozen sections were covered with one of the first antibodies mentioned above. As a secondary antibody, we used horseradish peroxidase-linked antirat immunoglobulin (Serotec). After visualization with 3,3′- diaminobenzidine (Dojin Chemical, Kumamoto, Japan) and nuclear staining with haematoxylin, sections were mounted with resin. After immunohistochemistry with F4/80, Oil red O stain was performed to detect lipid deposition in F4/80-positive cells.

RNA purification, cDNA synthesis and reverse transcription-polymerase chain reaction assay

For RNA extraction, animals were killed as described above, and the liver, spleen, lymph nodes, lung, thymus, stomach, small intestine, large intestine, kidney, heart and brain tissues were homogenized with Lysing Matrix D and FastPrep (Qbiogene, Carlsbad, CA, USA). They were then lysed with a QiaShredder (Qiagen, Valencia, CA, USA), and total RNA was purified from the lysate by using the RNeasy kit (Qiagen). RNA 1 µg was converted to cDNA by using the Omniscript RT kit (Qiagen) for first-strand cDNA synthesis. PCR primers for mouse MARCO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were custom synthesized (Invitrogen, Carlsbad, CA, USA) from the following sequences: MARCO: 5′-GAAACAAAGGGGACATGGG-3′ as the forward primer and 5′-TCCACACCTGCAATCCCTG-3′ as the reverse primer (277 bp), GAPDH: 5′-GGAAAGCTGTGGCGTTGGCGTGAT-3′ as the forward primer and 5′-CTGTTGCTGTAGCCGTATTC-3′ as the reverse primer (392 bp). cDNA was amplified in a 20-µl reaction mixture containing 0.2 mmol/l dNTPs, 1× PCR buffer, 2.5 U of HotStarTaq DNA polymerase, 0.5 µmol/l of 5′ and 3′ primers and RNase-free water (Qiagen). The reaction protocol was as follows: 15 min at 95 °C, 30 s at 94 °C and 30 s at 55 °C to 60 °C for 30 cycles with an iCycler (Bio-Rad Laboratories, Hercules, CA, USA). Amplified bands underwent electrophoresis on a 2.0% agarose gel and were scanned by a Gel Doc 2000 analyser (Bio-Rad Laboratories).

Endotoxin assay

Blood samples from the portal vein and inferior vena cava of C57BL/6, LDLR–/– and ApoE–/– mice were centrifuged at 20000 g for 10 min in room temperature. Before measurement of endotoxin, serum was diluted and heated at 75 °C for 10 min to inhibit the plasma inhibitor of endotoxin (Levin & Bang 1996). Serum endotoxin was detected via the Limulus Amebocyte Lysate test kit (Wako, Tokyo, Japan). Samples that showed aggregation after mixing with the reagents were judged to be positive and considered to contain endotoxin at a value more than 0.06 EU/ml. Through this assay, the samples were handled under pyrogen-free conditions.

Statistical analyses

Differences between groups were analysed by using Student's t-test and were considered statistically significant at P < 0.01.

Results

Serum levels of total cholesterol

Table 1 summarizes the profile of total serum cholesterol levels after 12 weeks of feeding of the high-fat diet or the normal diet. Even with normal diet, LDLR–/– and ApoE–/– mice showed higher serum cholesterol levels (LDLR–/– mice, 290 ± 81.1 mg/dl; ApoE–/– mice, 1093.0 ± 24.4 mg/dl) compared to C57BL/6 mice (16.7 ± 4.6 mg/dl). With high-fat diet, the serum cholesterol level of C57BL/6 increased four times higher (68.0 ± 9.2 mg/dl) than normal. In LDLR–/– mice, the serum cholesterol value (2466.7 ± 277.9 mg/dl) increased significantly, to about eight times higher than the value for mice fed the normal diet. The serum cholesterol level of ApoE–/– mice also showed a significant increase (1466.7 ± 58.4 mg/dl) after feeding of the high-fat diet.

Table 1.

Total cholesterol levels (mg/dl) in serum

Mouse strain Normal diet High-fat diet for 12 weeks
C57BL/6 16.7 ± 4.6 68.0 ± 9.2*
LDLR–/– 290.6 ± 81.1 2466.7 ± 277.9*
ApoE–/– 1093.0 ± 24.4 1466.7 ± 58.4*
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*

P < 0.01.

Serum levels of alanine transaminase

The serum ALT level was measured as a marker of liver injury. With the normal diet, serum ALT levels were about 15.7 ± 4.6 U/l in C57BL/6 mice, 18.7 ± 2.7 U/l in LDLR–/– mice and 25.7 ± 7 in ApoE–/– mice (Figure 1a–c). Twelve weeks of feeding on the high-fat diet induced a significant increase in the serum ALT level of about 4.3-fold (68.0 ± 9.2 U/l) in C57BL/6 mice and 2.8-fold (51.7 ± 6.0 U/l) in LDLR–/– mice compared with the values obtained after feeding the normal diet (Figure 1a,b). Although there was no statistical difference in corresponding ALT values for the ApoE–/– mice, the ALT level tended to increase with high-fat diet (Figure 1c).

Figure 1.

Figure 1

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Effect of a high-fat diet on serum alanine transaminase (ALT) levels. Blood samples were collected from age-matched control mice fed a normal diet (ND) and mice fed a high-fat diet (HFD) for 12 weeks. Data represent means ± SEM. n = 4 for each group. *P < 0.01 by Student's t-test.

Histopathological changes in the liver after feeding of the high-fat diet

Figure 2 shows histopathological changes in the liver of C57BL/6, LDLR–/– and ApoE–/– mice fed the normal or the high-fat diet for 12 weeks. No pathological changes were seen in the normal diet groups (Figure 2a–c). The high-fat diet induced fatty liver (Figure 2d–f) and ballooning degeneration of hepatocytes (Figure 2g–i) in all three strains of mice. Lipid staining confirmed lipid deposition in the liver (Figure 2j–l). Elastica Masson–Goldner stain revealed fibrosis around the central vein in high-fat diet groups (Figure 2m–o).

Figure 2.

Figure 2

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Histological features of livers from mice fed a normal diet and those fed a high-fat diet for 12 weeks. (a, d, g, j,m), C57BL/6 mice; (b, e, h, k,n), LDLR–/– mice; (c, f, i, l,o), ApoE–/– mice. (a–c), normal diet group; (d–o), high-fat diet group. In the high-fat diet group, fatty change of liver (d–f) and ballooning degeneration of hepatocytes (g–i) are seen. Haematoxylin and eosin staining. Oil red O stain confirms lipids deposition in the liver tissue (j–l). Elastica Masson–Goldner stain shows slight-to-mild fibrosis around the central vein. Black arrows: hepatocytes with ballooning degeneration. Original magnification: a–f, ×100; m–o, ×200; g–l, ×400.

Immunohistochemical detection of inflammatory cells

Although few neutrophils were seen in the livers obtained from the normal diet groups (Figure 3a–c), high-fat diet treatment induced distinct infiltration of neutrophils (Figure 3d–f) in all three mouse strains.

Figure 3.

Figure 3

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Immunohistochemical detection of Gr-1-positive granulocytes in the livers. (a, d), C57BL/6 mice; (b, e), LDLR–/– mice; (c, f), ApoE–/– mice fed a normal diet (a–c) or a high-fat diet (d–f). Original magnification, ×100.

In contrast to livers of the normal diet groups (Figure 4a–c), the numbers of F4/80-positive macrophages increased and showed foamy appearance after the high-fat diet (Figure 4d–f). Oil red O stain revealed lipid accumulation in these foamy macrophages (Figure 4g–i).

Figure 4.

Figure 4

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Immunohistochemical detection of F4/80-positive macrophages in livers from C57BL/6 mice (a, d, g), LDLR–/– mice (b, e, h) and ApoE–/– mice (c, f, i) fed a normal diet (a–c) or a high-fat diet (d–i). Double staining of immunohistochemistry with F4/80 and Oil red O stain (g–i). Black arrows: foamy macrophages double-stained with F4/80 and Oil red O stain. Original magnification: a–f, ×200; g–i, ×1000.

Detection of MARCO mRNA and protein

As the expression of MARCO is rapidly induced by endotoxin (van der Laan et al. 1997, 1999; Ito et al. 1999), MARCO expression in livers was examined by means of immunohistochemistry and reverse transcription-polymerase chain reaction assay (RT-PCR), on the assumption that endotoxin might be involved in the pathogenesis of NASH. Immunohistochemistry with ED31 antibody demonstrated no or only a few MARCO-positive cells in the livers of C57BL/6, LDLR–/– and ApoE–/– mice fed the normal diet (Figure 5a–c). In contrast, MARCO-positive cells were clearly detected in all three strains of mice fed the high-fat diet (Figure 5d–f). Expression of MARCO mRNA in various organs including spleen, lymph nodes, liver, lung, thymus, kidney, stomach, small intestine, large intestine, heart and brain from C57BL/6, LDLR–/– and ApoE–/– mice was analysed by RT-PCR and was compared with that of mice injected with LPS (Figure 6). In all mice fed the normal diet, constitutional expression of MARCO was observed in the spleen and lymph nodes, but no expression was observed in the liver, as have been shown previously (Elomaa et al. 1995; van der Laan et al. 1997, 1999; Takahashi et al. 2002). After feeding of the high-fat diet for 4 weeks, expression of MARCO mRNA was induced strongly in the liver and weakly in the lung in all three types of mice, whereas no mRNA induction was found for the other organs except spleen and lymph nodes (Figure 6). In contrast to high-fat diet treatment, LPS induced MARCO expression in many organs, including the brain (Figure 6).

Figure 5.

Figure 5

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Immunohistochemical detection of MARCO-positive cells in livers from C57BL/6 mice (a, d), LDLR–/– mice (b, e) and ApoE–/– mice (c, f) fed a normal diet (a–c) or a high-fat diet (d–f). Original magnification: ×200.

Figure 6.

Figure 6

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Expression of MARCO and control GAPDH mRNA in various organs from C57BL/6, LDLR–/– and ApoE–/– mice fed a normal diet (ND) or a high-fat diet (HFD) and from lipopolysaccharide (LPS)-treated C57BL/6 mice (LPS), as detected by RT-PCR.

Endotoxin assay

To evaluate the endotoxin levels in the portal and whole-body circulation, a semiquantitative endotoxin assay was performed. Positive reaction for endotoxin (more than 0.06 EU/ml) was detected in the portal vein but not in the inferior vena cava of ApoE–/– mice after the high-fat diet for 12 weeks, whereas no endotoxin was detected in ApoE–/– mice fed the normal diet (Figure 7a,b). Serum endotoxin was not detected in either the portal vein or the inferior vena cava in both high-fat and normal diet groups of LDLR–/– and C57BL/6 mice (data not shown).

Figure 7.

Figure 7

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(a) Detection of endotoxin in serum from the portal vein and inferior vena cava (IVC). Serum endotoxin levels in ApoE–/– mice fed an ND or an HFD and in C57BL/6 mice injected with LPS were determined via the Limulus Amebocyte Lysate assay as described in Materials and methods. n = 4 for each group. +, over 0.06 EU/ml of endotoxin level. (b) Endotoxin assay of serum of the samples obtained from two ApoE–/– mice fed an HFD. Both samples obtained from the portal vein showed aggregation, which indicates that the serum endotoxin content was higher than 0.06 EU/ml, whereas samples from the IVC showed no aggregation.

Discussion

Hyperlipidaemia has been considered one of the risk factors for NASH as well as obesity, diabetes and insulin resistance (García–Monzón et al. 2000; Dixon et al. 2001; Chitturi et al. 2002). In the present study, LDLR–/– and ApoE–/– mice fed the normal diet already showed significantly higher serum cholesterol levels than that of C57BL/6 mice fed the high-fat diet. These mice, however, showed neither significant elevation of serum ALT level nor histopathologic features of steatohepatitis. Whereas, long-term feeding of a high-fat diet induced steatohepatitis in LDLR–/–, ApoE–/– as well as C57BL/6 mice, as evidenced by the accumulation of lipids within hepatocytes, infiltration of neutrophils into the liver and increase of serum ALT levels regardless of the fact that the serum cholesterol levels were significantly different in three mouse strains. These findings suggest that daily intake of high-fat diet is an important factor for the development of NASH, whereas hyperlipidemic state alone is not enough to induce steatohepatitis.

In alcohol-related steatohepatitis, endotoxin from intestinal flora has been considered one of the important factors to cause hepatic inflammation (Bjarnason et al. 1984; Nanji et al. 1994; Adachi et al. 1995; Enomoto et al. 1998; Uesugi et al. 2002), though its role in NASH is still unclear. In this study, we have found that high levels of expression of MARCO were induced in the liver of all three mouse strains after high-fat diet. MARCO, a transmembrane protein, belonging to the class A scavenger receptor family, has been described as a receptor for bacteria (Elomaa et al. 1995; van der Laan et al. 1997, 1999; Elomaa et al. 1998; Ito et al. 1999; Sankala et al. 2002; Takahashi et al. 2002). Expression of MARCO is confined to macrophages in the marginal zone of the spleen and the marginal sinus of lymph nodes and is not detected in any other organs under normal steady conditions (Elomaa et al. 1995; van der Laan et al. 1997, 1999; Takahashi et al. 2002). However, it is rapidly induced on macrophages in the whole body including Kupffer cells of the liver after endotoxin injection or by septic shock (Elomaa et al. 1995; van der Laan et al. 1997, 1999). In the present study, high-fat diet-induced MARCO expression was almost exclusively restricted to the liver, although weak induction in the lung and constitutional expression in the spleen and lymph nodes were observed. These observations indicate the possibility that restricted expression of MARCO in the liver could be induced by the local exposure of liver macrophages to endotoxin in portal circulation.

To delineate the presence of endotoxin in the serum of portal vein and inferior vena cava, we performed semiquantitative endotoxin assay. Although no detectable level of endotoxaemia was observed in LDLR–/– and C57BL/6 mice, local endotoxaemia in portal circulation was detected in ApoE–/– mice fed a high-fat diet. Although the precise mechanism of portal endotoxaemia induced by high-fat diet is not clear, liver-restricted induction of MARCO in three mouse strains fed a high-fat diet and portal endotoxaemia in ApoE–/– mice fed a high-fat diet indicate the possible involvement of endotoxin in NASH pathogenesis. Accurate measurement of endotoxin levels in portal circulation and evaluation of hepatic MARCO expression in human cases will provide further insight into the pathogenesis of NASH.

Acknowledgments

We thank Takenobu Nakagawa, Makiko Tanaka and Junko Imura for technical and secretarial assistance. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports and Technology of Japan (12557023 to M. Takeya).

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