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. 2016 Sep 17;4(3):61–73. doi: 10.14252/foodsafetyfscj.2016012

Histopathological Analysis of Rat Hepatotoxicity Based on Macrophage Functions: in Particular, an Analysis for Thioacetamide-induced Hepatic Lesions

Jyoji Yamate 1,✉, Takeshi Izawa 1, Mitsuru Kuwamura 1
PMCID: PMC6989168  PMID: 32231908

Abstract

Hepatic macrophages play an important role in homeostasis. The functional abnormalities of hepatic macrophages primarily or secondarily influence chemically induced hepatotoxicity. However, the evaluation system based on their functions has not yet been established. Recently, a new concept (M1-/M2-macrophage polarization) was proposed; M1-macropahges are induced by INF-γ, and show high phagocytosis/tissue damage, whereas M2-macropahges are induced by IL-4 and play roles in reparative fibrosis by releasing IL-10 and TGF-β1. In hepatogenesis, CD68-expressing M1-macrophages predominantly exist in embryos; in neonates, in contrast, CD163-/CD204-expressing M2-macrophages appear along the sinusoids and mature as Kupffer cells. Activated Kupffer cells by liposome decrease AST and ALT values, whereas AST and ALT values are increased under Kupffer cells depleted with clodronate treatment. Since Kupffer cells may be involved in clearance of liver enzymes, macrophage condition should be taken into consideration when hepatotoxicity is analyzed. In TAA-induced acute hepatic lesions, INF-γ, TNF-α and IL-6 for M1-factors and IL-4 for M2-factors are already increased before histopathological change; the appearance of CD68-expressing M1-macrophages and CD163-expressing M2-macrophages follows in injured centrilobular lesions, and TGF-β1 and IL-10 are increased for reparative fibrosis. CD68-expressing M1-macrophages co-express MHC class II and Iba-1, whereas CD163-expressing M2-macrophages also express CD204 and Galectin-3. Under macrophage depletion by clodoronate, TAA-treated rat livers show prolonged coagulation necrosis of hepatocytes, and then develop dystrophic calcification without reparative fibrosis. The depletion of hepatic macrophages influences hepatic lesion development. Collectively, a histopathological analysis method for hepatotoxicity according to M1-/M2-macrophage polarization would lead to the refinement of hazard characterization of chemicals in food and feed.

Key words: M1-/M2-macrophage polarization, homeostasis, depletion, activation, immunophenotypes, hepatotoxicity, histopathological analysis method, rat

1. Introduction

Macrophages, which were found and named by Metchnikoff in 1892, are the most primitive cells; they possess properties both of amoebas having high wondering capacity and paramecia with activated englobement. They exist in common in all multicellular organisms. Fundamentally, macrophages belong to the mononuclear phagocyte system in mammals, and are divided into three types: exudate macrophages, resident macrophages (histiocytes) and antigen-presenting cells; these types differ in ontogeny, morphology, tissue distribution, and functions1–3) (Fig. 1). In normal tissues, specifically named macrophages exist, such as fetal macrophages with high phagocytic activity, Kupffer cells in the liver, alveolar macrophages, pulmonary intravascular macrophages whose functions resemble those of Kupffer cells, microglial cells/meningeal macrophages in the brain, abdominal/thoracic macrophages in the body cavity, osteoclasts in the bone tissue, interstitial dendritic cells in the connective tissues, Langerhans cells in the epidermis, and follicular/interdigitating follicular dendritic cells in the spleen and lymph nodes4–7). The last three types are regarded as antigen-presenting cells which mediate immune system in relation with Th1 and Th2 lymphocytes8,9).

Fig. 1.

Fig. 1

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General concepts of histogenesis of macrophages. At prenatal stages, macrophages generate from the yolk sac or liver hematopoiesis, and are known as primitive or fetal macrophages. After birth, macrophages consistently generate from hematopoietic stem cells in the bone marrow and then differentiate in the peripheral blood, through the monocytic lineage of GM-CFU, M-CFU, monoblasts, and monocytes. In connective tissues, there are macrophages named fixed macrophages (Kupffer cells and histiocytes) or antigen-presenting cells (dendritic cells and Langerhans cells), which are considered to generate from precursors originating at the prenatal stages. In pathological lesions, macrophages may be divided into three types; exudate macrophages, antigen-presenting cells and fixed macrophages. However, there is difficulty in determining the derivation and functions of infiltrated macrophages in pathological lesions. Therefore, the concept of M1-/M2-macrophage polarization is proposed (Fig. 2).

In damaged tissues, infiltrated macrophages display functions such as modulation/recruitment of inflammatory cells, killing of cells/microorganisms, debridement of necrotic tissues, and antigen presentation, as well as fibrogenic stimulation, by producing appropriate factors to induce these functions10–13). There are specifically-named macrophages in pathological lesions, based on their cell morphology; rod cells in the brain, fatty granular cells/gitter cells in encephalomalacia, scavenger cells., foamy macrophages, and hemosiderin-laden macrophages in hemorrhagic lesions, and epithelioid cells/multinucleated giant cells (such as Langhanse type and foreign body type which are seen mainly in granuloma lesions)1,4,6,7,14,15,16).

As mentioned above, although macrophages may be divided into three types, in pathological settings, macrophages exhibit phenotypes transitional/intermediate between exudate and resident macrophages11,12). In addition, antigen-presenting cells may be formed not only from dendritic cells but also from resident macrophages and blood monocyte-derived macrophages8,9). The three different types of macrophages can be interchangeable in their functions and morphology which may depend on microenvironmental conditions (Fig. 1). It is difficult for pathologists to recognize the derivation and functions of macrophages appearing in pathological conditions. Pathologically, Langerhans type giant cells may be induced by INF-γ, whereas foreign body type giant cells can be formed by IL-4; the former is functionally related to Th1 reaction, and the latter is regarded as Th2 immunity8,9). Recently, therefore, a new concept (M1-/M2-macrophagepolarization) was proposed for reactive macrophages in pathological conditions. M1-type is induced mainly by INF-γ, and shows high phagocytosis and cytotoxicity, whereas M2-type is induced mainly by IL-4 and plays roles in reparative fibrosis by releasing IL-10 and TGF-β1 (Fig. 2)17,18).

Fig. 2.

Fig. 2

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The concept of M1-/M2-macrophage polarization. Microenvironmental conditions in pathological lesions influence the properties of macrophages. Therefore, macrophages appearing in pathological lesions are divided into “classically activated macrophages (M1-macrophages)” and “alternatively activated macrophages (M2-macrophages)”. M1-macrophages are characterized by CD68 expression, whereas M2-macrophages mainly express CD163. The former macrophages are induced by IFN-γ, and produce pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, as well as IFN-γ itself. The latter are mainly induced by IL-4, and produce anti-inflammatory factors such as IL-10, IL-12 and TGF-β1. M1-macrophages enhance tissue damage by producing cytotoxic factors, whereas M2-macrophages participate in not only anti-inflammation, but also immune response and tissue repair/reparative fibrosis. M2-macrophages may be subdivided into M2a for anti-inflammation, M2b for immune response, and M2c for tissue repair. Generally, besides CD68, M1-macrophages express MHC class II and Iba-1, and appear at early stages of lesions; in addition to CD164, M2-macrophages possess CD204 and Galectin-3 antigens and take part in tissue remodeling at the advanced/repair stages. Interestingly, there are macrophages showing the shift from M1 to M2, or vice versa, according to the authors’ data with double-immunofluorescence with M1 and M2 maker antigens.

Hepatic macrophages have an important role in homeostasis and lesion development in the liver. The functional abnormalities of hepatic macrophages may primarily or secondarily influence chemically-induced hepatotoxicity (Fig. 3)19). However, the evaluation system based on their macrophage functions has not yet been established. Many chemicals are metabolized in the liver and, therefore, the pathogenesis of chemically-induced hepatotoxicity, particularly which may be related to functions of hepatic macrophages, should be investigated in toxicological pathology (Fig. 3).

Fig. 3.

Fig. 3

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General Pathogenesis of hepatotoxicity. Chemicals metabolized in the liver show different hepatotoxicity; the lesions may be characterized histopathologically by hepatocyte atrophy, degeneration, apoptosis and necrosis, as well as swelling. The lesions may be developed via the following three pathogenesis: A Direct injury to hepatocytes with the toxicity to cell membrane, cytoplasmic organelles or nucleic acid; B Injury by active metabolites generated by metabolizing enzymes (such as CYPs) in hepatocytes (enzyme-produced active metabolite-related injury); C Hepatic macrophage-induced/related-hepatotoxicity. In the hepatic macrophage-induced/related hepatotoxicity, the two mechanisms are present; C-1. Infiltrating macrophages can damage tissue by producing cytotoxic factors; C-2. Some macrophages appearing in injured areas may act as antigen-presenting cells under stimulation of chemicals themselves (enhancement or inhibition of hepatic macrophage functions) by ingesting cell debris (including DAMPs) of injured hepatocytes via MHC class II expression in relation to Th1/Th2 lymphocytes or Th17/Treg, leading to very complex immune-mediated hepatotoxicity such as allergic liver injury; the detailed mechanisms of T lymphocyte reaction in hepatotoxicity should be investigated further.

This review describes the importance of hepatic macrophages in liver homeostasis and hepatotoxicity, mainly based on the authors’ data. Issues on hepatic macrophages in mouse and human hepatotoxicity are beyond the scope of this review. Because rats have been widely used as an experimental animal in toxicity studies, the authors focus on hepatic macrophages in rat hepatotoxicity, particularly TAA-induced hepatic lesions which have been analyzed in the authors’ laboratory. Finally, it is proposed in this review that macrophages appearing in hepatic lesions induced by chemicals may be evaluated in rat toxicity studies: that is, the histopathological analysis based on macrophage functions may lead to the refinement of hazard characterization of chemicals in food and feed.

2. Immunophenotypical Detection of Rat Macrophages with Normal and Altered Functions

Rat macrophages can be detected with commercially available monoclonal antibodies, histopathologically. Mainly, six antibodies have been widely used to detect rat macrophages (Table 1). CD68 antigen expression is most commonly used for detection of blood monocytes and exudate macrophages in pathological lesions20,21); the antigen is located on the membrane of lysosomes, especially phagolysosome, of macrophages; thus, the amount of CD68 expression implies the extent of phagocytic activity20,22). CD163 antigen is expressed on cell surface of resident macrophages (Kupffer cells)20,23); the antigen is a glycoprotein, which belongs to scavenger receptor cysteine-rich group B family and functions as the scavenger receptor for hemoglobin-haptoglobin complexes23). Antibody to CD204 was generated against human type 1 scavenger receptor protein and CD204 expression is related to metabolism of oxidized low density lipoprotein24); in normal rat liver, CD204 is expressed in Kupffer cells existing along the sinusoid, similar to CD163expression25). MHC class II molecule may be expressed in dendritic cells and activated macrophages as the antigen-presenting cells21,26). Antibody to Iba1 is used usually to detect microglial cells in normal brain tissues, and Iba1 antigen plays some roles in inflammation such as migration, proliferation, and signal transduction of macrophages27,28). Galectin-3 was originally described as the Mac-2 antigen expressed on the surface of activated peritoneal macrophages29); functionally, the soluble factor of Galectin-3 may contribute to myofibroblast development in human and experimentally-induced hepatic fibrosis30,31). Galectin-3 may be expressed in activated macrophages with phagocytosis and its expression may be related to the induction of fibrosis through activation of hepatic stellate cells in liver lesions32,33).

Table 1. Information on primary antibodies which have been commonly used to detect rat macrophages in immunohistocemistry.

Antibody
(clone name)		 Type Fixative Dilution Pretreatment Source
CD68 (ED1) Mouse monoclonal PLP 1/500 100µg/ml Proteinase K, 10 min AbD Serotec, Oxford, UK
CD163 (ED2) Mouse monoclonal PLP 1/300 100µg/ml Proteinase K, 10 min AbD Serotec, Oxford, UK
CD204 (SRA-E5) Mouse monoclonal Zamboni’s
solution		 1/1000 MW in citrate buffer, 20 min Transgenic Inc., Kumamoto, Japan
MHC class II (OX6) Mouse monoclonal PLP 1/1000 MW in citrate buffer, 20 min AbD Serotec, Oxford, UK
Iba-1 Rabbit polyclonal Zamboni’s
solution		 1/1000 MW in citrate buffer, 20 min Wako Pure Chemical Industries, Osaka, Japan
Galectin-3 Rabbit polyclonal Zamboni’s
solution		 1/500 MW in citrate buffer, 20 min Santa Cruz Biotechnology, Santa Cruz, CA, USA
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PLP: periodate-lysine-paraformaldehyde; MW: microwave. Iba-1, Ionized calcium binding adaptor molecule 1.

3. Macrophages in Developing Rat Liver (hepatogenesis)

The majority of macrophages in developing rat liver at prenatal stages are of CD68-positive, indicative of prodigious phagocytic activity25,34); such macrophages are regarded as primitive or fetal macrophages1), which are considered to generate in York sac at the embryonic stage or liver hematopoiesis at the fetal stage. During prenatal development of rat liver, Kupffer cells expressing CD163 and CD204, and dendritic cells expressing MHC class II are fewer. After birth, however, CD68-expressing macrophages are gradually decreased, whereas macrophages expressing CD163, CD204 and MHC class II increase in number25). Kupffer cells and dendritic cells after birth may be related to maturation of the immune system and host defense25,35). Kupffer cells reside along the sinusoids of the hepatic cords, whereas dendritic cells are localized mainly in the Glisson’s sheath as an interstitial dendritic cell type19,25).

Hepatic macrophages show different functions, depending on stages of liver development; CD68-expressing fetal macrophages are most predominant in the prenatal stages, whereas in postnatal and adult stages, the common macrophages are Kupffer cells expressing CD163 and CD204, as well as MHC class II-expressing antigen-presenting cells25). The liver macrophages (Kupffer cells and antigen-presenting cells) in adults occupy approximately 20% of liver cell elements36). These macrophages should contribute to development of liver lesions induced by chemicals; however, their functional roles in hepatotoxicity have not been established (Fig. 3).

4. Hepatic Macrophages for Homeostasis

4–1.Activated Conditions of Hepatic Macrophages

Liposome, which is prepared from lipids and lipid mixtures with phospholipids, is one of the most efficacious and promising drug-carrier vehicles for intracellular delivery37). The liposome itself is a nontoxic, nondegradable and nonimmunogenic vehicle. Injected liposomes are rapidly phagocytized by hepatic macrophages38). The administration of empty liposomes can activate macrophages via a phagocytic stimulus39). Following empty liposome injection, macrophages expressing CD163 and CD68 are significantly increased in liposome-injected rats. CD163-expressing Kupffer cells are the most sensitive for the activation, and CD68 antigen expression indicates activated phagocytosis due to ingested liposomes. Although there is no abnormal change in liver histological structures, hepatocytes show an increased proliferating activity, demonstrable with proliferation marker immunostaining and by increase in gene profiles relating to “cell cycle.” In liposome-injected rats, AST and ALT levels are significantly decreased, and mRNA expression of MCP-1, IL-1β and TGF-β1 is increased. Collectively, hepatic macrophages activated by liposomes can influence liver homeostasis40).

4–2. Influences on Depletion of Hepatic Macrophages in Homeostasis

Dichloromethylene diphosphonate clodronate belongs to the family bisphosphonate used for treatment of osteolytic bone diseases and osteoporosis41). Once ingested by macrophages, the clodronate causes damage to macrophages via apoptosis, resulting in their depletion/disappearance42,43). Hepatic macrophages in rats are easily depleted by an intravenous injection of liposome-encapsulated clodoronate44,45). CD163-expressing Kupffer cells are almost completely depleted for long term, indicative of the most sensitive for clodoronate treatment (Fig. 4). Macrophages expressing CD68 are transiently reduced in number at the early stages after injection and then recovered gradually. In spite of hepatic macrophage depletion, no marked histological changes are seen in the liver, but the proliferating activity of hepatocytes is significantly increased, supported by changes of gene profiles relating to cell proliferation. The values of AST and ALT are significantly elevated (Fig. 4), and factors for macrophage induction/activation, such as MCP-1, CSF-1, IL-6 and IL-4, are increased transiently at early stages after the injection, whereas anti-inflammatory factors such as IL-10 and TGF-β1 remain significantly decreased under hepatic macrophage depletion43).

Fig. 4.

Fig. 4

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Depletion of hepatic macrophages. Hepatic macrophages are easily depleted in rats by an intravenous injection of liposome-encapsulated clodoronate (dichloromethylene diphosphonate clodronate). CD163-expressing Kupffer cells in the centrilobular (4-1) and periportal areas (4-2) are almost completely depleted, in contrast to controls (empty-liposome). AST and ALT levels are significantly elevated under hepatic macrophage depletion (4-3). CV, central vein; GS, Glisson’s sheath.

It is interesting to note that clodoranete treatment induces hepatic macrophage depletion without histopathological lesions, resulting in increased values of AST and ALT43). As mentioned above, AST and ALT values are decreased under hepatic macrophage activation24,40). In liver homeostasis, the conditions of hepatic macrophages influence AST and ALT values, suggesting that hepatic macrophages participate in clearance of these hepatic deviation enzymes17,46). In toxicological studies, if AST and ALT levels are increased or decreased without histopathological changes, the condition (increase or decrease) of hepatic macrophages should be analyzed by immunohistochemistry with CD163 antibody for M2-macrophages (mainly Kupffer cells). There would be chemicals capable of activating or inhibiting hepatic macrophage functions as an initial toxicity. Novel pathogenesis may be brought out in relation to hepatic macrophage functions40,43). The analysis of macrophage conditions in rat toxicity studies would lead to refinement of hazard characterization.

5. Hepatotoxicity Based on M1-/M2-macrophage Polarization

5–1.The Concept of M1-/M2-macrophage Polarization

The properties of macrophages are influenced by microenvironmental conditions. As mentioned above, recently, macrophages appearing in pathological lesions, where they may have complicated conditions depending on stages or tissues, are divided into “classically activated macrophages (M1-macrophages)” and “alternatively activated macrophages (M2-macrophages)” (Figs. 1 and 2)17,18,47). M1-macrophages develop under the influence of IFN-γ at early stages, and become an effecter that works in cell-mediated immunity as a combined response not only to IFN-γ itself but also to TNF-α, IL-6 and IL-1β11,18,47). M1-macrophages secrete pro-inflammatory cytokines and mediators, as well as cytotoxic factors. Factors produced by M1-macrophages are an important component of host defense after tissue injury, but they may cause extensive damage to host tissues by producing cytotoxic factors. M1-macrophages produce superoxide anions, oxygen radicals, and nitrogen radicals, thereby resulting in tissue destruction18).

By contrast, M2-macrophages appear in response to innate or adaptive signals. IL-4, produced mainly by macrophages themselves after tissue injury, is considered to be an important factor for induction and activation of M2-macrophages. IL-10, IL-12 and TGF-β, which are produced by M2-macrophages, contribute to anti-inflammation and deposition of extracellular matrices leading to reparative fibrosis18,47,48). M2-macrophages may be subdivided into three types: M2a for anti-inflammation, M2b for immune response, and M2c for tissue repair. It is difficult to identify such subtypes using immunohistochemical staining; therefore, such subtypes are not applicable in hepatotoxicity. Because of complicated functions, M2-macrophages are not always beneficial for the host. The high levels of IL-10 produced by activated M2-macrophages can predispose the host to infection, by reducing M1-macrophage functions49).

M1-and M2-macrophages are also related to tumor regression and progression, respectively; M1-macrophages inhibit tumor growth, whereas M2-macrophages promote tumor progression such as angiogenesis, invasion, and metastasis13,50).

5–2. Involvement of Macrophages in Hepatotoxicity

Chemicals such as medical drugs, agrochemicals, food additives, industrial products and toxins, which are produced artificially or generated naturally, are metabolized mostly in the liver (Fig. 3)51). Some chemicals with hepatotoxicity injure hepatocyte directly (cell membrane, cytoplasmic organelles or nucleic acid) with their toxicity, or the active metabolites generated by metabolizing enzymes (such as CYPs) present in hepatocytes may induce hepatocyte injury51,52); the injury may be characterized by atrophy, degeneration, apoptosis, necrosis, and swelling, and degeneration, as well as a mixture of these changes. These mechanisms (direct injury and enzyme-produced active metabolite-related injury) have been well investigated in the toxicology research field (Fig. 3). On the other hand, regardless the presence of hepatic macrophages (approximately 20% occupation) in the liver, there is little information on hepatic macrophage-related hepatocyte damage in hepatotoxicity. As aforementioned, macrophages play important roles in liver homeostasis and have heterogeneous functions depending on microenvironments40,43). Importantly, infiltrating macrophages can induce tissue injury by producing cytotoxic factors, indicating the presence of pathogenesis of hepatic macrophage-mediated hepatocyte injury, or the enhancement of chemical-induced hepatocyte injury by macrophages53). Some macrophages come to act as antigen-presenting cells under stimulation of chemicals themselves or by ingesting cell debris of injured hepatocytes; such antigen-presenting macrophages lead to very complex immune-mediated hepatotoxicity, which may be related to the occurrence of allergic liver injury (Fig. 3)54–56).

5–3.M1-/M2-macrophage Polarization in TAA-induced Acute Rat Liver Injury

Hepatotoxicity by TAA is caused by its active metabolite. TAA is metabolized to TAA-sulfoxide and further to TAA-S, S-dioxide (TASO2) by CYP2E1 in the liver. TASO2 covalently binds to and modifies cellular phosphatidylethanolamine lipids or protein lysine side chains51,57), resulting in hepatocellular injury, especially in the centrilobular area where CYP2E1 is the most abundant52,58). Using the acute hepatocyte injury model in rats injected with a sub-lethal dose of TAA, the lesion development is observed for 10 days after the injection11). Coagulation necrosis of hepatocytes is seen in the centrilobular area on days 1 and 2; then, fibrosis develops on days 3 and 5, and recovers gradually on days 7 and 10. CD68-expressing M1-macrophages and CD163-expressing M2-macrophages appear in the injured centrilobular area on days 1–3 with hepatocyte necrosis (Fig. 5). At hour 10, when hepatic lesions are not still developed and M1-/M2-macrophages do not appear, interestingly, IFN-γ, TNF-α, IL-1β, IL-6, and IL-4 are already increased, followed by increased expressions of IL-10 and TGF-β1 on days 1–3. According to M1-/M2-macrophage polarization concept59–61), IFN-γ, TNF-α, IL-1β and IL-6 are factors for M1-macrophage induction/activation; IL-4 is the most important factor for M2-macrophage induction/activation. In this acute model, because macrophages expressing CD204 and MHC class II already increase in the periportal regions and Glisson’s sheath without any lesion at hour 10, these factors for M1- and M2-macrophages might have been produced by CD204- and MHC class II-expressing macrophages pre-existing in the periportal/Glisson’s sheath area. Subsequent increase in IL-10 and TGF-β1, which are produced by activated M2-macrophages, are related to tissue repair/reparative fibrosis11).

Fig. 5.

Fig. 5

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Appearance of macrophages in TAA-induced hepatic lesions. The TAA-induced lesion develops in the centrilobular area as coagulation necrosis (HE stain). There are different macrophages expressing CD68 for M1-macrophages, CD163 for M2-macrophages or MHC class II for M1-macrophages in the lesion. Apparently, the cell morphology and distribution differ from each other.

In hepatotoxicity, collectively, CD204- and MHC class II-expressing macrophages pre-existing in the periportal area/Glisson’s sheath play important roles in subsequent M1-/M2-macrophage polarization in hepatotoxicity; the pre-existing macrophages take part in M1-macrophage induction at early stages and then M2-macrophage migration in reparative fibrosis11).

In the TAA-induced hepatic lesions, CD68-expressing M1-macrophages and CD163-expressing M2-macrophages appear almost simultaneously in the injured area; apparently, the cell morphology and distribution differ from each other (Fig. 5). The double immunofluorescence staining shows the shift of M1-macrophages to M2-macrophages, because of the presence of CD68/CD163-double positive macrophages (Fig. 2). MCP-1 (a chemoattractant) addition to cultured HS-P cells (a rat macrophage cell line) increases the cell numbers reacting to CD68 and CD163, whereas TGF-β1 treatment to HS-P cells decreases the number of CD68- and CD163-positive cells. At a minimum, these factors (MCP-1 and TGF-β1) may be involved in the shift of M1-and M2-macrophages in injured areas. MCP-1 is produced by activated hepatic stellate cells at early stages as a factor of M1-macrophage induction, and TGF-β1 is produced by M2-macrophages and influences M2-macrophage functions19,62). There is increasing evidence on interchangeability of M1- and M2-macrophages62,63). The mechanisms and significance should be investigated in further studies.

5–4. M1-/M2-polarization of Macrophages Expressing CD204, MHC Class II, Iba-1 or Galetin-3 in TAA-induced Rat Hepatotoxicity

In the TAA-induced acute hepatic lesions, there are macrophages showing double positive reaction to CD68/MHC class II, CD163/MHC class II, CD68/CD204, and CD163/CD20411,12); the predominant polarization analyses of MHC class II-positive macrophagesor CD204-expressing macrophages by double immunofluorescence analysis reveal that MHC class II-expressing macrophages and CD204-expressing macrophages are polarized towards CD68 M1-macrophages and CD163 M2-macrophages, respectively; that is, CD68-expressing M1-macrophages come to express MHC class II, presumably by taking up cell debris of injured hepatocytes, and may facilitate complement-mediated phagocytosis by secreting inflammatory factors47), whereas CD163-expressing M2-macrophages come to express CD204. CD204 expression is related to metabolism of oxidized low density lipoprotein24); similar to Kupffer cells expressing both CD163 and CD204 in normal rat livers25), CD163-expressing M2-macrophages in injured liver lesions can act as lipid-metabolizing cells. M2-macrophages are divided into M2a-, M2b- and M2c-types, showing different functions. It is interesting to investigate roles of M2-macrophages expressing CD163/CD204 in relation to tissue healing or progressive fibrosis (Fig. 2).

To find out the macrophage properties appearing in hepatotoxicity, immunophenotypical expressions of Iba1 and Galectin-3 were analyzed, in relation to CD68-expressing M1-macrophages and CD163-expressing M2-macrophages in centrilobular lesions induced in rat livers by a single injection of TAA11,12). In agreement with expression patterns of CD68- and CD163-macrophages, macrophages reacting to Iba1 and Galectin-3 simultaneously appear. Double immunofluorescence analysis for the predominant polarization shows that Iba1-and Galectin-3-positive macrophagesare polarized toward the M1-and M2-macrophages, respectively. Iba1 is associated with membrane ruffling and motility of cells27,28). Galectin-3 is a β-galactoside binding animal lectin, and regulates fibrogenesis, probably through TGF-β129,30). Iba1-and Galectin-3-expressing macrophages also participate in the pathogenesis of acute liver injury and reparative fibrosis in hepatotoxicity.

An M1-/M2-macrophage polarization paradigm would be useful for analyzing acute hepatotoxicity; CD68-expressing M1-macrophages can be characterized by expressions of MHC class II (antigen presentation) and Iba-1 (migration activity), whereas CD163-expressing M2-macrophages are considered to have functions to which CD204 (for lipid metabolism) and Galectin-3 (for fibrosis) could be related. Immunohistochemical analyses using these antibodies to antigens specific of M1- or M2-macrophages would be useful to investigate the stages and development of hepatic lesions induced by chemicals in toxicity studies11–13,15,16) (Figs. 2 and 3). The novel analysis methods for hepatotoxicity according to M1-/M2-macrophage polarization in rat toxicity studies would lead to refinement of the hazard characterization process.

5–5. Aggravated Lesion of TAA-induced Acute Liver Injury Underdepleted Hepatic Macrophages Depleted by Clodronate

To further determine the functions of macrophages in hepatotoxicity, hepatic macrophages were depleted by liposomal clodronate one day before a single administration of a sub-lethal dose of TAA45). In clodronate-treated rats, not only M1-macrophages expressing CD68 and MHC class II, but alsoM2-macrophages positive for CD163 and CD204 are markedly decreased. In clodronate-treated rats, interestingly, coagulation necrosis of hepatocytes due to TAA injection is prolonged, being accompanied with increased levels of hepatic enzymes (AST and ALT); reparative fibrosis following injury is incomplete, being replaced by dystrophic calcification in the injured area. These findings indicate that the depletion of hepatic macrophages aggravates liver damage due to TAA injection. Furthermore, inflammatory factors for M1-macrophages (MCP-1, IFN-γ, TNF-α, and IL-6) and fibrosis-related factors (TGF-β1, IL-10, MMP-2, and TIMP-1) for M2-macrophages are decreased, in agreement with decreased numbers of M1-/M2-macrophages, in clodoronate-treated rats; that is, hepatic macrophage depletion leads to abnormal macrophage functions, resulting in aggravated liver damage in hepatotoxicity45). It is thus clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity. Presumably, the persistent coagulation necrosis of hepatocytes results from lost phagocytosis, indicating importance of removal and clearance of cell debris by M1-macrophages. The aberrant wound repair associated with dystrophic calcification may be due to the depletion of M2-macrophages, because M2-macrophages play central roles in reparative fibrosis by producing fibrotic factors such as TGF-β1 and IL-10. The appropriate appearance of M1- and M2-macrophages is very important for tissue damage and subsequent reparative fibrosis in hepatotoxicity45). Analyzing functions of M1- and M2-macrophagesmay lead to the understanding of the mechanism of hepatotoxicity.

6. DAMPs and MHC Class II-expressing Antigen-presenting Macrophages in TAA-induced Acute Liver Injury

DAMPs are the endogenous danger signal molecules that are released by injured or necrotic hepatocytes, and activate innate immune system through TLRs. TLRs may be expressed mainly in antigen-presenting macrophages64,65). In addition to CD68-expressing M1-macophages and CD163-expressing M2-macrophages, there are macrophages with expression of MHC class II in lesions induced by TAA, showing differences in cell morphology and distribution (Fig. 5). In the liver, the relationships between DAMPs and TLRs have been investigated in ischemia/reperfusion mouse model and acetaminophen-induced liver injury mouse model66). DAMPs influencing lesion development consist mainly of HMGB-166,67), S-100 proteins68,69), and HSPs66). After released by injured or necrotic hepatocytes, these molecules play a role in enhancing liver inflammation (probably by stimulating M1-macrophages). On the contrary, some DAMPs take part in fibrosis; particularly, HMGB-1 induces liver fibrosis through activating TLR-4 on Kupffer cells, and S-100 A4 acts as a factor activating hepatic stellate cells which can produce extracellular matrices68,70). Hepatic stellate cells are stimulated by M2-macrophage-producing factors such as TGF-β1, and the cells can transform into myofibroblasts capable of producing abundant collagens resulting in fibrosis25,71,72). Thus, M2-macrophages relating to fibrosis may be induced by such DAMPs.

After a single administration of TAA, the occurrence of injured hepatocytes is accompanied by increases in HMGB-1/-2, S-100 A4 and HSP 70-276). Simultaneously, TLR-4 expression is up-regulated, whereas TLR-2, which is also a receptor of HMGBs, does not increase. Interestingly, the number of MHC class II-expressing macrophagesis increased in the injured hepatic lesions (Fig. 5), in concomitant with increased DAMPs and TLR-4. These findings suggest that DAMPs, particularly HMGB-1/-2 and S-100 A4, are associated with enhancing inflammation via activating TLR-4 on MHC class II macrophages in TAA-induced acute liver injury. Collectively, hepatocyte injury or necrosis at early stages may induce TLR-4-expressing MHC class II-expressing macrophages into the lesions, through DAMPs released by injured hepatocytes73). MHC class II-expressing macrophages are included mainly in M1-macrophages11,74). The relationship between liver injury and M1-macrophage functions through TLRs expression due to DAMP stimulations may be an interesting aspect of hepatotoxicity.

7. Conclusion

Toxicological evaluation of chemicals focusing on hepatic macrophages has not been conducted. It has been reported that hepatic macrophages influence liver homeostasis; particularly, the AST and ALT values are increased under hepatic macrophage depletion, whereas activated hepatic macrophages decrease these values. In addition, liver lesions in hepatotoxicity are aggravated under hepatic macrophage depletion. The hepatotoxicity may be histopathologically evaluated, based on macrophage functions, particularly the M1-/M2-macrophage polarization, which are analyzed immunohistochemically using antibodies specific for macrophage functions. Although further information with different chemicals is needed in future study, analyzing functions of hepatic macrophages may result in better understandings of mechanisms of hepatotoxicity, in part; hopefully, the novel analysis focusing on macrophage functions leads to the refinement of hazard characterization of chemicals in food and feed.

Acknowledgements

This work was supported by the captioned research program funded by the Food Safety Commission of Japan (FSCJ) (2014).

Abbreviations:ALT, alanine transaminase; AST, aspartate transaminase; CD, cluster of differentiation; IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; TAA, thioacetamide; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor; Th, T helper; Iba-1, ionized calcium binding adaptor molecule 1; CSF, colony stimulating factor; CYP, cytochrome P450; DAMPs, damageassociated molecular patterns; TLRs, toll-like receptors; HMGB, high mobility group box; HSPs, heat shock proteins; GM-CFU, granulocyte/macrophage-colony forming unit; M-CSF, macrophage-colony forming unit

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