Difference in the Mechanism of Iron Overload-Enhanced Acute Hepatotoxicity Induced by Thioacetamide and Carbon Tetrachloride in Rats.

Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.

The effect of lipopolysaccharide on liver homeostasis and diseases based on the mutual interaction of macrophages, autophagy, and damage-associated molecular patterns in male F344/DuCrlCrlj rats.

Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.

Macrophage pathology in hepatotoxicity.

The liver is the most important organ that metabolizes and detoxifies chemicals taken into the body. Therefore, there is always a risk of liver damage owing to the toxic effects of chemicals. The mechanisms of hepatotoxicity have been studied extensively and deeply based on toxic effects of chemicals themselves. However, it is important to note that liver damage is variously modified by the patho-biological reactions evoked mainly via macrophages. Macrophages appearing in hepatotoxicity are evaluated by the M1/M2 polarization; M1 macrophages promote tissue injury/inflammation, whereas M2 macrophages show anti-inflammatory action including reparative fibrosis. The "portal vein-liver barrier" regulated by Kupffer cells and dendritic cells in and around the Glisson's sheath may be related to the initiation of hepatotoxicity. In addition, Kupffer cells exhibit the two-sides of functions (that is, M1 or M2 macrophage-like functions), depending on microenvironmental conditions which may be raised in part by gut microbiota-derived lipopolysaccharide. Furthermore, damage-associated molecular patterns (DAMPs) (in particular, HMGB1) and autophagy (which degrades DAMPs) also play roles in the polarity of M1/M2 macrophages. The mutual relation of "DAMPs (HMGB-1)-autophagy-M1/M2 macrophage polarization" as the patho-biological reaction should be taken into consideration in hepatotoxicity evaluation.

Analyses of damage-associated molecular patterns, particularly biglycan, in cisplatin-induced rat progressive renal fibrosis.

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1ß, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Appearance of Heterogeneous Macrophages During Development of Isoproterenol-Induced Rat Myocardial Fibrosis.

Macrophages appearing in lesions are polarized toward M1 (for inflammation) and M2 (for anti-inflammation/fibrosis) types. We analyzed immunophenotypes of macrophages appearing in myocardial lesion in rats injected once with isoproterenol (10 mg/kg body weight). Inflammation following myocardial necrosis on day 1 was seen with a peak on days 3 and 5, and thereafter, reparative fibrosis developed on days 7 to 28. CD68+ M1 macrophages were seen in the early stages of injury and inflammatory on days 1 to 7, and thereafter, CD163+ M2 macrophages increased in the late stages of fibrosis on days 7 to 28. There was the polarization of M1 and M2 macrophages. The kinetics of macrophages reacting to Iba-1 and Galectin-3 was similar to that of M1 macrophages, indicating that Iba1- and Gal-3-positive macrophages might have functions of M1 type. Double immunofluorescence revealed that CD204- and MHC class II-positive macrophages are polarized toward M1 and M2 types, respectively. CCR2 messenger RNA expression is transiently elevated on day 1. Since CCR2 is a marker of blood monocytes, M1 macrophages might be recruited from blood monocytes. Collectively, macrophages expressing heterogeneous immunophenotypes participate in myocardial fibrosis. These findings would be useful for understanding the pathogenesis of myocardial fibrosis and analyzing myocardial toxicity.

Characterization of Immature Myofibroblasts of Stellate Cell or Mesenchymal Cell Origin in D-Galactosamine-Induced Liver Injury in Rats.

Lesions of D-galactosamine (D-GalN)-induced hepatotoxicity resemble those of human acute viral hepatitis. This study investigated hepatic mesenchymal cells including hepatic stellate cells (HSCs) and myofibroblasts in D-GalN-induced hepatotoxicity. Rats, injected with D-GalN (800 mg/kg body weight, once, intraperitoneally) were examined on post single injection (PSI) at 8 hours and days 1 to 5. Lesions consisting of hepatocyte necrosis and reparative fibrosis were present diffusely or focally within the hepatic lobules on PSI days 1 and 2, and then the injury recovered on PSI days 3 and 5. Myofibroblasts expressing vimentin, desmin, and α-smooth muscle actin (α-SMA) were present in the lesions. Double immunofluorescence showed that myofibroblasts reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, myofibroblasts reacting to vimentin, desmin, and α-SMA also co-expressed glial fibrillary acidic protein (GFAP), a marker of HSCs. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3 (both are markers of immature mesenchymal cells). Cells reacting to Thy-1, a marker for immature mesenchymal cells, also appeared in fibrotic lesions. In agreement with the myofibroblastic appearance, mRNAs of fibrosis-related factors (TGF-ß1, PDGF-ß, TNF-α, Timp2, and Mmp2) increased mainly on PSI days 1 and 2. Myofibroblasts with expression of various cytoskeletal proteins were present in diffuse or focal hepatic lesions, and they might be derived partly from immature HSCs and from immature mesenchymal cells.

Analyses of hemorrhagic diathesis in high-iron diet-fed rats.

Iron overload has been well recognized to cause oxidant-mediated cellular/tissue injury; however, little is known about the effects of iron overload on the blood coagulation system. We encountered an unexpected bleeding tendency in rats fed a high-iron diet in a set of studies using iron-modified diets. In this study, we investigated the mechanism of hemorrhagic diathesis induced by dietary iron overload in rats. Six-week-old F344/DuCrlCrlj male rats were fed a standard (containing 0.02% iron) or a high-iron diet (containing 1% iron) for 6 weeks and were then sampled for hematological, blood biochemical, coagulation, and pathological examinations. Serum and liver iron levels increased in rats fed the high-iron diet (Fe group) and serum transferrin was almost saturated with iron. However, serum transaminase levels did not increase. Moreover, plasma prothrombin time and activated partial thromboplastin time were significantly prolonged, regardless of the presence of hemorrhage. The activity of clotting factors II and VII (vitamin K-dependent coagulation factors) decreased significantly, whereas that of factor VIII was unaltered. Blood platelet levels were not influenced by dietary iron overload, suggesting that the bleeding tendency in iron-overloaded rats is caused by secondary hemostasis impairment. In addition, hemorrhage was observed in multiple organs in rats fed diets containing more than 0.8% iron. Our results suggest that iron overload can increase the susceptibility of coagulation abnormalities caused by latent vitamin K insufficiency.

Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)-Induced Rat Colonic Lesions.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)-induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.

Characterization of Macrophages and Myofibroblasts Appearing in Dibutyltin Dichloride-Induced Rat Pancreatic Fibrosis.

Macrophages and myofibroblasts are important in fibrogenesis. The cellular characteristics in pancreatic fibrosis remain to be investigated. Pancreatic fibrosis was induced in F344 rats by a single intravenous injection of dibutyltin dichloride. Histopathologically, the induced pancreatic fibrosis was divided into 3 grades (1+, 2+, and 3+), based on collagen deposition. Immunohistochemically, CD68-expressing M1 macrophages increased with grade and CD163-expressing M2 macrophages also increased later than M1 macrophage appearance. Double immunofluorescence showed that there were macrophages coexpressing CD68 and CD163, suggesting a possible shift from M1 to M2 types; similarly, increased major histocompatibility complex class II- and CD204-expressing macrophages were polarized toward M1 and M2 types, respectively. These findings indicated the participation of M1- and M2-polarized macrophages. Mesenchymal cells staining positive for vimentin, desmin, and α-smooth muscle actin (α-SMA) increased with grade. There were mesenchymal cells coexpressing vimentin/α-SMA, desmin/α-SMA, and glial fibrillary acidic protein (GFAP)/α-SMA; Thy-1-expressing immature mesenchymal cells also increased in fibrotic lesions. Because α-SMA expression is a reliable marker for myofibroblasts, α-SMA-expressing pancreatic myofibroblasts might be originated from GFAP-expressing pancreatic stellate cells or Thy-1-expressing immature mesenchymal cells; the myofibroblasts could simultaneously express cytoskeletal proteins such as vimentin and desmin. The present findings would provide useful information for analyses based on features of macrophages and myofibroblasts in chemically induced pancreatic fibrosis.

Seroprevalence of Antibodies Against Paracoccidioides Spp. in Captive Dolphins from Three Aquaria in Japan.

The skin disease paracoccidioidomycosis ceti occurs in several dolphin species globally. Infection by the unculturable fungi Paracoccidioides brasilensis or other Paracoccidioides spp. results in chronic cutaneous and granulomatous lesions. In this study we used immunohistochemistry to investigate the seroprevalence of antibodies to Paracoccidioides spp. in captive dolphins from three aquaria in Japan. We had previously reported that there were serological cross-reactions for Paracoccidioides spp. with related species in the order Onygenales. We hypothesized that the degree of serological cross-reactions for Paracoccidioides spp. might be lower in areas, such as Japan, where the fungal diseases coccidiodomycosis and paracoccidiodomycosis are not endemic. Sera from 41 apparently healthy dolphins, including 20 Atlantic bottlenose dolphins (BD: Tursiops truncatus), 6 Indo-Pacific bottlenose dolphins (IPBD: Tursiops aduncus), 2 F1 generation of a cross between BD and IPBD (F1), 3 Pacific white-sided dolphins (PWD: Lagenorhynchus obliquidens), 2 pantropical spotted dolphins (PSD: Stenella attenuata), 6 false killer whales (FKW: Pseudorca crassidens), and 2 rough-toothed dolphins (RTD: Steno bredanensis) were investigated. Sera from three dolphins with paracoccidioidomycosis ceti were used as a positive control. The yeast-form cells of Paracoccidioides spp. in the cutaneous tissue sample derived from the first Japanese paracoccidioidomycosis ceti case were used as the antigen for the immunohistochemistry. Of the 41 dolphins tested, 61.0% had antibodies against Paracoccidioides spp. This indicates that dolphins of several species in Japanese aquaria have likely been exposed to the pathogen Paracoccidioides spp.

Acetaminophen-Induced Rat Hepatotoxicity Based on M1/M2-Macrophage Polarization, in Possible Relation to Damage-Associated Molecular Patterns and Autophagy.

Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68+ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-g and TNF-α, whereas CD163+ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204+ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B+ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.

Participation of Somatic Stem Cells, Labeled by a Unique Antibody (A3) Recognizing both N-glycan and Peptide, to Hair Follicle Cycle and Cutaneous Wound Healing in Rats.

A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues.

Effects of dexamethasone on hepatic macrophages in normal livers and thioacetamide-induced acute liver lesions in rats.

Resident and infiltrative macrophages play important roles in the development of pathological lesions. M1/M2 macrophage polarization with respective CD68 and CD163 expression remains unclear in chemically induced liver injury. This study was aimed at investigating the influence of macrophages on normal and chemically induced liver injury. For this, dexamethasone (DX), an immunosuppressive drug, was administered in normal rats and thioacetamide (TAA)-treated rats. Liver samples were collected and analyzed with immunohistochemical methods. Repeated injections of DX (0.5 or 1.0 mg/kg BW) for 3, 7 and 11 days reduced the number of CD163 positive hepatic resident macrophages (Kupffer cells) in normal livers, while increasing AST and ALT levels. In TAA (300 mg/kg BW)-treated rats injected with DX (0.5 mg/kg BW) pretreatment, the number of M1 and M2 macrophages showed a significant decrease compared with that of TAA-treated rats without DX treatment. Additionally, reparative fibrosis resulting from hepatocyte injury induced by TAA injection was suppressed by DX pretreatment. Our data suggested that macrophages could influence not only normal hepatic homeostasis (reflected by AST and ALT levels) but also chemically induced hepatic lesion development (reduced reparative fibrosis).

Hepatic Myoepithelial Carcinoma in a Dog: Immunohistochemical Comparison With Other Canine Hepatic Carcinomas.

An 11-year-old female miniature Dachshund dog presented with a solid, soft, gray mass on the hepatic lateral left lobe. Histologically, the mass consisted of neoplastic proliferation of cells with round nuclei and eosinophilic and vacuolated cytoplasm arranged in alveolar, trabecular, and solid patterns. Immunohistochemically, the neoplastic cells were positive for pancytokeratin (CK AE1/AE3), CK5, CK14, vimentin, Sox9, and myoepithelial markers (α-smooth muscle actin, p63, and calponin). The morphological and immunohistochemical findings indicated a diagnosis of myoepithelial carcinoma. We conducted immunohistochemical studies on other representative canine hepatic tumors. Although the myoepithelial phenotype was not observed in the hepatocellular carcinoma, some tumor cells in cholangiocarcinoma showed immunohistochemical features of myoepithelium, suggesting that some neoplastic cells in cholangiocarcinoma may have the potential to differentiate into myoepithelial cells. To our knowledge, this is the first report in veterinary medicine of a hepatic carcinoma with a myoepithelial phenotype.

MicroRNAs: potential targets and agents of endocrine disruption in female reproduction.

MicroRNAs are short non-coding RNAs that have been widely recognized as key mediators in the epigenetic control of gene expression and which are present in virtually all cells and tissues studied. These regulatory molecules are generated in multiple steps in a process called microRNA biogenesis. Distinct microRNA expression patterns during the different stages of oocyte and embryo development suggest important regulatory roles for these small RNAs. Moreover, studies antagonizing specific microRNAs and enzymes in microRNA biogenesis pathways have demonstrated that interference with normal miRNA function leads to infertility and is associated with some reproductive abnormalities. Endocrine disrupting chemicals such as Bisphenol A (BPA) are synthetic hormone mimics that have been found to negatively impact reproductive health. In addition to their direct effects on gene expression, these chemicals are widely implicated in the disruption of epigenetic pathways, including the expression and activity of miRNAs, thereby altering gene expression. In this review, the roles of microRNAs during mammalian oocyte and embryo development are outlined and the different mechanisms by which endocrine disruptors such as BPA interfere with these epigenetic regulators to cause reproductive problems is explored.

The localization and distribution of cells labeled by a somatic stem cell-recognizing antibody (A3) in rat colon development; possible presence of a new cell type forming the intestinal stem cell niche.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma (MFH)-derived cloned cells, recognizes somatic stem cells (bone-marrow/hair follicle stem cells). We investigated the distribution of cells immunoreactive to A3 in the developing rat intestine (particularly, the colon), focusing on the ontogenic kinetics of A3-positive cells. In the rat intestine, A3 labeled spindle-shaped stromal cells localized in the submucosa and labeled endothelial cells of capillaries in the lamina propria forming villi in the early development stage. With development progression, A3-positive cells were exclusively localized around the crypts of the colon. Double immunofluorescence revealed that A3-positive cells around the crypts reacted to vimentin (for mesenchymal cells) and Thy-1 (for mesenchymal stromal cells) but not to α-SMA (for mesenchymal myofibroblastic cells) or CD34 (for hematopoietic stem cells), indicating that A3-positive cells around the crypts may have characteristics of immature mesenchymal cells. In addition, A3 labeled a few epithelial cells at the base of colon crypts. Furthermore, immunoelectron microscopy revealed that A3-positive cells lay inside myofibroblasts adjacent to the epithelium of the crypts. A3-positive cells were regarded as a new type of immature mesenchymal cells around the crypts. Collectively, A3-positive cells might take part in the stem cell niche in the colon, which is formed through epithelial-mesenchymal interaction.

Visualization of specific collagen-producing cells by Col1-GFP transgenic mice revealed novel type I collagen-producing cells other than fibroblasts in systemic organs/tissues.

Type I collagen is one of the most abundant proteins in mammals and plays important roles in maintaining the integrity of many tissues. Although fibroblasts are the main source of type I collagen, other cells also produce it; however, these cells are not well-defined owing to the lack of a specific marker. A transgenic (Tg) mouse line has been generated in which type I collagen-producing cells are labeled with enhanced green fluorescent protein (EGFP), which enables the monitoring of these cells without requiring an additional cell marker. This Tg mouse line has since been widely used to study type I collagen-producing cells and fibrosis; one study revealed that podocytes, which were not previously considered to produce type I collagen, expressed EGFP. This raises a question regarding the specificity of EGFP expression in this Tg mouse line. To exclude the possibility of non-specific EGFP expression in the existing Tg mouse line and specifically monitor type I collagen-producing cells, we generated a new Tg mouse line and histologically confirmed the specificity of EGFP expression throughout the body. Moreover, we explored type I collagen-producing cells other than fibroblasts and revealed for the first time that Leydig cells have the ability to produce type I collagen. Because of its highly specific and physiologically accurate expression, our new Tg mouse line will help to accurately elucidate not only type I collagen-producing cells in normal tissues but also the potential cells in fibrotic tissues, providing new insights into the pathology of fibrosis.

Macrophage Populations and Expression of Regulatory Inflammatory Factors in Hepatic Macrophage-depleted Rat Livers under Lipopolysaccharide (LPS) Treatment.

To investigate the significance of the appearance of hepatic macrophages and expression of inflammatory factors in normal and macrophage-depleted livers, hepatic macrophages were depleted with liposome (Lipo)-encapsulated clodronate (CLD; 50 mg/kg, i.v.) followed by lipopolysaccharide (LPS) administration (0.1 mg/kg, i.p.) in F344 rats (CLD + LPS). Vehicle control rats (Lipo + LPS) received empty-Lipo before LPS. The low dose of LPS did not result in microscopic changes in the liver in either treatment group but did modulate M1 and M2 macrophage activity in Lipo + LPS rats without altering repopulating hepatic macrophages in CLD + LPS rats. LPS treatment in Lipo + LPS rats dramatically increased the M1 (IL-1ß, IL-6, TNF-α, and MCP-1) but not M2 macrophage-related factors (IL-4 and CSF-1) compared to CLD + LPS rats. In the CLD + LPS rats, the M2 macrophage-related factors IL-4 and CSF-1 were elevated. In conclusion, low-dose LPS activated hepatic macrophages in rat livers without causing liver injury or stimulating repopulating hepatic macrophages. These data suggest that LPS may alter the liver microenvironment by modulating M1 or M2 macrophage-related inflammatory mediators and macrophage-based hepatotoxicity.

M1/M2-macrophage Polarization-based Hepatotoxicity in d-galactosamine-induced Acute Liver Injury in Rats.

d-galactosamine (d-GalN) is a well-known hepatotoxic agent that causes liver injury. Conversely, hepatic macrophages play a crucial role in maintaining liver tissue integrity. Macrophage functions were investigated in hepatic lesions induced by a single intraperitoneal injection of d-GalN (800 mg/kg body weight [BW]) in 6-week-old F344 rats. Blood and liver samples were examined at 8 hr and on 1, 2, 3, and 5 days postsingle injection (PSI). Hepatic lesions consisting of degeneration/sporadic foci of coagulation necrosis, inflammatory cell reaction, and reparative fibrosis were seen on PSI days 1 and 2, reflected by significantly increased serum levels of aspartate transaminase and alanine transaminase and upregulation of CD68 M1 (tumor necrosis factor-α, interleukin [IL]-6, and interferon-γ) and CD163 M2 (transforming growth factor-ß1, IL-10, monocyte chemoattractant protein-1, and IL-4) macrophage-related factors. Double immunofluorescence staining on PSI day 2 demonstrated that 82% of hepatic macrophages expressed of CD163/CD68 simultaneously; 65-75% of MHC class II macrophages showed co-expression of CD163 or CD68 and 95% CD204-expressing macrophages reacted to CD163 or CD68. These findings showed that both M1- and M2-macrophages contributed to the development of hepatic lesions induced by d-GalN and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization.