Kavindra Kumara Wijesundera

M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3

INTRODUCTION Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis. MATERIALS AND METHODS Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs. RESULTS Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1β, IL-6, and TNF-α) and M2-(TGF-β1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-β1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis. CONCLUSION Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.

Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1-10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.

Depletion of Hepatic Macrophages Aggravates Liver Lesions Induced in Rats by Thioacetamide (TAA).

Hepatic macrophages play crucial roles in hepatotoxicity. We investigated immunophenotypes of macrophages in liver injury induced in rats by thioacetamide (TAA; 300 mg/kg, intraperitoneal) after hepatic macrophage depletion; hepatic macrophages were depleted by liposomal clodronate (CLD; 10 ml/kg, i.v.) one day before TAA injection. Samples were obtained on post-TAA injection days 0, 1, 2, 3, 5, and 7. TAA injection induced coagulation necrosis of hepatocytes on days 1 through 3 and subsequent reparative fibrosis on days 5 and 7 in the centrilobular area, accompanied by increased numbers of M1 macrophages (expressing cluster of differentiation [CD]68 and major histocompatibility complex class II) and M2 macrophages (expressing CD163 and CD204) mainly on days 1 through 3. TAA + CLD treatment markedly decreased the numbers of M1 and M2 macrophages mainly on days 1 through 3; CD163+ Kupffer cells were most sensitive to CLD depletion. In TAA + CLD–treated rats, interestingly, coagulation necrosis of hepatocytes was prolonged with more increased levels of hepatic enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase) to TAA-treated rats; reparative fibrosis was incomplete and replaced by dystrophic calcification in the injured area, indicating the aggravated damage. Furthermore, in TAA + CLD–treated rats, inflammatory factors (monocyte chemoattractant protein [MCP]-1, interferon-γ, tumor necrosis factor-α, and interleukin-10) and fibrosis-related factors (transforming growth factor-β1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1) were decreased at messenger RNA levels, indicating abnormal macrophage functions. It was clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity.

Inflammatory regulation of iron metabolism during thioacetamide-induced acute liver injury in rats

Systemic iron homeostasis is tightly regulated by the interaction between iron regulatory molecules, mainly produced by the liver. However, the molecular mechanisms of iron regulation in liver diseases remain to be elucidated. Here we analyzed the expression profiles of iron regulatory molecules during transient iron overload in a rat model of thioacetamide (TAA)-induced acute liver injury. After TAA treatment, mild hepatocellular degeneration and extensive necrosis were observed in the centrilobular region at hour 10 and on day 1, respectively. Serum iron increased transiently at hour 10 and on day 1, in contrast to hypoferremia in other rodent models of acute inflammation reported previously. Thereafter, up-regulation of hepcidin, a central regulator of systemic iron homeostasis, was observed in hepatocytes on day 2. Expression of transferrin receptor 1 and ferritin subunits increased to a peak on day 3, followed by increases in liver iron content and stainable iron on day 5, in parallel with regeneration of hepatocytes. Histopathological lesions and hepatocellular iron accumulation disappeared until day 10. The hepcidin induction was preceded by activation of IL6/STAT3 pathway, whereas other pathways known to induce hepcidin were down-regulated. IL6 was expressed by MHC class II-positive macrophages in the portal area, suggestive of dendritic cells. Our results suggest that IL6 released by portal macrophages may regulate hepatocyte hepcidin expression via STAT3 activation during transient iron overload in TAA-induced acute liver injury.

Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions.

Ionized calcium binding adaptor molecule 1 (Iba1) is associated with membrane ruffling and motility of cells. Galectin-3 (Gal-3) is a β-galactoside binding animal lectin, and regulates fibrogenesis probably through transforming growth factor-β1. To evaluate macrophage properties, expressions of Iba1 and Gal-3 were investigated, in relation to macrophages expressing CD68 (ED1; reflecting increased phagocytosis) and CD163 (ED2; implying proinflammatory factor productions) in centrilobular lesions induced in rat livers with thioacetamide (TAA; 300 mg/kg body weight, once intraperitoneally). In agreement with expression patterns of CD68(+) and CD163(+) macrophages, cells reacting to Iba1 and Gal-3 were increased in numbers on post-injection (PI) days 1-5, peaking on day 2; thereafter, the positive cells gradually decreased to control levels until PI days 7 and 10. The increased expressions of Iba1 and Gal-3 were confirmed at mRNA levels by the RT-PCR. Double immunofluorescence staining on PI days 2 and 3 demonstrated Iba1 expression in 15-46% of CD68(+) and CD163(+) macrophages, and Gal-3 expression in 65-82% of CD68(+) and CD163(+) macrophages; Gal-3 expression was observed in 84-93% of Iba1(+) cells. Interestingly, Gal-3 was also expressed in a small number of α-smooth muscle actin-positive myofibroblasts in fibrotic lesions developed in injured centrilobular areas. These findings indicate that macrophages with various functions can participate in development of liver lesions and resultant fibrosis. Besides CD68 and CD163, Iba1 and Gal-3 immunohistochemistry for macrophages would be useful to analyze the pathogenesis behind developing hepatotoxicity.

M1-/M2-macrophage polarization in pseudolobules consisting of adipohilin-rich hepatocytes in thioacetamide (TAA)-induced rat hepatic cirrhosis

INTRODUCTION Liver steatosis is the most frequent liver disease and may further develop into non-alcoholic steatohepatitis (NASH), liver cirrhosis, and finally hepatocellular carcinoma. Adipophilin (Adp) is localized on lipid droplet membrane in cytoplasm, and its increased expression is related to development of steatosis and NASH. The relationship between M1-/M2-macrophage polarization and Adp-rich hepatocyte-consisting pseudolobules (PLs) was investigated in thioacetamide (TAA)-induced rat cirrhosis. MATERIALS AND METHOD F344 rats were injected twice weekly with TAA (100mg/kg bodyweight) and sacrificed at post-first injection (PFI) weeks 5, 10, 15, 20, 25 and 32. Macrophage immunophenotypes and Adp-containing hepatocytes were analyzed by single immunolabeling. Adp and M1-/M2-related factors were analyzed by real -time RT-PCR. RESULTS PLs consisting exclusively of Adp-containing hepatocytes (Adp-positive) and PLs consisting of few Adp-containing hepatocytes (Adp-negative) were clearly distinguishable at PFI week 20 onwards. The numbers of M1-macrophages (reacting to CD68 and Iba1) and M2- macrophages (reacting to CD163, CD204 and Gal-3) were considerably greater in Adp-positive PLs. Expressions for both M1 (TNF-α, MCP-1, and Iba1)- and M2 (IL-4, TGF-β1, Gal-3, and Hsp25)-related factors were markedly higher in Adp-positive PLs at PFI week 25. Interestingly, MHC class II-positive macrophages/dendritic cells were increased in Adp-positive clusters/foci at the early stages at PFI weeks 5 and 10, and the level was gradually decreased thereafter. CONCLUSIONS M1-/M2-macrophages may simultaneously participate in the pathogenesis of steatosis in TAA-induced cirrhosis through M1- and M2-related factors. MHC class II cells may be responsible for steatosis at early stages, suggesting different functions from the above M1-/M2-macropahges.

Analysis of glial fibrillary acidic protein (GFAP)-expressing ductular cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Glial fibrillary acidic protein (GFAP), a type III intermediate filament protein, is expressed in hepatic stellate cells (HSCs), the principal fibrogenic cell type in the liver. Further, GFAP could be a marker for hepatic progenitor cells (HPCs). In this study, the participation of GFAP-expressing cells in HPC expansion/ductular reaction was investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100mg/kg BW, twice a week) and examined at post-first injection weeks 5, 10, 15, 20 and 25. Fibrosis-related proliferation of ductular cells was observed as demonstrated by CK19 immunostaining. Some of these cells were stained with GFAP. No co-staining was observed between CK19 and α-smooth muscle actin (α-SMA; myofibroblast marker). There were proliferating ductular cells stained with α-fetoprotein or β-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors (Wnt2, Wnt4 and glypican-3). These results for the first time show the participation of GFAP-positive HPCs in ductular reaction in a chemically induced rodent model. Though the ductular cells were chaperoned by myofibroblasts, they show no direct evidence for epithelial to mesenchymal transition. These findings shed new light in understanding the roles of GFAP-expressing HPCs in liver cirrhosis and provide further evidence of interaction between newly-formed bile ductules and HSCs, suggesting that both cells could be in the common lineage of HPCs.

M1-/M2-macrophages contribute to the development of GST-P-positive preneoplastic lesions in chemically-induced rat cirrhosis

Glutathione S-transferase placental form (GST-P) expression in hepatocyte foci is regarded as a preneoplastic change in rats. We aimed to reveal the contribution of polarized macrophages in development of GST-P-positive pseudolobules (PLs) in chemically-induced rat cirrhosis. F344 rats were injected with thioacetamide (100mg/kg BW, twice a week, intraperitoneally). Macrophage immunophenotypes and expression of M1-/M2-related factors were analyzed by immunohistochemistry, real-time RT-PCR and laser microdissection. GST-P-positive foci/clusters were clearly observed at post-first injection week 15. GST-P-positive PLs were distinguishable at weeks 20-32. Microarray analysis revealed upregulation of preneoplastic genes in GST-P-positive PLs at week 32. M1 (CD68(+), Iba1(+))-and M2 (CD163(+), CD204(+), Gal-3(+))-macrophages were greater in number in the GST-P-positive PLs, whereas MHC class II-positive (M1) macrophage number was fewer in the GST-P-positive PLs. Expression of both M1 (IFN-γ, IL-1β, TNF-α, Iba1)- and M2 (IL-4, TGF-β1, IL-10)-related factors were higher in GST-P-positive PLs. Our results showed that both M1- and M2-macrophage populations contribute to the development of hepatic preneoplastic lesions. MHC class II-positive macrophages may be related to anti-tumor progression, since their kinetics showed reverse pattern to other macrophage phenotypes.

Spontaneous Extraskeletal Osteosarcoma in a Rabbit (Oryctolagus cuniculus): Histopathological and Immunohistochemical Findings

A spontaneously occurring subcutaneous mass in the left forelimb of a nine-year-old rabbit (Oryctolagus cuniculus) was examined histopathologically and immunohistochemically. Clinically, edema and hemorrhage were seen around the mass. No connection of the tumor mass to the appendicular skeleton was found. The tumor was arranged in a solid growth pattern and irregular bundles, and neoplastic cells were polygonal to spindle-shape. Osteoid (positive for osteocalcin) and multinucleated giant cells were diffusely or focally seen. Neoplastic cells were positive for vimentin, osterix and Ki-67, indicating the nature of osteoblasts with proliferating activity, but negative for α-smooth muscle actin, desmin or CD204. Based on these findings, a diagnosis of extraskeletal osteosarcoma was made, a very rare tumor both in laboratory and pet rabbits.