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.     

Immunophenotypical analysis of myofibroblasts and mesenchymal cells in the bleomycin-induced rat scleroderma,with particular reference to their origin

Cellular characteristics of myofibroblasts and its possible origin with mesenchymal stem cell nature in scleroderma remain to be investigated. We analyzed these cells in scleroderma induced in F344 rats by bleomycin (BLM) by immunolabeling using a panel of marker antibodies for cytoskeletons (vimentin, desmin, α-smooth muscle actin (α-SMA)) and stromal stem cells (Thy-1, A3). Skin samples were collected at 1, 2, 3, and 4 weeks after initiation of subcutaneous injections of BLM (100 μl of 1 mg/ml, daily). In double immunofluorescence, myofibroblasts reacting simultaneously to α-SMA, vimentin, and Thy-1 were seen in sclerotic lesions with a time-dependent increase. Mesenchymal cells in the perifollicular dermal sheath (PDS) displayed increased reactivity for Thy-1 and vimentin, but α-SMA expression did not increase in these cells. In double immunofluorescence, both myofibroblasts and pericytes in newly formed blood vessels in sclerotic lesions co-expressed α-SMA, vimentin and Thy-1, and the PDS cells and pericytes reacted simultaneously to A3, Thy-1 and vimentin. Desmin-positive cells were infrequently seen around the blood vessels. Based on these findings, the PDS cells and pericytes may be involved as possible progenitors of myofibroblasts in sclerotic lesions in the stromal stem cell lineage. Interestingly, increased number of TUNEL-positive apoptotic epithelial cells in the atrophied hair follicles significantly correlated with increase in immunohistochemical scoring of vimentin and Thy-1 in the PDS. Apoptosis in the hair follicle might have mediate the perifollicular fibrosis, resulting in extensive scleroderma. The present findings would provide new insights in the pathogenesis of BLM-induced scleroderma in terms of myofibroblasts and its origin.     

Amelioration of cisplatin-induced rat renal lesions by a cyclooxygenase (COX)-2 selective inhibitor

Cyclooxygenase (COX)-2, an inducible form of COX, plays important roles in inflammatory lesions. We investigated effects of a COX-2 selective inhibitor, NS-398, on cisplatin (CDDP)-induced rat renal lesions. As compared with rats injected with a single dose of CDDP (6 mg/kg; CDDP group), rats who were treated everyday with NS-398 (3 mg/kg) after the CDDP injection (inhibitor group), showed the declines of blood urea nitrogen and creatinine values, and the delay of the peak of regenerating renal epithelial cell number (demonstrable with 5′-bromo-2′-deoxyuridine immunohistochemistry); these findings suggested cytoprotective effects of the inhibitor. Furthermore, the numbers of ED1-immunopositive macrophages and α-smooth muscle actin (α-SMA)-immunopositive myofibroblasts were lower in the inhibitor group than in the CDDP group; mRNA expression of transforming growth factor-β1 (TGF-β1) was also decreased in the inhibitor group. Because the fibrotic area seen after CDDP injection were tended to decrease in the inhibitor group compared with the CDDP group, it was considered that the decreased number of infiltrating macrophages by the inhibitor might lead to the decreased production of TGF-β1, thereby resulting in the reduced number of α-SMA-positive myofibroblasts responsible for fibrosis. Collectively, although these differences between the CDDP and inhibitor groups were not always marked, the COX-2 inhibitor used in this study could ameliorate the CDDP-induced rat renal lesions.     

Slowly progressive cholangiofibrosis induced in rats by α-naphthylisothiocyanate (ANIT), with particular references to characteristics of macrophages and myofibroblasts

A progressive cholangiofibrosis was developed as an animal model in 6-week-old male F344 rats by repeated intraperitoneal injections of α-naphthylisothiocyanate (ANIT) for 19 weeks; liver samples were examined at post-first injection (PFI) weeks 3, 7, 10, 13, 16 and 19, focusing on characteristics of macrophages and myofibroblasts by immunohistochemical analyses. In the affected Glisson's sheath consisting of inflammatory cell infiltrates, bile duct proliferation and advancing fibrosis, the number of macrophages reacting to OX6 (recognizing MHC class II) increased consistently (PFI weeks 3–19), suggesting a central role of antigen presenting cells in the biliary fibrosis; macrophages reacting to ED1 (CD68, reflecting phagocytic activity) and ED2 (CD163, relating to proinflammatory factor production) showed a significantly increased number at PFI weeks 7–19 and PFI weeks 13–19, respectively. Interestingly, macrophages positive for SRA-E5 (CD204, reflecting lipid metabolism) increased at PFI weeks 7–19, and the appearance was limited in the sinusoids around the affected Glisson's sheath. Myofibroblasts appearing in the affected Glisson's sheath reacted to vimentin and desmin at early (PFI weeks 3–7) and mid (PFI weeks 10–13) stages, and then they came to strongly express α-smooth muscle actin at late stage (PFI weeks 16–19). This study shows that macrophages exhibit heterogeneous properties depending on stages and locations; in association with such macrophage populations, myofibroblasts expressing various cytoskeletons participate in cholangiofibrosis. These characteristics would be useful in evaluating the pathogenesis of possible cholangio-toxicants.     

Heterogeneity of macrophage populations and expression of galectin-3 in cutaneous wound healing in rats

The aim of this study was to investigate the properties of macrophages that infiltrated the sites of cutaneous wound healing in rats between 1 and 26 days post wounding (dpw). During the inflammation phase (1–3 dpw), ED1⁺ (CD68⁺) macrophages with enhanced lysosomal activity dominated. From 5 to 7 dpw there was formation of granulation tissue as indicated by the presence of myofibroblasts expressing α-smooth muscle actin. At this stage, ED2⁺ (CD163⁺) macrophages, capable of producing inflammatory factors, were dominant. The majority of ED1⁺ macrophages expressed galectin-3, a regulator of fibrosis. Corresponding to the increased numbers of ED1⁺ and ED2⁺ macrophages at 3–9 dpw, there was increased expression of genes encoding transforming growth factor-β1 (a major fibrogenic factor), monocyte chemoattractant protein-1 and colony stimulating factor-1. These macrophage-related factors might contribute to inflammation and formation of granulation tissue. OX6⁺ macrophages expressing class II molecules of the major histocompatibility complex became predominant in the healing stages (15–26 dpw), indicating important roles for antigen-presenting cells in tissue remodelling. The OX6⁺ macrophages were most likely derived from ED1⁺ macrophages. The results of this study show that infiltration of phenotypically- and functionally-distinct macrophage populations characterizes different stages of the wound healing process.     

Thy-1 expression,a possible marker of early myofibroblast development,in renal tubulointerstitial fibrosis induced in rats by cisplatin

Interstitial fibrosis is regarded as the common final pathway in chronic renal failure. Myofibroblasts play an important role in the renal fibrosis through producing extracellular matrices. In addition to expressions of cytoskeletons such as vimentin, desmin and α-smooth muscle actin (α-SMA), Thy-1 expression was investigated in cisplatin-induced rat renal interstitial fibrosis, to clarify the characteristics of myofibroblasts. Immunohistochemically, myofibroblasts in the renal fibrotic lesions reacted to vimentin, desmin and α-SMA in varying degrees, and the expression degrees were increased with advancing fibrosis. Vimentin expression was the greatest and the increased expression retained even in scar at end stages, whereas desmin and α-SMA expressions were almost completely decreased in scar. In double immunofluorescence, there were myofibroblasts reacting to both vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, indicating that renal myofibroblasts can simultaneously express different cytoskeletons. Thy-1 expression in renal myofibroblasts was increased according to progressing fibrosis; however, the increased expression was decreased in scar, similar to desmin and α-SMA expressions. Some myofibroblasts expressing Thy-1 reacted simultaneously to vimentin or desmin, but there were no cells reacting to both Thy-1 and α-SMA. Because well-differentiated myofibroblasts are characterized mainly by α-SMA expression and the pericytes (immature stromal stem cells) showed a positive reaction to Thy-1, renal myofibroblasts might be originated from immature mesenchymal cells through loosing Thy-1 expression. This study for the first time shows that renal myofibroblasts can variously exhibit such mesenchymal markers as vimentin, desmin, α-SMA and Thy-1; particularly, Thy-1 immunohistochemistry would be used to detect myofibroblasts at early stages in analyzing chemically induced renal lesions.