Vetnizah Juniantito

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.

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 Glissons 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 Glissons sheath. Myofibroblasts appearing in the affected Glissons 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.

Immunophenotypical Characterization of Macrophages in Rat Bleomycin-Induced Scleroderma

Scleroderma is a skin disorder characterized by persistent fibrosis. Macrophage properties influencing cutaneous fibrogenesis remain to be fully elucidated. In this rat (F344 rats) model of scleroderma, at 1, 2, 3, and 4 weeks after initiation of daily subcutaneous injections of bleomycin (BLM; 100 μl of 1 mg/ml daily), skin samples were collected for histological and immunohistochemical evaluations. Immunohistochemically, the numbers of cells reacting to ED1 (anti-CD68; phagocytic activity) and ED2 (anti-CD163; inflammatory factor production) began to increase at week 1, peaked at week 2, and decreased thereafter. In contrast, the increased number of cells reacting to OX6 (anti–MHC class II molecules) was seen from week 2 and remained elevated until week 4. α–Smooth muscle actin–positive myofibroblasts were increased for 4 weeks. Double labeling revealed that galectin-3, a regulator of fibrogenic factor TGF-β1, was expressed in CD68+, CD163+, and MHC class II+ macrophages and myofibroblasts. mRNA expression of TGF-β1, as well as MCP-1 and CSF-1 (both macrophage function modulators), were significantly elevated at weeks 1 to 4. This study shows that the increased number of macrophages with heterogeneous immunophenotypes, which might be induced by MCP-1 and CSF-1, could participate in the sclerotic lesion formation, presumably through increased fibrogenic factors such as galectin-3 and TGF-β1; the data may provide useful information to understand the pathogenesis of the human scleroderma condition.

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.

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.

Relationship of Cell Proliferating Marker Expressions with PGE2 Receptors in Regenerating Rat Renal Tubules after Cisplatin Injection

Cisplatin, an anticancer drug, is well known to have nephrotoxicity as an adverse effect. We investigated the expressions of cell cycle markers and prostaglandin E2 (PGE2) receptors (EP) in the affected renal tubules in rats injected with a single dose (6 mg/kg body weight) of cisplatin. On days 1–3 after dosing, the affected renal epithelial cells were almost desquamated, showing necrosis. On day 5 onwards, the renal tubules were rimmed by flattened or cuboidal epithelial cells with basophilic cytoplasm; BrdU-immunopositive cells began to significantly increase, indicating regeneration. Simultaneously, TUNEL-positive apoptotic cells were also seen. On days 1–5, cyclin D1-immunopositive cells were decreased with an increased expression in p21 mRNA, indicating G1 arrest in the cell cycle. The affected renal epithelial cells began to react to EP4 receptor, but not to EP2 receptor. Some EP4 receptor-reacting epithelial cells gave a positive reaction to BrdU or cyclin D1. Collectively, the affected renal tubules underwent various alterations such as necrosis, apoptosis, regeneration and G1 arrest; the aspects might be influenced by endogenous PGE2 through EP4 receptor.

Distribution of Cells Labelled by a Novel Somatic Stem Cell-recognizing Antibody (A3) in Pulmonary Genesis and Bleomycin induced Pulmonary Fibrosis in Rats

Stem cells play important roles in organogenesis and remodelling after tissue injury. A monoclonal antibody (A3) has been produced against rat somatic stem cells. The present study investigated the distribution of cells labelled by A3 in the lung of fetal, neonatal and adult rats, as well as in the lung of rats with bleomycin (BLM) induced pulmonary fibrosis. In developing fetal lungs, A3(+) interstitial cells were present around the bronchi/bronchioles and arterioles, while in neonatal and adult lungs, the A3 reactivity of the interstitial cells gradually disappeared and instead, vascular endothelial cells in alveolar capillaries and arterioles expressed A3. By double immunofluorescence labelling, the A3(+) interstitial cells also expressed vimentin (a mesenchymal marker) and CD34 (a marker of immature mesenchymal cells), indicating that the interstitial cells were immature mesenchymal cells concentrated in organs as precursors to cells of connective tissues. A3(+)endothelial cells were co-expressed RECA-1 (a marker of rat endothelial cells) and A3 was localized to the cell membrane and cytoplasm of these cells by immunoelectron microscopy. In BLM induced fibrotic lesions, there were many A3(+) cells, which also expressed vimentin or RECA-1 by dual immunofluorescence labelling. There were few CD34(+)/A3(+) double positive cells. No cells co-expressed A3 and α-smooth muscle actin (a marker of well-differentiated myofibroblastic cells). Although the detailed properties of cells labelled by A3 remain to be discovered, A3 would appear to be a useful marker of immature mesenchymal cells and vascular endothelial cells in developing lungs and in pulmonary fibrosis.

Rat hair follicle-constituting cells labeled by a newly-developed somatic stem cell-recognizing antibody: a possible marker of hair follicle development.

A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3-positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α-smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent post-progenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells.