Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.     

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II

BACKGROUND & AIMS: The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS: Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS: After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.     

Significance and therapeutic potential of endothelial progenitor cell transplantation in a cirrhotic liver rat model

BACKGROUND & AIMS: We investigated whether endothelial progenitor cell (EPC) transplantation could reduce established liver fibrosis and promote hepatic regeneration by isolating rat EPCs from bone marrow cells. METHODS: Recipient rats were injected intraperitoneally with carbon tetrachloride (CCl(4)) twice weekly for 6 weeks before initial administration of EPCs. CCl(4) was then readministered twice weekly for 4 more weeks, and EPC transplantation was carried out for these same 4 weeks. RESULTS: At 7 days in culture, the cells expressed Thy-1, CD31, CD133, Flt-1, Flk-1, and Tie-2, suggesting an immature endothelial lineage. Immunohistochemical analyses showed fluorescent-labeled, transplantation EPCs were incorporated into the portal tracts and fibrous septa. Single and multiple EPC transplantation rats had reduced liver fibrosis, with decreased alpha2-(I)-procollagen, fibronectin, transforming growth factor-beta, and alpha-smooth muscle actin-positive cells. Film in situ zymographic analysis revealed strong gelatinolytic activity in the periportal area, in accordance with EPC location. Real-time polymerase chain reaction analysis of multiple EPC-transplantation livers showed significantly increased messenger RNA levels of matrix metalloproteinase (MMP)-2, -9 and -13, whereas tissue inhibitor of metalloproteinase-1 expression was significantly reduced. Expression of hepatocyte growth factor, transforming growth factor-alpha, epidermal growth factor, and vascular endothelial growth factor was increased in multiple EPC-transplantation livers, while hepatocyte proliferation increased. Transaminase, total bilirubin, total protein, and albumin levels were maintained in EPC-transplantation rats, significantly improving survival rates. CONCLUSIONS: We conclude that single or repeated EPC transplantation halts established liver fibrosis in rats by suppressing activated hepatic stellate cells, increasing matrix metalloproteinase activity, and regulating hepatocyte proliferation.     

Colitis in mice lacking the common cytokine receptor gamma chain is mediated by IL-6-producing CD4+ T cells

BACKGROUND & AIMS: Mice that have a truncated mutation of the common cytokine receptor gamma chain (CR gamma -/Y) are known to spontaneously develop colitis. To identify the pathologic elements responsible for triggering this localized inflammatory disease, we elucidated and characterized aberrant T cells and their enteropathogenic cytokines in CR gamma -/Y mice with colitis. METHODS: The histologic appearance, cell population, T-cell receptor V beta usage, and cytokine production of lamina propria lymphocytes were assessed. CR gamma -/Y mice were treated with anti-interleukin (IL)-6 receptor monoclonal antibody to evaluate its ability to control colitis, and splenic CD4 + T cells from the same mouse model were adoptively transferred into SCID mice to see if they spurred the appearance of colitis. RESULTS: We found marked thickening of the large intestine, an increase in crypt depth, and infiltration of the colonic lamina propria and submucosa with mononuclear cells in the euthymic CR gamma -/Y mice, but not in the athymic CR gamma -/Y mice, starting at the age of 8 weeks. Colonic CD4 + T cells with high expressions of antiapoptotic Bcl-x and Bcl-2 were found to use selected subsets (V beta 14) of T-cell receptor and to exclusively produce IL-6. Treatment of CR gamma -/Y mice with anti-IL-6 receptor monoclonal antibody prevented the formation of colitis via the induction of apoptosis in IL-6-producing CD4 + T cells. Adoptive transfer of pathologic CD4 + T cells induced colitis in the recipient SCID mice. CONCLUSIONS: Colonic IL-6-producing thymus-derived CD4 + T cells are responsible for the development of colitis in CR gamma -/Y mice.     

Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners

BACKGROUND & AIMS: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. METHODS: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon gamma. RESULTS: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon gamma gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon gamma enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor-related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor-related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid-activated natural killer cells against activated hepatic stellate cells. CONCLUSIONS: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanisms, thereby ameliorating liver fibrosis.     

Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells

BACKGROUND & AIMS: Tumor desmoplasia is one of the representative histopathologic findings in ductal pancreatic adenocarcinoma. The aims of this study were to examine the cellular and molecular mechanisms of fibrogenesis associated with pancreatic adenocarcinomas. METHODS: Immunostainings were performed with human pancreatic adenocarcinomas (n = 27) and tumors induced in nude mice (n = 36) by subcutaneously injecting MiaPaCa2, Panc1, and SW850 with and without pancreatic stellate cells. Matrix-producing cells were isolated from pancreatic adenocarcinomas and compared with pancreatic stellate cells isolated from tissue of chronic pancreatitis. Paracrine stimulation of pancreatic stellate cells by carcinoma cells was studied regarding matrix synthesis (collagen and c-fibronectin on protein and messenger RNA level) and cell proliferation (bromodeoxyuridine incorporation). RESULTS: High numbers of desmin and alpha-smooth muscle actin-positive cells were detected in 26 of 27 pancreatic adenocarcinomas. Intense fibronectin and collagen stainings were associated with these cells. By using cytofilament stainings, gene expression profiling, and morphological examinations, the matrix-producing cells obtained by the outgrowth method from pancreatic adenocarcinomas were identified as pancreatic stellate cells. Supernatants of MiaPaCa2, Panc1, and SW850 cells stimulated proliferation and collagen type I and c-fibronectin synthesis of cultured pancreatic stellate cells. Preincubation of the carcinoma cell supernatants with neutralizing antibodies against fibroblast growth factor 2, transforming growth factor beta 1, and platelet-derived growth factor significantly reduced the stimulatory effects. Subcutaneous injection of carcinoma cells and pancreatic stellate cells induced fast-growing subcutaneous fibrotic tumors in nude mice. Morphometric analysis of carcinoma cells (cytokeratin stainings) showed a high density of carcinoma cells in these tumors. CONCLUSIONS: Pancreatic stellate cells strongly support tumor growth in the nude mouse model. The increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of pancreatic stellate cells by carcinoma cells.     

Hepatitis C virus core and nonstructural proteins induce fibrogenic effects in hepatic stellate cells

BACKGROUND & AIMS: The mechanisms by which hepatitis C virus (HCV) induces liver fibrosis are unknown. Hepatocytes secrete HCV proteins, which may interact with hepatic stellate cells (HSCs). Our aims were to investigate whether HCV proteins induce fibrogenic effects on HSCs. METHODS & RESULTS: Human-activated HSCs expressed messenger RNA (mRNA) for the putative HCV receptors CD81, LDL receptor, and C1q receptor as assessed by RT-PCR. Incubation of activated but not quiescent human HSCs with recombinant core and NS3 protein increased intracellular calcium concentration and reactive oxygen species production, as well as stimulated intracellular signaling pathways. Adenoviruses encoding core and nonstructural proteins (NS3-NS5) were used to express HCV proteins in HSCs. Expression of core protein increased cell proliferation in a Ras/ERK and PI3K/AKT dependent manner. In contrast, NS3-NS5 protein expression preferentially induced proinflammatory actions, such as increased chemokine secretion and expression of intercellular cell adhesion molecule type 1 (ICAM-1) through the NF-kappa B and c-Jun N-terminal kinase pathways. These effects were attenuated by antioxidants. Infection of freshly isolated rat HSCs with adenovirus-encoding core protein resulted in accelerated cell activation, as assessed by alpha-smooth muscle actin expression. Moreover, adenovirus-encoding core and NS3-NS5 proteins increased the secretion of bioactive TGF beta 1 and the expression of procollagen alpha1(I) in early cultured rat HSCs, as assessed by ELISA and RNase protection assay, respectively. CONCLUSIONS: HCV core and nonstructural proteins regulate distinct biologic functions in HSCs. A direct interaction between HCV proteins and HSCs may contribute to HCV-induced liver fibrosis.     

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin

BACKGROUND & AIMS: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS: When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.     

Isolation of mouse pancreatic ductal progenitor cells expressing CD133 and c-Met by flow cytometric cell sorting

BACKGROUND & AIMS: Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear. METHODS: Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity. RESULTS: CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133(+)CD34(-)CD45(-)Ter119(-) fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133(+)CD34(-)CD45(-)Ter119(-) cells. CONCLUSIONS: We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133(+) ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.     

Curcumin diminishes the impacts of hyperglycemia on the activation of hepatic stellate cells by suppressing membrane translocation and gene expression of glucose transporter-2

Diabetes is featured by elevated levels of blood glucose, i.e. hyperglycemia, which might be a risk factor for hepatic fibrogenesis in patients with non-alcoholic steatohepatitis. Hepatic stellate cells (HSCs) are the major effectors during hepatic fibrogenesis. This study was designed to evaluate impacts of high levels of glucose on HSC activation, assess roles of the phytochemical curcumin in attenuating the glucose impacts, and elucidate underlying mechanisms. In this report, levels of intracellular glucose were measured. Contents and gene expression of glucose transporter-2 (GLUT2) in cell fractions were examined. Levels of cellular glutathione and oxidative stress were analyzed. We observed that high levels of glucose induced cell proliferation, type I collagen production and expression of genes relevant to HSC activation, and elevated intracellular glucose levels in cultured HSCs. Curcumin eliminated the stimulatory impacts. Curcumin abrogated the membrane translocation of GLUT2 by interrupting the p38 MAPK signaling pathway. In addition, curcumin suppressed glut2 expression by stimulating the activity of peroxisome proliferator-activated receptor-gamma (PPARγ) and de novo synthesis of glutathione. In conclusion, hyperglycemia stimulated HSC activation in vitro by increasing intracellular glucose, which was eliminated by curcumin by blocking the membrane translocation of GLUT2 and suppressing glut2 expression. The latter was mediated by activating PPARγ and attenuating oxidative stress. Our results presented evidence to impacts of hyperglycemia on stimulating HSC activation and hepatic fibrogenesis, and provided novel insights into the mechanisms by which curcumin eliminated the hyperglycemia-caused HSC activation and potential therapeutic strategies for treatment of diabetes-associated hepatic fibrogenesis.