Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells

Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl(4))-induced damage. Rats treated with CCl(4) showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl(4) treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B(4) and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A(4), an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.     

Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury

Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.     

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCl4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin-6 (IL-6) gene expression was enhanced in liver and immunoreactive IL-6 was detected in infiltrating inflammatory cells. To delineate the role of IL-6 in this process, we treated IL-6-deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL-6-deficient than wild-type mice. Moreover, CCl4-induced expression of transforming growth factor beta1 and hepatocyte growth factor genes in liver was significantly reduced in IL-6-deficient mice. Thus, IL-6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines.     

Effect of pretreatment with artichoke extract on carbon tetrachloride-induced liver injury and oxidative stress.

Artichoke is a plant with antioxidant properties. In this study, we investigated the effect of artichoke extract pretreatment on carbon tetrachloride (CCl(4))-induced oxidative stress and hepatotoxicity. Rats were given artichoke leaf extract (1.5g/kg/day) by gavage for 2 weeks and after then CCl(4) (1ml/kg; i.p.) was applied. All rats were killed 24h after the CCl(4) injection. CCl(4) administration resulted in hepatic necrosis and significant increases in plasma transaminase activities as well as hepatic malondialdehyde (MDA) and diene conjugate (DC) levels in the liver of rats. Glutathione (GSH) and vitamin C levels decreased, but vitamin E levels increased in the liver of CCl(4)-treated rats. Hepatic superoxide dismutase (SOD) activities remained unchanged, but glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities decreased following CCl(4) treatment. In rats pretreated with artichoke extract, significant decreases in plasma transaminase activities and amelioration in histopathological changes in the liver were observed following CCl(4) treatment as compared to CCl(4)-treated rats. In addition, hepatic MDA and DC levels decreased, but GSH levels and GSH-Px activities increased without any change in other antioxidant parameters following CCl(4) treatment in artichoke-pretreated rats. The present findings indicate that in vivo architoke extract administration may be useful for the prevention of oxidative stress-induced hepatotoxicity.     

The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production

The monoclonal antibody ED2 is widely used to define macrophages (mphi) in the rat. We have recently identified the ED2 antigen as the rat CD163 glycoprotein. CD163 is a member of the scavenger receptor cysteine-rich group B (SRCR-B) family and functions as a scavenger receptor for hemoglobin-haptoglobin complexes. Moreover, CD163 has also been indicated as a marker for alternatively activated mphi. In the current study, we identify rat CD163/ED2-antigen as a marker for mature tissue mphi. Rat CD163 is constitutively expressed on most subpopulations of mature tissue mphi, including splenic red pulp mphi, thymic cortical mphi, Kupffer cells in the liver, resident bone marrow mphi and central nervous system perivascular and meningeal mphi, but is apparently absent from monocytes. Rat CD163 expression can be promoted by glucocorticoids, and this can be further enhanced by IL4. Finally, engagement of rat CD163 on peritoneal mphi induces the production of pro-inflammatory mediators, including NO, IL-1beta, IL-6 and TNF-alpha. Collectively, our findings identify rat CD163 as a broadly expressed macrophage scavenger receptor that may play a role in the activation of mphi during hemolytic and/or inflammatory conditions.     

Studies on the mechanism of glutathione prevention of carbon tetrachloride-induced liver injury.

The prior administration of reduced glutathione (GSH) partially prevents carbon tetrachloride (CCl4)-induced liver necrosis observed at 24 h after administration of the hepatotoxin. No prevention occurs when observations are made at 72 h. GSH pretreatment does not significantly modify the intensity of the covalent binding of CCl4 reactive metabolites to microsomal lipids or the intensity of the CCl4-induced lipid peroxidation process at either 1, 3 or 6 h after poisoning. GSH administration does not significantly prevent CCl4-induced cytochrome P-450 destruction or glucose 6 phosphatase activity depression. Pretreatment with GSH does not significantly modify the levels of CCl4 or i.p. administered CCl4 reaching the liver at 1, 3 or 6 h after intoxication. Pretreatment with GSH significantly prevents CCl4-induced decreases in body temperature. Results are interpreted as suggesting that GSH prevents CCl4-induced liver necrosis by changing the liver cell''s response to injury rather than by modification of early events of the process such as lipid peroxidation or covalent binding of reactive metabolites.     

Liver precursor cells increase hepatic fibrosis induced by chronic carbon tetrachloride intoxication in rats

Hepatic fibrosis, the major complication of virtually all types of chronic liver damage, usually begins in portal areas, and its severity has been correlated to liver progenitor cells (LPC) expansion from periportal areas, even if the primary targets of injury are intralobular hepatocytes. The aim of this study was to determine the potential fibrogenic role of LPC, using a new experimental model in which rat liver fibrosis was induced by chronic carbon tetrachloride (CCl(4)) administration for 6 weeks, in combination with chronic acetylaminofluorene treatment (AAF), which promotes activation of LPC compartment. Treatment with CCl(4) alone caused a significant increase in serum transaminase activity as well as liver fibrosis initiating around central veins and leading to formation of incomplete centro-central septa with sparse fibrogenic cells expressing α-smooth muscle actin (αSMA). In AAF/CCl(4)-treated animals, the fibrogenic response was profoundly worsened, with formation of multiple porto-central bridging septa leading to cirrhosis, whereas hepatocellular necrosis and inflammation were similar to those observed in CCl(4)-treated animals. Enhanced fibrosis in AAF/CCl(4) group was accompanied by ductule forming LPC expanding from portal areas, αSMA-positive cells accumulation in the fibrotic areas and increased expression of hepatic collagen type 1, 3 and 4 mRNA. Moreover, CK19-positive LPC expressed the most potent fibrogenic cytokine transforming growth factor-β (TGFβ) without any expression of αSMA, desmin or fibroblast-specific protein-1, demonstrating that LPC did not undergo an epithelial-mesenchymal transition. In this new experimental model, LPC, by expressing TGFβ, contributed to the accumulation of αSMA-positive myofibroblasts in the ductular reaction leading to enhanced fibrosis but also to disease progression and to a fibrotic pattern similar to that observed in humans.     

Role of Kupffer cells in developing streptococcal cell wall granulomas. Streptococcal cell wall induction of inflammatory cytokines and mediators.

Hepatic granulomas are induced by intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats. Kupffer cells rapidly clear SCW from the blood, and the authors examined Kupffer cells further for a role in SCW-hepatic inflammation. Isolated Kupffer cells cultured with SCW secreted high levels of tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), transforming growth factor beta (TGF beta), and prostaglandin E2 (PGE2). SCW transiently induced increased steady-state levels of IL-1 beta and TNF alpha mRNA; in contrast, constitutive expression of TGF beta 1 mRNA in Kupffer cells was not affected by SCW. Low concentrations of SCW induced the accumulation of intracellular IL-1 and TGF beta bioactivity, with intracellular IL-1 bioactivity remaining high through at least 72 hours of culture. Kupffer cells isolated 1, 7, and 21 days after SCW injection did not express IL-1 beta or TNF alpha mRNA greater than control levels and exhibited marked hyporesponsiveness to secondary in vitro stimulation with SCW or LPS. SCW transiently induces Kupffer cells to secrete a variety of soluble mediators that contribute to hepatic inflammation by inducing leukocyte recruitment and activation and fibroproliferation. The transient nature of the Kupffer cell response and the hyporesponsiveness to secondary stimulation may be a mechanism by which the hepatic inflammation is negatively regulated.     

Transglutaminase type II plays a protective role in hepatic injury

The up-regulation of "tissue" transglutaminase (TG2) gene has been shown to occur in various pathologies and can lead to severe liver injury; however, its role in the onset of liver damage has not yet been clarified. To address this issue, we have used two experimental settings: carbon tetrachloride (CCl(4))-induced liver injury in wild-type and TG2 knockout mice; and liver biopsies obtained from a large cohort of hepatitis C virus (HCV)-infected patients. Mice lacking TG2 failed to clear the hepatic necrotic tissue formed in response to prolonged CCl(4) exposure (5 weeks) and 60% of them died before the end of the treatment. By contrast, wild-type mice were able to recover after the toxic insult. CCl(4)-treated TG2 null mice showed a derangement of the hepatic lobular architecture and a progressive accumulation of extracellular matrix (ECM) components and inflammatory cells which were not observed in the liver of control animals. Consistent with this protective role, we observed that TG2 levels were much higher (up to 15-fold) during the initial stages of liver fibrosis in HCV-infected individuals (METAVIR = F2) compared with uninfected controls, in which the enzyme protein localized in the hepatocytes facing the periportal infiltrate. By contrast, the enzyme levels decreased in the advanced stages (METAVIR = F3 and F4) and their localization was limited to the ECM. Our data demonstrate that TG2 plays a protective role in the liver injury by favoring tissue stability and repair.     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor.

Signaling by tumor necrosis factor type 1 receptor (TNFR-1) is required for the initiation of liver regeneration after partial hepatectomy. Using knockout mice that lack either TNFR-1 or TNFR-2, we determined whether signaling through TNF receptors is important for liver injury and hepatocyte proliferation induced by carbon tetrachloride (CCl4). Lack of TNFR-1 inhibited hepatocyte DNA synthesis after CCl4 injection. At 44 hours after the injection, replication of hepatocytes in TNFR-1 was 50% to 90% lower than in wild-type (WT) animals, depending on the dose injected. In WT animals, hepatocyte replication was essentially completed by 4 days after CCl4 injection, but replication at a low level persisted in TNFR-1 mice for at least 2 weeks. TNFR-1 knockout mice had little detectable NF-kappa B and STAT3 binding during the first 5 hours after CCl4, high plasma TNF, and reduced levels of plasma interleukin (IL)-6 and liver IL-6 mRNA. Injection of IL-6 30 minutes before CCl4 administration corrected the deficiency of hepatocyte replication at 44 hours and restored STAT3 binding to normal levels. In contrast, mice lacking TNFR-2 did not differ significantly from WT mice in NF-kappa B and STAT3 binding, IL-6 and TNF levels, or hepatocyte replication. Although AP-1 binding was induced in WT TNFR-1 and TNFR-2 knockout mice, binding in TNFR-2 knockouts was lower than in WT mice. C/EBP binding was much lower in TNFR-1 and TNFR-2 knockout mice than in WT mice. As assessed by morphological analysis and alanine aminotransferase levels, the acute injury caused by CCl4 appeared to be similar in the three groups of animals, but subsequent regeneration was impaired in mice lacking TNFR-1. We conclude that a TNFR-1 signaling pathway involving NF-kappa B, IL-6, and STAT3 is an important component of the hepatocyte mitogenic response induced by CCl4 injury in mouse liver.     

Macrophage Populations in Different Stages of Induced Hepatic Metastases in Rats: an Immunohistochemical Analysis

Macrophages play a role in the host defence against cancer. Little is known about changes in macrophage populations during early metastatic growth. To evaluate the distribution, number and phenotype of macrophages in the development of hepatic metastases in a rat model (Wag/Rij rats and syngeneic CC531 colon carcinoma cell line), an immunohistochemical study was performed with the monoclonal antibodies ED1 (monocytes, and all macrophages), ED2 (resident tissue macrophages, like Kupffer cells) and ED3 (a subpopulation of macrophages which may play a role in the recruitment of lymphocytes). OX19 and Hisl4 were used to identify lymphocytes. In this study a new monoclonal antibody CC52 is described, which recognizes the CC531 tumour cell line. Liver metastases were induced by injection of CC53I colon carcinoma cells into a mesenteric vein. Rats were killed at various intervals. Results show three major macrophage populations during hepatic tumour growth: (1) on day 3, infiltrates are observed around the micrometastases, which contain mainly newly recruited macrophages (ED1⁺ and ED2⁻); (2) after 7 days, ED3-positive (ED3 ⁺) macrophages together with T lymphocytes are found in the infiltrates; (3) an increase in the number of ED2-positive (ED2⁺) Kupffer cells is observed in the liver parenchyma after 14 days. In conclusion, the present results suggest that various populations of macrophages, newly recruited (ED1⁺) as well as resident Kupffer cells (ED2⁺), are involved in the immune response against tumour cell deposits in the liver.     

Reactive oxygen intermediates and eicosanoid production by Kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4

OBJECTIVE AND DESIGN: The aim of the present study was to characterize during acute and chronic liver injury induced by CCl4, macrophage phenotypes and whether a change in reactive oxygen intermediates (ROI) and eicosanoids production by Kupffer cells (KC) was observed. MATERIAL AND METHODS: Liver steato-necrosis and cirrhosis were induced in rats after 3 weeks and 9 weeks of CCl4 intoxication, respectively. Monocytes and tissue macrophages were identified by immunohistochemical study using monoclonal antibodies ED-1 and tissue macrophages using the antibody ED-2. The release of ROI and eicosanoids in response to the phorbol ester TPA (protein kinase activator) and to the calcium ionophore A23187 was assessed in cultivated cells. RESULTS: As compared to healthy controls, livers of rats with steato-necrosis or cirrhosis exhibited a significant increase of ED-1 and ED-2 positive cells. Only KC from rats with liver steato-necrosis were found to have higher A23187, TPA + A23187 or opsonized zymosan induced ROI production than healthy controls (p < 0.01). After TPA + A23187 or opsonized zymosan stimulation, KC from both rats with steato-necrosis or cirrhosis produced more TxB2 and leukotrienes and less PGE2 as compared to healthy controls (p <0.05). CONCLUSIONS: These results suggest an influx of monocytes into the liver during acute and chronic injury induced by CCl4. Functional changes of this inflammatory infiltrate have been demonstrated with an increase of ROI production only in the early stage of liver injury whereas a rise in KC leukotriene production and an imbalance between cytoprotective and cytotoxic prostanoids were observed at all stages of liver disease.     

Appearance of ductular hepatocytes in rat liver after bile duct ligation and subsequent zone 3 necrosis by carbon tetrachloride.

Intrahepatic biliary cell plasticity was investigated in a rat model that combined prior bile ductular cell hyperplasia after bile duct ligation with subsequent CCl4-induced hepatonecrosis. Morphometric analysis of histologic liver sections from rats at 4 to 6 weeks after bile duct ligation and 3 to 5 days after CCl4 demonstrated the total section area to be occupied by near-equal amounts of hyperplastic bile ductular tissue area and hepatonecrotic area. Of particular significance was the unique presence, albeit infrequent, of newly appearing hepatic cell cholangioles composed of both biliary epithelial cells and one or more 'ductular hepatocytes' exclusively within the hyperplastic bile ductular tissue area of liver sections from the bile-duct-ligated/CCl4-treated rats, but not observed in control liver sections. This finding is compatible with the possibility of a 'transdifferentiation' of some hyperplastic biliary epithelial cells into 'ductular hepatocytes' in response to an extreme hepatic injury.     

In vivo biologic and immunohistochemical analysis of interleukin-1 alpha, beta and tumor necrosis factor during experimental endotoxemia. Kinetics, Kupffer cell expression, and glucocorticoid effects.

Using a model of sepsis induced by parenteral challenge of mice with bacterial lipopolysaccharide (LPS), the authors analyzed the in vivo expression of interleukin-1 (IL-1) alpha,beta and tumor necrosis factor (TNF). Both TNF and IL-1 alpha,beta were detected in hepatic sinusoidal macrophages (Kupffer cells), immunohistochemically. Kinetic analysis showed a clear sequence of synthesis. Tumor necrosis factor was produced first, reaching maximal expression at 1 hour after LPS challenge, then rapidly disappeared. IL-1 beta followed, reaching maximal expression at 2 to 3 hours, then dropped off by 6 hours. Interleukin-1 alpha expression reached a peak at 6 hours and had disappeared by 18 hours. Analysis of serum bioactivity also revealed sequential expression that correlated with immunohistochemical findings. Tumor necrosis factor was maximal at 1 hour and IL-1 at 6 hours. The IL-1 bioactivity was not due to interleukin-6 (IL-6), as this was depleted from specimens by immunoabsorption. Also IL-6 bioactivity reached maximal levels at 3 hours, earlier than IL-1. Pretreatment with 4 mg/kg dexamethasone significantly decreased Kupffer cell expression of TNF and IL-1 alpha (about 80% and 60% suppression, respectively) but had less effect on IL-1 beta expression (about 30% suppression). Accordingly, serum levels of TNF were suppressed by 75% while serum IL-1 was decreased by 39%, indicating differential sensitivity of these cytokines to glucocorticoids. Endogenous corticosteroid levels increased as TNF levels decreased, supporting the contention that glucocorticoids regulate TNF synthesis. In contrast, IL-1 levels rose concurrently with corticosterone. These data indicate a sequential activation of cytokine gene expression in vivo, which may be critical to the cascade of events leading to septic shock, and provide evidence that Kupffer cells are a major source of cytokines in endotoxemia. Finally, the differential sensitivity of cytokine expression to glucocorticoids may in part explain the inadequacy of the latter in the treatment of sepsis.     

Exploring an animal model of amodiaquine-induced liver injury in rats and mice

Amodiaquine (AQ) is associated with a relatively high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. A previous study reported that a combination of high dose AQ and glutathione (GSH) depletion led to liver injury. However, the characteristics of this toxicity were very different from AQ-induced liver injury in humans. We developed a model of AQ-induced liver injury with characteristics similar to the injury in humans by treating mice with lower doses of AQ for several weeks. In this study we found that not only did GSH depletion not increase AQ covalent binding to hepatic proteins at this lower dose, but also it paradoxically prevented the liver injury. We extended the model to rats and found AQ treatment led to a mild delayed onset liver injury that resolved despite continued treatment with AQ. Immunohistochemistry indicated the presence of Kupffer cell activation, apoptosis and hepatocyte proliferation in the liver. There was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1 and TGFβ, but a decrease in leptin. Coincident with the elevated serum ALT, the number of liver CD4⁺ T-cells, IL-17 secreting cells and T_H17/T_reg cells increased at Week 3 and decreased during continued treatment. Increases in NK1.1+ cells and activated M2 macrophages were also observed during liver injury. These results suggest that the outcome of the liver injury was determined by the balance between effector and regulatory cells. Co-treatment with cyclosporin prevented AQ-induced liver injury, which supports an immune mechanism. Retinoic acid (RA), which has been reported to enhance natural killer (NK) cell activity, exacerbated AQ-induced liver injury. These results suggest that AQ-induced IDILI is immune mediated and the subsequent adaptation appears to represent immune tolerance.     

Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages

Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-β1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.     

Interaction between monensin and CCl4 after chronic treatment: dolichol and retinol content of rat liver sinusoidal cells

The aim of this investigation was to study retinol, which is known to decrease in hepatic stellate cells during fibrogenesis, and dolichol, which influences membrane fluidity and decreases in liver injury, in freshly isolated liver parenchymal and nonparenchymal cells after intoxication of rats with CCl4 combined with the ionophore monensin for 3, 5 and 7 weeks. To study the interrelationship between dolichol and vitamin A transport, a load of vitamin A was given to batches of rats 3 days before sacrifice. Monensin did not modify the action of CCl4 in hepatocytes. On administration of CCl4 and CCl4-monensin, dolichol decreased independently of vitamin A load, while retinol increased, especially when a load of vitamin A was given to rats 3 days before sacrifice. Hepatocytes appeared to no longer be able to export or metabolize vitamin A. In a subfraction of hepatic stellate cells (Ito-1 cells) dolichol always decreased, while retinol was no longer stored after each treatment; dolichol and retinol showed the same behavior but the decrease was more pronounced in monensin after vitamin A load and after 3 weeks. These data support the hypothesis that by modulating membrane characteristics, dolichol might be involved in intracellular or intercellular retinol transport and that altered transport between hepatocytes and Ito-1 cells might accompany liver injury. The data regarding another subfraction, Ito-2 cells, partly resemble those for the Ito-1 fraction and are in agreement with the heterogeneity of hepatic stellate cells. In Kupffer and sinusoidal endothelial cells, dolichol and retinol content was not homogeneous and was only slightly altered after the treatments. Monensin and CCl4 are not interactive. Although both drugs alter membrane lipids, their association allows some sinusoidal cell responses to be differentiated.     

Increased serum type IV collagen peptide in carbon tetrachloride-treated rats

We developed a competitive enzyme-immunoassay for serum type IV collagen peptide as a marker of fibrogenesis, and examined the relationship between serum type IV collagen peptide and hepatic disorder in CCl4-treated rats. The rats were treated for 8 weeks and signs of liver damage began to appear from about week 2. With the progression of these signs to liver fibrosis, type IV collagen increased in the fibrous septa and especially in the perisinusoidal walls, where the increase was manifested as development of a real basement membrane beneath the sinusoidal endothelial cells. In CCl4-treated rats, serum type IV collagen peptide significantly increased with the progression of liver fibrosis. When CCl4 administration was stopped, the collagen peptide rapidly decreased without any rebound rise. An intimate relationship was found between the production of serum type IV collagen peptide and liver prolyl hydroxylase activity and the amount of collagen deposited in the liver. These results suggest that serum type IV collagen peptide will be a useful biochemical marker for the early detection of fibrogenesis in the liver.