Danko Batusic

Mast cells distribution in human liver disease and experimental rat liver fibrosis. Indications for mast cell participation in development of liver fibrosis

BACKGROUND/AIMS The development of liver fibrosis due to chronic liver diseases is thought to be mediated by inflammatory cells releasing fibrogenic mediators that activate fat-storing cells (Ito-cells). Recently, the involvement of mast cells in fibrogenesis has been suggested. We studied the distribution of these cells in normal human liver and human nonfibrotic and fibrotic liver disease as well as in normal rat liver and acutely and chronically injured rat liver (CCl4 model). METHODS Mast cells were identified by histochemical and immunohistochemical methods. The immunoreactivity of liver and comparatively of rat peritoneal mast cells to the serpins alpha1-antitrypsin, alpha1-antichymotrypsin and antithrombin III was also studied. RESULTS In normal human and rat liver, mast cells were rarely found in portal tracts, and there was no change in cell numbers in nonfibrotic human or acutely injured rat livers. In contrast, cirrhotic human and rat livers contained numerous mast cells in the portal tracts and the fibrous septa. They exhibited strong immunoreactivity to the serpins, as did rat peritoneal mast cells. CONCLUSIONS The results indicate that in the late stages of liver fibrogenesis, mast cells may be involved by displaying protease inhibitory activity in the fibrotic septa.

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury.

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl4. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1+ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1+ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl4-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue.

Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis

Abstract Christiansen, H., Batusic, D., Saile, B., Hermann, R. M., Dudas, J., Rave-Frank, M., Hess, C. F., Schmidberger, H. and Ramadori, G. Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis. Radiat. Res. 165, 318–325 (2006). The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.

Prospero-related homeobox 1 (Prox1) is a stable hepatocyte marker during liver development, injury and regeneration, and is absent from "oval cells"

The aim of this study was to analyse the changes of Prospero-related homeobox 1 (Prox1) gene expression in rat liver under different experimental conditions of liver injury, regeneration and acute phase reaction, and to correlate it with that of markers for hepatoblasts, hepatocytes, cholangiocytes and oval cells. Gene expression was studied at RNA level by RT-PCR, and at protein level by immunohistochemistry. At embryonal stage of rat liver development (embryonal days (ED) 14–16) hepatoblasts were found to be Prox1+/Cytokeratin (CK) 19+ and α-fetoprotein (AFP)+, at this stage Prox1−/CK19+/AFP- small cells (early cholangiocytes?) were identified. In fetal liver (ED 18–22) hepatoblasts were Prox1+/CK19−/AFP+. CK7+ cholangiocytes were detected at this stage, and they were Prox1−/AFP−. In the adult liver hepatocytes were Prox1+/CK19−/CK7−/AFP−, cholangiocytes were CK19+ and/or CK7+ and AFP−/Prox1−. In models of liver damage and regeneration Prox1 remained a stable marker of hepatocytes. After 2-acetyl-aminofluorene treatment with partial hepatectomy (AAF/PH) the amount of Prox1 specific transcripts was low in the liver, when CK19 and AFP gene expression was high, and at no time point AFP+/CK19+ “oval cells” were found to be Prox1+. However, a few Prox1+/CK19+ and a few Prox1+/CK7+ cells were identified in the liver of AAF/PH-animals, which may represent precursors of hepatocytes, or a precancerous state.

Identification of genes specific to “oval cells” in the rat 2-acetylaminofluorene/partial hepatectomy model

Under certain conditions liver regeneration can be accomplished by hepatic progenitor cells (“oval cells”). So far, only few factors have been identified to be uniquely regulated by the “oval cell” compartment. Using macroarray analysis in a rat model of oval cell proliferation (treatment with 2-acetylaminofluorene and partial hepatectomy, AAF + PH), we identified 12 differentially expressed genes compared to appropriate control models (AAF treatment and sham operation or AAF treatment alone). Further analysis in models of normal liver regeneration (ordinary PH) and acute phase response (turpentine oil-treated rats) revealed that three out of 12 genes (thymidine kinase 1, Jun-D and ADP-ribosylation factor 4) were not affected by the hepatic acute phase reaction but similarly overexpressed in both “oval cell”-dependant and normal liver regeneration. We characterized Jun-D and ADP-ribosylation factors as novel factors upregulated in oval cells and in non-parenchymal liver cells of normally regenerating livers. However, two out of 12 differentially expressed genes were specifically expressed in oval cells: ras-related protein Rab-3b and Ear-2. On protein level, Rab-3b was increased in total liver homogenates and demonstrated only in clusters of oval cells. We postulate that Ear-2 and Rab-3b may represent novel regulatory factors specifically activated in “oval cells”.

Changes of hepatic lactoferrin gene expression in two mouse models of the acute phase reaction

Lactoferrin (Ltf), an iron binding glycoprotein, is a pleiotropic molecule whose serum concentration increases under acute phase conditions. The physiological roles of this protein have been well elucidated, but the source and serum regulation of Ltf gene expression have not been investigated in detail as part of the acute phase reaction (APR). In the current work, the changes in hepatic Ltf-gene-expression during turpentine oil- (TO-) or LPS-induced APR were investigated. Ltf was upregulated at both the mRNA and protein levels in the liver of TO- and LPS-treated wild type (WT) mice. The pattern of induction however was different in both animal models indicating distinctive signalling patterns resulting in an acute phase reaction. Cytokines are the core regulators of APR. Among the major cytokines, IL-6 is an important signalling molecule, which also regulates iron homeostasis in response to an inflammatory situation. In this study, the administration of IL-6 induced Ltf gene expression in the liver of WT mice, in murine hepatocytes and in hepa 1-6 cells. Ltf-gene-expression was upregulated also in the liver of TO- and LPS-treated IL-6 knockout (KO) mice. The increase in serum Ltf after LPS injection was greater than after TO-injection both in WT and IL-6-KO mice. To evaluate the contribution of other acute phase cytokines in the regulation of Ltf-gene-expression in the liver, both in vitro and in vivo studies with IL-1β, TNF-α, or IFN-γ were performed. The results demonstrate that TNF-α and IFN-γ also upregulated Ltf-gene-expression, while IL-1β has no role in the regulation of Ltf-gene-expression.

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.