Kyrylo Tron

Upregulation of heme oxygenase-1 gene by turpentine oil-induced localized inflammation: involvement of interleukin-6.

Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4–6 h after TO administration. HO-1 protein levels in the liver were elevated starting from 6–12 h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.

Cytokine-induced neutrophil chemoattractant-1 is released by the noninjured liver in a rat acute-phase model

The source of serum cytokine-induced neutrophil chemoattractant (CINC-1) and consequences of its presence in the tissue of synthesis have not been clearly elucidated under acute-phase situation. To pursue this question, turpentine oil (TO) was intramuscularly injected into rats, and RNA and local protein levels of acute-phase cytokines and of CINC-1 were studied in the TO injected gluteal muscle, as well as in noninjured muscle, in the liver, kidney, lung and spleen. The serum levels of acute-phase mediators and of CINC-1 were measured together with total leukocyte subpopulations. Recruitment of inflammatory cells in muscle and in the other organs was investigated by quantitative immunohistochemical methods. The effect of acute-phase mediators, including interferon gamma (IFN-γ) on the synthesis of CINC-1 in cultured hepatocytes was also investigated at the RNA and protein level. We found that the sera of the TO-treated rats contained elevated levels of IL-6, IL-1β and CINC-1. Increased serum levels of IFN-γ were also observed not only in the injured muscle but also and to a higher extent in the liver. However, while neutrophils and mononuclear phagocytes were found in the injured muscle, no inflammatory cells were detected at the non-‘inflamed’ site, namely, the liver or in the other organs. In vitro, treatment of cultured hepatocytes with IL-1β led to elevated CINC-1 gene expression. This was true to a lesser extent upon IL-6 and tumor necrosis factor (TNF-α) exposure. Interestingly, IFN-γ did not effect CINC-1 gene expression. These results indicate that CINC-1 behaves as an acute-phase protein and its expression is inducible in hepatocytes. However, CINC-1-production in the liver does not lead to recruitment of inflammatory cells into the organ.

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.

Expression and regulation of the insulin-like growth factor axis components in rat liver myofibroblasts

Apart from hepatic stellate cells (HSC), liver myofibroblasts (MF) represent a second mesenchymal cell population involved in hepatic fibrogenesis. The IGF system including the insulin‐like growth factors I and II (IGF‐I, ‐II), their receptors (IGF‐I receptor, IGF‐IR; IGF‐II/mannose 6‐phosphate receptor, IGF‐II/M6‐PR), and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF. Methods: Cultures of MF from passages 1 to 4 (P1–4) were studied. IGFBP secretion was analyzed by [125I]‐IGF‐I ligand and immunoblotting. IGF‐I, IGF‐IR, IGF‐II/M6‐PR, and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Results: MF from P1 to 4 constitutively expressed mRNA transcripts specific for IGF‐I, IGF‐IR, and IGF‐II/M6‐PR. In MF, biosynthesis of IGFBP‐3 and ‐2 was observed that was stimulated by IGF‐I, insulin, and transforming growth factor β (TGF‐β), whereas platelet‐derived growth factor (PDGF‐BB) revealed inhibitory effects. IGF‐I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP‐2 or ‐3 with IGF‐I diminished the mitogenic effect of IGF‐I on MF whereas preincubation of MF with IGFBP‐2 or ‐3 further potentiated the IGF‐I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of fibrogenesis during acute and chronic liver injury.

Decrease of PECAM-1-gene-expression induced by proinflammatory cytokines IFN-γ and IFN-α is reversed by TGF-β in sinusoidal endothelial cells and hepatic mononuclear phagocytes

Background and aimThe mechanisms of transmigration of inflammatory cells through the sinusoids are still poorly understood. This study aims to identify in vitro conditions (cytokine treatment) which may allow a better understanding of the changes in PECAM (platelet endothelial cell adhesion molecule)-1-gene-expression observed in vivo.Methods and resultsIn this study we show by immunohistochemistry, that there is an accumulation of ICAM-1 (intercellular cell adhesion molecule-1) and ED1 positive cells in necrotic areas of livers of CCl4-treated rats, whereas there are few PECAM-1 positive cells observable. After the administration of CCl4, we could detect an early rise of levels of IFN-γ followed by an enhanced TGF-β protein level. As shown by Northern blot analysis and surface protein expression analysed by flow cytometry, IFN-γ-treatment decreased PECAM-1-gene-expression in isolated SECs (sinusoidal endothelial cells) and mononuclear phagocytes (MNPs) in parallel with an increase in ICAM-1-gene-expression in a dose and time dependent manner. In contrast, TGF-β-treatment increased PECAM-1-expression. Additional administration of IFN-γ to CCl4-treated rats and observations in IFN-γ-/- mice confirmed the effect of IFN-γ on PECAM-1 and ICAM-1-expression observed in vitro and increased the number of ED1-expressing cells 12 h after administration of the toxin.ConclusionThe early decrease of PECAM-1-expression and the parallel increase of ICAM-1-expression following CCl4-treatment is induced by elevated levels of IFN-γ in livers and may facilitate adhesion and transmigration of inflammatory cells. The up-regulation of PECAM-1-expression in SECs and MNPs after TGF-β-treatment suggests the involvement of PECAM-1 during the recovery after liver damage.

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”.

Temporal and spatial expression of IGF-I and IGFBP-1 during acute-phase response induced by localized inflammation in rats

OBJECTIVE The acute-phase response (APR), a cytokine-induced defense reaction of the body that enhances the innate immunity mechanisms directed to eliminate the noxious agent and restrict the area of damage, is accompanied by numerous alterations of the IGF axis. The liver is a central organ of both the IGF system and the APR because it releases most of IGF-I and IGFBP-1 in the circulation and is the main target organ for acute-phase-cytokines such as IL-6. METHODS In the current work the expression of IGF-I and IGFBP-1 was studied in the liver and extrahepatic tissues in a rat model of localized inflammation induced by intramuscular injection of turpentine oil (TO). The mRNA expression of IGF-I and IGFBP-1 was determined by Northern blot analysis and quantitative RT-PCR. Circulating levels of IGF-I and IGFBP-1 were evaluated by radioimmunoassay and [(125)I]-IGF-I ligand blotting, respectively. RESULTS Administration of TO to the rats led to a significant reduction of IGF-I gene expression in the liver and spleen. These changes were accompanied by a reduction of serum IGF-I concentrations to approximately 50% of levels observed in control rats. In contrast to IGF-I, IGFBP-1 mRNA expression was rapidly elevated in the livers of TO-treated rats. IGFBP-1 transcripts were already detectable at 30 min after TO injection and reached their maximal levels by 6h. IGFBP-1 gene expression was also increased in the kidneys. This elevation, however, was delayed and less prominent than in the liver. CONCLUSIONS Our data demonstrate that localized inflammation induced by intramuscular TO injection is accompanied not only by decreased IGF-I but also by increased IGFBP-1 gene expression explaining at least in part the catabolic changes of metabolism observed during the acute-phase response.