Amniotic-fluid-derived mesenchymal stem cells overexpressing interleukin-1 receptor antagonist improve fulminant hepatic failure

Uncontrolled hepatic immunoactivation is regarded as the primary pathological mechanism of fulminant hepatic failure (FHF). The major acute-phase mediators associated with FHF, including IL-1β, IL-6, and TNF-α, impair the regeneration of liver cells and stem cell grafts. Amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) have the capacity, under specific conditions, to differentiate into hepatocytes. Interleukin-1-receptor antagonist (IL-1Ra) plays an anti-inflammatory and anti-apoptotic role in acute and chronic inflammation, and has been used in many experimental and clinical applications. In the present study, we implanted IL-1Ra-expressing AF-MSCs into injured liver via the portal vein, using D-galactosamine-induced FHF in a rat model. IL-1Ra expression, hepatic injury, liver regeneration, cytokines (IL-1β, IL-6), and animal survival were assessed after cell transplantation. Our results showed that AF-MSCs over-expressing IL-1Ra prevented liver failure and reduced mortality in rats with FHF. These animals also exhibited improved liver function and increased survival rates after injection with these cells. Using green fluorescent protein as a marker, we demonstrated that the engrafted cells and their progeny were incorporated into injured livers and produced albumin. This study suggests that AF-MSCs genetically modified to over-express IL-1Ra can be implanted into the injured liver to provide a novel therapeutic approach to the treatment of FHF.     

Interleukin-1 receptor antagonist modulates the early phase of liver regeneration after partial hepatectomy in mice

Background

Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.

Methods

We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.

Results

At 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.

Conclusion

IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury.

The cytokine IL-6 plays a significant role in liver regeneration in conjunction with additional growth factors (HGF, TNF-alpha, and TGF-alpha). Many IL-6 effects depend on a naturally occurring soluble IL-6 receptor (sIL-6R). Here, the chimeric protein hyper-IL-6, constructed from the human IL-6 protein fused to a truncated form of its receptor, was found to have superagonistic IL-6 properties, and as such, enhanced liver cell regeneration. Hyper-IL-6 reversed the state of hepatotoxicity and enhanced the survival rates of rats suffering from fulminant hepatic failure after D-galactosamine administration. The hyper-IL-6 protein has a significant potential for use in the treatment of severe human liver diseases.     

Production of cytokines by immature erythroid cells derived from human embryonic liver

It is well known that regulatory interactions between hematopoietic and lymphoid cells are mediated by different mediators. The cells of erythroid lineage are not an exception and have a regulatory effect on hemato- and immunopoiesis that can be mediated through the production of cytokines i.e. by soluble factors - a universal mechanism for cell regulation in hematopoietic and immune systems. It has been previously shown that erythroid progenitor cells from mice express mRNA of cytokines such as IL-1 alpha and beta, IL-4, IL-6, IFN-gamma, GM-CSF and TGF-beta. In this report we present the results of the production of the main immunoregulatory cytokines by erythroid cells derived from human embryonic liver. It was revealed that the cell population enriched with erythroid progenitors, isolated from human fetal liver, can produce IL-1 beta, IL-2, IL-4, IL-6. The levels of production of cytokines by immature erythroid progenitor cells is compared to the levels of corresponding cytokines produced by mitogen-stimulated peripheral blood mononuclear cells. The production of these cytokines changed quantitatively under the effect of erythropoietin, and are correlated with the expression of differentiation markers of erythroid cells such as AG-EB and Glycophorin A. The role of cytokine production by erythroid cells in hemato- and immunopoiesis and the mechanisms of self-regulation of proliferation and differentiation of erythroid progenitor cells is discussed.     

Induction of hepatocyte mitosis in intact adult rat by interleukin-1 alpha and interleukin-6

In the adult rat the liver is normally quiescent, but it proliferates rapidly in response to partial hepatectomy (PH). A hepatectomized rat is subjected to stress by the operation. We have examined the effects of acute phase cytokines. To investigate the mediation of hepatocyte growth, recombinant human interleukin-1 alpha (IL-1) and interleukin-6 (IL-6) were injected into male rats. Administration of IL-1 or IL-6 followed by NH4Cl and glucagon could induce hepatocyte mitosis 30 h after the first injection. This activity was lost when interleukins were exposed to 90 degrees C for 30 minutes. NH4Cl augmented the effects of IL-1 and IL-6. These results suggest that the IL-1 and IL-6 are important mediators of liver regeneration after PH. We present a hypothesis for the triggering mechanism of hepatocyte proliferation.     

The roles of signaling pathways in liver repair and regeneration

The liver has remarkable regeneration potency that restores liver mass and sustains body hemostasis. Liver regeneration through signaling pathways following resection or moderate damages are well studied. Various cell signaling, growth factors, cytokines, receptors, and cell types implicated in liver regeneration undergo controlled hypertrophy and proliferation. Some aspects of liver regeneration have been discovered and many investigations have been carried out to identify its mechanisms. However, for optimizing liver regeneration more should be understood about mechanisms that control the growth of hepatocytes and other liver cell types in adults. The current paper deals with the possible applicability of liver regeneration signaling pathways as a target for therapeutic approaches and preventing various liver damages. Furthermore, the latest findings of spectrum-specific signaling pathway mechanisms that underlie liver regeneration are briefly described.     

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

小鼠肝部分切除模型

小鼠肝部分切除模型是在经典的大鼠模型基础上发展起来的。随着显微外科技术的发展和小鼠腹部手术围手术期管理的完善,快速、可靠、重复性高的小鼠模型得以建立。因为成本较低,且存在大量为研究肝切除后肝再生的转基因小鼠,小鼠已经成为研究肝再生的重要动物模型。肝再生的调控相当复杂,且不是由单一因素控制的,因此需要多种类的转基因小鼠进行肝切除后肝再生研究来明确各因子在肝再生过程中的确切作用与机制。通过小鼠肝切除后肝再生的研究,目前已证实的参与调控肝再生的细胞因子和生长因子有:肝再生增强因子(ALR)、肿瘤坏死因子α(TNFα)、白介素6(IL-6)、白介素1(IL-1)、肝细胞生长因子(HGF)、去甲肾上腺素(NE)、卵泡抑素(FS)、转化生长因子α(TGF-α)、转化生长因子β-1(TGF-β-1)等。     

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.     

A Model of Liver Regeneration

The network of interactions underlying liver regeneration is robust and precise with liver resections resulting in controlled hyperplasia (cell proliferation) that terminates when the liver regains its lost mass. The interplay of cytokines and growth factors responsible for the inception and termination of this hyperplasia is not well understood. A model is developed for this network of interactions based on the known data of liver resections. This model reproduces the relevant published data on liver regeneration and provides geometric insights into the experimental observations. The predictions of this model are used to suggest two novel strategies for speeding up liver mass recovery and a strategy for enabling liver mass recovery in cases where a resection leaves <20% of the liver that would otherwise result in complete loss of liver mass.     

Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the alpha1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor beta-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.