Nonparenchymal Cell Fraction Research Articles

Alpha-syntrophin deficiency protects against non-alcoholic steatohepatitis associated increase of macrophages, CD8+ T-cells and galectin-3 in the liver

Non-alcoholic steatohepatitis (NASH) is characterized by immune cell infiltration. Loss of the scaffold protein alpha-syntrophin (SNTA) protected mice from hepatic inflammation in the methionine-choline-deficient (MCD) diet model. Here, we determined increased numbers of macrophages and CD8+ T-cells in MCD diet induced NASH liver of wild type mice. In the mutant animals these NASH associated changes in immune cell composition were less pronounced. Further, there were more γδ T-cells in the NASH liver of the null mice. Galectin-3 protein in the hepatic non-parenchymal cell fraction was strongly induced in MCD diet fed wild type but not mutant mice. Antioxidant enzymes declined in NASH liver with no differences between the genotypes. To identify the target cells responsive to SNTA loss in-vitro experiments were performed. In the human hepatic stellate cell line LX-2, SNTA did not regulate pro-fibrotic or antioxidant proteins like alpha-smooth muscle actin or catalase. Soluble galectin-3 was, however, reduced upon SNTA knock-down and increased upon SNTA overexpression. SNTA deficiency neither affected cell proliferation nor cell death of LX-2 cells. In the macrophage cell line RAW264.7 low SNTA indeed caused higher galectin-3 production whereas release of TNF and cell viability were normal. Moreover, SNTA had no effect on hepatocyte chemerin and CCL2 expression. Overall, SNTA loss improved NASH without causing major effects in macrophage, hepatocyte and hepatic stellate cell lines. SNTA null mice fed the MCD diet had less body weight loss and this seems to contribute to improved liver health of the mutant mice.

The importance of inter-individual Kupffer cell variability in the governance of hepatic toxicity in a 3D primary human liver microtissue model

The potential for nanomaterial (NM) translocation to secondary organs is a realistic prospect, with the liver one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting and/or troublesome (i.e. short life-span reduced metabolic activity, lacking important cell populations, high inter-individual variability, etc.). Building on previous work, this study utilises a 3D human liver microtissue (MT) model (MT composed of mono-culture of hepatocytes or two different co-culture MT systems with non-parenchymal cell (NPC) fraction sourced from different donors) to investigate the importance of inter-donor variability of the non-parenchymal cell population in the overall governance of toxicological response following exposure to a panel of NMs. To the best of our knowledge, this is the first study of its kind to investigate inter-donor variability in hepatic NPC population. The data showed that the Kupffer cells were crucial in dictating the overall hepatic toxicity following exposure to the materials. Furthermore, a statistically significant difference was noted between the two co-culture MT models. However, the trend for particle-induced biological responses was similar between the co-cultures (cytotoxicity, cytokine production and caspase activity). Therefore, despite the recognition of some discrepancies in the absolute values between the co-culture models, the fact that the trends and patterns of biological responses were comparable between the multi-cellular models we propose the 3D liver MT to be a valuable tool in particle toxicology.

Effect of cryopreservation on the appearance and liver function of hepatocyte-like cells in cultures of cirrhotic liver of biliary atresia.

Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of biliary atresia (BA) may contain stem/progenitor cells, and clusters of hepatocyte-like cells appear via hepatocyte growth factor/c-Met signaling in primary cultures of NPCs. BA is a rare and serious liver disease, and procurement of BA cells is difficult. Therefore, cryopreservation of BA liver cells is an unavoidable challenge. In this study, we examined the appearance and liver function of hepatocyte-like cells in cultures of BA liver-derived NPC fractions after cryopreservation for 1 or 6mo using a chemically defined cryopreservation solution, STEM-CELLBANKER. Although a decrease in cell viability was observed in recovered cells after 1mo of cryopreservation, clusters of hepatocyte-like cells appeared in the culture of cells that had been cryopreserved for 1 or 6mo, similar to non-cryopreserved cells. In addition, these hepatocyte-like cells expressed hepatocyte-related mRNAs and demonstrated albumin production and glycogen storage. The present results suggest that hepatic stem/progenitor cells in NPC fractions may be efficiently cryopreserved, as demonstrated by the appearance of hepatocyte-like cells that show various hepatic functions even after cryopreservation. This study may lead to future BA cell therapy using the patient's own cells.

Isolation, characterization and cold storage of cells isolated from diseased explanted livers.

Livers discarded after standard organ retrieval are commonly used as a cell source for hepatocyte transplantation. Due to the scarcity of organ donors, this leads to a shortage of suitable cells for transplantation. Here, the isolation of liver cells from diseased livers removed during liver transplantation is studied and compared to the isolation of cells from liver specimens obtained during partial liver resection. Hepatocytes from 20 diseased explanted livers (Ex-group) were isolated, cultured and stored at 4°C for up to 48 hours, and compared to hepatocytes isolated from the normal liver tissue of 14 liver lobe resections (Rx-group). The nonparenchymal cell fraction (NPC) was analyzed by flow cytometry to identify potential liver progenitor cells, and OptiPrep™ (Sigma-Aldrich) density gradient centrifugation was used to enrich the progenitor cells for immediate transplantation. There were no differences in viability, cell integrity and metabolic activity in cell culture and survival after cold storage when comparing the hepatocytes from the Rx-group and the Ex-group. In some cases, the latter group showed tendencies of increased resistance to isolation and storage procedures. The NPC of the Ex-group livers contained considerably more EpCAM+ and significantly more CD90+ cells than the Rx-group. Progenitor cell enrichment was not sufficient for clinical application. Hepatocytes isolated from diseased explanted livers showed the essential characteristics of being adequate for cell transplantation. Increased numbers of liver progenitor cells can be isolated from diseased explanted livers. These results support the feasibility of using diseased explanted livers as a cell source for liver cell transplantation.

Analysis of soluble factors in conditioned media derived from primary cultures of cirrhotic liver of biliary atresia.

Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and β chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-β1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.

Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

Isolation and Culture of Single Cell Types from Rat Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

Origin of myofibroblasts in the fibrotic liver in mice.

Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP(+) cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl4 liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.

Collagenolytic Potential of Rat Liver Myofibroblasts

Rat liver myofibroblasts (MFB) were isolated by repeated passaging of nonparenchymal liver cell fraction. They were cultured on polystyrene Petri dishes, on fibrin or on type I collagen gels for 5 days. Quantitative RT-PCR, Western blotting, zymography and immunocytochemistry were used to study differences in cell morphology and protein expression. MFB were large and spread on plastic substrate, with prominent alpha-smooth muscle (alpha-SMA) fibres. They turned much smaller and elongated on collagen which was accompanied by the rearrangement of the cytoskeleton and a decrease in alpha-SMA and beta-actin content. Collagen gel induced the expression of a group of metalloproteinases (MMP-2, -3, -9, -13), on mRNA and protein level which resulted in the degradation of the gel. This response was accompanied by changes in the mRNA expression of cytokines of TGF-beta family, CTGF and interleukin-6, as well as of osteopontin and thrombospondin-2 that are involved in metalloproteinases (MMPs) regulation. The expression of MMPs substrates, collagen types I, IV and XII did not change or decreased. The effects of fibrin gels on MFB were milder than those of collagen. MFB assumed to deposit collagen and other ECM components in fibrotic liver, besides hepatic stellate cells, also possess a great collagenolytic potential.

Ly6C + monocytes are extrahepatic precursors of hepatic stellate cells in the injured liver of mice

We previously reported that hepatic stellate cells (HpSCs) are of hematopoietic origin in liver injury. However, the immediate precursors of HpSCs remain unknown. This study was conducted to elucidate whether terminally differentiated blood cells can differentiate into HpSCs. We adoptively transferred a variety of cells isolated from enhanced green fluorescent protein (EGFP)-transgenic mice into carbon tetrachloride (CCl(4))-treated nontransgenic mice twice weekly for 2 weeks. We examined the presence of EGFP(+) HpSCs in the injured liver using immunofluorescence analysis. Monocytes, neutrophils, eosinophils, B cells, or T cells from EGFP mice were transferred into CCl(4)-treated mice. Thirty percent of EGFP(+) cells in the livers of mice given Ly6C(high)c-kit(-) monocytes were negative for CD45, but were positive for glial fibrillary acidic protein, desmin, CD146, ADAMTS13, and α-smooth muscle actin, well-known markers of HpSCs. EGFP(+)CD45(-) cells were predominantly positive for glial fibrillary acidic protein. Although 48% of EGFP(+) cells were positive for procollagen type I, half of them were CD45(-). In the livers of mice given neutrophils, eosinophils, B cells, or T cells, all of the EGFP(+) cells were CD45(+). The majority of EGFP(+) cells in the nonparenchymal cell fraction purified from the livers of mice given Ly6C(high)c-kit(-) monocytes contained lipid droplets and were positive for glial fibrillary acidic protein, desmin, ADAMTS13, and procollagen type I. When Ly6C(+) monocyte-depleted peripheral blood total nucleated cells were adoptively transferred into CCl(4)-treated mice, we found no EGFP(+)CD45(-) cells in the liver. These results suggest that Ly6C(+) monocytes can become HpSCs in the injured liver.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

Beneficial Paracrine Effects of Cannabinoid Receptor 2 on Liver Injury and Regeneration†

The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions. CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

In Vitro Differentiation of Liver Progenitor Cells Derived from Healthy Dog Livers

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.

Differential Effects of GdCl3‐ or MDP Treatment on Rat Liver Microcirculation and Gene Expression in the Hepatic Non‐Parenchymal Cell Fraction in LPS Shock

Dichloromethylenebisphosphonate (MDP) and gadolinium chloride (GdCl(3)) are substances frequently used for experimental depletion of Kupffer Cells (KC) in models of endotoxin shock. The aim was to determine whether depletion of KC through pretreatment with GdCl(3) or MDP alters the hepatic microcirculation during lipopolysaccharide (LPS)-induced shock in rats and to test if there are substance-specific differences. Rats received either MDP or GdCl(3) or saline prior to induction of LPS shock. Hepatic microcirculation was evaluated by intravital microscopy (sinusoidal diameter, sinusoidal bloodflow, leukocyte adhesion), and the gene expression in the hepatic non-parenchymal cell fraction was determined by RT-PCR. GdCl(3) pretreatment prevented sinusoidal narrowing but did not restore sinusoidal blood flow and did not normalize leukocyte-endothelial interaction time after LPS. In contrast, MDP pretreatment improved hepatic microcirculation consistently for all parameters measured compared to GdCl(3) pretreated animals. In the non-parenchymal cell fraction, eNOS gene expression was preserved and gene expression of TNF-alpha was blocked after MDP but not after GdCl(3) application prior to LPS shock. The results show that GdCl(3) and MDP cannot be used equivalently for experimental KC depletion in the condition of LPS-induced shock. These findings should be taken into consideration in studies that evaluate the role of Kupffer cells in models of endotoxin-induced shock.

Putative Human Liver Progenitor Cells in Explanted Liver

Background/Aims: Hepatocyte progenitors have frequently been cultured from rodents but reports from human liver are rare. Methods: Non-parenchymal cell fraction isolated from 19 explant livers (removed at orthotopic liver transplantation for acute or chronic liver disease) and histologically normal human liver was cultured. Results: Proliferating epithelioid colonies were identifiable after 2–3 weeks culture as a very rare event (<1 per million cells plated) expressing mRNAs and protein antigens of mixed hepatocytic/biliary phenotype. Colony survival could be prolonged by transduction of the catalytic sub-unit of telomerase. Hepatocyte growth factor, epidermal growth factor and oncostatin M did not further enhance hepatocytic differentiation. The expression of markers associated with hepatocyte precursor status was investigated by flow cytometry. Cells expressing the stem cell-associated markers CD133 and CD117 were identified at low frequency. The proportion of cells expressing the integrin CD49f was higher in diseased liver than in normal liver, but the proportion expressing the hepatocyte growth factor receptor c-met was lower. Successful enrichment of plated populations for progenitors was not achieved. Conclusion: Although there is clear histological evidence of hepatocyte precursors in human explant livers, predictable culture of such cells with differentiation toward mature hepatocyte phenotype remains elusive.

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

The ability of the bone marrow cells to differentiate into liver, pancreas, and other tissues led to the speculation that these cells might be the source of adult stem cells found in these organs. The present study analyzed whether the bone marrow cells are a source of hepatic oval cells involved in rat liver regeneration induced by 2-acetylaminofluorene (2-AAF) and 70% partial hepatectomy (PHx). Three groups of mutant F344 dipeptidyl peptidase IV-deficient (DPPIV(-)) rats were required for the study. Groups A and B received the mitotic inhibitor monocrotaline, followed by male F344 (DPPIV(+)) bone marrow transplantation. Next, group A received PHx only, while group B received the 2-AAF/PHx required for the oval cell activation. The last group C was used to analyze the effects of monocrotaline on transplanted bone marrow cells. These rats underwent transplantation with bone marrow cells and were then treated with monocrotaline. Subsequently, the animals were treated with 2-AAF/PHx. In group A, DPPIV(+) hepatocytes were found in the liver. Group B showed that approximately 20% of the oval cell population expressed both donor marker (DPPIV) and alpha-fetoprotein, and some differentiated into hepatocytes. In contrast, animals in group C failed to significantly induce oval cells with the donor DPPIV antigen. In addition, X/Y-chromosome analysis revealed that fusion was not contributing to differentiation of donor-derived oval cells. Our results suggest that under certain physiologic conditions, a portion of hepatic stem cells might arise from the bone marrow and can differentiate into hepatocytes.

Liver sinusoidal endothelial cells are insufficient to activate T cells.

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45(-)) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcgammaRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4(+) or CD8(+) TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.

Immunomodulatory Role of Kupffer Cell in Liver Allografts

Liver transplantation is widely used as a treatment for end-stage disease. Liver allografts seem privileged compared with other solid organ grafts for a number of reasons. Successful transplantation can be performed across major histocompatibility complex (MHC) disparities, and the is the only solid organ that, when transplanted, can protect other co-transplanted syngeneic organs from immunological tissue destruction [1]. A proportion of patients with stable function can have immunosuppression reduced or withdrawn completely [2,3]. The reasons for this liver effect are not understood. Recently, there has been interest in lymphocyte apoptosis that occurs in OLT animal models. Allograft tolerance can be readily induced in experimental animals, particularly rodents, and spontaneous tolerance of mismatched grafts occurs in some train combinations [4]. In a spontaneously tolerant murine model of transplantation, the development of tolerance was associated with a higher rate of apoptosis of cells in the portal inflammatory infiltrate [5]. Sharland et al. [6], in a rat model, also showed that tolerant animals had higher numbers of apoptotic cells; they demonstrated by double staining that these were T lymphocytes. Kupffer cells (KC), the resident macrophage population in the liver, are found within the sinusoidal lumen, adhering to the sinusoidal endothelial cells (LSEC). KC, which comprise one of major populations (20%) of the hepatic nonparenchymal cell fraction (NPC), can directly interact with passenger leukocytes and thus may play a role in immunomodulation and the induction of tolerance [7]. Following transplantation, donor KC not only migrate into the recipient lymph nodes, but also can be quickly replaced by recipient-derived monocytes [8,9]. Thus, KC are uniquely positioned for regulation of the T cell response in the liver. Our previous studies have shown that KC play an immunomodulatory role as manifested by inhibiting T lymphocyte proliferation in response to alloantigen stimulation. The purpose of this study was to quantify the effects of transplantation on KC immunomodulatory function, focusing specifically on Fas ligand (FasL) expression and their role in allo-reactive T cell apoptosis in chronic accepted and acutely rejected hepatic allografts.

Tolerogenic role of Kupffer cells in allergic reactions.

Drug-induced allergic reactions (DIARs), including allergic hepatitis, cutaneous reactions, and blood dyscrasias, are unpredictable and can be life threatening. Although current studies suggest that DIARs are caused by immunogenic drug-protein adducts, it remains unclear what factors determine the susceptibility to DIARs. We hypothesized that most individuals may be resistant to DIARs in part because they become immunologically tolerant to drug-protein adducts in the liver, an organ with tolerogenic properties. Because animal models of DIARs are elusive, we tested this hypothesis using a murine model of 2,4-dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity reaction that is mediated by immunogenic 2,4-dinitrophenylated (DNP)-protein adducts. Intravenous pretreatment of mice with DNP-BSA led to its accumulation in hepatic Kupffer cells (KC) and induced immunological tolerance to subsequent DNCB sensitization. Tolerance could be abrogated by prior depletion of KC or induced in naïve mice by transferring a T cell-depleted, KC-enriched fraction of liver nonparenchymal cells from mice tolerized 1 month earlier by DNP-BSA pretreatment. These findings implicate KC as a primary and sustained inducer of tolerance against DNP-protein adducts and suggest a similar role in modulating allergic reactions against drug-protein adducts. Perhaps genetic and/or environmental factors affecting the activities of these cells may play a role in determining individual susceptibility to DIARs.