Primary Rat Kupffer Cells Research Articles

Time-dependent degradation of carbon nanotubes correlates with decreased reactive oxygen species generation in macrophages.

Introduction and objective: With the increase in carbon nanotube-based products on the commercial market, public concern regarding the possible toxicity of these nanomaterials has attracted much attention. Although previous studies found no obvious toxicity related to carbon nanotubes (CNTs), their safety has not been established because long-term evaluation is still needed. In vitro assays are used to understand the toxicity of nanomaterials. However, the data published so far were generated in short-term assays in which cells are continuously exposed to CNTs. Therefore, the objective of this study is to quantitatively assess the relative long-term cytotoxicity and degradation of CNTs after uptake by macrophages.Methods: We used macrophage cell line of RAW 264.7 and primary rat Kupffer cells to investigate macrophage uptake of CNTs as well as their quantity changes up to a relatively late time point after uptake (7 days) by measuring optical absorbance in the near infrared region and Raman spectra of CNTs in the cell lysates. The time-dependent cytotoxicity was evaluated by measuring reactive oxygen species (ROS), glutathione, cell viability, and caspase 3/7 activity in 1–7 days.Results: CNTs were degraded by approximately 25–30% within first 4 days after uptake; however, and no additional degradation occurred for the remainder of the 7-day test period. Generation of ROS by macrophages decreased as CNT degradation occurred, returning to control levels by Day 7. In the meantime, the glutathione level gradually recovered over time. There were no changes in cell viability or caspase 3/7 activation during CNT degradation.Conclusion: These results confirm that degradation of CNTs by macrophages is associated with ROS generation. The data also suggest that CNT cytotoxicity decreases as they are degraded.

Kupffer Cells Survive Plasmodium berghei Sporozoite Exposure and Respond with a Rapid Cytokine Release.

The liver stage of the Plasmodium life cycle features sporozoite traversal of the liver sinusoidal barrier through Kupffer cells (KCs) followed by invasion of hepatocytes. Little is known about the interaction of Plasmodium sporozoites with KCs, the liver-resident macrophages. Previous reports suggest KCs do not mount a pro-inflammatory response and undergo cell death following this interaction. Our work explores this interaction using primary rat KCs (PRKCs) and Plasmodium berghei sporozoites. We analyzed PRKC culture supernatants for markers of an immunological response through cytokine arrays. Additionally, cell wounding and death were assessed by monitoring lactate dehydrogenase (LDH) levels in these supernatants and by live/dead cell imaging. We found that PRKCs mount an immunological response to P. berghei sporozoites by releasing a diverse set of both pro- and anti-inflammatory cytokines, including IFNγ, IL-12p70, Mip-3α, IL-2, RANTES, IL-1α, IL-4, IL-5, IL-13, EPO, VEGF, IL-7, and IL-17α. We also observed no difference in LDH level or live/dead staining upon sporozoite exposure, suggesting that the KCs are not deeply wounded or dying. Overall, our data suggest that sporozoites may be actively modulating the KC’s reaction to their presence and altering the way the innate immune system is triggered by KCs.

Phospholipid-Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability.

The success of nanoparticulate formulations in drug delivery depends on various aspects including their toxicity, internalization, and intracellular location. Vesicular assemblies consisting of phospholipids and amphiphilic block copolymers are an emerging platform, which combines the benefits from liposomes and polymersomes while overcoming their challenges. We report the synthesis of poly(cholesteryl methacrylate)- block-poly(2-(dimethylamino) ethyl methacrylate) (pCMA- b-pDMAEMA) block copolymers and their assembly with phospholipids into hybrid vesicles. Their geometry, their ζ-potential, and their ability to adsorb onto polymer-coated surfaces were assessed. Giant unilamellar vesicles were employed to confirm the presence of both the phospholipids and the block copolymer in the same membrane. Furthermore, the cytotoxicity of selected hybrid vesicles was determined in RAW 264.7 mouse macrophages, primary rat Kupffer cells, and human macrophages. The internalization and lysosomal escape ability of the hybrid vesicles were confirmed using RAW 264.7 mouse macrophages. Taken together, our findings illustrate that the reported hybrid vesicles are a promising complementary drug delivery platform for existing liposomes and polymersomes.

The cellular and proteomic response of primary and immortalized murine Kupffer cells following immune stimulation diverges from that of monocyte-derived macrophages.

Kupffer cells (KCs) are the first line of defense in the liver against pathogens, yet several microbes successfully target the liver, bypass immune surveillance, and effectively develop in this tissue. Our current, albeit poor, understanding of KC-pathogen interactions has been largely achieved through the study of primary cells, requiring isolation from large numbers of animals. To facilitate the study of KC biology, an immortalized rat KC line 1, RKC1, was developed. We performed a comparative global proteomic analysis of RKC1 and primary rat KCs (PRKC) to characterize their respective responses to lipopolysaccharide-mediated immune stimulation. We identified patent differences in the proteomic response profile of RKC1 and PRKC to lipopolysaccharide. We observed that PRKC upregulated more immune function pathways and exhibited marked changes in cellular morphology following stimulation. We consequently analyzed the cytoskeletal signaling pathways of these cells in light of the fact that macrophages are known to induce cytoskeletal changes in response to pathogens. Our findings suggest that KCs respond differently to inflammatory stimulus than do monocyte-derived macrophages, and such data may provide insight into how pathogens, such as the malaria parasite, may have evolved mechanisms of liver entry through KCs without detection.

Macrophage‐derived hedgehog ligands promotes fibrogenic and angiogenic responses in human schistosomiasis mansoni

Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.

Toll-like receptor-4 mediates obesity-induced non-alcoholic steatohepatitis through activation of X-box binding protein-1 in mice

BackgroundNon-alcoholic fatty liver disease is an obesity-related chronic liver disorder ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which may progress to liver fibrosis and cirrhosis.ObjectiveTto investigate the role of...

Carbon monoxide induces hypothermia tolerance in Kupffer cells and attenuates liver ischemia/reperfusion injury in rats

Ischemia/reperfusion (I/R) injury in liver grafts, which is initiated by cold preservation and is augmented by reperfusion, is a major problem that complicates graft quality, posttransplant patient care, and outcomes of liver transplantation (LT). Kupffer cells (KCs) play important roles in I/R injury; however, little is known about their changes during cold preservation. We examined whether a pretreatment with carbon monoxide (CO), a cytoprotective product of heme degradation, could influence KC activity during cold storage and protect liver grafts against LT-induced I/R injury. In vitro, primary rat KCs were stimulated for 24 hours under hypothermic conditions (4°C, 20% O(2)), with lipopolysaccharide, or under hypoxic conditions (37°C, 5% O(2)) with or without a CO pretreatment. When rat KCs were exposed to hypothermic conditions, they produced reactive oxygen species (ROS), but they did not produce tumor necrosis factor α (TNF-α) or nitric oxide. The preincubation of KCs with CO up-regulated heat shock protein 70 (HSP70) and inhibited ROS generation. When liver grafts from donor rats exposed to CO (250 ppm) for 24 hours were transplanted after 18 hours of cold preservation in University of Wisconsin solution, HSP70 expression increased in these grafts versus control grafts, and serum aspartate aminotransferase and alanine aminotransferase levels as well as necrotic areas and inflammatory infiltrates were significantly reduced after LT. CO-pretreated liver grafts showed less up-regulation of TNF-α and inducible nitric oxide synthase messenger RNA (mRNA) and reduced expression of proapoptotic B cell lymphoma 2-associated X protein mRNA, cleaved caspase-3, and poly(adenosine diphosphate ribose) polymerase. In conclusion, the pretreatment of donors with CO ameliorates LT-associated I/R injury with increased hepatic HSP70 expression, particularly in the KC population.

Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

Methyl palmitate: Inhibitor of phagocytosis in primary rat Kupffer cells

Kupffer cells are involved in phagocytosis and known to release biologically active mediators during early events of liver injury. Such functional properties of Kupffer cells can be modulated by methyl palmitate (MP). Therefore, efficacy of MP to modulate Kupffer cell function was evaluated in cultured primary Kupffer cells from rat liver. Phagocytic activity of Kupffer cells was measured by their capacity to phagocytize latex beads and the release of TNF-α, IL-10, IL-6, nitric oxide, and PGE2 was determined in cell culture medium after incubating the cells with various concentrations of MP for 24 h followed stimulation with lipopolysaccharide (LPS) for 6 h. To understand the mechanism of phagocytosis, we investigated the hydrolysis of MP, and determine ATP levels and activity of NF-κB in MP-inhibited Kupffer cells. A significant decrease was observed in phagocytosis. Phagocytosis evaluated at 0.5 mM MP was found to be time-dependent with a maximum decrease of 49% at 6 h. Exposure of Kupffer cells to MP followed by LPS stimulation showed a dose-dependent decrease in phagocytosis and reduced the release of TNF-α, IL-10, nitric oxide, and PGE2 but not of IL-6 levels in the supernatant as compared to the control. While ATP levels were unchanged, the nuclear factor NF-κB (p65) activity was inhibited in Kupffer cells treated with MP after LPS stimulation (35.6 RLU versus 49.6 RLU in control). Hydrolysis of MP was found to be time-dependent; maximum concentration of MP and palmitic acid (hydrolysis products) in the cell being at 3 and 6 h, respectively. In general, MP appears to reduce phagocytosis and levels of TNF-α, IL-10, nitric oxide, and PGE2 without affecting ATP levels and is probably mediated by NF-κB. This in vitro model is useful for detailed mechanistic studies of inhibition of phagocytosis by MP and other fatty acid esters.

4-hydroxynonenal decreases interleukin-6 expression and protein production in primary rat Kupffer cells by inhibiting nuclear factor-kappaB activation.

Kupffer cells have been documented to play an important role in the early events of liver injury and regeneration by releasing biologically active mediators such as interleukin-6 (IL-6). 4-Hydroxy-trans-2-nonenal (4-HNE), a major end product of lipid peroxidation, has multiple cytotoxic effects and is implicated in chemical-induced liver injury. Consequently, the purpose of this study was to evaluate the ability of 4-HNE to modulate IL-6 production in isolated primary rat Kupffer cells. 4-HNE (0.1-10 microM) reduced both lipopolysaccharide (LPS)-induced IL-6 protein production and mRNA levels. The role of nuclear factor-kappaB (NF-kappaB) in IL-6 induction was elucidated using Kupffer cells transduced in vitro with a recombinant adenovirus containing a IkappaBalpha super-repressor resistant to phosphorylation and degradation (Ad5IkappaB). Using this system, LPS-induced IL-6 protein production was inhibited by 65% in Ad5IkappaB-infected cells. The treatment of Kupffer cells for 1 h with 4-HNE followed by stimulation for 1 h with LPS (500 ng/ml) resulted in a concentration-dependent decrease in NF-kappaB activation. Similarly, decreased NF-kappaB activity in these cells paralleled a reduction in IkappaBalpha mRNA levels. Furthermore, upon LPS stimulation, 4-HNE stabilized IkappaBalpha, which corresponded to a decrease in phosphorylated IkappaBalpha. At lower 4-HNE concentrations (0-5 microM), interactions between p65 and IkappaBalpha proteins were maintained as detected by immunoprecipitation-immunoblot analyses. In conclusion, these data suggest that 4-HNE inhibits IL-6 production in rat Kupffer cells by preventing activation of the NF-kappaB pathway and suppressing IkappaBalpha phosphorylation. These results have functional implications in that 4-HNE may interfere with the ability of Kupffer cells to produce cytokines proposed to play an important role in liver regeneration.

Lung and liver injury following hepatic ischemia/reperfusion in the rat is increased by exogenous lipopolysaccharide which also increases hepatic TNF production in vivo and in vitro.

Hepatic ischemia/reperfusion (I/R) results in tumor necrosis factor (TNF) release. Kupffer cells (KC) are one source of this TNF. This study investigates the effects of hepatic I/R combined with lipopolysaccharide (LPS) on the lung and liver injury that follow hepatic I/R and on hepatic release of TNF, epithelial neutrophil activating protein (ENA-78), and macrophage inflammatory protein-2 (MIP-2). The effects of these experimental conditions on TNF production by primary rat KC in vitro were also investigated. Rats were subjected to hepatic I/R alone, hepatic I/R + LPS, sham laparotomy alone, or sham laparotomy + LPS and pulmonary MPO, pulmonary microvascular permeability, hepatic neutrophil influx, hepatic injury, and hepatic TNF, ENA-78, and MIP-2 production were measured. These experiments demonstrated that hepatic I/R in conjunction with LPS results in a more severe lung and liver injury and increased hepatic TNF, ENA-78, and MIP-2 release. The effects of these experimental conditions on rat KC TNF production demonstrated that hepatic I/R + LPS results in a more significant release of TNF as compared to LPS alone or I/R alone. Hepatic I/R plus LPS results in a more severe lung and liver injury and is likely secondary to a more significant and prolonged release of TNF by KC. This may provide a mechanism for development of multiple organ system failure in some patients undergoing hepatic resection, hepatic transplantation, complex vascular operations, or in the setting of hypovolemic shock. Portal endotoxemia related to mesenteric venous congestion or other systemic insults may have a significant impact on post-operative complications and recovery in the setting of a local or global hepatic I/R injury.

Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes

Nonparenchymal cells, particularly Kupffer cells, might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. This intercellular communication via the exchange of soluble factors was investigated in primary rat Kupffer cells and hepatocytes. Freshly isolated rat Kupffer cells were seeded onto cell culture inserts and cocultured with 5 day old serum-free rat hepatocyte monolayer cultures at a ratio of 1:1 for 2 days. Hepatocyte cultures, Kupffer cell cultures or cocultures were treated with 0.1 ng/ml–10 μg/ml lipopolysaccharide (LPS). Within this concentration range, no significant toxicity was observed in either cell type. In LPS-exposed cocultures, tumor necrosis factor α (TNFα) levels rose up to 5 ng/ml within 5 h; nitric oxide (NO) levels increased up to 70 μM within 48 h of treatment, both in a dose-dependent fashion. The release of negative (albumin) and positive (α1-acid-glycoprotein) acute phase proteins from the hepatocytes was strongly down- and up-regulated, respectively. The simultaneous treatment of the cocultures with phenobarbital and LPS (10 ng/ml) or 3-methylcholanthrene and LPS (10 ng/ml) resulted in a strong down-regulation (85%) of the phenobarbital-induced cytochrome P450 (CYP) isoform CYP2B1 in the hepatocytes whereas the 3-methylcholanthrene-induced isoform CYP1A1 was only weakly affected (15%). This specific down-regulation of CYP2B1 was mediated exclusively by TNFα, released from the Kupffer cells. It was not linked with NO release from or inducible NO synthase activity in the hepatocytes. The TNFα release was not affected by the two xenobiotics. Acetaminophen tested in these cocultures showed no direct interaction with the Kupffer cells. The use of liver cell cocultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

Lipopolysaccharide (LPS) processing by Kupffer cells releases a modified LPS with increased hepatocyte binding and decreased tumor necrosis factor-alpha stimulatory capacity.

Normal physiological clearance of gut-derived endotoxin lipopolysaccharide [LPS] has been described previously; initially, there is uptake by Kupffer cells (KC), then release of modified LPS, followed by hepatocyte uptake. Previous work in our laboratories indicated that LPS is structurally modified with loss of carbohydrate prior to its release by KC. In this study, we functionally characterize KC modified LPS. KC-modified 125I-LPS was prepared from primary rat KC. Escherichia coli 0127:B8 native 125I-LPS or KC-modified 125I-LPS (40 ng) was incubated for 1 hr with 1 x 10E6 primary hepatocytes. The binding of KC-modified LPS was 4.33-fold higher than native LPS (P = 0.0024). Binding analysis studies were conducted to determine the region of KC-modified LPS responsible for enhanced hepatocyte binding. KC-modified Salmonella minnesota LPS was competed with 100-fold excess native or mutant (Ra, Rc, Rd, or Re) strains of LPS or Lipid A with no decrease to hepatocyte binding. S. minnesota-native 125I-LPS was compared with KC-modified 125I-LPS in a study to assess induction of tumor necrosis factor (TNF)-gamma by rat peritoneal macrophages. Native or KC-modified 125I-LPS (100 ng) was presented to 1 x 10E7 peritoneal macrophages for 6 hr. TNF-alpha was measured in supernatants using the WEHI-164 cytotoxicity assay. Native LPS induced 5.7-fold higher TNF-alpha levels than KC-modified LPS (P < 0.0001). The above data suggest that structural alterations in KC-modified LPS are accompanied by functional alterations resulting in enhanced hepatocyte binding and decreased TNF-alpha release. The latter result implies that an early step in LPS detoxification occurs in the KC in which LPS is modified to prevent elicitation of biologically active cytokines.