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Bård Smedsrød

Publications and source records attributed to Bård Smedsrød.

16 recordsLinked to original sources
PubMed2006

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells.

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (Øynebråten et al.(2000) J. Leukocyte Biol. 67; 183-188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [3H] in the acetyl groups of N-acetyl galactosamine residues, [14C] in the pyranose rings, or [35S] in the sulfate groups were identified as[3H]-acetate, [14C]-lactate and [35S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with 125I in the tyrosine residues, or biosynthetically with [35S] or [3H] in the sulfate or acetyl groups, respectively, showed that 125I appeared more rapidly in the medium than [35S]-sulfate and [3H]-acetate. Judging from the speed of appearance of free 125I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains. Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.

Acetates↗
PubMed2005

Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides.

BACKGROUND/AIMS: Bacterial DNA and synthetic oligonucleotides containing unmethylated motifs have become the focus of many studies due to their ability to activate cells of the innate immune system through interaction with Toll-like receptor 9 (TLR9). This study was undertaken to investigate if and how CpG-oligonucleotides (CpGs) activate liver sinusoidal endothelial cells (LSECs), known to be the main site of clearance of DNA-oligonucleotides from the circulation. METHODS: Expression of TLR9 was analyzed by RT-PCR and immunohistochemistry. Production of IL-1beta and IL-6 was measured by ELISA. RESULTS: Here we show for the first time that mouse LSECs express TLR9 mRNA and protein. Moreover, our findings suggest that CpGs are first taken up by LSECs by scavenger receptor(s)-mediated endocytosis, and then join TLR9 in the lysosomal compartments. Furthermore, we found that uptake of CpGs in LSECs results in the activation of transcription factor NF-kappaB and secretion of IL-1beta and IL-6. CONCLUSIONS: The presence of functional TLR9 in LSECs emphasizes the importance of these cells in the innate defense mechanisms of the liver.

Animals↗
PubMed2005

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding.

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.

Amino Acid Sequence↗
PubMed2005

Long-term preservation of high endocytic activity in primary cultures of pig liver sinusoidal endothelial cells.

Together with Kupffer cells, liver sinusoidal endothelial cells (LSECs) constitute the most powerful scavenger system in the body. However, studies on LSEC function are hampered by the fact that the cells lose their scavenger ability and start deteriorating after a few days in culture. The purpose of the present study was to improve the conditions of cultivation to prolong the survival of pig LSECs in vitro. We used the high capacity receptor-mediated endocytosis of soluble waste molecules as a marker for functionally intact cells in the cultures. Compared with two commercially-, and two other media specifically designed for use with either SECs or hepatocytes from rat, our newly developed serum-free medium, DM 110/SS, devoid of any components of animal origin, was superior in maintaining the endocytic activity. Of six growth factors studied for their effect on endocytosis, basic fibroblast, and recombinant epidermal, but not vascular endothelial growth factor, were found to be most beneficial. After 8 days in DM 110/SS, LSECs maintained endocytosis via the scavenger receptor, mannose receptor, collagen alpha-chain receptor and the Fc-gamma receptor. All endocytosed ligands, except for aggregated IgG were degraded in 8-day-old cultures. Using the new medium, the cells endocytosed ligands for up to 20 days, and survived for at least an additional 10 days, albeit without the high endocytic activity typical of intact LSECs. Importantly, DNA synthesis in prolonged cultures of LSECs was observed only when maintained in DM 110/SS medium. In conclusion, we describe a protocol for the maintenance of LSECs in culture for the longest period yet reported.

Animals↗
PubMed2004

Scavenger properties of cultivated pig liver endothelial cells.

BACKGROUND: The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC. RESULTS: Endocytosis of five 125I-labelled molecules: collagen alpha-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of alpha-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells. CONCLUSION: Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.

Journal Article↗
PubMed2003

Liver sinusoidal endothelial cells represents an important blood clearance system in pigs.

BACKGROUND: Numerous studies in rats and a few other mammalian species, including man, have shown that the sinusoidal cells constitute an important part of liver function. In the pig, however, which is frequently used in studies on liver transplantation and liver failure models, our knowledge about the function of hepatic sinusoidal cells is scarce. We have explored the scavenger function of pig liver sinusoidal endothelial cells (LSEC), a cell type that in other mammals performs vital elimination of an array of waste macromolecules from the circulation. RESULTS: 125I-macromolecules known to be cleared in the rat via the scavenger and mannose receptors were rapidly removed from the pig circulation, 50% of the injected dose being removed within the first 2-5 min following injection. Fluorescently labeled microbeads (2 &mgr;m in diameter) used to probe phagocytosis accumulated in Kupffer cells only, whereas fluorescently labeled soluble macromolecular ligands for the mannose and scavenger receptors were sequestered only by LSEC. Desmin-positive stellate cells accumulated no probes. Isolation of liver cells using collagenase perfusion through the portal vein, followed by various centrifugation protocols to separate the different liver cell populations yielded 280 x 107 (range 50-890 x 107) sinusoidal cells per liver (weight of liver 237.1 g (sd 43.6)). Use of specific anti-Kupffer cell- and anti-desmin antibodies, combined with endocytosis of fluorescently labeled macromolecular soluble ligands indicated that the LSEC fraction contained 62 x 107 (sd 12 x 107) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors. CONCLUSIONS: We show here for the first time that pig LSEC, similar to what has been found earlier in rat LSEC, represent an effective scavenger system for removal of macromolecular waste products from the circulation.

Journal Article↗
PubMed2003

Structure and liver cell expression pattern of the HFE gene in the rat.

BACKGROUND/AIMS: Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver. METHODS: Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells). RESULTS: The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes. CONCLUSIONS: The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism.

Animals↗
PubMed2003

Lipoteichoic acid is a potent inducer of cytokine production in rat and human Kupffer cells in vitro.

BACKGROUND: Kupffer cells have been proposed to be a major cellular origin of pro-inflammatory mediators in sepsis. However, the cytokine response of Kupffer cells to gram-positive bacteria and their endotoxins peptidoglycan (PepG) and lipoteichoic acid (LTA) has never previously been studied. MATERIALS AND METHODS: Primary cultures of rat and human Kupffer cells were exposed to live Staphylococcus aureus (S. aureus) (4.0 x 10(1) to 4.0 x 10(7) CFU/mL culture medium), as well as highly purified PepG and LTA (0-100 microg/mL). Lipopolysaccharide (LPS) at 1 microg/mL was used for control. In parallel experiments, whole blood obtained from the same rats was stimulated in a similar manner. Accumulation of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma or culture supernatants were assessed by enzyme immuno assays. TNF-alpha and IL-6 mRNA were analyzed by real-time RT-PCR. RESULTS: PepG and LTA, as well as live S. aureus, induced the production of TNF-alpha and IL-6 in Kupffer cells from both species in a time- and dose-dependent manner. Whereas PepG was a more potent inducer of TNF-alpha and IL-6 in whole blood, the opposite seemed to be the case in Kupffer cells. In fact, a 100-fold lower concentration of LTA (1 microg/mL) than of PepG (100 microg/mL) was sufficient to induce a substantial production of both TNF-alpha and IL-6 in the Kupffer cells. TNF-alpha and IL-6 mRNA were induced correspondingly. CONCLUSION: Our results support the contention that gram-positive bacteria may activate cytokine production in Kupffer cells during bacteremia and suggest that LTA is important in this interaction.

Animals↗
PubMed2003

Shooting HARE.

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Animals↗
PubMed2002

Scavenger endothelial cells of vertebrates: a nonperipheral leukocyte system for high-capacity elimination of waste macromolecules.

Studies over the last two decades have shown that mammalian nonmacrophagic liver endothelial cells clear the blood from numerous physiological and foreign waste macromolecules, such as polysaccharides and proteins released during extracellular matrix turnover, intracellular macromolecules, modified serum proteins, and bacterial and fungal proteins [Smedsrød, B., Pertoft, H., Gustafson, S. & Laurent, T. C. (1990) Biochem. J. 266, 313-327]. These macromolecules are released daily in gram-amounts in a normal human body and are effectively taken up and degraded by the liver endothelial cells. Recent studies show that bony fishes harbor a similar system of specialized nonmacrophagic scavenger endothelial cells in either kidney [Smedsrød, B., Gjøen, T., Sveinbjørnsson, B. & Berg, T. (1993) J. Fish Biol. 42, 279-291] or heart [Sørensen, K. K., Melkko, J. & Smedsrød, B. (1998) J. Exp. Biol. 201, 1707-1718], but not in liver. Using specific and extremely effective endocytosis, these fish scavenger endothelial cells function as their mammalian counterpart to eliminate soluble waste macromolecules from the circulation. We show here that species from all seven vertebrate classes carry a population of nonmacrophagic scavenger endothelial cells that efficiently eliminate an array of circulating waste macromolecules. Thus representing an important part of the vertebrate innate immune system, these scavenger endothelial cells display the following distribution in the different vertebrate classes: Gills in Agnatha and Chondrichtyes; heart or kidney in Osteichtyes; and liver in Amphibia, Reptilia, Aves, and Mammalia.

Animals↗
PubMed2002

Serum is a rich source of ligands for the scavenger receptor of hepatic sinusoidal endothelial cells.

The present study was prompted by findings in our laboratory showing that serum effectively inhibits scavenger receptor (SR)-mediated endocytosis in hepatic sinusoidal endothelial cells (SEC). Experiments with SEC in vitro showed that the presence of 20% human serum inhibited endocytosis of SR ligands, 125I-formaldehyde treated bovine serum albumin (FSA) and 125I-nidogen, by 30 and 50%, respectively, whereas pre-heated foetal bovine serum (10%) inhibited endocytosis of 125I-FSA by as much as 56%. Human, bovine and rat serum had similar inhibitory effect on endocytosis in SEC. Fractionation of foetal bovine and human serum on anion exchange chromatography demonstrated that the inhibitory principle co-purified with macromolecules of high negative charge. The serum fraction that most effectively inhibited SR-mediated endocytosis of 125I-FSA did not affect mannose receptor-mediated endocytosis of 125I-mannan to the same extent. Trap-labelled negatively charged serum fraction administered intravenously to rats was eliminated almost exclusively by liver, with a blood decay of 50% over the first 3 min after injection. Isolation of liver cells showed that the populations of Kupffer cells and SEC contained 39 and 61% of liver radioactivity 30 min after injection of trap-labelled negatively charged fractions prepared from pre-heated ('complement inactivated') foetal bovine sera. These findings suggest that the process of serum formation from native blood generates appreciable amounts of macromolecules that compete specifically with the SR for endocytosis in SEC. The inhibitory power of pre-heated serum is particularly great. For this reason pre-heated serum should be used with caution in studies of SR in SEC.

Animals↗
PubMed2002

The physiological scavenger receptor function of hepatic sinusoidal endothelial and Kupffer cells is independent of scavenger receptor class A type I and II.

This study was undertaken to determine the role of scavenger receptor class A type I and II (SR-Al/II) in the physiological scavenger function of hepatic sinusoidal endothelial cells (SEC) and Kupffer cells (KC). Following intravenous administration of radiolabelled SR-ligands, [advanced glycation end (AGE)-products, N-terminal propeptide of type III procollagen (PIIINP) and formaldehyde treated serum albumin (FSA)] in SR-AI/II-deficient and wild-type mice, radioactivity was removed equally rapidly from the circulation of both types of mice. The major site of uptake was the liver. Separation of liver cells showed that the population of SEC and KC were responsible for approximately 55 and approximately 25% of the uptake. There was no difference in plasma clearance, organ distribution or cell distribution in SR-Al/Il-deficient and wild-type mice. Experiments performed to determine the specificity of endocytosis in cultured SEC showed that uptake of radiolabelled SR-ligands (AGE-protein, PIIINP or FSA) was inhibited equally well by unlabelled FSA and AGE-protein in SEC from receptor deficient and wild-type mice. We conclude from these findings that SR-Al/lI is of minor importance in the plasma clearance of physiological as well as foreign SR-ligands.

Animals↗
PubMedDate not supplied

Osteoprotegerin is expressed in colon carcinoma cells.

BACKGROUND: Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family, is produced by various cell types and tissues and plays a key role in the physiological regulation of osteoclast differentiation and activity. Also, OPG is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing factor (TRAIL). In the present study we investigated whether the human colon cancer cell lines HT-29 and SW-480 produce and secrete OPG in vitro. MATERIALS AND METHODS: Expression of OPG mRNA was examined by RT-PCR. OPG protein was analysed by ELISA assay and immunostaining methods. The effect of OPG secretion on TRAIL-mediated apoptosis was also investigated. RESULTS: By RT-PCR, it was demonstrated that mRNA transcripts for OPG were produced by both cell lines. By ELISA analysis, OPG was detected in the culture medium; and treatment of cells with proinflammatory cytokines TNF-alpha and IL-1beta increased OPG secretion significantly. Tumor xenografts in nude mice also were shown to express OPG by immunohistochemistry. When RANKL, which selectively binds OPG, was added to cell cultures along with recombinant TRAIL, apoptosis was shown to increase significantly. CONCLUSION: These data indicate that OPG may be involved in tumorigenesis and the progression of colon cancer.

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