High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

Studies on uptake and intracellular processing of infectious pancreatic necrosis virus by Atlantic cod scavenger endothelial cells.

Previous work in our group has identified the scavenger endothelial cells (SECs) of heart endocardium in cod, Gadus morhua L., as the major site for elimination of both physiological and foreign macromolecular waste from the circulation. The present study was undertaken to establish the role of cod SECs in the clearance of virus. We focused on infectious pancreatic necrosis virus (IPNV) as it is a well-known virus with a broad host range, and causes significant economic losses in the salmon industry. Our results showed that cod SEC cultures infected by the IPNV produce high titres of new virus. Ligand-receptor inhibition experiments suggested that the virus did not enter the cells through any of the major endocytosis receptors of cod SECs. Yet, the infection lowered the capacity of the cells to endocytose ligands via the scavenger receptor. Inhibitors of receptor recycling and vesicle acidification did not affect virus infectivity. The finding that SEC cultures prepared from 25% of the cod produced high titres of IPNV without being infected in the laboratory, suggests that SECs of cod may serve as reservoirs for IPNV in persistently infected cod.

The effect of cyclooxygenase inhibitor diclofenac on experimental murine colon carcinoma.

BACKGROUND: Cyclooxygenase-2 (Cox-2) has been found to be overexpressed in several types of human cancers and its role in tumorigenesis has been proposed. The aim of this study was to investigate the effects of the cyclooxygenase inhibitor diclofenac on the growth of murine C-26 colon carcinoma cells. MATERIALS AND METHODS: Expression of Cox-2 mRNA and protein was examined by RT-PCR analysis and immunohistochemistry, respectively. By using MTT-assay, we examined the effects of diclofenac at various concentrations on the growth of C-26 cells in vitro. The effect of diclofenac on the growth of the C-26 tumor in syngeneic mice was also investigated. RESULTS: By RT-PCR, Cox-2 mRNA was detected in C-26 cells. Cox-2 protein was localized to C-26 cells and treatment with diclofenac resulted in apoptotic cell death in a dose-dependent manner. Diclofenac administered in drinking water resulted in growth inhibition of C-26 tumor in mice and correlated with plasma levels of both PGE2 and TXB2. CONCLUSION: Our data show that diclofenac may be a potential agent for the prevention and treatment of human colon cancer.

Advanced glycation end products impair the scavenger function of rat hepatic sinusoidal endothelial cells.

AIMS/HYPOTHESIS: We have previously reported that advanced glycation end products are eliminated from the circulation mainly by scavenger receptor-mediated uptake in hepatic sinusoidal endothelial cells. Our experiments showed that the degradation of AGE-modified protein after endocytosis in hepatic sinusoidal endothelial cells occurs slowly compared with that of other scavenger receptor ligands. The aim of this study was to investigate further the mechanism whereby AGE-modified protein affects the important scavenger function of hepatic sinusoidal endothelial cells. METHODS: Primary cultures of hepatic sinusoidal endothelial cells were pre-incubated with unlabelled ligand, unbound ligand was washed off, and the endocytic capacity was measured by addition of radiolabelled ligand, and immune electron microscopy. RESULTS: Pre-incubation with unlabelled AGE-modified bovine serum albumin reduced subsequent endocytosis of radiolabelled scavenger receptor ligands AGE-modified bovine serum albumin, formaldehyde-treated serum albumin, oxidized low density lipoprotein and acetylated low density lipoprotein by 50, 56, 32 and 20%, respectively. Non-scavenger receptor-mediated endocytosis was not affected by pre-exposure to AGE-modified protein. Pre-incubation with a number of non-AGE-ligands did not affect subsequent endocytosis via any of the major endocytosis receptors in hepatic sinusoidal endothelial cells. Incubation in fresh medium for 6 h after pre-exposure to AGE-modified protein almost completely restored normal scavenger receptor-mediated endocytic activity. Quantitative immune electron microscopy showed that the amount of a newly described scavenger receptor for AGE-modified protein is reduced after pre-incubation with AGE-modified protein. Subcellular fractionation showed that pre-incubation with AGE-modified protein delays intracellular transport of scavenger receptor ligands. CONCLUSION/INTERPRETATION: Endocytosis of AGE-modified protein leads to loss of scavenger receptors and delayed intracellular transport in hepatic sinusoidal endothelial cells.

Tumor necrosis factor-related apoptosis-inducing ligand induces apoptosis in human articular chondrocytes in vitro.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is produced by immune cells and by mediating apoptosis, TRAIL plays an important role in tumor surveillance. TRAIL binds four different membrane-bound receptors: DR4, DR5, DcR1, and DcR2. The DR4- and DR5-receptors mediate apoptosis, whereas the others do not. We demonstrated by reverse transcriptase-polymerase chain reaction and flow cytometry that, in vitro, normal human articular chondrocytes express the receptors mediating apoptosis (DR4 and DR5) and one of the decoy receptors (DcR2). Also, we demonstrated that chondrocytes were subjected to cell death within few hours after challenge with TRAIL and that cytotoxicity was dose-dependent. Treated cells had apoptotic morphology accompanied by active caspase-3 immunoreactivity. These data indicate that normal human articular chondrocytes are susceptible to TRAIL-mediated apoptosis, which otherwise is typical for transformed cells, and also that death receptors and their respective ligands may have a crucial role in cartilage generation and destruction.

Re-evaluation of the reticulo-endothelial system.

By injecting lithium carmine (Lit-car) into living animals, Ribbert (1904) and Kiyono (1914) showed that specific staining occurred in some cells in various organs. Kiyono termed those cells "histiocytes" which consisted of free amoeboid cells and cells of reticulo-endothelium. Aschoff (1924) introduced the concept 'reticulo-endothelial system (RES)' for the collection of cells having in common the property of vital staining. Van Furth (1972) proposed the term "mononuclear phagocyte system (MPS)" advocated that RES be replaced by MPS. As a consequence we presently suffer from a "reticulo-endothelial confusion", based on the delusion that cells other than macrophages are not members of the RES. The point that most clearly illustrates this is the fact that the Lit-car-laden cells in the body are named macrophages in modern textbooks of histology. To clarify the confusion, we re-examined the vital staining at light and electron microscopic levels and analyzed the scavenger cells using biochemical methods. Injected Lit-car was actively endocytosed by sinusoidal endothelial cells in the liver and reticular cells in the sinus of lymph nodes. Of note, uptake of the dye was comparatively much lower in macrophages/monocytes. Our findings indicate the existence of scavenger endothelial cells and reticular cells in blood and lymphatic circulations respectively. We name these two systems "the scavenger RES" collectively.

Mannose-receptor-mediated clearance of lysosomal alpha-mannosidase in scavenger endothelium of cod endocardium.

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua). Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.

Specific endocytosis and catabolism in the scavenger endothelial cells of cod (Gadus morhua L.) generate high-energy metabolites.

The catabolic fate of circulating hyaluronan and the proteoglycan chondroitin sulphate (CSPG) was studied in the Atlantic cod (Gadus morhua L.). Distribution studies using radio-iodinated ligand demonstrated that CSPG was rapidly eliminated from the blood by the endocardial endothelial cells (EECs) of the heart atrium and ventricle. The presence of excess amounts of hyaluronan or CSPG inhibited uptake of [125I]hyaluronan into cultured atrial EECs (aEECs) by 46% and 84%, respectively. Neither formaldehyde-treated serum albumin (FSA) nor mannose inhibited this uptake. The presence of excess amounts of CSPG and hyaluronan inhibited uptake of [125I]CSPG by 90% and 42%, respectively, suggesting that aEECs express a specific hyaluronan binding site that also recognizes CSPG. FSA inhibited endocytosis of [1251]CSPG by 65%, indicating that CSPG is also recognized by the scavenger receptor. Approximately 17% and 57% of added [125I]hyaluronan and 15% and 65% of the added [125I]CSPG were endocytosed after 1 and 24h, respectively. High-performance liquid chromatographic analyses of the spent medium after endocytosis of hyaluronan and CSPG serglycin labelled biosynthetically with 3H in the acetyl groups identified labelled the low-molecular-mass degradation products as [3H]acetate, indicating that aEECs operate anaerobically. These findings suggest that acetate released from cod EECs following catabolism of endocytosed hyaluronan and CSPG represents a high-energy metabolite that may fuel cardiomyocytes.

Interferon-gamma modulates TRAIL-mediated apoptosis in human colon carcinoma cells.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by immune cells and has been shown to play an important role in tumor surveillance due to its ability to induce apoptosis in various transformed cells. Interferon gamma (IFN-gamma), a multipotent cytokine with broad stimulatory effects on anti-tumoral immune reactions, may exert its cytotoxic activity directly on tumor cells or indirectly via stimulation of effector cells. This study was designed to determine the effect of IFN-gamma on TRAIL-mediated apoptosis in human colon carcinoma cell lines. MATERIALS AND METHODS: Cytotoxicity was assessed by MTT-assay. Expression of death receptors was measured by reverse transcriptase polymerase chain reaction. Apoptosis was assessed by caspase-8 immunoblot, DNA fragmentation and morphological studies. RESULTS: Treatment with TRAIL resulted in detectable cytotoxicity within 5 hours and was enhanced in a dose-dependent manner. When cells were pretreated with IFN-gamma, the cytotoxic effect of TRAIL increased significantly. Treated cells showed a typical apoptotic morphology that was accompanied by internucleosomal cleavage of DNA. Up-regulation of caspase-8 expression and activation were detected as a result of pretreatment with IFN-gamma and subsequent apoptosis mediated by TRAIL. CONCLUSION: Our results demonstrated that IFN-gamma sensitises human colon carcinoma cells to TRAIL-mediated apoptosis, partly by elevated caspase-8 expression.

Scavenger-receptor-mediated endocytosis of lipopolysaccharide in Atlantic cod (Gadus morhua L.).

The mechanism of elimination of blood-borne Vibrio salmonicida lipopolysaccharide (LPS) from Atlantic cod (Gadus morhua L.) was studied. The anatomical distribution of LPS was determined using both morphological and radiotracing methods. Immunohistochemistry performed on tissue specimens after injection of LPS disclosed that the endocardial endothelial cells (EECs) represented the cellular site of uptake in heart. Co-injection of trace amounts of [(125)I]LPS together with excess amounts of formaldehyde-treated albumin (FSA), a ligand for the scavenger receptor, significantly inhibited the accumulation of the radiotracer in heart only. Studies on purified monolayer cultures of atrial EECs showed that fluorescein-labelled LPS was taken up in structures reminiscent of endosomal/lysosomal vesicles. Incubation of cultures with [(125)I]LPS together with excess amounts of FSA, fucoidan and dextran sulphate, molecules known to compete for endocytosis via the scavenger receptor, reduced uptake of the probe by 80 %. Mannan, a ligand for the mannose receptor, did not compete for uptake. Kinetic studies on the uptake and degradation of [(125)I]LPS in cultured atrial endocardial cells revealed no degradation after 48 h of culture. In conclusion, we have shown that the EECs of cod remove V. salmonicida LPS from the circulation by scavenger-receptor-mediated endocytosis.

Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A.

Previous studies with peritoneal macrophages obtained from macrophage scavenger receptor class A (MSR-A) knock-out mice showed that the endocytic uptake of advanced glycation end products (AGE) by macrophages was mediated mainly by MSR-A. However, it is controversial whether the endocytic uptake of intravenously injected AGE proteins by liver sinusoidal endothelial cells (LECs) is similarly explained by receptor-mediated endocytosis via MSR-A. The present study was conducted to compare the capacity to endocytose AGE proteins in LECs and peritoneal macrophages obtained from MSR-A knock-out and littermate wild-type mice. The endocytic degradation capacity of MSR-A knock-out LECs for AGE-BSA was indistinguishable from that of wild-type LECs, whereas that of MSR-A knock-out peritoneal macrophages for AGE-BSA was decreased to 30% of that in wild-type cells. Similarly, the endocytic degradation of MSR-A knock-out LECs for acetylated low-density lipoprotein (acetyl-LDL) did not differ from that of wild-type LECs, whereas the endocytic degradation of acetyl-LDL by MSR-A knock-out peritoneal macrophages was less than 20% of that in wild-type cells. Furthermore, formaldehyde-treated serum albumin (f-Alb), a ligand known to undergo scavenger-receptor-mediated endocytosis by LECs, was effectively taken up by MSR-A knock-out LECs at a capacity that did not differ from that of wild-type LECs. Moreover, the endocytic uptake of AGE-BSA by LECs was effectively competed for by unlabelled f-Alb or acetyl-LDL. These results indicate that the scavenger-receptor ligands AGE proteins, acetyl-LDL and f-Alb are endocytosed by LECs through a non-MSR-A pathway.

Selective initial in vivo homing pattern of 5T2 multiple myeloma cells in the C57BL/KalwRij mouse.

One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are unique. To investigate this, in vivo homing kinetics of murine 5T2MM cells shortly after injection were assessed in bone marrow, liver, spleen, lungs, heart, intestines, kidney and testis by tracing of radiolabelled cells, by immunostaining of isolated cells and by polymerase chain reaction analysis. We demonstrated the presence of 5T2MM cells in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. We here demonstrate that the specific in vivo localization of the 5T2MM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone marrow, spleen and liver, and a selective survival and growth of these tumour cells in the bone marrow and spleen but not in the liver.

Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver.

This study was undertaken to determine the fate of the circulating chondroitin sulfate proteoglycan serglycin. The human monocytic cell line THP-1 was cultured under serum-free conditions in the presence of [35S]sulfate. The conditioned medium was harvested and 35S-macromolecules were purified by Q-Sepharose anion-exchange chromatography and Superose 6 gel chromatography. After labeling with 125I, the purified material was treated with chondroitinase ABC and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. A major band with mr of approximately 14 kDa appeared, consistent with the core protein of serglycin. The identity of the proteoglycan was confirmed by amino-terminal amino acid sequencing. Purified serglycin, labeled either with [35S]sulfate or 125I and fluorescein isothiocyanate, was injected intravenously into rats. The blood content of radiolabeled serglycin fell by 50% from 1 to 2.4 min after injection, indicating an initial t1/2 of 1.4 min or shorter. Approximately 90% of the recovered radioactivity was localized in the liver, 5% in the blood, and 5% altogether in urine, kidneys, and spleen about 30 min after injection. Isolation of liver cells at the same time point showed that 70% of the radioactivity was taken up by the sinusoidal scavenger endothelial cells, and 23 and 7% by the hepatocytes and Kupffer cells, respectively. When excess amounts of unlabeled hyaluronan was coinjected with radiolabeled serglycin, the elimination of serglycin was significantly inhibited, indicating that the hyaluronan receptor on the sinusoidal scavenger endothelial cells is responsible for the elimination of serglycin.

Characterization of a hyaluronan receptor on rat sinusoidal liver endothelial cells and its functional relationship to scavenger receptors.

Hyaluronan is a widely distributed extracellular component of connective tissue with several mechanical and cell biological functions. The serum level of hyaluronan is elevated in rheumatic and liver diseases and in certain malignancies. The major route of hyaluronan clearance from the blood is via the liver, taken up predominantly by sinusoidal liver endothelial cells. We have purified a novel hyaluronan binding protein from liver that also has an affinity for the N-terminal propeptide of type I procollagen, a physiological scavenger receptor ligand. A polyclonal antibody raised against the protein was found to inhibit the binding and degradation of hyaluronan as well as two scavenger receptor ligands by cultured sinusoidal liver endothelial cells. Immunostaining of nonpermeabilized liver cells and liver sections showed that the antibody specifically stains the surface of sinusoidal liver endothelial cells. After pretreatment with monensin to block the recirculation of endocytic receptors, the immunostaining was specifically associated with early endosomes of these cells. Thus, this rat sinusoidal liver endothelial cell hyaluronan receptor shares functional properties with the scavenger receptor family, a group of proteins shown to play a key role in the uptake of atherogenic lipids and other waste products from the tissues.

Early events of hepatic metastasis formation in mice: role of Kupffer and NK-cells in natural and interferon-gamma-stimulated defense.

Surgical manipulation of a tumor may result in increased influx of tumor cells into the systemic and portal circulation and give rise to formation of metastases. In addition, major surgery has been reported to cause profound immunosuppression. In an attempt to increase the host-antitumor immune mechanisms following surgery we have studied the effect of preoperative administration of interferon-gamma, related to the antimetastatic effects of Kupffer cells (KC) and natural killer cells (NK-cells) in the early phase of liver metastasis formation. Colon carcinoma cells were injected into the superior mesenteric vein of syngeneic mice and after 17 days metastases were quantified by weight, number, and uptake of [125I]iododeoxyuridine. Unstimulated control mice developed 10.5 surface nodules per liver 17 days following injection of colon carcinoma cells into the superior mesenteric vein of syngeneic mice. This figure was only 2.6 in mice stimulated with a single dose of 1000 IU IFN-gamma 4 h prior to inoculation of tumor cells. Administration of GdCl3, which is reported to deplete and block the function of Kupffer cells, 24 h prior to tumor cell inoculation resulted in a 5-fold tumor mass increase relative to control. Injection of anti-asiolo-GM1 antiserum, which eliminates the hepatic NK-cells, induced a 10-fold increase in tumor mass. These results indicate an important early antimetastatic function of hepatic NK-cells and KC and that presurgical administration of IFN-gamma may be important for eliminating circulating tumor cells and inhibiting development of residual tumors.

Transport of residual endocytosed products into terminal lysosomes occurs slowly in rat liver endothelial cells.

Receptor-mediated endocytosis of circulating collagen is a major physiological scavenger function of the liver endothelial cell and an important catabolic event in the complete turnover of this abundant connective tissue protein. In the present study, transport of collagen through the endocytic pathway was investigated in cultured liver endothelial cells. Collagen conjugated to fluorescein isothiocyanate, to allow detection of the ligand by fluorescence and immunoelectron microscopy, was found sequentially in three different organelles that compose the basic degradative endocytic pathway of eukaryotic cells: early endosomes, late endosomes, and terminal lysosomes. Early endosomes were identified as vesicles positive for early endosome antigen 1 (EEA1). Late endosomes were distinguished as structures positive for the late endosomal/lysosomal marker rat lysosomal membrane glycoprotein 120, but negative for EEA1 and lysosomally targeted BSA-gold. Lysosomes were defined by their content of BSA-gold, injected 24 hours before isolation of cells. Coated pits and coated vesicles mediated an extremely rapid internalization. Shortly after internalization and during the first 20 minutes, ligand was found in early endosomes. From 20 minutes on, ligand started to appear in late endosomes (23%), and by 2 hours the transfer was largely complete (82.5%). Only 2.5% of ligand was transferred to the lysosomes after 2 hours, and this number slowly increased to 21% and 53% after 6 and 16 hours, respectively. We conclude that 1) EEA1 is a useful marker for tracing early events of endocytosis in liver endothelial cells; 2) in contrast to the rapid internalization, transit of internalized ligand through early sorting endosomes generally takes from 20 minutes to 2 hours; and 3) exit from the late endosomes is very slow, requiring several hours.

Uptake of lithium carmine by sinusoidal endothelial and Kupffer cells of the rat liver: new insights into the classical vital staining and the reticulo-endothelial system.

Sinusoidal cells in the rat liver were studied in vivo and in vitro using the original vital staining with lithium carmine, which has contributed much to the development of the concept of the reticulo-endothelial system. Immunohistochemical and electron-microscopic studies revealed that the dye-incorporating cells were sinusoidal endothelial cells, Kupffer cells, and monocytes. The endothelial cells took up much more dye than did the Kupffer cells and bulged largely into the sinusoidal lumen. Electron microscopy revealed that small particles of lithium carmine were associated with coated vesicles of endothelial cells and ruffled membranes of Kupffer cells. In the endothelial cells, these particles were present in various concentrations within vacuolated structures and condensed in the lysosomes forming large aggregates of lithium carmine lumps. These lumps showed crystalline structures, within which the size of the individual particle was up to 30 nm in width and 50 nm in length. A few endothelial cells containing abundant dye underwent degeneration, and some were taken up by Kupffer cells. Liver endothelial cells isolated from lithium carmine-administered rats endocytosed fluorescence-labeled collagen. Isolated endothelial cells from normal rat liver, when cultured with lithium carmine, did not take up any dye, and their endocytosis of formaldehyde-treated albumin was inhibited dose-dependently. We conclude that in the liver, endothelial cells, but not Kupffer cells, predominantly take up lithium carmine. Furthermore, we propose the existence of a generalized cell system based on its vital staining capacity.

Scavenger-receptor-mediated endocytosis in endocardial endothelial cells of Atlantic cod Gadus morhua.

Scavenger receptors are multifunctional integral membrane proteins that mediate the endocytosis of many different macromolecular polyanions and also participate in host defence reactions and cell adherance. In Atlantic cod (Gadus morhua L.), two intravenously injected scavenger receptor ligands, [125I]tyramine-cellobiose-labelled formaldehyde-treated serum albumin (125I-TC-FSA) and 125I-labelled N-terminal propeptide of type I procollagen (125I-PINP), distributed mainly to the heart. Cellular uptake was visualized by injections of fluorescently labelled FSA (FITC-FSA), which was recovered in discrete vesicles in endocardial endothelial cells of both heart chambers. Studies in vitro showed that radioiodinated FSA and PINP were endocytosed and degraded very efficiently by cultured atrial endocardial endothelial cells. Moreover, uptake of 125I-FSA was Ca2+-independent. Out of a range of unlabelled ligands, only the scavenger receptor ligands FITC-FSA, polyinosinic acid and, to a varying extent, FSA, acetylated low-density lipoprotein (AcLDL) and PINP, were able to compete with radioiodinated FSA, PINP or AcLDL for uptake in isolated endocardial cells. From our findings, we conclude that the endocardial endothelial cells are major carriers of scavenger receptors in cod. In addition, our results strengthen the hypothesis that these cells in cod play the same important function as that established for the scavenger endothelial cells of the mammalian liver.

Insulin enhances macrophage scavenger receptor-mediated endocytic uptake of advanced glycation end products.

Hyperglycemia accelerates the formation and accumulation of advanced glycation end products (AGE) in plasma and tissue, which may cause diabetic vascular complications. We recently reported that scavenger receptors expressed by liver endothelial cells (LECs) dominantly mediate the endocytic uptake of AGE proteins from plasma, suggesting its potential role as an eliminating system for AGE proteins in vivo (Smedsrod, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567-573). In the present study we examined the effects of insulin on macrophage scavenger receptor (MSR)-mediated endocytic uptake of AGE proteins. LECs expressing MSR showed an insulin-sensitive increase of endocytic uptake of AGE-bovine serum albumin (AGE-BSA). Next, RAW 264.7 cells expressing a high amount of MSR were overexpressed with human insulin receptor (HIR). Insulin caused a 3.7-fold increase in endocytic uptake of 125I-AGE-BSA by these cells. The effect of insulin was inhibited by wortmannin, a phosphatidylinositol-3-OH kinase (PI3 kinase) inhibitor. To examine at a molecular level the relationship between insulin signal and MSR function, Chinese hamster ovary (CHO) cells expressing a negligible level of MSR were cotransfected with both MSR and HIR. Insulin caused a 1.7-fold increase in the endocytic degradation of 125I-AGE-BSA by these cells, the effect of which was also inhibited by wortmannin and LY294002, another PI3 kinase inhibitor. Transfection of CHO cells overexpressing MSR with two HIR mutants, a kinase-deficient mutant, and another lacking the binding site for insulin receptor substrates (IRS) resulted in disappearance of the stimulatory effect of insulin on endocytic uptake of AGE proteins. The present results indicate that insulin may accelerate MSR-mediated endocytic uptake of AGE proteins through an IRS/PI3 kinase pathway.

Inhibition of establishment and growth of mouse liver metastases after treatment with interferon gamma and beta-1,3-D-glucan.

The purpose of this study was to investigate the combined antitumor effect of aminated beta-1,3-D-glucan (AG) and interferon-gamma (IFN-gamma) in an experimental liver metastasis model. Liver metastases were established by inoculation of C-26 colon carcinoma cells into the superior mesenteric vein of syngeneic mice. Treatment of mice started 24 hours after inoculation of tumor cells by daily intravenous injections of either AG, IFN-gamma, or a combination of both for a duration of 6 days. The resultant liver metastases were then quantified after an additional period of 11 days. Combination of IFN-gamma and AG inhibited the growth of liver metastases almost entirely. IFN-gamma was also very efficient, while AG alone did not exert any significant antitumor effect. These results, along with histological studies from mice receiving AG and IFN-gamma, indicated that activation and recruitment of liver macrophages may be a part of the mechanism responsible for the inhibition of metastatic growth observed in this study.