Bård Smedsrød

Separation and Characterization of Liver Cells

Publisher Summary This chapter discusses the separation and characterization of liver cells. The methods of preparation of isolated liver cells involve treatment with enzymes, separation by centrifugation, and selective substrate attachment. The other developed method involving collagenase perfusion, isopycnic centrifugation in Percoll, and selective substrate attachment is rapid and gives high-yield and high-purity preparations of functionally intact cells. A rapid and frequently used method for assessing cell viability is based on the capacity of cells to exclude trypan blue. However, this method gives an indication only of the integrity of the plasma membrane. Cells that exclude trypan blue may prove to be nonviable when tested for functional or morphological properties. Light microscopy forms the basis of most methods of identification of liver cells. Electronic cell counters are certainly useful, but only visual inspection with the aid of a hemocytometer and a phase-contrast microscope give information about the presence of aggregates, cell debris, and PC-derived particles. Cytochemical staining for endogenous peroxidase is the most frequently used means to positively distinguish KC from LEC and SC in the rat.

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.

Old Age and the Hepatic Sinusoid

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up‐regulation of von Willebrand factor and ICAM‐1 with reduced expression of caveolin‐1. These changes have been termed age‐related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age‐related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age‐related diseases. Anat Rec, 291:672–683, 2008.

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

BackgroundNumerous 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.Results125I-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 μm 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 × 107 (range 50–890 × 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 × 107 (sd 12 × 107) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors.ConclusionsWe 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.

The scavenger endothelial cell: a new player in homeostasis and immunity

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

Age-related changes in the hepatic microcirculation in mice.

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

Aminoterminal Propeptide of Type III Procollagen is Cleared from the Circulation by Receptor-Mediated Endocytosis in Liver Endothelial Cells

Intravenously administered [125I]-labelled bovine aminoterminal propeptide of type III procollagen ([125I]-BPIIINP) had a half-life in blood of about 2 minutes. Low molecular weight degradation products appeared in the circulation about 5 minutes after injection. BPIIINP coupled to [125I]-labelled tyramine cellobiose ([125I]-TC-BPIIINP) was administered intravenously to determine the cellular site of uptake. TC is non-degradable and is therefore accumulated intralysosomally. With this ligand I could show that PIIINP is taken up mainly by the liver endothelial cells (LEC), with very low uptake in other types of liver cells and at extrahepatic sites. Studies on binding and endocytosis of labelled PIIINP in cultures of purified populations of liver cells can be summarized as follows: 1) Uptake and degradation were observed mainly in LEC; 2) PIIINP associated with Kupffer and parenchymal cells, but degradation was very low; 3) Serum was not required for binding of PIIINP to LEC; 4) Binding was specific, that is, other ligands, such as collagen type III, hyaluronan, chondroitin sulfate, formaldehyde-treated albumin, and mannose, that are recognized by distinct receptors on LEC, did not compete with PIIINP for binding; 5) BPIIINP, TC-BPIIINP, and rat PIIINP (RPIIINP) were recognized with the same specificity by LEC; 6) BPIIINP bound to LEC with high affinity (dissociation constant = 1 nM), and about 4.2 x 10(5), 3.2 x 10(5), and 1.6 x 10(5) molecules of BPIIINP, TC-PIIINP, and R-PIIINP, respectively were bound per cell; 7) PIIINP could not be degraded by conditioned medium from cultured Kupffer cells; 8) Leupeptin, which is a strong inhibitor of lysosomal collagenolysis, only weakly inhibited degradation of PIIINP; 9) Binding and endocytosis of PIIINP was not Ca++-dependent; 10) Agents that inhibit the endocytic machinery inhibited uptake and degradation of PIIINP. In conclusion, the present results suggest that PIIINP is rapidly eliminated from the circulation by receptor-mediated endocytosis in LEC.

The mannose receptor on murine liver sinusoidal endothelial cells is the main denatured collagen clearance receptor

The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180‐kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose‐receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild‐type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild‐type mice, and denatured collagen uptake in wild‐type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007.)

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.

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.