Biomedical subjects
Publications and source records attributed to D L Knook.
Rats kept under germfree conditions showed lower specific lysosomal enzyme activities in liver endothelial cells, but not in Kupffer cells.
The clearance of five radioactively labeled test substances--polyvinylpyrrolidone, colloidal albumin, antimony sulfur colloid, endotoxin, and heparin--by the reticuloendothelial system was studied after i.v. injection of these substances into rats. The participation of parenchymal, Kupffer, and endothelial liver cells could be determined after isolation and purification of these cell classes. Only endotoxin was almost exclusively taken up by Kupffer cells. All other substances were also taken up by both endothelial and parenchymal cells. From the rate of uptake, expressed as the endocytic index, it appeared that all substances, with the exception of polyvinylpyrrolidone, were taken up by adsorptive endocytosis. Although the specific rate of uptake by parenchymal cells was always slower than that shown by both Kupffer and endothelial cells, the total contribution of parenchymal cells to the clearance of polyvinylpyrrolidone, antimony sulfur colloid, and heparin by the liver was even greater than that of the other two cell classes. The results demonstrate that nonphagocytosing parenchymal and endothelial cells make an important contribution to the clearance of the various test substances which, in addition, is highly dependent on the nature of the substance used. This necessitates a revision of present concepts of clearance by the reticuloendothelial system.
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1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.
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Sinusoidal cells in the rat liver react intensively for G6DPH activity after appropriate incubation (Rieder et al. 1978). After isolation and purification of the sinusoidal Kupffer and endothelial cells, it was demonstrated that Kupffer cells exhibit a 5-8 times higher G6PDH activity on a per cell basis by comparison with endothelial cells, while the specific G6PDH activity was 3-4 times higher in Kupffer cells. The Kupffer cells can be divided into two groups which differ significantly in G6PDH activity calculated on a per cell basis. In histochemical studies, G6PDH can be used as a marker for Kupffer cell identification.
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Isolated non-parenchymal cells from the rat liver were separated by centrifugal elutriation into two fractions containing structurally intact Kupffer and endothelial cells with purities of over 90% in both fractions. These two cell types were then examined by transmission and scanning electron microscopy. It is concluded that Kupffer and endothelial cells are readily distinguished under the scanning electron microscope on the basis of their different surface features. Kupffer cells show ridges and ruffles while endothelial cells have microvilli and blebs and lack ruffles. As in earlier studies, transmission electron micrographs show that Kupffer cells are larger, have a smaller nucleus--cytoplasm ratio and contain lysosomes while endothelial cells are smaller, have a higher nucleus-cytoplasm ratio and show extensive sieve-plates and fenestrations.
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The cellular basis of the age-related decline in the functional capacity of many mammalian organs is still poorly understood. In this paper, the rat liver is presented as a promising model for studying cellular phenomena underlying organ ageing. The recent development of methods for isolation and purification of parenchymal, Kupffer and endothelial cells from the rat liver makes possible the comparison of functional and metabolic changes in the intact liver with changes in distinct liver cell classes isolated from rats of various age groups. An attempt has been made to correlate age changes in some important liver-specific functions, such as bromsulophthalein uptake and albumin synthesis, at the organ and at the cellular level. To compare cellular ageing phenomena in long-lived cells (parenchymal cells) and short lived cells (Kupffer and endothelial cells) from the same organ, the role of lysosomes in cellular ageing processes was investigated, with secial reference to the functioning of the lysosomal enzyme cathepsin D. The specific cathepsin D activity in Kupffer cells was abour 3 times higher than in endothelial cells and about 20 times higher than in parenchymal cells. The enzyme activity in the latter cell type showed a significant increase with age.
Highly purified suspensions of parenchymal, endothelial and Kupffer cells were prepared from the rat liver. The respective roles of these cell classes in the degradation of proteins was investigated by analysing the cellular distribution of two lysomal proteases. The specific arginine naphthylamidase activity was 2 times higher in Kupffer cells compared with the nearly equal activities in endothelial and parenchymal cells. The specific activity of the important endopeptidase cathepsin D in endothelial and Kupffer cells was about 12 and 36 times higher, respectively, than the activity in parenchymal cells. These results are in agreement with an important role of Kupffer and endothelial cells in the degradation of proteins and protein containing material of exogenous origin.
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Parenchymal and nonparenchymal cells were isolated from the livers of female BN/BiRij rats, aged 3, 12, 24 and 30-35 months, by means of enzymatic techniques. About 70% of the cells in the nonparenchymal cell suspensions were endothelial cells and 25% were Kupffer cells. More than 90% of the isolated parenchymal, Kupffer and endothelial cells were viable as judged by trypan blue exclusion and ultrastructural appearance. The age-related changes in the specific activities of the lysosomal enzymes acid phosphatase, beta-galactosidase, cathepsin D and arylsulphatase B in parenchymal and nonparenchymal cells showed no correlated behavior. The most prominent change was observed for the cathepsin D activity in parenchymal cells, which nearly triples during the lifespan of the rat. A comparison of the activities obtained with homogenates of the whole liver and with parenchymal and nonparenchymal cells revealed that aging changes in lysosomal enzyme activities in homogenates should be carefully interpreted, since opposite patterns of change were often observed in the activities in parenchymal cells and in nonparenchymal cells.