Kupffer cells were found to be distributed over zone 1 (periportal), zone 2 (midzonal), and zone 3 (perivenous) of the rat liver acinus in a ratio of 4:3:2. After pronase digestion of the liver, two purified tractions of Kupffer cells could be obtained by centrifugal elutriation. Liver zone marking with methylene blue prior to cell isolation revealed that one fraction contained Kupffer cells from the periportal area; the other consisted mainly of such cells from the midzonal and perivenous areas. Periportal Kupffer cells were larger and showed higher lysosomal enzyme activities on a per cell basis as compared with midzonal and perivenous Kupffer cells. The immediate uptake of 0.31-micrometer. latex particles during a 2-minute perfusion of the liver was mainly accomplished by periportal Kupffer cells. The higher phagocytic activity of these cells was independent of the direction which latex was flushed through the liver. Seven days after in vivo administration of latex particles, at least 80 per cent of all Kupffer cells contained latex, but again periportal Kupffer cells showed a higher phagocytic activity in that they accumulated a relatively larger number of particles per cell. Latex phagocytosis increased the activity of cathepsin D in all Kupffer cells. Titration with pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than pepstatin revealed that periportal Kupffer cells contained many more cathepsin D molecules than midzonal and perivenous Kupffer cells. Latex particle uptake stimulated an increase in the molecular activity of cathepsin D in periportal cells as well as in the number of cathepsin D molecules in perivenous and midzonal cells. Kupffer cells show a functional heterogeneity that is related to their position in the liver acinus. Kupffer cells with a high endocytic activity, large and heterogeneous lysosomes, and high lysosomal enzyme activities are found in the periportal zone. This zonal heterogeneity can be reduced after phagocytosis of a triggering dose of latex.

Rats kept under germfree conditions showed lower specific lysosomal enzyme activities in liver endothelial cells, but not in Kupffer cells.

Purified suspensions of highly viable parenchymal, endothelial and Kupffer cells were prepared from the livers of young (3 months of age) and old (33-34 months of age) female rats. In the cell suspensions, the activities of the multiple forms of acid phosphatase were determined with the substrates 4-methylumbelliferyl phosphate, 1-naphthyl phosphate and p-nitrophenyl phosphate. Most specific enzyme activities increased during aging, but the preference to hydrolyse any of the three substrates did not change. The small age-related changes in effects of the inhibitors fluoride and alloxan were not uniform for the the three liver cell types. The pattern for multiple forms of acid phosphatase obtained after isoelectric focusing did not change with age for Kupffer cells, while one of the two major forms of the enzyme almost disappeared in endothelial cells from old rats. With age, a very pronounced heterogeneous pattern was observed for the enzyme forms in the long-lived parenchymal cells. The increase in heterogeneity in multiple forms of acid phosphatase in aging parenchymal cells may be the result of post-translational modifications.

The activities of nine key lysosomal enzymes were determined in isolated parenchymal cells and in isolated Kupffer and endothelial cells purified by centrifugal elutriation. Compared with parenchymal cells, all specific lysosomal enzyme activities were much higher in Kupffer and endothelial cells. Kupffer cells showed a specific enrichment in acid lipase, β-glucuronidase, cathepsin D and aminopeptidase B, whereas the specific activities of acid phosphatase, acid DNAse, β-acetylglucosaminidase and arylsulphatase B were much higher in endothelial cells as compared with Kupffer cells; β-galactosidase was equally present in both cell classes. The distribution of lysosomal enzyme activities reflects the role of Kupffer and endothelial cells in the clearance and degradation of serum lipoproteins and glycoproteins.

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.

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.

Purified suspensions of highly viable parenchymal, endothelial, and Kupffer cells were prepared from rat liver. In the liver cell classes, total activities of acid phosphatase were determined with 4-methylumbelliferylphosphate, 1-naphthylphosphate, and p-nitrophenylphosphate. The specific enzyme activities were different for each type of cell and, even within one cell class, the enzymes showed different conversion rates for the three substrates. These results indicate the presence of multiple forms of acid phosphatase enzymes in each cell class. The inhibiting effects of tartrate, fluoride, and alloxan on the acid phosphatase activities were investigated. Depending on the substrate used, the inhibitors inactivated the enzymes at different rates, which also indicates the presence of multiple forms of acid phosphatase enzymes in the liver cell classes. By means of an isoelectric focusing technique, acid phosphatase enzymes could be separated on the basis of their differences in isoelectric points. One form with an isoelectric point around 4 is found in Kupffer cells, whereas another form with an isoelectric point of about 7 is found in parenchymal cells. Endothelial cells possess both forms. These findings suggest a specificity in the function of this lysosomal enzyme in each cell class.

Non-parenchymal cell suspensions were prepared from rat livers by three different methods based on a collagenase, a pronase and a combined collagenase-pronase treatment. The highest yield of Kupffer and endothelial cells was obtained with the pronase treatment. Attempts were made for a further purification of these cells by Metrizamide density gradient centrifugation after preferentially loading lysosomal structures in Kupffer cells with Triton WR 1339, Jectofer®, Neosilvol®, Zymosan or colloidal carbon. After loading with Triton WR 1339 or Jectofer®, highly purified endothelial cell suspensions were obtained, but the final Kupffer cell preparations were contaminated with about 20% of endothelial cells. Kupffer and endothelial cells purified in this way showed an altered ultrastructure and contained increased activities of the lysosomal enzymes acid phosphatase, arylsulphatase B and cathepsin D. As an alternative procedure for the purification of Kupffer and endothelial cells, a method based on centrifugal elutriation was employed. With this procedure, highly purified preparations of Kupffer or endothelial cells with a well preserved ultrastructure were obtained. Compared with endothelial cells, purified Kupffer cells had a three times higher cathepsin D activity, whereas the arylsulphatase B activity was three times higher in endothelial cells. The high cathepsin D activity in Kupffer cells could be nearly completely inhibited by the specific cathepsin D inhibitor pepstatin, which excludes a possible contribution to this activity by proteases endocytosed during the isolation of the cells.

Isolated non-parenchymal cells from rat liver were separated by centrifugal elutriation into two fractions consisting of structurally intact Kupffer and endothelial cells with purities of 91 and 95%, respectively. Purified Kupffer and endothelial cells showed nearly equal specific activities for the lysosomal enzyme acid phosphatase, whereas the specific activity of cathepsin D was about 3 times higher in Kupffer cells. It was calculated that a significant amount of the cathepsin D activity in the liver is present in the Kupffer cells.

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.

Intact and viable parenchymal and non-parenchymal liver cell preparations were isolated by enzyme perfusion techniques from young and old rats. The distribution of the lysosomal enzymes acid phosphatase, beta-galactosidase, cathepsin D, acid DNAse, and arylsulphatase B over parenchymal and non-parenchymal cells was determined. In addition, morphological and morphometric changes which occur in parenchymal cells with age were investigated. All lysosomal enzymes studied are present in both cell classes, but non-parenchymal cells possess much ligher activities per mg protein than do parenchymal cells. This phenomenon is most pronounced for cathepsin D with a 13-times higher specific activity in non-parenchymal cells. Electron microscopic observations demonstrated that the lysosomal activities in non-parenchymal cells can be attributed mainly to the large and numerous lysosomal structures in Kupffer cells. Parenchymal cells from old rats have higher lysosomal enzyme activities per mg protein than do hepatocytes from young rats. This observation is in agreement with the general increase with age in the cytoplasmic volume fraction occupied by lysosomal structures in parenchymal cells. In general, non-parenchymal cells show no increase in specific enzyme activities with age. The results obtained suggest an increase in the heterogeneity--in both appearance and enzyme content--of the lysosomal structures in parenchymal cells with age.