Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Cysteine metabolism in periportal and perivenous hepatocytes: Perivenous cells have greater capacity for glutathione production and taurine synthesis but not for cysteine catabolism

Summary.  Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine dioxygenase, cysteinesulfinate decarboxylase, γ-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [³⁵S]cysteine, total cysteine catabolism ([³⁵S]taurine plus [³⁵S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity in periportal cells, 16% of the total catabolism of [³⁵S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [³⁵S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but γ-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells. Received January 15, 2002 Accepted February 4, 2002 Published online September 4, 2002 Acknowledgements This work was supported by the National Research Initiative Competitive Grants Program/United States Department of Agriculture Competitive Research Grant 02-37200-7583. Authors' address: Dr. Martha H. Stipanuk, Division of Nutritional Sciences, 227 Savage Hall, Cornell University, Ithaca, NY 14853-6301, U.S.A., E-mail: mhs6@cornell.edu     

Human and Murine High Endothelial Venule Cells Phagocytose Apoptotic Leukocytes

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0–6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosisin vivo.Mice were treated with a natural corticosterone (4-pregnene-11β,21-diol-3,20-dione) at levels detected during stress or malnutrition (93–180 μg serum cortisol/dl). At 4–12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Acinar heterogeneity of the epidermal growth factor receptor in the liver of male rats

Epidermal growth factor is cleared from the circulation by the liver, forming a very steep portal-to-central sequestration gradient. It was unknown whether this was due to the position within the liver acinus or whether it was due to functional differences in the hepatocytes. Experiments were undertaken to elucidate the lobular distribution and heterogeneity of the epidermal growth factor receptor in rat liver. Immunocytochemistry showed a predominantly higher staining density over periportal localized hepatocytes. Receptor binding studies with isolated, cultured hepatocytes, enriched in periportal or perivenous located cells, were performed. Our data revealed high- and low-affinity binding sites with a kd of 26 pM and 0.87 nM, respectively, for periportal hepatocytes. The high-affinity receptors were restricted to the periportal hepatocytes only, whereas the number of low-affinity receptors showed a 3 to 4-fold concentration gradient between both cell populations.     

Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules

The in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.     

Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules

Summary The in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.     

Zonation of the metabolic action of vasopressin in the bivascularly perfused rat liver

Predominance of the vasopressin binding capacity in the hepatic perivenous area leads to the hypothesis that the metabolic effects of the hormone should also be more pronounced in this area. Until now this question has been approached solely by experiments with isolated hepatocytes where an apparent absence of metabolic zonation was found. We have reexamined this question using the bivascularly perfused liver. In this system periportal cells can be reached in a selective manner with substrates and effectors via the hepatic artery when retrograde perfusion (hepatic vein --> portal vein) is done. The action of vasopressin (1-10 nM) on glycogenolysis, initial calcium efflux, glycolysis and oxygen uptake were measured. The results revealed that the action of vasopressin in the liver is heterogeneously distributed. Glycogenolysis stimulation and initial calcium efflux were predominant in the perivenous area, irrespective of the vasopressin concentration. Oxygen uptake was stimulated in the perivenous area; in the periportal area it ranged from inhibition at low vasopressin concentrations to stimulation at high ones. Lactate production was generally greater in the perivenous zone, whereas the opposite occurred with pyruvate production. Analysis of these and other results suggests that at least three factors are contributing to the heterogenic response of the liver parenchyma to vasopressin: a) receptor density, which tends to favour the perivenous zone; b) cell-to-cell interactions, which tend to favour situations where the perivenous zone is amply supplied with vasopressin; and c) the different response capacities of perivenous and periportal cells.     

肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的γ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异生的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的内源性酮体生成比门静脉周围肝细胞高１．３倍。给予能明显刺激酮体生成的辛酸盐,肝静脉周围肝细胞的酮体生成仅略为增加。我们的结果证实,在基础和刺激的条件下,葡萄糖的异生在门静脉周围肝细胞中优先,而酮体生成仅在肝静脉周围肝细胞占微弱的优势。     

Endogenous Tumor Necrosis Factor α Unmasks the Binding Sites for N-Acetylglucosaminyl-(β1-4)-N-Acetylmuramyl-Alanyl-D-Isoglutamine on the Surface of Melanoma Cells

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 ± 200 per cell) and affinity (K _d= 10 ± 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitrowith GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 ± 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor (TNF-). The mechanism of the TNF- action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient. Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state.

Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the long-term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P less than 0.01) in gluconeogenesis in periportal than in perivenous cells (93 +/- 16 versus 30 +/- 14 nmol/h per mg of protein; 72% versus 30% increase), but short-term incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of beta-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes.     

Histochemical and immunohistochemical localization of hexokinase isoenzymes in normal rat liver

Summary Histochemical and immunohistochemical procedures have been used to examine the localization of three of the four hexokinase isoenzymes present in the liver of fed female Wistar rats. Distinctive distribution patterns were found for hexokinase type I and glucokinase but hexokinase type II was not detectable. Hexokinase type I was identified in sinusoidal cells and in bile duct epithelia, nerves and arteries in the portal triad. Glucokinase, the major isoenzyme, was confined to parenchymal cells where it was present in much higher amounts in perivenous compared with periportal hepatocytes. Staining within these two zones was not homogeneous and each had a mosaic appearance caused by the presence of a few hepatocytes containing little or no glucokinase amongst the majority of darkly stained cells in perivenous areas and a few darkly stained cells amongst the majority of unstained cells in periportal areas. Hence, hepatocytesin situ are a strikingly heterogeneous population of cells. Their metabolic status cannot be controlled simply by the differential supply of oxygen, substrates and hormones to different regions of the liver acini as proposed in the metabolic zonation model. Phenotypic differences may exist between cells within a given metabolic zone which influence their ability to respond to different environmental conditions.     

Hepatocyte heterogeneity in the metabolism of carbohydrates.

Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.     

Sex differences of the influence of T3 on the topical distribution of phosphoenolpyruvate carboxykinase activity in the liver acinus

Summary Phosphoenolpyruvate carboxykinase (PEPCK) activity was investigated in livers of triiodothyronine (T₃) treated male and female rats with special regard to its intraacinar localization. In untreated controls of both, male and female rats, the activity was heterotopically distributed within the acinus with highest values in the periportal zone, and with lowest values in the perivenous zone. This periportal to perivenous activity gradient revealed to be under the influence of T₃. Application of T₃ resulted in a relative increase of PEPCK activity which was much greater in the livers of females than in males. The extent of T₃-induced augmentation of PEPCK activity was dependent on the intraacinar position. In both sexes greatest relative activation was found in the perivenous zone. In female animals, the perivenous activity of T₃ treated livers was comparable to that observed in the periportal zone of controls.     

Zonation of the action of ethanol on gluconeogenesis and ketogenesis studied in the bivascularly perfused rat liver

Zonation of the actions of ethanol on gluconeogenesis and ketogenesis from lactate were investigated in the bivascularly perfused rat liver. Livers from fasted rats were perfused bivascularly in the antegrade and retrograde modes. Ethanol and lactate were infused into the hepatic artery (antegrade and retrograde) and portal vein. A previously described quantitative analysis that takes into account the microcirculatory characteristics of the rat liver was extended to the analysis of zone-specific effects of inhibitors. Confirming previous reports, gluconeogenesis and the corresponding oxygen uptake increment due to saturable lactate infusions were more pronounced in the periportal region. Arterially infused ethanol inhibited gluconeogenesis more strongly in the periportal region (inhibition constant = 3.99 ± 0.22 mM) when compared to downstream localized regions (inhibition constant = 8.64 ± 2.73 mM). The decrease in oxygen uptake caused by ethanol was also more pronounced in the periportal zone. Lactate decreased ketogenesis dependent on endogenous substrates in both regions, periportal and perivenous, but more strongly in the former. Ethanol further inhibited ketogenesis, but only in the periportal zone. Stimulation was found for the perivenous zone. The predominance of most ethanol effects in the periportal region of the liver is probably related to the fact that its transformation is also clearly predominant in this region, as demonstrated in a previous study. The differential effect on ketogenesis, on the other hand, suggest that the net effects of ethanol are the consequence of a summation of several partial effects with different intensities along the hepatic acini.     

Immunohistochemical study of ontogeny and phylogeny of a terminalN-acetylglucosamine cluster antigen

Summary In this work an immunohistochemical method was used to study the ontogeny and phylogeny of a terminalN-acetyglucosamine (GlcNAc) cluster antigen which is an epitope(s) of highly branchedN-linked oligosaccharides terminating in GlcNAc residues. The ontogenic studies demonstrated that expression of the antigen is developmentally regulated in lymphocytes, epithelia cells of endodermal origin and kidney mesangial cells of the chicken. The antigen was found in several other avian species studied, namely, the Japanese quail, duck, goose and turkey. Furthermore, the distribution of the antigen in all these species was similar. In adult animals, it was found in bursal and thymic lymphocytes, macrophages, spleen reticulum cells, epithelial cells of the intestine and bronchioles and capillary endothelial cells. The antigen was also detected in epithelial cells of the gastrointestinal tract of several lower vertebrates studies: the amphibian (frog), reptile (chameleon) and fish (rainbow trout). It was undetectable in various organs of the human, African green monkey, calf, pig, rat and guinea-pig, but was found in the intestinal epithelial cells of ten mouse strains. It is likely that biosynthetic processing leading to the formation of highly branchedN-linked glycans terminating in GlcNAc residues is conserved during evolution in birds and other lower vertebrates.     

Modulation of cell surface expression of liver carbohydrate receptors during in vivo induction of apoptosis with lead nitrate

Cell surface expression of carbohydrate receptors (i.e. mannose and galactose receptors) and phagocytosis of apoptotic cells by sinusoidal liver cells was studied. Binding sites and phagocytic activity were quantified at different time intervals (1, 3, 5, 7, 9, 11, 13, 15, 20, 30, 40 and 60 days) after the in vivo administration to rats of a potent liver mitogen, lead nitrate, that also induces apoptosis. The number and distribution of binding sites was receptor and cell-type dependent during the days following the metal injection. The use of competing saccharides in inhibition uptake experiments suggests that sinusoidal liver cells actively phagocytose apoptotic hepatocytes and circulating apoptotic cells by using both receptors. In particular, Kupffer cells at 5 and 15 days after the lead nitrate injection are very active in internalizing apoptotic cells (two- to threefold control). However, phagosomes containing apoptotic hepatocytes are often seen inside the cytoplasm of parenchymal and endothelial cells.