Lysosomal degradation of cell organelles. II. Ultrastructural analysis of uptake and digestion of intravenously injected microsomes and ribosomes by Kupffer cells.

Rough and smooth microsomes, "mixed" or total microsomes, and ribosomes were isolated from one single rat liver and subsequently injected intravenously into a series of inbred rats. The uptake and the degradation of the injected organelles by Kupffer cells were followed by means of electron microscopic analysis. By 1 minute after injection, microsomes were seen attached to the surface of Kupffer cells separated by a gap of 200 to 300 A. No attachment to hepatocytes, fat-storing cells, or endothelial cells was seen. By 5 and 10 minutes, most microsomes were phagocytosed and sequestered in large numbers within single membrane-enclosed vacuoles or phagosomes. The engulfment proceeded by two mechanisms: (1) most frequently, flaplike processes of cytoplasm embraced aggregates of microsomes, concomitant with the formation of indention of the cytoplasm; (2) occasionally, single microsomal profiles were taken up by bristle-coated endocytic vacuoles. Ribosomes were also seen penetrating into the wormlike structures (micropinocytosis vermiformis) at the cell surface. At 30 minutes after injection, clear signs of alteration were noted starting with vesicle aggregation, clumping, and elongation of the microsomal profiles. The ribosomes were quickly stripped from their microsomal membranes and marginated to the inside of the vacuoles but separated from the limiting membrane by a distance of 200 to 300 A. By 1 and 2 hours, disruption of the vesicles into membrane fragments and formation of dense material in and between the profiles occurred. By 8 hours it was difficult to recognize the degradation products as membrane derivatives. The digestive vacuoles retained their size at this time interval. Typical pentalaminar structures were observed. By 14 to 24 hours the digestive vacuoles became electron lucent and appeared to shrink, and in addition to containing various types of granular material, many were laden with lipid-like droplets presumed to be conglomerates of phospholipid remnants. Rough microsomes, when compared to smooth microsomes, gave rise to more granular material within the digestive vacuoles. Ribosomes were still identifiable 24 hours after injection, indicative of a somewhat slower rate of degradation. Accumulation of various types of lipid-like droplets in the "residual bodies" was typical after microsomal injections. It is concluded that although microsomes appear to be phagocytosed at a quicker rate than mitochondria, they are digested within the lysosomal apparatus of the Kupffer cells at a somewhat slower rate. This especially seems to be the case for ribosomes. Heterophagy of microsomes is one source of residual bodies.     

Lysosomal degradation of cell organelles. III. Uptake and disappearance in Kupffer cells of intravenously injected isotope-labeled mitochondria and microsomes in vivo and in vitro.

14C-leucine and 3H-glycerol-labeled microsomes and mitochondria were intravenously injected into a series of highly inbred rats. The uptake and disappearance of the organelles were followed in a crude liver lysosomal fraction and in serum. Approximately half of the injected dose was recovered in the liver, and only smaller amounts were found in lungs, kidneys, spleen, and heart. The clearance in serum was more rapid for microsomes (t1/2, 5 to 15 minutes) than for mitochondria (t1/2 30 to 60 minutes). Both organelles showed a biphasic type of disappearance curve consistent with the two-phase theory of phagocytosis: attachment and engulfment. The estimated half-life for mitochondria of the liver was in the range of 3 to 4 hours, whereas that of the microsomes was considerably longer, or 8 hours. There was an increase of trichloroacetic acid-soluble material in the crude lysosomal fraction up to 2 hours after injection of glycerol-labeled microsomes, whereas the peak was reached at 60 minutes after 14C-leucine labeling. In vitro hydrolysis rate of hydrolysis. Experiments with Kupffer cells previously labeled with Thorotrast and biochemical assay of hydrolysis indicated that there was a lag phase of approximately 10 to 20 minutes before the phagosomes gained acid hydrolases, presumably by fusion with lysosomes. It is concluded at somewhat different rates. The remnants from lipid degradation, in comparison with protein degradation, seem to remain for a longer period within the lysosomal apparatus. These results are compatible with the concept that lysosomes represent an important, and at the present the only well defined locus for organelle turnover.     

Non-immunological cell death of intravenously injected murine tumour cells.

Most DBA mastocytoma and Sarcoma 180 cells trapped in the lungs of mice after i.v. injection died within 7 h. Rates of cell death were similar for both tumour cell lines. Rates of tumour cell death were unrelated to whether the cells were allogeneic or syngeneic, induced platelet aggregation or not, had different patterns of subsequent tumour growth, or were injected in varying numbers. Cell death was by coagulative necrosis, not apoptosis. Sarcoma 180 tumour cells were quickly localized in the lung and enclosed in platelet aggregates which remained, with degranulation, until the time of tumour cell death. However, platelet aggregation did not appear to play a role in tumour cell killing. The prevention of platelet aggregation by pretreatment of mice with an anticoagulant had little effect on the rate of death of tumour cells in the lung. Mastocytoma tumour cells did not cause platelet aggregation, yet died in the lung at similar rates to Sarcoma 180 cells. The killing of tumour cells in the lung did not appear to be cell-mediated. No mononuclear cells were seen in the vicinity of tumour cells and the type of cell death was not that associated with cell-mediated killing. The tumour cells did not die within 6 h of being injected into the peritoneal cavity. It is suggested that a nonspecific non-immunological process results in the death of intravenously injected tumour cells in the lung. This process was not affected by differing oxygen levels in the inhaled gas.     

Uptake and degradation of glycogen by Kupffer cells.

Rat liver glycogen was isotopically labeled with [¹⁴C] glucose and isolated. The isolated glycogen was injected intravenously into a series of rats and its vascular clearance, uptake and degradation in liver was analyzed by means of labeling and ultrastructural techniques. Injected glycogen was quickly removed from serum with a $\text{t}\text{1}\text{2}$ of less than 15 min. Glycogen particles, identified in the electron microscope, were never seen to attach to the surface of Kupffer cells or hepatocytes. These particles appeared to be taken up by Kupffer cells by nonspecific pinocytosis “fluid endocytosis” e.g., as a solute with engulfed liquid. By 10 min the particles were present within single membrane bound vacuoles of Kupffer cells. At this early time point, the vacuoles did not seem to have fused with preexisting prelabeled secondary lysosomes containing ferritin. At later time points, glycogen particles were seen intermingled with ferritin. By 1, 2, and 4 hr, increasing numbers of vacuoles containing granular material believed to represent glycogen were observed. These vacuoles often showed extensive enlargement (“swelling”). By 24 hr, most glycogen particles had disappeared and granular material was prresent only in occasional lysosomes which no longer appeared swollen. The estimated half-life for glycogen in Kupffer cell lysosomes was in the range of 12 to 16 hr. This is considerably longer than for membrane proteins and lipids introduced into Kupffer cell lysosomes by means of heterophagy. Because of possible reutilization of isotope it was difficult to define the half-life of glycogen more exactly. It is concluded that glycogen is degraded in Kupffer cell lysosomes, although at a relative slow rate, in comparison with the capability of lysosomal hydrolases to digest proteins and lipids. This conclusion is in line with the general notion that glycogen degradation takes place in the cell sap and is not primarily associated with any particular organelle.     

The functional development of the reticulo-endothelial system: I. The uptake of intravenously injected particles by foetal rats

A study has been made of the uptake of various particles by the reticulo-endothelial system of foetal rats. It was shown that the rate of uptake varied both with the age of the foetus and with the particles used. This indicated that the phagocytic properties of this system develop progressively during foetal life.     

Lysosomal degradation of cell organelles. IV. Heterophagocytosis and acute inflammation in liver after intravenous injection of isolated liver-cell plasma membranes.

Isolated liver-cell plasma membranes were injected intravenously into a series of highly inbred rats. The uptake and digestion by Kupffer cells were followed by ultrastructural analysis in order to evaluate the capacity of lysosomes to degrade cellular membranes. By 1 to 5 min following administration, clusters of plasma membranes appeared in the sinusoidal lumens. Invaginations of the Kupffer-cell surfaces in combination with flap-like processes embraced the aggregates and formed endocytic vacuoles. Fibrinous deposits and platelet accumulation were sometimes observed at the surface of the Kupffer cells. At later time points (10 min to 2 hr) an increased number of plasma-membrane derivatives with trilaminar structures still recognizable was observed in large digestive vacuoles. In some focal areas, an acute inflammatory response was noted in the sinusoids often surrounding clusters of fibrin deposits, trapped erythrocytes, and aggregates of plasma membranes and platelets. At 8 hr there was a clearance from some digestive vacuoles of identifiable membranes with the appearance of dense homogeneous material; plasma membranes were still present in the sinusoids together with degranulating and degenerating leukocytes also phagocytizing plasma membranes. By 24 hr most of the inflammatory response had subsided. Many Kupffer cells resembled those in control animals although the digestive vacuoles or residual bodies contained numerous lipid-like droplets presumed to be remnants of membrane lipid digestion. By 2–5 days the Kupffer cells gradually became indistinguishable from the controls and the micropinocytosis vermiformis reappeared. It is concluded that plasma membranes after being phagocytized are digested within lysosomes. In contrast to mitochondria and microsomes (Glaumann et al., 1975ab; Glaumann and Trump, 1975), intravenous injections of plasma membranes gave rise to focally occurring thrombi which were surrounded by an acute transient inflammatory response.     

Comparative studies of endotoxin uptake by isolated rat Kupffer and peritoneal cells.

The process of uptake of endotoxin by cells of the reticuloendothelial system is of current interest. Rabbit peritoneal macrophages have been used to study macrophage-endotoxin interactions and have suggested a receptor-mediated process. It is generally believed that the site of in vivo endotoxin clearance is the liver and that this clearance involves the Kupffer cell population. In the current report, the uptake characteristics of iodine-125-labeled Salmonella minnesota lipopolysaccharide (LPS) were compared in both isolated rat Kupffer cells and elicited rat peritoneal cells. Both types of cells were isolated from male Sprague-Dawley rats fed a semisynthetic AIN-76 5% saturated-fat diet either by peritoneal lavage for peritoneal cells or by collagenase perfusion followed by purification on a 17.5% metrizamide gradient for Kupffer cells. Hot phenol water-extracted S. minnesota LPS was labeled with iodine by the chloramine-T method following a reaction with methyl-p-hydroxybenzimidate. The in vitro uptake of [125I]LPS by Kupffer cells was unsaturable up to concentrations of 33.33 micrograms/ml, while peritoneal cells became saturated at between 16.67 and 25 micrograms of LPS per ml. Uptake by both types of cells could be inhibited by a 10-fold excess of unlabeled LPS. Kinetic experiments demonstrated that Kupffer cells were unsaturable after 60 min of incubation, while peritoneal cells were saturable after 40 min of incubation. Pretreatment with 75 mM colchicine inhibited uptake by peritoneal cells but not Kupffer cells, while pretreatment with 12 mM 2-deoxyglucose inhibited uptake by Kupffer cells but not peritoneal cells. These results are consistent with a process of receptor-mediated endocytosis for peritoneal cells, while Kupffer cells may internalize endotoxins by absorptive pinocytosis. These results suggest that studies of peritoneal cell-endotoxin interactions do not accurately describe the physiologic process within the liver, the major site for the clearance of gut-derived endotoxins.     

Lysosomal alterations in hypoxic and reoxygenated hearts. I. Ultrastructural and cytochemical changes.

Rabbit hearts perfused under hypoxic conditions underwent progressive subcellular damage, which becomes irreversible by one hour. During the first 20 minutes of perfusion, minor dilation of mitochondria and condensation of nuclear chromatin were the only salient features of cell injury. By 40 minutes moderate mitochondrial swelling was evident in hypoxic myocytes. Moreover, an increase in degenerating mitochondria and autophagic vacuoles was apparent. Reperfusion after either 20 or 40 minutes of hypoxia restored contractility, and injured myocytes underwent a cellular repair process that involved a dramatic increase in lysosomal autoplagy. One hour of hypoxia yielded irreversibly injured myocytes. Upon reoxygenation, some of these cells displayed typical changes of necrosis, but others apparently underwent an abortive repair process involving the formation of large, probably nonfunctional lysosomes. These observations suggest that lysosomal autophagy is important in the efforts at repair that cardiac cells initiate during and after hypoxia.     

Studies on mobilization of Kupffer cells in mice: II. Mobilization mediated by necrotic tissue injected intraperitoneally

Systemically acting mobilizing factors liberated from the necrotic centrilobular zone are suggested as a possible cause of the dramatic mobilization of Kupffer cells seen in mice following CCl₄ intoxication. The present work shows that Kupffer cell mobilization occurs in mice carrying necrosing Ehrlich ascites tumour cells intraperitoneally, but that other fixed reticulo-endothelial phagocytes (spleen and bone marrow) are not mobilized by the systemic effects of necrotic tissue.     

血清对分离枯否细胞细菌脂多糖摄取过程的影响

本文应用单克隆抗细胞抗细菌多糖抗体，利用免疫荧光及激光共聚焦扫描技术，观察了分离培养的枯否细胞有无血清条件下对二种不内发子结构细菌脂多糖摄取过程的影响，无血清状态下，枯否细胞加入Ｓ型脂多糖（Ｓ－ＬＰＳ）及Ｒ型脂多糖（Ｒｄ－ＬＰＳ）培育５ｍｉｎ后，其胞闪内抗脂多糖荧光强度显著高于空白对照水平。且Ｓ－ＬＰＳ培育后胞浆内荧光强度增高幅度高于Ｒｄ－ＬＰＳ培养后的增高幅度，１０％小牛血清状态下，Ｓ－ＬＰＳ培     

Ultrastructural characteristics of duodenal somatostatinomas. I. Somatostatinoma cells and Schwann-like cells

This report describes the general ultrastructural characteristics of somatostatinoma cells and Schwann-like cells in two cases of duodenal somatostatinomas. Duodenal somatostatinoma cells had common findings to endodermal cells, i.e. the granules of exocrine type, intracytoplasmic canaliculi, microvilli, small clear vesicles and others. This may suggest that endocrine cells of the gut are of endodermal origin. On the other hand, a few Schwann-like cells were discovered in the tumor nests. Schwann-like cells surrounding the nest of tumor cells were characterized by multilayered piled up cytoplasm, abundant microfilaments and others. This fact suggests that somatostatinoma has some relationship to the nerve.     

Cytoplasmic vacuoles of Rous virus transformed cells are organelles involved in cation uptake.

Cytoplasmic vacuoles induced during transformation of cells by Bryan strain Rous sarcoma virus (RSV-BH) have been studied using the cationic dye, neutral red(NR). Both the rate of uptake and the accumulation of NR are greater in RSV-BH transformed cells than non-transformed cells however, uptake was greater in vacuolated than in non-vacuolated cells, whether or not they were transformed. The NR was incorporated into pre-existing vacuoles in the absence of cytoplasmic staining, suggesting the existence of direct channels from the cell surface to the vacuoles. Other low mol. wt. cationic dyes could also be incorporated into vacuoles, although those with branched structures or cationic weights greater than 330 were excluded. No anionic dyes were incorporated. Infection of cells with a virus mutant, RSV-BH-Ta, induces temperature-dependent vacuolization. After a shift to the vacuole-permissive temperature, vacuoles developed at different rates and with morphological variations with different cations. Vacuoles which had formed in the presence of several cations, (K+, Rb+, tris+, choline+) failed to disappear when cells were incubated at a temperature sufficient to revert vacuoles formed in Na+-containing medium. No short-term effects of Cl-replacements (Br-, I-, or SO2-4) on vacuolization or reversal were observed. The results suggest that these vacuoles are organelles involved in cation uptake. A possible function for these organelles in RSV-BH induced malignancy is discussed.     

Biochemical identification of cutaneous leishmanias by analysis of kinetoplast DNA. I. Ultrastructural and buoyant density analysis

Ultrastructural analysis has been carried out on three Leishmania isolates which are proven causal agents of human cutaneous Leishmaniasis, L. tropicamajor, L. aethiopica and a unidentified species, Leishmania SP48. No significant differences in submicroscopic morphology have been found in thin-sectioned organisms from the three isolates. Extensive plate cristae have been observed within the mitochondria and connections between the rim of the kinetoplast nucleoid and the inner mitochondrial membrane noted.Kinetoplast DNA (kDNA) has been isolated from these isolates and from L. tarentolae and examined by protein monolayer spreading and darkfield electronmicroscopy. The basic molecular arrangement of isolated kDNA in the form of 5 μm networks of 0.28-0.3 μm mini-circles with long looped DNA in the interior and at the periphery of networks is similar in all isolates. Minor differences between L. aethiopica and SP48 compared with L. tropicamajor have been observed. The kDNAs of L. aethiopica and SP48 are identical morphologically.Buoyant density analysis has shown that kDNA from L. aethiopica and SP48 have identical values and these are different from the value for L. tropicamajor. The finding of similar buoyant densities for kDNA from L. tropicamajor and L. tarentolae also imply a sequence homology by this criteria which is refuted by the results given in the following paper.The results given in this and the following paper (Arnot, D.E. and Barker, D.C. (1981) Mol. Biochem. Parasitol. 3, 47–56 indicate that the unknown Leishmania SP48 is very closely related to, if not identical with, L. aethiopica. This finding is consistent with the clinical and ecological facts known for the organism SP48.     

Increased calcium uptake by muscle mitochondria of cold-acclimated rats.

Skeletal muscle mitochondria of cold-acclimated rats have an altered morphology that is related to the occurrence of nonshivering thermogenesis. The transport of calcium by these mitochondria was studied in a search for an alteration in an energy-dissipating mechanism which might be related to the altered morphology and to the altered mode of thermogenesis in the cold-acclimated animal. The rates of calcium uptake, of calcium-stimulated respiration, and of state 4 respiration after calcium uptake were increased in the altered mitochondria. The capacity to accumulate calcium without phosphate was increased, whereas with phosphate all the calcium was removed from the medium and no difference in total uptake was seen. Spontaneous release of calcium was greater but sodium-induced release was unchanged. No effect of cyclic AMP or prostaglandin E1 on release of calcium was seen. The increase in rate of calcium uptake occurred gradually during the first 3-5 wk of acclimation to cold. The results are considered to give some support to the hypothesis that adaptive changes in the mitochondrial calcium transport cycle in skeletal muscle occur during acclimation to cold.     

Mutations affecting immunoglobulin light chain secretion by myeloma cells. I. Functional analysis by cell fusion.

Two clones of MOPC 315 cells have been selected which synthesize but do not secrete Ig lambda light chains. These clones were analyzed by fusion with a cell line synthesizing and secreting kappa chains. Conditions were established for recovery at high frequency (approximately 10(-3)) of spontaneously fused, viable hybrid cells. The resulting hybrid cell lines synthesized both kappa and lambda chains but secreted only kappa chains. Hybrid cells produced by fusion of a lambda-secreting clone of MOPC 315 with the kappa-secreting cell line were also isolated and shown to synthesize and secrete both kappa and lambda chains. These results suggest that the nonsecretion of lambda chains was not due to a defect the secretion mechanism of the variant cells. A more likely alternative is that the lambda chains in the variant cell lines were structurally altered to a form which could not be secreted.     

Interaction of mistletoe lectin I with Kupffer cells and endothelial cells of mouse liver

The in vitro-formation of blebs by endothelial and Kupffer cells of the mouse liver after treatment with the toxic lectin I from mistletoe are demonstrated by means of scanning electron microscopy. The interaction of toxic lectins with nonparenchymal liver cells can be of importance concerning the elimination of these lectins in vivo.