Early atherogenesis in the White Carneau pigeon. III. Lipid accumulation in nascent foam cells.

The role of lysosomes in aortic atherogenesis in White Carneau pigeons was examined by means of acid phosphatase cytochemistry. Foam cells were the major constituent of nascent atherosclerotic lesions in pigeons fed a 0.5% cholesterol diet for either 5 or 10 weeks. Seventy-four percent of foam cell lipid from animals at 5 weeks was in cytoplasmic droplets. The remaining lipid appeared in secondary lysosomes. After 10 weeks of cholesterol feeding, lysosomal lipid accounted for 73% of the lipid volume. The lipid accumulation correlated with increases in both size and number of lysosomes. An average of 2.4 lysosomes per 10(4) cu mu of cytoplasm was observed at 5 weeks. This value doubled by 10 weeks. The average lysosome diameter also increased between 5 and 10 weeks from 2.2 mu to 5.75 mu. Concomitantly, the complexity of lysosomes increased from simple, spherical organelles at 5 weeks to complex, multichambered organelles at 10 weeks. In contrast, lipid storage within cytoplasmic lipid droplets did not change either in size or in number. These observations suggest that by 5 weeks lipid storage within cytoplasmic droplets was maximized, and continued increases in lipid stores occurred predominantly through lysosomal loading.     

Lysosomal alterations during coronary atherosclerosis in the pigeon: correlative cytochemical and three-dimensional HVEM/IVEM observations

Lysosomal changes have been implicated as one of the major factors contributing to the progression and complications of atherosclerosis, and recently foam cell formation has been correlated with increases in several acid hydrolases. To explore at the subcellular level relationships among lesion progression, cellular lipid accumulation, and lysosomal change, atherosclerotic lesions from hypercholesterolemic White Carneau pigeons have been studied through combined ultrastructural cytochemistry and stereo (three-dimensional) high-voltage electron microscopy. Lysosomal enzyme activity in the prelesion intima and in foam cells of early lesions was in discrete lysosomes of macrophage foam cells. Foam cell lipid at the early stages was primarily (72%) in cytoplasmic droplets, which formed a three-dimensional network with the small (0.25-0.8 microM in diameter), reaction-positive lysosomes suspended at the vertices of a cytoplasmic lattice that delineated individual lipid pools. Concomitant with lesion progression and increasing complexity, foam cell lysosome number, size, and complexity increased. The complexity was characterized by lysosome lipid accumulation (60% of cell lipid) and the fusion of lysosomes to form multilobulated organelles in which the acid phosphatase reaction product typically was circumferential to the lysosomal lipid core. The involvement of lysosomes climaxed in the more advanced region of lesions with foam cells in which the bulk of cytoplasmic volume was occupied by large (15-20 microM in diameter), multicompartmental, lipid-containing lysosomes. It is suggested that this progressive involvement of lysosomes is responsible for cell and tissue necroses characteristic of advanced lesions.     

Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester.

The subcellular sites of accumulation of cholesterol and cholesteryl esters in rabbit atheromatous cells, were investigated by morphologic and biochemical techniques. Electron microscopy of lipid-filled cells in situ in atheromatous aortas of cholesterol-fed rabbits revealed lipid accumulation in the cytoplasm as lipid droplets and within lysosomes in the form of lipid globules, membranous whorls, and crystals. When such cells were isolated from the rabbit aortas by enzymic digestion, and then treated with Flickinger's aldehyde fixative containing 0.2 per cent digitonin, characteristic digitonide-lipid complexes ("spicules") were observed in discrete sites of the cytoplasm distinct from the cytoplasmic droplets. If these cells were first stained cytochemically for acid phosphatase and then treated with digitonin-aldehyde fixative, enzyme reaction product was found associated with the spicules indicating that the lysosomes of the atheromatous cells possess digitonin-reactive lipid. Subcellular fractionation of isolated rabbit aortic foam cells by sucrose density gradient centrifugation demonstrated the coequilibration of most of the intracellular unesterified cholesterol with low density lysosomes. Some cholesteryl ester was also associated with low density lysosomes, although most was found in a lipid droplet fraction of very low density. Together the results indicate that in rabbit atheromatous cells, lysosomes are the site of accumulation of intracellular cholesterol in excess of that structurally associated with membranes and that both cytoplasmic droplets and lysosomes are depot sites for cholesteryl esters.     

Subcellular fractionation of arterial smooth muscle cells laden with lipid following incubation with low density lipoproteins and chloroquine

Lipid accumulation was induced in pig aortic smooth muscle cells in culture by incubating them with LDL and the lysosomotropic agent chloroquine, as an in vitro model of lipid accumulation in atherosclerosis. The cells were homogenized and subjected to a conventional low-speed centrifugation to prepare a postnuclear supernatant. This supernatant was then subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. This showed that in normal smooth muscle cells most of the cholesterol was in the plasma membrane. When postnuclear supernatants of cells that had been incubated with LDL, either with or without chloroquine, were applied to the density gradient, there was no change in the distribution of cholesterol. The distribution of cholesterol became denser, however, when postnuclear supernatants of cells that had been incubated with chloroquine alone were studied, whereas the equilibrium density of the plasma membrane was not increased to the same extent. The cholesterol in these postnuclear supernatants was probably contained in both the plasma membrane and in the lysosomes. The distribution of chloroquine in the density gradients was consistent with a lysosomal localization. Incubation with chloroquine alone preferentially reduced the activity of the lysosomal component of β-glucuronidase compared to its endoplasmic reticulum component. The ratio of the activity of the cytosolic to the mitochondrial forms of malate dehydrogenase in cells incubated with chloroquine, either with or without LDL, was also decreased. When the cells were incubated with LDL and chloroquine together, lipid droplets, at least some of which were membrane-bound, and large autophagic vacuoles developed in the cells. When homogenates of these cells were subjected to low-speed centrifugation to prepare a postnuclear supernatant, all of the large autophagic vacuoles were sedimented into the nuclear fraction, together with most of the cholesteryl ester and chloroquine and most of the acid hydrolase activities. Therefore whole homogenates of smooth muscle cells, rather than postnuclear supernatants, were applied to the sucrose density gradients. When the cells had been incubated with LDL and chloroquine together, most of the cholesteryl ester and chloroquine and part of the nonesterified cholesterol were then found to be associated with lysosomes. These lipid-laden lysosomes probably correspond to the autophagic vacuoles seen in the cells by electron microscopy. The density of the lipid-laden lysosomes was increased considerably compared to the lysosomes of normal cells. This may have been due to either the high chloroquine content of the lysosomes, to an enhanced permeability of the lysosomal membrane to sucrose, or to the presence of partially degraded cell organelles within the lysosomes due to the autophagy caused by chloroquine. The distributions of cholesterol and cholesteryl ester had a small shoulder of low density, which may possibly correspond to the lipid droplets seen within the cells by electron microscopy. Thus, when large-scale lipid accumulation is induced in smooth muscle cells by incubating them with LDL and chloroquine together, the large majority of the cholesterol and cholesteryl ester that accumulates in the cells is contained within the autophagic vacuoles rather than within the lipid droplets.     

Macrophages in brain tumours induced transplacentally by N-ethyl-N-nitrosourea in rats: an electron-microscope study.

The fine structure of macrophages has been studied in experimental brain tumours induced transplacentally in BD-IX rats by a single intravenous injection of 30 mg of N-ethyl-N-nitrosourea per kg of body weight on the 15th day of gestation. The tumours, depending on their localisation and size, caused various lesions in the brain, namely axonal degeneration, loss of myelin, oedema, haemorrhage and cell necrosis. The tumours and the resulting alterations elicited a strong reaction by macrophages: activation of microglial cells in situ and infiltration of the brain by leucocytes, chiefly by monocytes. Since both microglial cells and monocytes underwent morphological changes, it was difficult, or impossible, to establish the origin of these reacting cells. In a few cases, however, microglial cells and monocytes could be distinguished; this indicated that microglial cells were still being activated and leucocytes were still entering the brain. Various stages of activity of macrophages have been described: the number of lysosomes and cytoplasmic inclusions were thought to indicate activation, phagocytosis and repletion. Activation is characterised by an increase of lysosomes and frequent cell divisions. Phagocytic activity is accompanied by the appearance of inclusions which varied in different lesions: protein-like material in oedema, remnants of erythrocytes in haemorrhages and myelin-lamellae with lipid droplets in demyelination. These various inclusions were frequently present in the same cell, since the different lesions not uncommonly occurred at the same time. In the stage of repletion macrophages contained mainly lipid droplets and unidentifiable debris in their abundant cytoplasm and thus corresponded to the compound granular corpuscles.     

TWO MALE SIBLINGs' CASES OF SPECIFIC LIPIDOSIS FOUND BY ELECTRON MICROSCOPY

Biopsy materials obtained from cervical lymph nodes and liver tissues of two male siblings, 5 years and 10 months old and 1 year and 11 months old, were studied electron microscopically. The results were summarized as follows: 1) Marked accumulation of lipid droplets, probably neutral fat, and cholesterol clefts in reticulum cells of lymph node, Kupffer cells and hepatic cells was the most prominent feature in these biopsies. 2) These lipids were found in both lysosomes and cytoplasmic matrices. Many of the lysosomes were considered to be autolysosomes or autophagosomes, because they contained fibrillar components, endoplasmic reticulum, glycogen particles and degraded organelles. It was suggested that these lipids might have been synthesized in lysosomes. 3) This disorder seems not to be included in the category of inborn lysosomal disease, and is considered as an unclassified one.     

The stereology of pulmonary alveolar macrophages after prolonged experimental exposure to tobacco smoke.

Morphometric analysis was performed on alveolar macrophages obtained by bronchopulmonary lavage of rats exposed to tobacco smoke for periods up to 60 consecutive days. Smoke dosage levels were adjusted so as to be comparable to those of human smokers. Size distributions were obtained for the lavaged macrophage populations of treated and age-matched control animals following 30 and 60 days of smoke exposure. Electron microscopic stereology was used to quantitate alterations in the ultrastructure of the same macrophage preparations. Several significant changes were observed in macrophage morphology following exposure to tobacco smoke, with an estimated mean cell volume more than twice that of controls following 60 days of smoking. After 30 and 60 days of exposure there was a 10- and 16-fold increase, respectively, in the volume density of cytoplasmic lipid inclusions. The surface to volume ratio of the cells and the lysosomal volume density were reduced in macrophages from smoke-exposed animals. A possible relationship between the incidence of lysosomes and the accumulation of cytoplasmic lipid in treated cells is discussed.     

Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.     

Lysosome lipid storage disorder in NCTR-BALB/c mice. II. Morphologic and cytochemical studies.

Electron-microscopic and cytochemical studies were carried out on tissues of NCTR-BALB/c mice. These mice are affected with a neurovisceral genetic disorder involving excessive tissue accumulation of lipid. Distinctive polymorphic intracellular inclusions, bounded by a membrane and containing lamellated bodies, were found in many cells of liver, spleen, lung, kidney, intestine, lymph nodes, and brain. The inclusions transformed reticuloendothelial cells into massive foam cells. Acid phosphatase cytochemical studies performed on sections of liver demonstrated that the inclusions were lysosomes. Fixation of liver in the presence of digitonin produced "spicules" in the inclusions characteristic of digitonin-cholesterol complexes. Clefts of cholesterol crystals were seen in the inclusions in liver, spleen, and lung. We conclude that the NCTR-BALB/c mice are affected by a lysosome lipid storage disease and that cholesterol is a major storage product.     

Arterial prostaglandins and lysosomal function during atherogenesis. II. Isolated cells of diet-induced atherosclerotic aortas of rabbit

This report validates and expands further the interpretation of our findings on prostaglandins and lysosomes in rabbit aortic homogenates (see paper I of this series) to enzymatically isolated and separated aortic cell populations during atherogenesis. Evidence is provided by which isolated arterial cells may be considered representative of in situ increases of diseased aortic tissue prostaglandin I2 and E2 levels, as well as lysosomal acid hydrolase activities and total cholesterol content based on DNA. Increasing latency of aortic lysosomal N-acetyl-beta-glucosaminidase activity was confirmed and correlated with increasing severity of atherosclerosis, in parallel to increasing levels of prostaglandin I2 but not increasing levels of prostaglandin E2. Ultrastructural observations also confirmed aortic intracellular lipid accumulation within lysosomes and as lipid droplets. Consistent with these relationships, separated low density, lipid-filled aortic cells were especially increased in total (197%) and latent (15%) lysosomal acid hydrolase activities, catalase activity (274%), total cholesterol (151%), and in both prostaglandin I2 (67%) and E2 (325%) levels based on DNA, as compared to control aortic cells or more normal-appearing high-density diseased aortic smooth muscle cells; high-density diseased aortic cells were increased in prostaglandin E2 but similar in latent acid hydrolase activity compared to control aortic cells. Since the total cholesterol content of rabbit atherosclerotic aortas was evidenced more intracellularly (75%) than extracellularly (25%) in this study, the association of increased prostaglandin I2 and E2 levels with low-density lipid-filled cells suggest the participation of these prostaglandins in the genesis of aortic foam cells during arterial lipid accumulation in rabbit atherosclerosis. The association of increasing prostaglandin I2 levels and increasing latent lysosomal N-acetyl-beta-glucosaminidase activities also implicates a possible relationship between this prostaglandin and lysosomal membranes of aortic cells, either primary or secondary to intralysosomal lipid accumulation.     

Eosinophilic granular cells in a cryptorchid testis

Intratubular cells with numerous refractile eosinophilic cytoplasmic granules were identified in a cryptorchid testis from a 17-year-old boy. The staining properties of the granules and the ultrastructural features were studied. The accumulation of lipid or lysosomal inclusions accounted for the observed cytoplasmic granules. These granular cells were probably altered Sertoli's cells, the exact nature and significance of which need further study.     

Ultrastructural localization of peroxidase in atherosclerotic lesions of pigeons.

Atherosclerotic lesions are known to have metabolic alterations which are associated with progressive lipid accumulation. Among the changes, lysosomal enzyme activity has been extensively characterized and at the ultrastructural level has been correlated with the amount of foam cell lipid. In a fashion paralleling lysosomal change, artery wall peroxidase activity is also altered during disease progression. The present study focuses upon the ultrastructural localization of peroxidase activity in atherosclerotic lesions of the aorta and coronary arteries from White Carneau pigeons fed a cholesterol-supplemented (0.3%) diet for 3 years. This resulted in fibrous lesions, rich in smooth muscle cells. The birds were necropsied by perfusion fixation, and peroxidase cytochemistry was carried out using the diaminobenzidine reaction. Peroxidase activity was found within endothelial cells and smooth muscle cells in both the media and intima, but cytochemically demonstrable activity was not found in macrophage foam cells. Peroxidase was localized within the nuclear envelope and endoplasmic reticulum, especially within cells that had lipid inclusions. The degree of peroxidase positivity varied within and among the arteries. In nonlesion regions of the aorta 20% of medial smooth muscle cells was peroxidase positive; the value for coronary artery smooth muscle cells was less. The peroxidase activity within aortic lesions was increased with 44% of intimal smooth muscle cells being positive. Notably, 85-90% of the lipid-containing intimal smooth muscle cells were positive. In contrast, intimal smooth muscle cells in the coronary artery lacked peroxidase reaction product, even in cells containing lipid. We conclude from these studies that aortic lesions contain a cytochemically differentiated subset of lipid-containing, peroxidase-positive smooth muscle cells; but coronary lesions lack a comparable subset of smooth muscle cells.     

Lipid storage disease: Part II. Ultrastructural pathology of lipid storage cells in sphingolipidoses

The ultrastructural pathology of the stored materials in lipid storage cells, particularly of macrophagic nature, in various disorders of sphingolipidosis was investigated. Cell morphology of the lipid storage cells was largely divided into two groups; one had peculiar cell morphology, such as Gaucher cells or globoid cells, and the other showed the appearance of foam cells. These cytological characteristics of the lipid storage cells were closely related to the ultrastructural configuration of lipid storage inclusions. By transmission electron microscopy, the fundamental structures of the stored materials were classified into two types; tubular and lamellar. The tubular structures were formed by accumulation of ceramide or monohexosyl ceramide, whereas the lamellar structures were formed by accumulation of larger sphingolipids than monohexosyl ceramide. These tubular structures were proven to consist of multilayers of lamellae, which are considered fundamentally similar to the lamellar structures. Almost all the lipid storage inclusions are considered to be of lysosomal origin, because of their encirclement by a single unit membrane and localization of acid phosphatase activity, and participation of heterophagic or autophagic mechanisms as for the development of the inclusions may be noted. Besides, the occurrence of secondary lipid storage was pointed out in some disorders of sphingolipidosis.     

Oncocytic adrenal cortical tumor with cytoplasmic inclusions and hyaline globules

Adrenal cortical tumors, particularly oncocytic tumors, have been reported to contain a variety of intracytoplasmic and intramitochondrial inclusions. Oncocytic cortical tumors can also morphologically mimic pheochromocytomas. We report an unusual, partially oncocytic cortical neoplasm with nesting architecture, intranuclear inclusions, and hyaline globules reminiscent of pheochromocytoma, together with numerous, small, brightly eosinophilic, periodic acid-Schiff-positive cytoplasmic inclusions and typical cytoplasmic lipid droplets. Ultrastructural study revealed oncocytes containing numerous mitochondria with intramitochondrial crystals and lipid droplets. Immunohistochemistry and immunoblots were utilized to further characterize the tumor. Immunohistochemistry demonstrated immunoreactivity of both the eosinophilic inclusions and the hyaline globules for adipose differentiation-related protein (ADRP), which is one of a group of proteins associated with storage of neutral lipids in many cell types. Immunoblots confirmed the presence of ADRP and demonstrated an imbalance between ADRP and perilipin, another neutral lipid-associated protein, in tumor tissue compared to normal adrenal cortex. The findings suggest that mitochondrial dysfunction in oncocytic cortical tumors may lead to abnormal processing of proteins related to the lipid-storing functions of the adrenal cortex, resulting in unusual cytoplasmic inclusions and extracellular globules resembling the globules in pheochromocytomas. The finding of ADRP as a constituent of inclusions in adrenal cortical tumors has not been previously reported.     

Induction of fatty streak-like lesions in vitro using a culture model system simulating arterial intima.

In this study a two-compartment culture model of arterial intima was used for the in vitro induction of fatty streaklike lesions. The apparatus consisted of upper and lower compartments separated by a human amnion membrane stretched between them. Human umbilical vein endothelial cells (HUVECs) were cultured to confluence on the stromal surface of the amnion membrane. Maximal migration of blood mononuclear cells (MCs) through the HUVEC monolayer in response to a f-Met-Leu-Phe gradient was observed at 10(-8) mol/l; the migration was 3.29 times greater than that observed under the condition of random migration (control). In the study of MC transformation into lipid-laden cells in the amnion membrane (foam cell formation in 'arterial intima'), 10(6) MCs were incubated, in the presence of freshly prepared low-density lipoprotein (LDL; 100 microgram/ml). The lipid loading of MCs was time dependent. After 12 hours' incubation, 39% of the MCs that migrated into the amnion membrane contained a small number of lipid droplets, whereas the remaining 61% showed no lipid droplets. Only 1.7% of the cells contained a high number of lipid droplets in the cytoplasm and took on the appearance of foam cells. With time, the number of lipid-laden cells and the amounts of intracytoplasmic lipid droplets gradually increased. At 72 hours after incubation, 65.4% of the MCs were loaded with lipid droplets, and 20.9% of them, an eightfold increase over 12 hours of incubation, showed a foamy cell appearance. Because MCs consist of 70% monocytes and 30% lymphocytes, about 93% of the monocytes were filled with lipid after a 72-hour incubation. Ultrastructural examination showed that lipid-laden cells took on macrophage characteristics, such as wide and heterogeneous cytoplasm, indented nuclei, and abundant lysosomes. A minority of the MCs in the amnion were considered lymphocytes; they had scanty cytoplasm, round nuclei with abundant heterochromatin, no lysosomes, and no lipid vacuoles. In conclusion, the formation of an in vitro fatty streaklike lesion is demonstrated, and this is reminiscent of in vivo human atherogenesis.     

LIPID STORAGE DISEASE : PART II

The ultrastructural pathology of the stored materials in lipid storage cells, particularly of macrophagic nature, in various disorders of sphingolipidosis was investigated. Cell morphology of the lipid storage cells was largely divided into two groups; one had peculiar cell morphology, such as Gaucher cells or globoid cells, and the other showed the appearance of foam cells. These cytological characteristics of the lipid storage cells were closely related to the ultrastructural configuration of lipid storage inclusions. By transmission electron microscopy, the fundamental structures of the stored materials were classified into two types; tubular and lamellar. The tubular structures were formed by accumulation of ceramide or monohexosyl ceramide, whereas the lamellar structures were formed by accumulation of larger sphingolipids than monohexosyl ceramide. These tubular structures were proven to consist of multilayers of lamellae, which are considered fundamentally similar to the lamellar structures. Almost all the lipid storage inclusions are considered to be of lysosomal origin, because of their encirclement by a single unit membrane and localization of acid phosphatase activity, and participation of heterophagic or autophagic mechanisms as for the development of the inclusions may be noted. Besides, the occurrence of secondary lipid storage was pointed out in some disorders of sphingolipidosis. ACTA PATHOL. JPN. 35 : 385–408, 1985.     

Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.     

Lysosomal degradation of cell organelles. I. Ultrastructural analysis of uptake and digestion of intravenously injected mitochondria by Kupffer cells.

An experimental model comparable to autophagocytosis is presented to evaluate lysosomal degradation of biologic membranes in vivo. This design takes advantage of the efficient phagocytic capacity of Kupffer cells. For this purpose, mitochondria were isolated from one rat liver (inbred strain) and subsequently injected intravenously into a series of rats, which were sacrificed at various time points. The uptake and intralysosomal digestion were examined by electron microscopy. Mitochondria were seen in the sinusoids and attached to the surface of Kupffer cells 1 to 3 minutes after injection. One or two mitochondrial profiles were trapped in cavelike structures often embraced by long hyaloplasmic pseudopods. By 5 and 10 minutes, the mitochondria were in single membrane-bound phagosomes within the Kupffer cells and early signs of mitochondrial alterations were present, e.g., loss of cristae and condensation. At 30 minutes and 1 hour the mitochondria were seen in large vacuoles which contained up to 20 profiles in a single section. The mitochondria showed evident signs of digestion such as membrane fragmentation and flocculent densities. By 4 hours and especially by 8 hours, most digestive vacuoles were laden with flocculent membrane debris. By 24 hours, the Kupffer cells showed multiple lysosomal structures which were irregular in shape and small. These often contained a number of lipid-like droplets of various electron densities and pentalaminar structures. By 2 and 5 days, the Kupffer cells had returned to their normal appearance although the lysosomal apparatus was still prominent. No uptake of mitochondria was seen in endothelial cells, fat-storing cells, or hepatocytes. It is concluded that lysosomes have an efficient capacity to digest mitochondria. Some lipid remnants are not completely or slowly degraded by Kupffer cell lysosomes but remain in "residual bodies" for up to 1 or 2 days.     

Significance of lysosomes in the morphogenesis of coronaviruses

Summary Virus-containing electron-dense membrane-bound cytoplasmic bodies are described in tracheal epithelial cells of chickens infected with Infectious Bronchitis Virus and in intestinal epithelial cells of swine infected with Porcine Epidemic Diarrhea Virus.Using silver-methenamine staining, phosphotungstic acid staining and acid phosphatase enzyme cytochemical staining of ultra-thin sections, these bodies were shown to be virus-containing secondary lysosomes and residual bodies.The accumulation of viral particles in the lysosomes is suggested to possibly represent an intracellular defense mechanism. However, no morphological alterations were found indicating a destruction of the viruses by the lytic lysosomal enzymes.With 4 FiguresPart of this work was presented as a thesis before the faculty of Veterinary Medicine at the State University of Gent.     

Ultrastructural lesions of ovine GM1 gangliosidosis.

Ovine GM1 gangliosidosis, an inherited disease of sheep with deficiencies of beta-galactosidase and alpha-neuraminidase, storage of GM1 ganglioside, asialo-GM1 and neutral long chain oligosaccharides in the brain, autosomal recessive inheritance, and histopathologic lesions typical of lysosomal storage diseases, has been described recently. Selected tissues from two sheep with the condition and an age-matched control were examined by transmission electron microscopy to characterize the ultrastructural lesions. In all central and peripheral neurons, the majority of the cytoplasmic space was occupied by membrane-limited enlarged bodies judged to be lysosomes, with a resultant displacement of normal organelles. The neuronal lysosomes usually contained stacks and concentric whorls of lamellae of stored material with a periodicity of 25 to 75 nM. Individual lamellae consisted of fine, multilayered (three to 10, and occasionally more) bands. Less commonly, enlarged neuronal lysosomes contained fibrillogranular or electron dense material. Central nervous system microglia and peripheral nervous system satellite cells had less extensive storage of similar material within enlarged lysosomes, whereas oligodendrocytes, astrocytes, and Schwann cells were relatively unaffected. Hepatocytes and renal epithelial cells also had storage of less quantity than neurons, but within even larger lysosomes. In contrast to neuronal storage material, visceral storage consisted of vesicles containing fibrillogranular or electron dense material within a mostly electron lucent matrix with only occasional lamellae. Kupffer cells and macrophages from bone marrow were affected similarly to but less severely than hepatocytes and renal epithelial cells, whereas hematopoietic cells and chondrocytes were unaffected. Both neuronal and visceral storage were evident, but the neuronal storage was much more extensive.(ABSTRACT TRUNCATED AT 250 WORDS)