AUTOPHAGIC VACUOLES PRODUCED IN VITRO : II. Studies on the Mechanism of Formation of Autophagic Vacuoles Produced by Chloroquine

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1–2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.     

Polysaccharide Complexes in Full-Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone-Induced Maturation

Immature full-grown oocytes of Pleurodeles waltlii contain large amounts of small electron-dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24–36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22–24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9–10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.     

Cytoplasmic vacuoles and bodies of the osteoclast

Summary Cytoplasmic vacuoles and bodies in the osteoclast (rat) were studied by electron microscopy. The vacuole-like structures (0.03–5 in diameter) may be classed as a) vacuoles b) coated vacuoles and c) invaginations. The cytoplasmic bodies vary in size from 0.02–3 in diameter and these may similarly be classed as a) light cytoplasmic bodies, b) dense cytoplasmic bodies, c) coated cytoplasmic bodies and d) cytoplasmic bodies containing inclusions. Both the cytoplasmic vacuoles and the bodies are limited by a triple layered membrane of about 91 Å in thickness. Their relationship to the lysosomal system and the role of this system in the osteoclast is discussed.This research was supported by the Danish Research Council. Grant no. 512–727 and 512–819.     

Electron Microscopy of Tissue Culture Cells Infected with Brucella abortus

Thin sections of hamster kidney tissue cultures were examined by electron microscopy over a 7-day period after infection with Brucella abortus 3183. Numerous bacteria and structures resembling L-forms were present both intracellularly and extracellularly after the first 24 hr of infection. Most intracellular microorganisms were enclosed by a cytoplasmic membrane, but in a few instances no limiting membrane was detected. After 4 to 7 days, fewer microorganisms were present, and most normal-appearing bacteria were intracellular, particularly in antibiotic-treated cultures. Structures typical of Brucella L-forms were extracellular at the latter time intervals. Several structures were observed in cells from infected cultures whose relationship to the infecting organisms is not known. These consisted of various membranous structures within cytoplasmic vacuoles, myelin-like structures surrounding occasional intracellular organisms, and small bodies present within vacuoles and extracellularly. The latter structures observed throughout the experimental period appeared to occur more frequently as the duration of the infection increased.     

ELECTRON MICROSCOPY OF THE HUMAN SYNOVIAL MEMBRANE

The structure of the lining cells at the surface of the synovial membrane facing the joint cavity has been studied by electron microscopy. The long cytoplasmic processes of these cells appear to be oriented toward the surface of the membrane, where they overlap and intertwine. The matrix of the lining cells contains dense material but no fibers with the periodicity of collagen. The lining cells are divided into two cell types or states of activity on the basis of their cytoplasmic contents. Type A is more numerous and contains a prominent Golgi apparatus, numerous vacuoles (0.4 to 1.5 microns in diameter) containing varying amounts of a dense granular material, many filopodia, mitochondria, intracellular fibrils, and micropinocytotic-like vesicles. Type B contains large amounts of ergastoplasm with fewer large vacuoles, micropinocytotic-like vesicles, and mitochondria. The probable functions of these cells are discussed in the light of current knowledge of the metabolism and function of the synovial membrane.     

Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function

Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 m (mean ± SEM) compared with 2 ± 0.12 m after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 m, p < 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.     

ULTRASTRUCTURE OF MUCOCYSTS AND PELLICLE OF TETRAHYMENA PYRIFORMIS

Tetrahymena pyriformis GL was fixed with glutaraldehyde and/or OsO₄ for a study of cytoplasmic ultrastructure. Many small vacuoles 0.05 to 0.5 µ in diameter were found to contain each a dense particle enveloped by a limiting membrane. This membrane is continuous with the membrane of the vacuole. The particles are irregular in shape and size, but similar to the mucocysts in the appearance of the matrix. It is suggested that they are the first morphologically distinguishable stages in the development of mucocysts. In the course of this development, amorphous material becomes crystalline with a longitudinal period of 150 A and a lateral period of 100 A. The mature mucocysts are rather uniform in size and have a spheroidal shape. During discharge, the crystalline pattern disappears and the mucocysts assume a spherical configuration. The inner limiting membrane of a mucocyst seems to disintegrate during the process of discharge while the outer membrane becomes continuous with the outermost pellicular membrane; the inner pellicular membrane is continuous with the cytoplasmic membrane. Rows of few to 15 or more microtubules were found either between the cytoplasmic membrane and the ectoplasmic layer (longitudinal fibrils) or underneath the ectoplasmic layer (transverse fibrils). The outer and inner pellicular membranes are uniformly spaced and connected by "cross-bridges." Details of these structures are described.     

The elastic cartilage in the normal rat epiglottis

Summary Chondrocytes of the rat epiglottis contain large amounts of glycogen and lipids, which often make the cells resemble fat cells. The content of lipids is interpreted as being related to the function of the cells. The membranes of some of the large vacuoles are stained with ruthenium red. The cells give rise to long cytoplasmic processes. As in hyaline cartilage the intercellular substance consists of a fine network containing proteoglycan granules together with thicker cross striated fibers. Furthermore elastic fibers are found, consisting of amorphous and microfibrillar parts. In the matrix, both lysosome-like granules and more or less empty vesicles are observed. Accumulations of a finely particulate electron dense material and of a translucent amorphous material containing membrane bound granules are found in some lacunae situated in the outer part of the cartilage. These accumulations are possibly related to the development of collagenous and elastic fibers.     

Phagocytic and motile properties of endothelial cells measured magnetometrically: effects of endotoxin

Endothelial cells lining the vasculature share some properties with macrophages and neutrophils in that they can take up material from the blood and are known to migrate, particularly during wound healing. We observed that endothelial cells isolated from bovine pulmonary arteries ingested magnetic iron oxide particles during culture in vitro. Using a non-optical, magnetometric method, we examined motions of magnetic-particle containing intracellular vacuoles. We demonstrated that these organelles move within endothelial cells, but at a slower rate than phagosomes within macrophages. Magnetometry was used to show that incubation with endotoxin (10 micrograms/ml) for 4 hr resulted in a decrease in cytoplasmic movement; yet the fluidity of the cytoplasm was increased, as measured by intracellular particle response to forced motion. We conclude that intracellular magnetic probe particles can detect vesicular motion in endothelial cells, and that endotoxin exposure can affect endothelial cells directly, altering their physical properties; these alterations precede ultrastructural evidence of cell death.     

Absorption of intraarticularly injected horseradish peroxidase in synoviocytes of rat synovial membrane: an ultrastructural-cytochemical study

The ability of type A and type S synoviocytes to absorb horseradish peroxidase (HRP) and the intracellular fate of this tracer were studied by electron microscopic cytochemistry. Different concentrations of HRP (0.1-5 mg/ml) were injected into the left knee joint of rats and at intervals ranging from 1 min to 24 hr after injection the synovial membrane was fixed and incubated for HRP. Type A synoviocytes showed a striking ability to absorb HRP at low concentrations. At 1 and 5 min after injection reaction product was localized in coated pits and coated vesicles (110 nm) as well as in smooth-walled vesicles, vacuoles, and tubules. At 15 min to 4 hr postinjection the lysosomal system became increasingly loaded with reaction product. At 24 hr after injection reaction product had disappeared. At higher concentrations of HRP similar observations were made in the A cells, but reaction product was still apparent in lysosomes at 24 hr postinjection. With respect to type S synoviocytes no reaction product was detected within these cells at any time interval after injection of low concentrations of HRP. However, at 5 min after injection of higher concentrations of HRP reaction product was localized in smooth vesicles and vacuoles mainly restricted to the large cytoplasmic processes facing the joint cavity. At 30 min to 4 hr postinjection the lysosomal system became progressively more loaded with HRP reaction product. At 24 hr after injection reaction product still remained in the lysosomal system. The present findings that type A and type S synoviocytes showed major differences with respect to endocytic capacity and cellular structures involved in absorption of HRP support the interpretation that the A and S cells represent two distinct types of cells and further suggest that endocytosis in these two types of cells serve different functions.     

HEMOGLOBIN UPTAKE BY RAT HEPATOCYTES AND ITS BREAKDOWN WITHIN LYSOSOMES

The peroxidatic activity of hemoglobin permitted visualization of its uptake by rat hepatocytes by means of the Graham-Karnovsky 3,3'-diaminobenzidine (DAB) procedure. Lysosomes were visualized by their acid phosphatase, β-glucuronidase, and glucosaminidase activities. When large doses of rat, cow, or human hemoglobin are intravenously injected, or when hemoglobinemia is induced by injection of distilled water, DAB-positive hemoglobin is engulfed by pinocytosis. Pinocytotic vacuoles become digestive vacuoles ("phagolysosomes") by fusion with lysosomes of the dense body type that have moved from their pericanalicular position. By 16–24 hr after even massive amounts of hemoglobin (400 mg/100 g), the protein is barely demonstrable in hepatocytes. At the lowest doses of injected hemoglobin (15 mg/100 g body weight), DAB-positive vacuoles are demonstrable only in the Kupffer cells.     

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediately after 3 hr feeding period. 2. The size of the autophagic vacuole and lysosome (dense body) compartments increased in both types of cells during 2-72 hr fasting parallel with decreasing serum insulin levels. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells.

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, '78) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.     

Intracellular transport of horseradish peroxidase in the absorptive cells of goldfish hindgut in vitro,with special reference to the cytoplasmic tubules

Summary This study was undertaken to determine whether the numerous cytoplasmic tubules (CT) in the apical cytoplasm of goldfish hindgut absorptive cells are directly involved in the endocytotic transport of macromolecules into the cells, or whether they are derived from the intracellular membrane components. The absorptive cells were exposed to horseradish peroxidase (HRP)-containing medium in organ culture and subsequently fixed and prepared for electron microscopy. Analysis revealed that 5 sec after exposure, many vesicular structures, including coated vesicles, were labelled with reaction product whereas almost all CT were negative. After a 1-min exposure, reaction product was detected in about 11 % of the CT, and thereafter, the percentage increased to about 95% after 15 min exposure. As labelled CT increased in number, the number of densely labelled vacuoles with attached CT also increased. CT connected to vacuoles with a peripheral margin of dense reaction product were always HRP-positive, whereas those connected to vacuoles which were not distinctly labelled were themselves also devoid of HRP reaction product. This indicated that the labelling of CT was closely associated with the labelling of the inner surface of the vacuolar membrane. These results indicate that CT are probably formed by a budding off from these vacuoles, rather than being directly involved in endocytosis.     

Plasma membrane shedding and colloid vacuoles in hyperactive human thyroid tissue

The ultrastructural appearance of colloid vacuoles, considered to be a typical sign of hyperactivity in the human thyroid gland, was studied in human thyroid tissue transplanted to nude mice and in human thyroid tissue fixed directly after surgical removal in patients with thyrotoxicosis. Transplanted normal thyroid tissue and toxic diffuse goiter (TDG) tissue was fixed by vascular perfusion with glutaraldehyde 5 or 12 weeks after transplantation. Light microscopic quantification showed that daily injections for 2 weeks of a gamma globulin fraction of patient sera containing thyroid-stimulating immunoglobulins (TSI) greatly increased the number of colloid vacuoles in both types of transplants. The vacuoles were mainly located in the periphery of the follicle lumen, giving the colloid a scalloped appearance. Electron microscopy of TSI-exposed tissue revealed, in addition to colloid vacuoles, the presence of large amounts of membrane material in the follicle lumen. Only sparse amounts of intraluminal membrane material were present in controls. The colloid vacuoles were almost invariably associated with such membrane material, which lined the border between the vacuole and the surrounding colloid. The intraluminal material consisted of spherical and elongated formations, each structure limited by a triple-layered membrane and often containing a dense interior. The elongated structures were often of the same dimensions as microvilli. The apical surface of follicle cells in TSI-exposed tissue expressed numerous microvilli, of which many showed a similar dense interior as the intraluminal membrane structures. The intraluminal membranes frequently showed, like the apical plasma membrane of the follicle cells, a positive reaction for peroxidase. Organelles, such as mitochondria, lysosomes or rough endoplasmic reticulum, were not encountered among the intraluminal membrane structures. These observations indicate that the intraluminal membrane material is derived from the apical plasma membrane of the follicle cells, presumably by shedding of microvilli. A similar association between colloid vacuoles and membrane material was also found in thyroid tissue from patients with thyrotoxicosis fixed directly at operation. It is suggested that the presence of membrane material in the follicle lumen precipitates the formation of colloid vacuoles in hyperactive thyroid tissue. The possible involvement of intraluminal membrane material in the development of microsomal autoantibodies in Graves’ disease, i.e. exposure and presentation of thyroid microsomal antigen (identical to thyroperoxidase) to the immune system, is discussed.     

OBSERVATIONS ON A TRANSIENT PHASE OF FOCAL SWELLING IN DEGENERATING UNMYELINATED NERVE FIBERS

This study describes the nature and time-course of a swelling phase during the degeneration of unmyelinated nerve fibers, as observed in highly organized cultures of rodent sensory ganglia. Observations were made on nerve fascicles after they were cut and during nutritional deprivation. About 12 hr after nerve transection, large, clear vacuoles appear throughout fascicles distal to the cut. These vacuoles are most numerous at 24 hr and then gradually subside; after 48 hr, only small granules mark the severed fascicles. Electron microscopy shows that the vacuoles are, in fact, massive focal dilations of unmyelinated axons. Similar focal dilations in unmyelinated axons are observed if cultures are not refed for 5–7 days; under these conditions glucose concentrations fall below 20 mg/100 ml and degenerative changes begin to appear in neuronal somas. If the gas-tight assembly is opened and the culture refed, there is rapid disappearance of axonal dilations (usually within 1 hr) and recovery of many of the damaged neurons. Cooling (4°C) prevents this reversal, suggesting that an active process is involved. It is postulated that the swellings result from the failure of active axolemmal ion-pumping mechanisms prior to loss of selective permeability in the axon membrane. The reasons for the focal nature of the swellings is unknown. A literature review indicates that a phase of focal swelling has frequently been observed during the degeneration of unmyelinated nerve fibers in vivo.     

Electron Microscopic Study of the Phagocytosis Process in Lung

Diluted India ink was instilled into the nasal cavity of mice and the lungs of some animals were fixed with osmium tetroxide at various intervals after one instillation. The lungs of other animals were fixed after 4, 7, 9, 16, or 18 daily instillations. The India ink was found to be phagocytized almost exclusively by the free alveolar macrophages. A few particles are occasionally seen within thin portions of alveolar epithelium, within the "small" alveolar epithelial cells, or within occasional leukocytes in the lumina of alveoli. The particles are ingested by an invagination process of the plasma membrane resulting in the formation of intracellular vesicles and vacuoles. Ultimately large amounts of India ink accumulate in the cell, occupying substantial portions of the cytoplasm. The surfaces of phagocytizing macrophages show signs of intense motility. Their cytoplasm contains numerous particles, resembling Palade particles, and a large amount of rough surfaced endoplasmic reticulum. These structures are interpreted as indicative of protein synthesis. At the level of resolution achieved in this study the membranes of this reticulum appear as single dense "lines." On the other hand, the plasma membrane and the limiting membranes of vesicles and of vacuoles often exhibit the double-line structure typical of unit membranes (Robertson, 37). The inclusion bodies appear to be the product of phagocytosis. It is believed that some of them derive from the vacuoles mentioned above, and that they correspond to similar structures seen in phase contrast cinemicrographs of culture cells. Their matrix represents phagocytized material. Certain structures within this matrix are considered as secondary and some of these structures possess an ordered form probably indicative of the presence of lipid. The possible origin and the fate of alveolar macrophages are briefly discussed.     

Biogenesis of multilamellar bodies via autophagy

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.     

Plasma membrane shedding and colloid vacuoles in hyperactive human thyroid tissue

The ultrastructural appearance of colloid vacuoles, considered to be a typical sign of hyperactivity in the human thyroid gland, was studied in human thyroid tissue transplanted to nude mice and in human thyroid tissue fixed directly after surgical removal in patients with thyrotoxicosis. Transplanted normal thyroid tissue and toxic diffuse goiter (TDG) tissue was fixed by vascular perfusion with glutaraldehyde 5 or 12 weeks after transplantation. Light microscopic quantification showed that daily injections for 2 weeks of a gamma globulin fraction of patient sera containing thyroid-stimulating immunoglobulins (TSI) greatly increased the number of colloid vacuoles in both types of transplants. The vacuoles were mainly located in the periphery of the follicle lumen, giving the colloid a scalloped appearance. Electron microscopy of TSI-exposed tissue revealed, in addition to colloid vacuoles, the presence of large amounts of membrane material in the follicle lumen. Only sparse amounts of intraluminal membrane material were present in controls. The colloid vacuoles were almost invariably associated with such membrane material, which lined the border between the vacuole and the surrounding colloid. The intraluminal material consisted of spherical and elongated formations, each structure limited by a triple-layered membrane and often containing a dense interior. The elongated structures were often of the same dimensions as microvilli. The apical surface of follicle cells in TSI-exposed tissue expressed numerous microvilli, of which many showed a similar dense interior as the intraluminal membrane structures. The intraluminal membranes frequently showed, like the apical plasma membrane of the follicle cells, a positive reaction for peroxidase. Organelles, such as mitochondria, lysosomes or rough endoplasmic reticulum, were not encountered among the intraluminal membrane structures. These observations indicate that the intraluminal membrane material is derived from the apical plasma membrane of the follicle cells, presumably by shedding of microvilli. A similar association between colloid vacuoles and membrane material was also found in thyroid tissue from patients with thyrotoxicosis fixed directly at operation. It is suggested that the presence of membrane material in the follicle lumen precipitates the formation of colloid vacuoles in hyperactive thyroid tissue. The possible involvement of intraluminal membrane material in the development of microsomal autoantibodies in Graves' disease, i.e. exposure and presentation of thyroid microsomal antigen (identical to thyroperoxidase) to the immune system, is discussed.