Iron storage in macrophages and endothelial cells. Histochemistry, ultrastructure, and clinical significance.

1 hour after i. v. infusion of colloidal iron in iron deficient subjects uniform phagosomal iron granules were observed in macrophages and endothelial cells of several organs. 7 to 10 days later transformation into ferritin coould be visualized in macrophages only. Now, these cells showed diffuse iron staining of the cytoplasm due to dispersed ferritin molecules. Polymorphous lysosomes contained densely packed particles from still unchanged ferric hydroxide to paracristalline ferritin. The macrophageal iron was mobilizable in few days to several weeks. The univorm lysosomal iron granules of endothelial cells disappeared after 1 to 2 years. Endothelial iron siderosis without previous i. v. iron application was a frequent finding in pernicious anaemia and iron overload of diverse origin.     

An experimental study on the pathway of iron transfer from macrophages to erythrocytes in rat liver

In order to reveal the pathway of iron release from macrophages, a 59Fe-labelled ferric hydroxide-potassium polyvinyl sulfate complex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with activated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the 59Fe-PVS injection. Histochemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the nonparenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron compounds and that the iron is supplied for erythroid cell proliferation.     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Endocytic pathways and time sequence of lysosomal transfer of macromolecules in cultured mouse peritoneal macrophages

Summary A double-labeling protocol was used to study endocytic pathways and lysosomal transfer of exogenous macromolecules in cultured mouse peritoneal macrophages. After pulse-chase labeling of lysosomes with horseradish peroxidase (visualized cytochemically), the cells were exposed to native, anionic ferritin for 0–45 min at 37° C and then analysed by transmission electron microscopy. The results show that ferritin binds to the plasma membrane, accumulates in coated pits, and is rapidly taken up in small, smooth-surfaced endocytic vesicles. The latter carry the ferritin molecules directly to lysosomes, recognized by their peroxidase labeling, or fuse with each other to form larger endocytic vacuoles (endosomes) which in turn fuse with and empty their content into lysosomes. The first signs of transfer of ferritin into the lysosomes were seen after 5–10 min of exposure and after 25–30 min most of the lysosomes were labeled. Union of ferritin-labeled and other lysosomes was also noted, suggesting that the contents of the lysosomes were spread within the lysosomal compartment by fusion-fission processes. It is concluded that a multiplicity of structures is involved in the uptake and intracellular transport of exogenous macromolecules in macrophages and that the time sequence of lysosomal transfer of the interiorized material is highly variable.     

Detection of a Fine Lamellar Gridwork After Degradation of Ocular Melanin Granules by Cultured Peritoneal Macrophages

In the present study we investigated by electron microscopy whether melanin granules derived from choroidal melanocytes and retinal pigment epithelium of cattle could be degraded in the phagolysosomes of cultured murine macrophages. It was found that degradation of ocular melanin is possible by the lysosomes of these macrophages. During degradation of the melanin granules an internal gridwork of fine concentric, highly ordered membranes, 3-4 nm thick, became visible. These membranes may represent remnants of the melanin polymer in the original melanosome or may result from self-assembly of degradation products. Early-stage melanosome-like structures also appeared during digestion of these melanin granules. Melanin granules that seemed to break down into smaller fragments without any visible internal structure were also observed.     

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.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phagocytized intracellular microsporidian blocks phagosome acidification and phagosome-lysosome fusion

This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITC)-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.     

Functional morphology of phagocytosing alveolar macrophages. Long-term electron microscopic and X-ray microanalytical investigations on the rat model

A single instillation of 1 ml iron dextran (containing 191.3 mg iron(III)hydroxide and 200 mg dextran) was administered under anaesthesia by a polyvinyl catheter into the lower lobe of the right lung in one hundred 4-week-old wistar rats. The animals were killed at intervals ranging between 1 min and 4 weeks. The lower lobe of the right lung was examined by light and electron (transmission and scanning) microscopy. In addition, X-ray microanalyses were performed on tissue sections in the transmission and scanning electron microscopes. The process of phagocytosis of iron dextran by alveolar macrophages can be subdivided into three stages, which we have termed the "phase of attachment" (from 1 to 5 min), followed by the "phase of phagocytosis" (from 5 to 20 min) and finally the "resident macrophage stage" (from 1 to 24 h). X-ray microanalysis shows a high phosphorus content even if iron dextran is concentrated on the surface of macrophages. Phagocytosis of particles between 15 and 40 A in size occurs within minutes, the particles being engulfed in phagosomes, which form as double-layered invaginations of the cell membrane into the interior of the cell. The fusion of phagosomes with lysosomes produces phagolysosomes (type 2 lysosomes) in which iron dextran is broken down into lamellar residual bodies. In these lamellar bodies X-ray microanalysis shows that in addition to abundant iron, there is a high phosphorus content, which may indicate the involvement of surfactant. Only 1 h after instillation, free particles of iron dextran can no longer be demonstrated in the alveoli, although a proportion of the iron dextran remains in resident macrophages (pulmonary tissue macrophages) and some is also found in splenic macrophages.     

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.     

Structure of frataxin iron cores: an X-ray absorption spectroscopic study

X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.     

Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.     

Phagocytized Intracellular Microsporidian Blocks Phagosome Acidification and Phagosome-Lysosome Fusion

This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITQ-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles

Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.     

AN ELECTRON MICROSCOPIC CYTOCHEMICAL STUDY OF MACROPHAGES DURING UTERINE INVOLUTION

Acid phosphatase was localized at the fine structural level in rat endometrial phagocytes during the period of postpartum involution. These cells showed intense phagocytotic and pinocytotic activities, which were accompanied by the development of abundant lysosomes. Phagosomes acquired their enzymatic complement by fusion with lysosomes; the same appeared to be true in the case of pinocytotic vesicles, but, because of the small size of these vesicles, this point could not be established with certainty. Digestion within some phagolysosomes led to the formation of electron-lucent vacuoles containing solubilized products. Other phagolysosomes showed accumulation of lipid residues in the form of droplets and myelin figures, and the structures acquired the appearance of residual bodies. In many macrophages, overfeeding led to the formation of unusually large numbers of phagolysosomes, which occupied almost the entire cytoplasm with exclusion of other cell organelles. In these cells the presence of abundant lead deposits, apparently free in the cytoplasm suggested an intracytoplasmic release of hydrolases.     

Electron Microscopy of Hemosiderin: Presence of Ferritin and Occurrence of Crystalline Lattices in Hemosiderin Deposits

Injections of hemoglobin were given to rats in order to produce hemosiderosis, and selected hemosiderin granules in sectioned cells of proximal convoluted tubules were studied by means of electron microscopy. When examined at high resolution, many of the dense particles that were present in hemosiderin granules proved to have the structure that characterizes the iron hydroxide micelles of molecular ferritin. In some hemosiderin deposits the dense particles formed lattices similar to those present in sections of crystalline ferritin. Such ordered arrangement of dense particles was encountered inside as well as outside of the cytoplasmic organelles for which the name "siderosomes" has been proposed previously, and which may be derived from mitochondria. Study of hemosiderin granules in hepatic parenchymal and reticuloendothelial cells of human beings yielded similar results. The findings confirm the inference that ferritin is a component of hemosiderin, and they indicate that some of the so called hemosiderin granules are crystals of ferritin.     

Unmasking and redistribution of lysosomal sulfated glycoconjugates in phagocytic polymorphonuclear leukocytes

Rabbit heterophil and human neutrophil primary granules contain sulfated glycosaminoglycans (GAGs) and acid phosphatase, which can be readily stained in immature but not mature lysosomes. To determine whether this loss of staining represents masking of reactive components or removal of these components, we examined rabbit heterophils to see if high-iron diamine (HID)-reactive sulfate and acid phosphatase staining reappears in phagocytic vacuoles. Rabbit heterophils, obtained by peritoneal lavage, were incubated in vitro with latex beads or Pseudomonas aeruginosa for 15-60 min. Pre-embedment HID staining was enhanced in thin sections of unosmicated specimens with thiocarbohydrazide and silver proteinate (TCH-SP). Phagocytosis of latex beads or bacteria was progressively more prominent with time. Primary granules that were degranulated or in the process of degranulating into phagocytic vacuoles demonstrated intense sulfate staining with large (13 +/- 7 nm) HID-TCH-SP stain deposits. Smaller (6 +/- 1 nm) HID-TCH-SP stain deposits were present in tertiary granules, which were less frequently observed degranulating into phagosomes. Acid phosphatase staining was most intense during early phagolysosome formation. HID-TCH-SP staining was also observed in extracellular degranulated lysosomal matrices and on the surface of many peritoneal heterophils. These results indicate that loss of sulfate staining in mature heterophil granules is the result of masking by intragranular substances rather than of removal, and that these components may be unmasked during phagocytosis and/or redistributed to the cell surface after exocytosis.     

In Vivo and In Vitro Localization of Leishmania within Macrophage Phagolysosomes: Use of Colloidal Gold as a Lysosomal Label1

The interaction of Leishmania with lysosomes within macrophages in vivo has been investigated. Lysosomes labeled with colloidal gold in vivo fused with phagocytic vacuoles containing Leishmania amastigotes within the macrophages of infected footpad tissue of BALB/c mice. This localization of Leishmania within macrophage phagolysosomes in vivo is the first confirmation for any obligate intracellulaire protozoon that parasite-lysosome interactions in vitro occur in vivo.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.