In vivo monastral blue-induced lamellar-bodies in lysosomes of pulmonary intravascular macrophages PIMs of bovine lungImplications of the surface coat.код для вставкиСкачать
THE ANATOMICAL RECORD 234:223-239 (1992) In Vivo Monastral Blue-Induced Lamellar-Bodies in Lysosomes of Pulmonary lntravascular Macrophages (PIMs) of Bovine Lung: Implications of the Surface Coat ONKAR S. ATWAL, KANWAL J. MINHAS, B A U I T S. GILL, AND PRAHLAD S. SANDHU Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada ABSTRACT We previously reported that the pulmonary intravascular macrophages (PIMs) of sheep, goat, and calf lung contained a heparin and a lipolytic lipase sensitive surface coat by using tannic acid as a component of paraformaldehyde-glutaraldehyde-basedfixative. The implication of this sensitivity was that the surface coat was predominantly comprised of lipoprotein-like substance. In this study we report that monastral blue (MB) used as a vascular tracer interacted with the coat globules and lost its original particulate appearance. Its precise localization in the PIMs was in combination with altered macromolecules of the surface coat in the form of lipid droplets, which conformed to the conventional view of neutral lipids. In contrast, pigment particles examined in their native state resembled metalic particles as electron-dense eliptical rods. The lipid droplets were subsequently internalized through endocytic route and found their access into the lysosomal compartments of PIMs at the electron microscopic level. Lamellar bodies (LLBs) arose from the lysosomal matrix after the entry of lipid droplets in the secondary lysosomes. Acid phosphatase activity was located in secondary lysosomes as well as in endosomes. These observations suggest that coat granules of the PIMs acted as a carrier of exogenous MB particles to deliver the complex to the lysosomal compartment where partial digestion lead to the formation of lamellar bodies. The implications of MB (cationic dye) as a vascular tracer for studying phagocytic index of PIMs in the light of their coat and the rapid development of LLBs are discussed. It is proposed that MB by initially combining with the surface coat provokes mobilization of intracellular lipid pools. In this way metabolism of vasoactive lipid in the PIMs is stimulated to influence the dynamics of pulmonary circulation in the calves. o 1992 Wiley-Liss, Inc. Key words: Pulmonary intravascular macrophages, Bovine, Monastral blue, Lysosomes, Lamellar bodies, Lipidosis Monastral blue (copper phthalocyanine-CuPc) is a aqueous suspension of copper phthalocyanine in 0.85% highly coloured water-insoluble pigment that can be sodium chloride solution. It is assumed that this susconverted into water-soluble dye by introducing solu- pension is neither toxic nor metabolized and is easily bilizing groups like sulphonic acid, carboxylic acid, and identified in tissue sections in the form of intensely chloromethyl into its molecule (Pearse, 1985). One of blue inclusions within the phagocytic cells. It is also its soluble derivatives such as Alcian blue has been claimed that MB particles can be easily identified at employed as a specific stain for mucins (Steedman, the electron microscopic level (Joris et al., 1982; Alber1950). Luxol Fast blue, which is an amine salt of sul- tine and Staub, 1986; Staub, 1989). Several studies have provided experimental proof phonated CuPc, is used in lipid histochemistry. According to Pearse (1985), CuPc technique lacks specificity that a functional population of pulmonary intravascuwithin the broad groups of lipids, even though these lar macrophages (PIMs) is present in some animals, particularly the ruminants (Atwal and Saldanha, moieties are stained with a considerable success. In recent years monastral blue (MB) is sought as a 1985; Warner and Brain, 1986; Brain, 1988; Atwal et vascular tracer dye in the absence of biological ink al., 1989). A recent study has described morphologic (Joris et al., 1982).In the past, India ink as a biological ink was used especially to study the kinetics of phagocytic system (Bowden and Adamson, 1978). Monastral Received April 26, 1991; accepted February 17, 1992 blue with a dye component of -90% is available as 3% 0 1992 WILEY-LISS, INC. 224 O.S. ATWAL ET AL. features of human PIMs that appeared in samples of lung tissue from patients undergoing thoracotomies for excision of non-infectious diseases (Dehring and Wismar, 1989). In general, PIMs satisfy major morphological, cytochemical, and functional criteria for macrophages including the most important criterion of marked capacity for phagocytosis (Warner and Brain, 1984; Miyamoto et al., 1988; Sorokin and Hoyt, 1987; Atwal et al., 1989). Our recent study by using tannic acid as a component of paraformaldehyde-glutaraldehyde-based fixative revealed the presence of an electron-dense coat on the surface of the cell membrane of the PIMs in sheep, goat, and cattle. The coat was organized in the form of a linear chain of spherical globules with a consistent periodicity created by the intervening translucent space between the individual granules. The surface coat disappeared after heparin infusion as well as after enzymatic digestion with lipolytic lipase in vitro, suggesting that the surface coat was probably lipoprotein (LDL) in nature (Atwal et al., 1989; Jassal, 1989). In phagocytic cells, the glycocalyx is somewhat complex and is subdivided into layers parallel to the cell membrane and influences endocytosis (Emeis and Barderoo, 1980;Emeis, 1976).In our study, the linear globular dense coat of the PIMs was not membrane bound but was separated by a translucent gap (lamina lucida) of an approximate width of 35-39 nm from the outer leaflet of the cell membrane (Atwal et al., 1989). The present study was designed to investigate the morphology of phagocytosis of monastral blue (MB)by the PIMs of bovine lung in perfused tissues in vivo primarily to ascertain if the hypothetical LDL-coat of the PIMs had any influence on phagocytosis of tracer particles. The LDL particle has been suggested as a carrier by which t o introduce therapeutic agents into cells, where the complex is accumulated as a substrate in the lysosomal fraction (Kreiger et al., 1979; Poznasky et al., 1989). In this work, we demonstrate that MB dye particles undergo mutual alterations in shape and electron density by complexing with granules of the surface-coat of the PIMs of the bovine lung. The altered globules of the coat appeared very much like lipid droplets a t the surface and were subsequently internalized by the PIMs. Furthermore, endosomes carrying these lipid droplets fused with the lysosomes, where after hydrolysis the majority of the lipid droplets were converted into lamellar bodies. MATERIALS AND METHODS Animals Thirteen healthy male calves, primarily of dairy breeds, ranging from 4 months to 1 year of age were used in this study. Cattle were purchased from the local breeding farms and from Elora Research Station farm of the University of Guelph. Animals were acclimatized to the controlled isolated housing conditions for 4-6 days. Seven animals received 0.2 ml/kg bw of 3% particle suspension of monastral blue (Sigma Chemical Co.) in sodium chloride by a slow iv injection (rate not determined) and allowed to circulate for 2-3 rnin (4 animals) and 15-20 rnin (3 animals). Six animals received the same amount of normal saline intravenously, which was allowed to circulate for 2-20 min. All animals were overdosed with pentobarbital sodium. Clinical Symptoms During the experiment, calves were monitored for respiration and clinical signs indicative of pulmonary discomfort immediately after MB injection. Fixation and tissue preparation for electron microscopy The lungs were fixed after cannulating the trachea; 1,000-3,000 ml of fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.2 M HCl-Na cacodylate buffer, pH 7.4) was introduced through a tracheal cannula, and fixation in situ was carried out for 30 min. After fixation in situ, specimens were collected from the cranial, middle, and caudal lobes of right lung and diced into small pieces of about 1 mm3, and fixation was continued by immersion in the same fixative for 2 hr. Hepatic tissue was fixed only by immersion in the same fixative and for the same duration. Tissue taken from all three lobes of the right lung was postfixed for 90 min in 1.5% OsO, in 0.1 M HC1Na-cacodylate buffer (pH 7.4). Staining en bloc with 0.5% tannic acid in 0.1 M HC1-Na-cacodylate buffer was carried out for 30 min at room temperature. All tissues thus prepared were dehydrated in ethanol and propylene oxide and finally embedded in Jembed 812 resin (J.B.EM Services). Thick sections were stained with toluidine blue-basic fuchsin and were viewed in a Zeiss universal microscope. Photomicrographs were taken with Kodak Ektachrome 160 Tungsten films. Ultrathin sections were stained with both lead citrate and uranyl acetate. The stained sections were examined with JEOL-100s microscope a t 80 kV. Histochemical acid phosphatase assay Tissue was fixed with a solution of paraformaldehyde (2.0%) and glutaraldehyde (2.5%)in 0.1 M cacodylate buffer (pH 7.4) for 1 hr at 4°C. The tissue was washed with cacodylate buffer (pH 7.4) and rinsed twice with acetate buffer (pH 5.0) before incubation in the medium containing P-glycerophosphate and lead nitrate (pH 7.4). The tissue was subsequently postfixed, dehydrated, and embedded as above. RESULTS Clinical Signs Clinical signs of respiratory distress characterized by mild dyspnea appeared immediately after intravenous administration of MB in all animals of the treated group. Within 1-2 min, mild dyspnea developed into severe panting, which was later on accompanied by coughing, micturition, and general discomfort. The acute symptoms subsided within 20 min. Ultrastructureof PIMs (relationship between coat-globules and endocytic pathway) Detailed description of ultrastructural properties of PIMs of calf, sheep, and goat and their surface coat are described in separate studies (Atwal et al., 1989; Jassal, 1989). However, the present focus is on the coat globules and their relationship with organelles associated with endocytic pathway, more specifically with coated pits and vesicles. After using tannic acid as a component of paraformaldehyde-glutaraldehyde based fixative, subsequent staining revealed the presence of an electron-dense coat on the surface of the PIMs (Fig. LAMELLAR BODIES AND DYE IN PULMONARY INTRAVASCULAR MACROPHAGES 225 Fig. 1. A portion of a PIM of control lung shows a complete layer of surface coat comprised of electron dense globules farrows). Endocytic vesicles (V) contain material of the same morphology, as the coat globules. Lysosomes (Lys), mitochondria (M) and coated pit (small arrow) are depicted. F’t-platelet; E-endothelium; AS-alveolar space. Uranyl acetate and lead citrate staining. x 20,000. 1).I n contrast, no such coat was seen on the surface of alveolar macrophages (AM) (Fig. 2). The coat was organized in the form of a linear chain of spherical globules, with a consistent periodicity created by the intervening translucent space between individual globules. In situations where the coat was missing on the surface of PIMs, the coat globules were found internalized and appeared in different compartments of endocytic pathway such a s coated pits, coated vesicles, and endosomes (Figs. 2,3). These organelles were found missing in the case of alveolar macrophages. There was also a striking difference between the morphology of mitochondria of AM and PIM. In the later case, mitochondria1 matrix was prominently electron dense coupled with narrow cristae. The rich matrix may signify the presence of fatty acid oxidation enzymes in the PIMs. The mitochondria of AM, in contrast, showed a conventional appearance by having prominent cristae and moderate amount of intervening matrix. In several instances, there appeared to be one to one relationship between 226 O.S. ATWAL ET AL. Fig. 2.A longitudinal TEM view of capillary of a calf lung shows a PIM lying against endothelium (El of thick side of the interalveolar septum. The coat globules are absent from the surface and are lysing instead in the endocytic vesicles (large arrowheads). Mitochondria (M) of the PIM contain more electron dense matrix in contrast to the mitochondria1 matrix of alveolar macrophage (AM). Several coated pits (small arrowheads) and coated vesicles (arrows) are also seen in the PIM, whereas they are absent in the alveolar macrophage (AM). AS-alveolar space. Uranyl acetate and lead citrate staining. x 10,000. LAMELLAR BODIES AND DYE IN PULMONARY INTRAVASCULAR MACROPHAGES 227 Fig. 3.A high power view of an edge of a PIM shows several coated pits (arrows), isolated coated vesicle (arrowhead) and endosomes carrying globular units of internalized surface coat (open arrows). Plaques (short arrows) on structures similar to multivesicular body (MVB) and endosome reminiscent of the coating on coated pits and coated vesicles are also depicted. There appears to be one to one relationship between individual coat globule and individual coated pit and coated vesicle. Uranyl acetate and lead citrate staining. x 75,000. the individual coat globules and individual coated pits and coated vesicles (Fig. 3). Tracks of microtubules in bundle form existed wherever coated vesicles and endosomes were conspicuously distributed in the area adjacent to Golgi complex and lysosomes (Fig. 4). Light Microscopic Morphology of MB Uptake by PIMs and sections (1-2 pm), prior to ultrathin sectioning and stained with toluidine blue-basic fuchsin, showed inclusions of similar size and intense bluish hue in the PIMs and Kupffer cells. Kupffer Cells Ultrastructure of Phagocytosed MB Particles by Kupffer Cells Monastral blue (MB) was localized in the PIMs and Kupffer cells within 2-20 min of iv injection. Plastic Kupffer cells were easily recognized by their characteristic shape of radiating cytoplasmic processes, which 228 O.S. ATWAL Fig. 4. A TEM view of a PIM includes Golgi complex (C) with associated coated vesicles (arrowheads), a large tract of microtubules (double arrows) and lysosomes (Lys). A few coat globules are seen on the surface (arrows) but the majority of such globules are internalized ET AL. in the endosomes (Endos). Note medium electron-dense matrix of a mitochondrion. E = endothelium. Uranyl acetate and lead citrate staining. x 46,875. LAMELLAR BODIES AND DYE IN PULMONARY INTRAVASCULAR MACROPHAGES 229 Fig. 5. Portion of a Kupffer cell (kupff) shows several lysosomes (Lys) decorated with MB granules (arrowheads).Uranyl acetate and lead citrate staining. x 27,600. gave the cell a stellate shape, especially when processes were thick and contained several organelles like lysosomes, mitochondria, RER, light membranous vacuoles, and an occasional microbody. Phagocytosis of MB particles created distinct bulges of the cell body into the sinusoid. The individual tracer particles were easily recognized against the less dense matrix of the lysosomes. Each particle was rounded in appearance and retained its original shape even after 20 min of iv injection. In several instances the tracer particles filled the entire matrix of the lysosomes. There was no evidence of presence of the surface coat in Kupffer cells nor was there any adsorption of dye particles at the surface (Fig. 5). Effect of MB Particles on the Surface Coat of PlMs The MB particles that labeled the matrix of lysosomes of Kupffer cells as discrete, spherical, electrondense particles were instead seen in the form of electron-lucent oval bodies at the surface of the PIMs. 230 0,s.ATWAL ET AL. Fig. 6. A high magnification TEM view of a portion of PIM of a calf treated with monastral blue. Altered (lipid droplets; arrowheads) and usual globules (arrows) of the surface coat as well as endosomes (Endos) carrying lipid droplets are shown. Uranyl acetate and lead citrate staining. x 75,000. These electron-lucent bodies assumed the same spatial relationship with the cell membrane by replacing some of the globular units of the surface coat. After 2 min of MB injection, the majority of the globules of the coat had undergone modification not only in their staining affinity for tannic acid but in their shape as well and displayed an electron-lucent component that resembled the conventional appearance of neutral lipids. From their usual rounded (globular) shape, coat globules were converted into somewhat oval forms (Fig. 6). The individual lipid droplet was comprised of an electrondense crescentic line and an inner homogeneous electron-lucent core. The electron-dense line resembled a limiting membrane that could not be confirmed at higher magnifications. Modified globules would form sizable aggregates especially in the area of ruffling of cell membrane presumably the place of origin of large endocytic vesicles at 15-20 min post-MB injection (Fig. 7). At 2 min after MB injection, many usual coat globules were still seen at the surface as well as in distinct small endosomes after internalization. In Vitro Ultrastructure of MB Particles By electron microscopy on sections of plastic embedded pellets, the pigment particles appeared as electrondense rods that resembled metallic rods in their stain- LAMELLAR BODIES AND DYE IN PULMONARY INTRAVASCULAR MACROPHAGES 231 Fig. 7. A PIM of a calf treated with MB shows internalization of lipid droplets at the coated pits (double arrows). The smooth membrane rufflings and aggregations of altered and nonaltered coat globules are also depicted (arrowheads). Endosomes (thick double arrows) contain a mixture of droplets of vesicles. Uranyl acetate and lead citrate staining. x 50,000. ing properties. The electron-lucent component, which was consistently present in particles on the surface as well as in the phagolysosomes of the PIMs, was not noticeable in in vitro preparations. Large spherical amorphous masses, probably comprised of aggregated particles of the pigment after fixation with tannic acid, were seen in all sections examined by transmission electron microscopy (Fig. 8). Endocytosis of Modified Surface Coat At 2 min of exposure, cellular structures involved in the uptake and processing of lipid droplets could be 232 O.S. ATWAL ET AL. Fig. 8. A TEM view of dye particles after sections of pellet prepared from 3% suspension of monastral blue. Uranyl acetate-lead citrate staining. X 25,000. identified as coated pits, vesicles connected to plasma membrane by a neck, and small curved vacuoles representing early precursor of endosomes. A few nonreactive globules of the coat still showed at the surface after 2 min of exposure to MB particles. After longer exposure to MB, all altered coat globules (lipid droplets) were internalized in different compartments of the endocytic pathway. Most often, early endosomes were located near the cell periphery and presented a few lipid droplets within their limiting membrane. After longer exposure, lipid droplets were seen more and more in larger endosomes and prelysosomal vacuoles. Peripheral endosomes contained a chain of intraluminal lipid droplets closely associated with the inner face of the endosomal membrane, suggesting that the altered LDL-granules remained bound to receptors during this process. In some calves, MB particles triggered widespread membrane ruff ling and filipod formation, LAMELLAR BODIES AND DYE I N PULMONARY INTRAVASCULAR MACROPHAGES which was followed by complete internalization of the altered coat-globules as early as 3 min after exposure to MB particles. Lysosomal degradation of lipid droplets resulted in the formation of lamellar bodies (LLBs). The membranous material was arranged in concentric layers with a periodicity of 4-5 nm. A limiting membrane surrounded the whole lysosomallamellar body complex as early as 2-3 min post-MB injection. After extensive scrutiny of PIMs from different lobes and several tissue blocks of MB treated animals, only lysosomes showed manifest alterations of this kind, wheras other cell organelles remained unaffected. Accumulated osmiophilic LLBs with concentric disposition were noticed in more than one lysosomal center of the cell. Such lamellar structures were already detected in cells exposed to MB for 2-3 min in almost 60% of the animals injected. Some animals showed these alterations after a longer exposure to monastral blue (Figs. 9-11). 233 fixation with glutaraldehyde and osmic acid (Ghadially, 1989). Joris et al., (1982) while experimenting with the suitability of MB as a vascular tracer, showed dye particles as electron-lucent, rod-shape bodies similar to the present lipid droplets inside the lumen of small veins of skeletal muscles. In contrast, pigment particles examined in the native state were electrondense rods quite identical to the ultrastructural forms observed in in vitro preparations during the present study. In another separate study, aggregates of electron-dense amorphous mass of MB particles were shown in the vascular lumen and inside the marginated monocyte of pancreatic venules (Majno et al., 1987) different than the lipoid inclusions in the PIMs of bovine lungs. It appears that MB particles assume different morphologic forms in terms of their shape, size and electron density under different in vitro and in vivo conditions. The use of tannic acid alone or in combination with paraphenylenediamine allows reliable ultrastructural discrimination of lipid vesicles and Acid Phosphatase Cytochemistry lipid droplets (Katz, 1980; Kruth, 1984; Simionescu et Acid phosphatase activity was located in the lyso- al., 1986; Guyton and Klemp, 1988). The vesicular lipsomes. Heavy lead phosphate precipitates were ob- ids consist of phospholipids and unesterified cholesterol served in secondary lysosomes after the endosomes had with 2% or less cholesterol ester by weight and are merged with the main body of the lysosomal particles. surrounded by membrane showing bilayer structure. Peripheral endosomes also showed lead precipitate in a In contrast, droplets are predominantly neutral lipids, concentric position around the individual lipid droplets which implies mostly cholesterol esters (Katz, 1980). corresponding to the electron-dense line encircling the Guyton and Klemp (1988),by using tannic acid in comlipid core (Fig. 12). Acid phosphatase positive material bination with paraphenylenediamine in their study of resembling lead phosphate precipitate in the endo- atherosclerotic lesions, successfully discriminated besomes was primarily located at the periphery of sec- tween vesicular lipids (surrounded by a bilayer of mulondary lysosomes. In contrast, empty vesicles and en- tilamellar membranes) and lipid droplets. The droplets zyme positive myelinoid membranes, a forerunner of consisted of a single electron-dense line that encircled actual LLB formation, were seen in the inner zones of a homogeneous core of electron-lucent material. Similarge secondary lysosomes. Nonspecific lead precipitate larly, the MB-treated PIMs in the present study conwas observed on the apical plasmalemma of alveolar tained lipid droplets that were comprised of a homogetype I and I1 cells. In control tissue, stained without neous electron-lucent interior and surrounded by a P-glycerophosphate, similar staining with lead nitrate thicker electron-dense line. was seen in the alveolar epithelium. In a topologic sense, the present study showed lipid droplets a t two places. First, the lipid droplets apDISCUSSION peared at the surface and then were subsequently inThe present morphologic study demonstrates local- ternalized via coated pits into the endosomal and lysoization of modified MB particles in the PIMs of calf soma1 structures, where finally enzymatic degradation lung following their interaction with the globular units led to the formation of lamellar bodies. Ultrastructural of the surface coat. The light microscopic observations studies have provided evidence that cell membrane enat 2, 15, and 20 min postinjection showed distribution gulfing response in the ingestion process is a very of MB in association with the PIMs and Kupffer cells in localized phenomenon, where exogenous particles bindlarge granules of similar size and bluish color inten- ing at the cell membrane is segmental during phagosity. The clearance seemed to be complete from the cytosis (Griffin and Silverstein, 1974). In PIMs, the circulation a t these time intervals, since no intravas- surface visualization did not represent the classical piccular deposits were observed 15-20 min after intrave- ture of particle binding as a prelude to phagocytosis but nous injection of MB particles. However, the precise instead closely resembled the globular units of the coat localization of MB at the electron microscopic level was in terms of their spatial relationship with the cell proven to be of a different morphology in the PIMs and membrane along the entire cell boundary of the PIM. Kupffer cells. In Kupffer cells the pigment was recog- In contrast, in the Kupffer cells, particles were never nized exquisitely against the lysosomal matrix in the seen at the surface prior to their sequestration within form of round discrete particles of 40-45 nm in size. the phagolysosomes. We consider this as an indirect This is the given size range of MB particles in the lit- evidence that MB interacted with the coat globules as erature (Majno et al., 1987; Desemone et al., 19901, a stepwise activity before its entry into the endosomal whereas in the PIMs the counter part of blue color in- vesicles by way of coated pits. In the majority of the clusions of light microscopy was represented by large PIMs, the first step was recognized in every static EM aggregates of altered granules of the surface coat in- view of the cells in the form of lipid droplets, which at termixed with the dye. The altered granules depicted times represented 70-80% of the units of linear chain the conventional appearance of neutral triglycerides as at the surface of the macrophages. This transitional observed under the electron microscope by optimum stage of lipid droplet formation at the surface perhaps 234 O.S. ATWAL ET AL. Fig. 9. A PIM contains a few secondary lysosomes (Lys), one showing a developing lamellar body (LLB). Lamellar body arises from the lysosomal matrix. A coated pit (large arrowhead) is seen initiating endocytosis of lipid droplets (small arrowheads). Endosomes (arrows) are seen merging with the lysosome. Several lipid droplets are shown within the lysosomal compartment (white arrowheads). Calf treated with monastral blue. AS-alveolar space; M-mitochondria. Uranyl acetate and lead citrate staining. x 37,500. represented a dynamic activity of a certain duration probably catalyzed by enzymatic reactionb). In conventional terms, the transition perhaps represented the adhesion stage, “a condition sine qua non,” the first step prior t o phagocytosis (van Oss et al., 1984).This stage was of a prolonged duration in the case of PIMs as compared to Kupffer cells, which were given the same period of exposure to injected dye particles and were simultaneously monitored at the EM level along with the pulmonary intravascular macrophages. LAMELLAR BODIES AND DYE IN PULMONARY INTRAVASCULAR MACROPHAGES 235 Fig. 10. Lamellar bodies (LLB) developed in secondary lysosomes (Lys).Lipid droplets are seen sequestered into lysosomal compartment (small arrow). Big arrows point to endosomes. M-mitochondria. Uranyl acetate and lead citrate staining. x 36,800. Although phagocytosis of MB by PIMs and Kupffer cells took place within a set of identical micromedia environment, with the possible exception of a low particle concentration within the hepatic sinusoids, the fine morphology of the dye particles within the lysosoma1 matrix nevertheless was quite different in these cells. This difference is important enough to emphasize the ubiquity of the surface coat of the PIMs which trapped and influenced the rod-shape particles prior to their internalization by the PIMs. The spherical particle%-incontrast, were spared to be picked up rapidly by Kupffer cells, without any well-defined adhesion stage. The lamellar bodies (LLBs) arose from the homogeneous lysosomal matrix probably as a sequelae to the entry of lipid droplets into the lysosomal compartment. It is significant to point out that such lamellar bodies were not seen even after 1h r of intravenous injection of sonicated magnetic iron oxide in the phagosomes of 236 O.S. ATWAL ET AL. Fig. 11. Lysosomal-lamellar body (Lys-LLB) complex and endosome (arrows) of a PIM in MB treated calf. E = endothelium. Uranyl acetate-lead citrate staining. x 50,000. LAMELLAR BODIES AND DYE I N PULMONARY INTRAVASCULAR MACROPHAGES 237 Fig. 12. A TEM view of a PIM demonstrates acid phosphatase activity in a large lysosomal complex enclosed by a limiting membrane (large arrowheads). Several lipid droplets (small arrowheads) are seen within the limiting membrane. Lead phosphate deposits are located a t three (1,2, 3) potential sites of LLB development (compare with Fig. 10). Uranyl acetate-lead citrate staining. x 20,000, sheep PIMs, where the iron particles remained inert in phospholipases A and C resulting in the accumulation their native form (Warner and Brain, 1986). In our of phospholipids within lysosomes. For more details a current studies, the use of cationized ferritin as a mul- recent review article (Reasor, 1989) provides compretivalent tracer agent failed to show the formation of hensive explanations for the development and accumuLLBs 20-30 min postvascular perfusion ofjugular vein lation of LLBs induced by a large group of drugs usuin the goats (unpublished data). According to the model ally described under the name of cationic amphiphilic proposed by Reasor (Reasor, 1989), LLBs develop after drugs. the entry of extracellular material into the cell by The present study presents evidence for the first time phagocytosis. In the present situation, the model is ex- that MB, which was used in the past as a tracer subemplified by the entry of lipid droplets possibly via stance, reacted with the hypothetical LDL-coat of the receptor-mediated endocytosis and finally sequestered bovine PIMs leading to the formation of lipid-droplets into more than one lysosomal compartment (Hruban, at the surface during a short duration of 2-3 min after 1984). It has been proposed by several workers that MB perfusion. Subsequently, lipid droplets were interLLBs are formed because of drug-induced impairment nalized predominantly a t the coated pits (receptor-mein the metabolism of sequestered-polar lipids, particu- diated endocytosis) and through the endocytic pathway larly by those drugs that fall under the category of found their way into the lysosomes. Uptake also apcationic amphiphilic drugs (CADS).The LLB-lysosomal peared to occur via phagocytosis because cell memcomplexes are regarded secondary lysosomes, formed brane ruffling and filipods surrounding the aggregates by the storage of polar lipids, primarily phospholipids, of lipid droplets were also observed in addition to enwhich as a substrate are not fully degraded (Matsu- docytosis at the coated pits. The ultimate end result waza and Hostetler, 1980; Hostetler, 1984). It is theo- was the development of LLBs, an outcome of partial rized that CADSinhibit the activities of both lysosomal digestion of lipid droplets inside the lysosomes (Axline 238 O.S. ATWAL ET AL. and Cohn, 1970) within 2-3 min of MB administration. It appears to be a rapid process from the time the altered coat-globules were a t the coated pits and subsequently internalized for transport to the lysosomal compartment. The rapidity with which LLB formation was accomplished in the PIMs suggests that surface receptors may have a high affinity for lipid droplets. Once internalized at the coated pits, the droplets developed a rapid association with endocytic structures of the PIM to reach lysosomes in such a short time. The coated pits can mediate a n endocytic event within a few seconds to a minute in eukaryotic cells (Pastan and Willingham, 1985). Following receptor-mediated endocytosis, LDL are directed to lysosomes and degraded within the same time frame (Brown and Goldstein, 1983). It has been proposed that some dissociation and early sorting of ligand and receptors also takes place in endosomes, a n activity that depends upon low pH of 5.0-5.5 inside the endosomes for optimum hydrolytic activity (Amara et al., 1989; Dunn et al., 1989). In recent years, the standard definition of endosomes as prelysosomal compartments lacking lysosomal hydrolases has been challenged by some workers (Roederer et al., 1987; Diment et al., 1988). Based on biochemical and immunocytochemical evidence, early endosomes and peripheral vesicles of alveolar macrophages contain proteolytic enzymes like cathepsin B and D (Rodman et al., 1990). As a n analogy to this property of alveolar macrophages, the present cytochemical presence of acid phosphatase in endosomes may be a physiological indicator of cell activation, following stimulation initiated by receptor-mediated endocytosis of lipid droplets in pulmonary intravascular macrophages. Acid phosphatase had frequently been used as a “marker enzyme” for lysosomes by electron microscopists. It is assumed that lysosomes also contain other acid hydrolases. This presumption has been verified in some cases by correlation of cytochemical findings with findings on cell fractionation (Holtzman, 1989). It is therefore quite possible that hypothetical LDLcoat after its modification locally, under the oxidizing influence of Cu+ of monastral blue (copper phthalocyanine), was recognized by receptors of the PIM and subsequently internalized on a somewhat larger scale. There are several reports in literature suggesting that hydrolysis of phosphatidylcholine (PC) and proteins in LDL occurs under the oxidizing influence of cupric ions (Steinbrecher et al., 1984; McLean and Hageman, 1989). Reduction in the electron density of the coat, including change in the shape of coat globules and subsequent LLB formation in the lysosomes, indicates that MB is toxic to PIMs and perhaps other pulmonary cells. In this case, MB may not be a suitable tracer for studying kinetics of phagocytosis by pulmonary intravascular macrophages. Similar doubts were expressed by Albertine and Staub (1986) while discussing the basic criteria of suitability of MB as a tracer. In their study MB immediately caused systemic arterial hypotension, pulmonary arterial hypertension, and bronchoconstriction after single intravascular administration of MB in sheep. These detrimental side effects were partially blocked by indomethacin-a cyclooxygenase inhibitor, suggesting the possible role of metabolites of arachidonic acid mediating these responses. They showed lo+ calization of MB particles in the PIMs, therefore emphasizing the importance of PIMs as primary mediators of hernodynamic changes. We conclude that repertoire of above functional and structural properties of PIMs and Kupffer cells, which are directly related to their anatomic site and location along the vascular tree, have led to the present dichotomy in the ultimate morphology of the dye particles within these cells. The ingestion of lipid droplets and subsequent development of lamellar bodies in the lysosomes as a n index of flux of intracellular lipid pool perhaps signify the extent of functional changes in the PIMs, especially related to increased metabolism of vasoactive lipids and their secretion in the pulmonary circulation (Bertram et al., 1989; Montgomery and Cohn, 1989; Staub, 1989; Decker, 1990). A variety of cells can be stimulated as a result of activation of PIMs in this manner. We have observed marked thrombocytopenia during our current studies of PIMs in horses (unpublished data) following iv injection of MB. Ultrastructural analysis of pulmonary tissue showed largescale phagocytosis of platelets by pulmonary intravascular macrophages. 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