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In vivo monastral blue-induced lamellar-bodies in lysosomes of pulmonary intravascular macrophages PIMs of bovine lungImplications of the surface coat.

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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
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada
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%
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.
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.
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.
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
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.
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
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
in the endosomes (Endos). Note medium electron-dense matrix of a
mitochondrion. E = endothelium. Uranyl acetate and lead citrate
staining. x 46,875.
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.
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-
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
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,
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).
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
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.
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
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.
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
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,
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.
In conclusion, the present effects of MB on the surface coat and PIM as a whole may provide a suitable
model for experimental pathology of pulmonary intravascular macrophages with wider implications of their
role in the pathophysiology and general defense mechanisms of the lung in ruminants and horses.
The authors thank Mrs. Melva McGregor and Cathy
Young for typing the manuscript. This investigation
was supported by grants from the Ontario Ministry of
Agriculture and Food (Red Meat Program) and the National Scientific and Engineering Research Council of
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intravascular, pulmonaria, induced, blue, surface, bodies, lysosomes, coat, monastral, bovine, macrophage, lamellae, pims, vivo, lungimplications
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