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From the Second Department of Pathology*
and the
Department of Biochemistry,
Kumamoto
University School of Medicine, Kumamoto, Japan; and the Department of
Biochemistry,
Dartmouth Medical School,
Hanover, New Hampshire
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, specific polyclonal antibodies against the ACAT-1 protein from humans or mice have been produced.14-16 In human cells and tissues, these antibodies recognize the ACAT-1 protein as a single 50-kd protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.14 Using an antibody against the human ACAT-1 protein, DM10, we performed immunoblot analysis and ACAT enzyme activity assay and found that human monocyte-derived macrophages expressed high levels of ACAT-1 protein and high ACAT enzyme activity during the early stage of monocyte/macrophage differentiation.17 We also demonstrated that the ACAT-1 protein was amply present in macrophages, but not in smooth muscle cells, within the atherosclerotic lesions of human aorta.17 Other than the atherosclerotic lesions, little is known about the distribution of ACAT-1 protein in normal human organs at the cellular level, particularly in those that play important roles in cholesterol homeostasis. At the single cell level, using the specific anti-ACAT-1 antibody (DM10) for immunofluorescent microscopy, Chang et al reported in human melanoma cells that ACAT-1 is mainly located in endoplasmic reticulum.14 Using a similar method, Khelef et al reported in murine macrophages that a minor portion of ACAT-1 protein resides in membranes other than the endoplasmic reticulum16 ; the non-rough endoplasmic reticulum localization of ACAT-1 may be the trans-Golgi network.18 The distribution of ACAT-1 at the ultrastructural level in any cell type has not been reported yet.
In the present study, we examined by immunohistochemistry the distribution and localization of ACAT-1 in various normal human tissues. In addition, to investigate the intracellular localization of ACAT-1 at both the protein and cellular levels, we performed immunoblot analysis, immunoelectron microscopy, and immunofluorescent microscopy of cultured human macrophages before and after cholesterol loading by treating cells with acetylated low density lipoprotein (AcLDL).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For immunohistochemistry, tissue specimens were obtained from various organs and tissues of 8 autopsy cases (6 males and 2 females, 41 to 75 years old) within 4 hours postmortem. These specimens were fixed in an ice-cold 2% periodate-lysine-paraformaldehyde fixative for 6 hours and washed with phosphate-buffered saline, pH 7.2, containing a graded series of sucrose (10, 15, and 20%). To prevent ice crystal formation, the specimens were immersed in 0.01 mol/L phosphate-buffered saline containing 20% sucrose and 10% glycerol for 30 minutes and embedded in OCT compound (Miles, Elkhart, IN). These embedded materials were frozen and cut sequentially into 5-µm-thick sections with a cryostat (HM 500 M; MICROM, Waldorf, Germany).
Preparation of AcLDL
Low density lipoprotein (LDL, d = 1.091 to 1.063) was isolated by sequential ultracentrifugation from normolipidemic human plasma, dialyzed in 0.15 mol/L NaCl and 1 mmol/L ethylenediamine tetraacetic dihydrate, pH 7.4 (Nacalai Tesque, Kyoto, Japan), and treated with acetic anhydride to prepare AcLDL as described previously.19
Cell Culture
Monocytes were collected from peripheral blood of healthy volunteers according to the method described elsewhere, with a minor modification.20 Briefly, mononuclear leukocytes were segregated from peripheral blood using the Ficoll/Hypaque gradient centrifugation method, resuspended in RPMI1640 (Nissui Pharmaceutical Co., Tokyo, Japan) containing 10% autologous serum or 10% fetal calf serum, and plated in culture chambers (Lab-Tek Chamber Slide, Nalge Nunc International, Naperville, IL) for 2 hours. After nonadherent cells were removed by gently washing with culture medium, adherent monocytes were cultured for 7 days to induce differentiation and maturation into macrophages. Cultured macrophages were further incubated for 3 more days with a culture medium containing 100 µg/ml of AcLDL. After AcLDL treatment, foam cell transformation was confirmed by observing the accumulation of lipid droplets under phase-contrast microscopy.
Antibodies
Specific polyclonal rabbit antibody for human ACAT-1, DM10, was generated and affinity-purified as described elsewhere.14 The antibody recognizes the first 131 amino acids residues in N-terminal region of the human ACAT-1. To confirm the formation of mature macrophages derived from cultured peripheral monocytes, we used a mouse anti-human macrophage monoclonal antibody, AM-3K,21 generated in our laboratory. To detect rough endoplasmic reticulum by immunofluorescent and immunoelectron microscopy, we used a goat polyclonal antibody N-20 raised against a human 78-kd glucose-regulated protein, GRP 78 (Santa Cruz Biotechnology, Santa Cruz, CA).
Immunohistochemistry
To detect the expression of ACAT-1 in the normal human tissues, we performed the indirect immunoperoxidase method with a minor modification as described previously.17 Briefly, after inhibition of endogenous peroxidase activity according to the method of Li et al,22 the frozen sections were incubated with 5% normal donkey serum for 20 minutes and sequentially reacted with DM10 diluted 1:200 as primary antibody at room temperature for 1 hour. The sections were rinsed 5 times with ice-cold 0.01 mol/L phosphate-buffered saline, pH 7.2, and incubated with peroxidase-labeled anti-rabbit immunoglobulin F(ab')2 (Amersham, Little Chalfont, UK) diluted 1:100 as second antibody. After washing, the peroxidase activity was visualized as black color with a solution containing Ni, Co, and 3,3'-diaminobenzidine (Dojin Chemical Co., Kumamoto, Japan),23 and the sections were stained with Methylgreen and mounted with Malinol (Mutoh Chemical Co., Tokyo, Japan). To assure the immunoreactive specificity of DM10 in each tissue, control stainings were done in the same manner, omitting the primary antibody. In each case, the control stainings provided only a very weak positivity on background.
Immunoblotting
Immunoblot analysis for cultured macrophages with or without AcLDL treatment was performed as described elsewhere.17 Briefly, the 1 x 107 cells seeded in 10-cm dishes were washed several times with phosphate-buffered saline, stored at -80°C, and dried in monolayers for up to 7 days. The frozen cell monolayers were thawed and extracted with 0.1 ml of 10% sodium dodecyl sulfate per dish. Cells were scraped and sheared, using syringes with 25-gauge needles. Protein concentrations of cellular extracts were determined by the method of Lowry et al.24 Samples were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunoblotting. The primary antibody (DM10) was used at a final concentration of 0.5 µg/ml.
Immunoelectron Microscopy
Immunoelectron microscopy was performed according to the same method as described previously.21 Briefly, cultured macrophages with or without AcLDL treatment were fixed with 4% periodate-lysine-paraformaldehyde and 0.1% glutaraldehyde (Nacalai Tesque, Kyoto, Japan) in phosphate-buffered saline, pH 7.2, at 4°C for 20 minutes. After washing with phosphate-buffered saline and treating with 0.005% saponin containing phosphate-buffered saline for 10 minutes, the cells were stained by the immunoperoxidase method, using DM10 or N-20 as primary antibody. After visualization with 3,3'-diaminobenzidine for 5 minutes, the cells were postfixed with 1% osmium tetroxide at 4°C for 30 minutes. After rinsing, the cells were dehydrated with a graded series of ethanol and embedded in Epok 812. Ultrathin sections were made by an ultramicrotome MT7000 ULTRA (RMC Inc., Tucson, AZ) and observed under an electron microscope H-7500 (Hitachi, Tokyo, Japan).
Immunofluorescent Double Staining
Immunofluorescent double staining with DM10 and N-20 was used to confirm the localization of ACAT-1 in rough endoplasmic reticulum. Briefly, cultured human macrophages with or without AcLDL treatment were fixed in 2% periodate-lysine-paraformaldehyde solution at 4°C for 20 minutes and rinsed with phosphate-buffered saline containing 0.005% saponin. The cells were incubated with 5% normal donkey serum and 0.1% Triton X-100 for 20 minutes and reacted to primary antibodies containing 200-fold-diluted DM10 with 20-fold-diluted N-20 for 1 hour at room temperature. After rinse, the cells were incubated with secondary antibody sequentially, FluoroLink Cy3-labeled donkey anti-goat IgG(H+L) (Amersham, Little Chalfont, UK) diluted 1:1000 was reacted to the cells, and FluoroLink Cy2-labeled goat anti-rabbit IgG(H+L) (Amersham) were further applied to the cells for 1 hour at room temperature after washing out the former fluorescent labeled antibody. The specimens were mounted using Dako fluorescent mounting medium (Dako, Carpinteria, CA) after washing out nonreacted secondary antibody and observed by a confocal laser scanning microscope (FLUOVIEW, Olympus, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
summarizes the distribution of
ACAT-1 in various organs and tissues of autopsy cases as revealed by
immunohistochemistry, using the specific anti-ACAT-1 antibodies DM10.
Immunoreactivity was demonstrated in various cells and tissues. The
specificity of immunoreactivity using DM10 was demonstrated previously
by immunoblot analysis: Lee et al reported that a single specific
signal at 50 kd was detected in human liver, adrenal glands, and
kidneys as well as in macrophages.25
In the digestive
tract, immunoreactivity was expressed in the epithelia of the small
intestines and gastric fundic glands, mesothelial cells, and smooth
muscle cells. In the small intestine, absorptive epithelia expressed
ACAT-1, whereas the goblet cells and Paneth cells showed a very weak
immunoreactivity (Figure 1, A and B)
.
Negative control staining (without incubation with the first
antibodies) in specimens obtained from small intestine, liver, adrenal
glands, and neurons confirmed that the immunosignals were specific for
ACAT-1 (Figure 1C)
. Myenteric ganglion cells also showed a weak
reactivity (data not shown). A very weak immunoreactivity was found in
the squamous epithelium of esophagus and in the absorptive epithelial
cells of the large intestine (Figure 1D)
, though these regions are not
involved in cholesterol absorption. Kupffer cells in the liver
expressed a marked immunoreactivity for ACAT-1; hepatocytes also
stained positive with DM10, whereas the epithelial cells of bile ducts
showed a much weaker immunoreactivity (Figure 1, E and F)
. No
immunoreactivity was found in endothelial cells of the hepatic
sinusoids (Figure 1F)
. Marked immunoreactivity for ACAT-1 was found in
steroid hormone-producing cells including the adrenal cortical cells,
Leydig cells in the testis, and granulosa cells in the ovary (Figure 2, A and B)
. In contrast, other types of
endocrine cells that are not involved in steroid hormone production
showed only an extremely weak immunoreactivity.
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, in the
lymphatic sinuses of the lymph nodes, and in the red pulp of spleen,
expressed an intense immunoreactivity to the ACAT-1 antibodies.
Dendritic cells and endothelial cells of lymphatic sinuses in lymph
nodes indicated immunoreactivity to DM10 (Figure 2D)
and epidermal
Langerhans cells were also positively stained by the antibodies (Figure 2E)
. Control experiments showed that macrophages in these tissues were
positively stained with the anti-macrophage antibody AM-3K, while
dendritic cells were not stained by AM-3K (data not shown). In the
skin, the most prominent signals were observed in the sebaceous glands,
while the eccrine sweat glands did not show
immunoreactivity (Figure 2F)
. Neurons and their dendrites express
ACAT-1 in the cerebral cortex, basal ganglia, cerebellar cortex,
pontine nucleus, and spinal cord (Figure 2, G and H)
, but neural glia
showed no immunoreactivities. In data not shown, DM10-positive
immunoreactivities were also found in the tubular epithelial cells of
kidneys, transitional epithelia of urinary tracts, cardiomyocytes,
alveolar or bronchial epithelium, and bronchial glands in the
respiratory tract, epithelial cells of the prostate and thyroid
follicles, and pancreatic acinar and ductal epithelial cells. Immunoblot Analysis of ACAT-1 in Cultured Human Macrophages with or without Cholesterol Loading
Monocytes collected from peripheral blood of normal
healthy human volunteers were incubated in culture for 7 days.
Immunostaining using the macrophage-specific antibody AM-3K confirmed
that at this stage, all of the cells were differentiated into mature
macrophages (data not shown). Modified LDL, such as AcLDL, are known to
induce cholesteryl ester accumulation in macrophages and cause
subsequent foam cell formation.20
To investigate whether
AcLDL induces any changes in the ACAT protein content in human
macrophages, we used DM10 as the primary antibody to perform immunoblot
analysis. The results showed that a single 50-kd protein, corresponding
to the human ACAT-1 protein,25
was detected in sodium
dodecyl sulfate-polyacrylamide gel electrophoresis; the intensity of
the 50-kd protein signal remained the same in cells with or without
AcLDL for 3 days (Figure 3)
.
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Immunoelectron microscopy was used to study the subcellular
localization of ACAT-1 in macrophages. Without cholesterol loading,
immunoreactivities against DM10 were found in the membranes of
intracellular organelles, especially in rough endoplasmic reticulum and
the nuclear membrane (Figure 4A)
;
however, no immunoreactivity was detectable in the Golgi complexes
(Figure 4B)
. After AcLDL treatment, immunoreactivity could be found in
small vesicles with diameter of 80 to 150 nm, in addition to the
tubular endoplasmic reticulum structure (Figure 4, C and D)
. Based on
the results of examining 50 or more individual electron micrographs, we
estimated that approximately 30 to 40% of the total immunoreactivity
appeared in these small vesicles after the transformation of cultured
macrophages into foam cells. Under this condition, despite careful
examinations, we have still failed to detect any immunoreactivity in
the Golgi complexes (data not shown).
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, a
result consistent with the earlier findings implying that ACAT-1 is
mainly distributed in the endoplasmic reticulum.16
When
anti-GRP 78 antibody, a specific marker for endoplasmic reticulum, was
used, similar patterns were demonstrated (Figure 5B)
.26,27
By double immunofluorescent staining, the extensive colocalization of
ACAT-1 and GRP 78 was demonstrated (Figure 5C)
. After cholesterol
loading, the extent of colocalization of ACAT-1 and GRP 78 remained
extensive and essentially unaltered (Figure 5, D
-F).
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. After cholesterol
loading, numerous immunoreactive small vesicles appeared throughout the
cytoplasm of the cells (Figure 6B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In macrophages, our current result indicated that there is no change in the ACAT-1 protein content when the cells are transformed into foam cells (by loading cells with cholesterol via AcLDL treatment). This result is consistent with previous studies in various cells and tissues supporting the notion that the main mechanism for increase in ACAT enzyme activity by cholesterol influx is probably due to the allosteric property of the ACAT-1 enzyme in combination with an increase in intracellular cholesterol trafficking toward the endoplasmic reticulum.1,28,29 The electron microscopic studies in macrophages described in this study report ACAT-1 localization at the ultrastructural level for the first time, demonstrating that the ACAT-1 protein is located mainly in the rough endoplasmic reticulum. This result is consistent with previous biochemical studies using other cell types, suggesting that ACAT-1 is an integral membrane protein that functions in the rough endoplasmic reticulum.1,30-32 In an earlier study using immunofluorescence microscopy, Khelef et al demonstrated that a minor portion of ACAT-1 in mouse macrophages is distributed in Golgi complexes in addition to its main location in the endoplasmic reticulum.16 In human macrophages, however, we have examined numerous electron micrographs (more than 50) but have failed to detect any positive signals in the Golgi complexes. This discrepancy may result from species difference used in our current study and the studies by Khelef et al.16 When human macrophages were loaded with cholesterol by treating with AcLDL, we still could not detect any ACAT-1-positive immunoreactivity in the Golgi complexes. Instead, we found that the ACAT-1 signals were present in tubular endoplasmic reticulum as well as in the small-sized vesicles, 80 to 150 nm in diameter; these small vesicles also stained positive for GRP 78, known as a specific marker for endoplasmic reticulum. In addition, our double immunofluorescent staining revealed that the ACAT-1 signals colocalized extensively with the GRP 78 signals; the extent of colocalization between ACAT-1 and GRP 78 did not change before and after cholesterol loading. Overall, our findings raise the possibility that, at least in human macrophages, cholesterol loading may induce an accelerated endoplasmic reticulum vesiculation process to produce certain novel small vesicles enriched in the ACAT-1 protein from rough endoplasmic reticulum. The vesiculation process, if it exists, may increase the accessibility of ACAT-1 protein to intracellular free cholesterol.
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Acknowledgements |
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Footnotes |
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Accepted for publication September 4, 1999.
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s). Circulation 1998, 97(suppl I):311 (abstr)
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