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RED CELLS
Failure of red blood cell maturation in mice with defects in the high-density
lipoprotein receptor SR-BI
Teresa M. Holm, Anne Braun, Bernardo L. Trigatti, Carlo Brugnara, Masa Sakamoto, Monty Krieger, and Nancy C. Andrews
Mammalian erythrocytes undergo a
unique maturation process in which they
discard their nuclei and organelles and
assume a flexible biconcave shape. We
found that altered plasma lipoprotein metabolism can profoundly influence these
events. Abnormal erythrocyte morphology was observed in hypercholesterolemic mice lacking the high-density lipoprotein receptor SR-BI. This was
exacerbated by feeding mice a highcholesterol diet or, more dramatically, by
inactivating the apolipoprotein E gene.
Erythrocytes from SR-BIⴚ/ⴚ/apolipoprotein Eⴚ/ⴚ mice and SR-BIⴚ/ⴚ mice that
were fed cholesterol had markedly increased membrane cholesterol. Their
morphology appeared immature, with
macrocytosis, irregular shape, and large
autophagolysosomes. Autophagolysosomes from SR-BIⴚ/ⴚ/apolipoprotein Eⴚ/ⴚ
erythrocytes were expelled when the
erythrocytes were transfused into wildtype animals or incubated in vitro with
normolipidemic serum or the cholesterolsequestering agent methyl cyclodextrin.
We propose that autophagocytosis and
phagolysosome expulsion are essential
steps in erythroid maturation and that
expulsion is inhibited in the presence of
markedly increased cellular cholesterol.
(Blood. 2002;99:1817-1824)
© 2002 by The American Society of Hematology
Introduction
Red blood cells are descended from multipotential hematopoietic
progenitor cells in the bone marrow. Lineage commitment establishes an erythroid differentiation program in which differential
gene expression leads to high-level hemoglobin production, nuclear
condensation and expulsion from the cell, and the shutdown of
most cellular functions. The erythroid precursors generated by the
expulsion of nuclei from bone marrow erythroblasts are called
reticulocytes. Early reticulocytes, which are rarely found in the
peripheral blood, are irregular in shape and contain cytoplasmic
organelles and polyribosomes.1 As reticulocyte maturation
progresses, RNA, mitochondria, and ribosomes are degraded;
protein synthesis ceases; and cell-surface transferrin receptors
(Trfrs) disappear. Finally, cytoskeletal remodeling transforms reticulocytes into compact, biconcave, discoid corpuscles devoid of
organelles and translational apparatus. The earliest steps in erythroid commitment and differentiation have been the subject of
extensive study (reviewed in Bungert and Engel2). However,
maturational events subsequent to nuclear expulsion have been
only partially characterized by morphologic studies.1,3,4 Some
patients with severe anemia syndromes, especially those whose
spleen has been removed, have a small fraction of circulating
erythrocytes containing autophagosomes.1 Because these autophagosomes contain mitochondria and ribosomes that are absent
from mature erythrocytes, it has been proposed that they represent
intermediates in the final stages of red cell maturation.1 Furthermore, splenic macrophages have been postulated to participate in
the removal of autophagosomes from such precursors, but the
mechanism of autophagosome extraction in the bone marrow was
not addressed.1 We show here that hypercholesterolemic mice with
abnormal lipoproteins due to inactivation of the high-density
lipoprotein (HDL) receptor SR-BI gene (SR-BI⫺/⫺)5 have an
unexpected defect in late erythroid maturation.
SR-BI mediates cellular uptake of cholesterol and plays a key
role in the transport of cholesterol from peripheral tissues to the
liver by HDL (reviewed in Krieger6). During studies of the role of
SR-BI in atherosclerosis,7 SR-BI⫺/⫺ mice were bred to mice that
were homozygous for a null mutation in the gene encoding
apolipoprotein E (apoE). ApoE⫺/⫺ mice also have defects in
lipoprotein metabolism that result in hypercholesterolemia and the
early, spontaneous development of atherosclerosis.8-10 The plasma
cholesterol concentration of double-knockout SR-BI⫺/⫺/apoE⫺/⫺
mice is 2-fold higher than that of apoE⫺/⫺ mice. These doubleknockout mice have unusually large, HDL-like lipoprotein particles and they develop severe atherosclerosis at an accelerated
pace,7 resulting in coronary artery disease and early death.29
In the current study, we observed that SR-BI⫺/⫺/apoE⫺/⫺ mice
were anemic and had unusual red blood cell morphology. The
erythrocytes had abnormally high-cholesterol content and resembled proposed intermediates in erythroid differentiation in that
they had irregular shapes and an accumulation of large intracellular
phagolysosomes. Similar but less severe morphological defects
were observed in the erythrocytes from chow-fed SR-BI⫺/⫺ singleknockout mice. Feeding the SR-BI⫺/⫺ single-knockout mice a
cholesterol-enriched diet dramatically exacerbated these abnormalities.
From the Division of Hematology/Oncology, Children’s Hospital; the
Department of Pediatrics, Harvard Medical School; and the Howard Hughes
Medical Institute, Boston, MA; and the Department of Biology, Massachusetts
Institute of Technology, Cambridge, MA.
Howard Hughes Medical Institute.
Reprints: Nancy C. Andrews, HHMI/Hematology, Enders 720, Children’s
Hospital, 300 Longwood Ave, Boston, MA 02115; e-mail: nandrews@enders.
tch.harvard.edu.
Submitted August 10, 2001; accepted October 25, 2001.
Supported by grants from the National Institutes of Health (M.K. and N.C.A.).
A.B. was a European Molecular Biology Organization and Human Frontiers
Science Program postdoctoral fellow. B.L.T. was a Medical Research Council
of Canada postdoctoral fellow. N.C.A. is an Associate Investigator of the
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
1817
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1818
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
HOLM et al
The large intracellular phagolysosomes in the erythroid cells from
the double-knockout mice were expelled both in vivo, when the
abnormal cells were transfused into the circulation of normolipidemic wild-type control mice, and in vitro, when the cells were
incubated with normolipidemic serum or the cholesterol-sequestering agent methyl cyclodextrin. Thus, the abnormal lipoprotein
metabolism and intrareticulocyte accumulation of excess cholesterol in SR-BI⫺/⫺ mice appears to induce a reversible block in the
expulsion of autophagolysosomes from reticulocytes. We postulate
that autophagocytosis and cholesterol-sensitive phagolysosome
expulsion are important steps in normal erythropoiesis that can be
interrupted when red cell lipid homeostasis is perturbed.
Methods
Animals
We housed mice in the barrier facilities at Children’s Hospital (Boston,
MA) and the Massachusetts Institute of Technology (Cambridge, MA) and
maintained them on standard mouse diet unless otherwise specified. We
originally purchased homozygous apoE⫺/⫺ mice on a C57BL/6 background8,9 and hemoglobin-deficit (hbd) mutant mice from the Jackson
Laboratory (Bar Harbor, ME). SR-BI⫺/⫺, apoE⫺/⫺, and SR-BI⫺/⫺/apoE⫺/⫺
mice were generated as previously described.5,7 We performed most
experiments on 5- to 6-week-old animals. However, a second cohort of 4- to
5-week-old SR-BI⫺/⫺ male mice and wild-type age- and sex-matched
controls was placed on a 1% cholesterol diet for 3 months prior to analysis.
We determined mouse genotypes by polymerase chain reaction analysis of
tail DNA as previously described.7 For bone marrow transplantations, we
irradiated mixed background (129 ⫻ C57BL/6) apoE⫺/⫺ mice using a
Gammacell 40 (12 Gy), and we injected them retro-orbitally with 106 bone
marrow cells harvested from the femurs and tibias of either SR-BI⫺/⫺/
apoE⫺/⫺ mice or littermate apoE⫺/⫺ mice. Bone marrow replacement was
assessed by SR-BI–genotype analysis of CD11b-expresssing cells harvested
from the mice receiving transplants 3 months after transplantation. Only the
mutant SR-BI allele was detected in cells prepared from the apoE⫺/⫺ mice
receiving bone marrow from SR-BI⫺/⫺/apoE⫺/⫺ mice, suggesting a high
degree of chimerism (data not shown). Red blood cell morphology was
analyzed 5 to 6 months after transplantation.
Erythrocyte analysis
Blood was obtained by retro-orbital bleeding or direct cardiac puncture. We
determined complete blood counts and erythrocyte measurements (mean
corpuscular volume [MCV] and mean corpuscular hemoglobin) using an
automated ADVIA 120 analyzer (Bayer Diagnostics, Tarrytown, NY). We
analyzed at least 4 animals to determine average values for every
measurement. We fixed peripheral blood smears in 100% methanol for 7
minutes, stained them with Wright-Giemsa (Fisher Diagnostics, Pittsburgh.
PA) for 7 minutes, and washed them 4 times with fresh distilled water for 1
minute each before visualizing by means of an Olympus BX50 microscope
(Lake Success, NY). We visualized fixed, unstained smears with Differential Interference Contrast (DIC) optics (Zeiss Axioplan microscope [Thornwood, NY] and Chroma Optical [Chroma Technology, Brattleboro, VT]
custom filter sets). We captured images digitally and analyzed them using
Adobe PhotoShop (San Jose, CA). We prepared 200 ␮L peripheral blood
for transmission electron microscopy as described previously.11
fluorescent cleavage product. We visualized fluorescence by confocal
imaging on a Kr/Ar Zeiss Axio Vert s100 microscope.
Flow cytometry
Trfr was detected with a phycoerythrin-conjugated monoclonal anti-Trfr
antibody (Pharmingen, San Jose, CA), and RNA was detected with thiazole
orange (Becton Dickinson, San Jose, CA) as previously described.12 SR-BI
was detected with a polyclonal rabbit anti–SR-BI antibody13 and a 488
fluorophore–labeled antirabbit antibody (Molecular Probes). We detected
circulating erythroid cells at all stages of maturity with a phycoerythrinconjugated antimouse TER-119 antibody (Pharmingen).
Induced reticulocytosis
Reticulocytosis was induced in wild-type mice by daily removal of 0.3 to
0.5 mL blood for a period of 7 days, maintaining the hematocrit above 20%.
Iron dextran (5 mg) (Sigma) was administered intraperitoneally on day 3.
Final reticulocyte counts in these animals ranged from 45% to 55%.
In vitro incubation of blood cells
We washed red blood cells once with phosphate-buffered saline (PBS) and
once with minimum essential medium (BioWhittaker) augmented with 1
g/L glucose (5.5 mM), 25 mM Hepes, 20% fetal calf serum, 2 mM
L-glutamine, 200 U/mL penicillin, and 200 ␮g/mL streptomycin, (GibcoBRL, Grand Island, NY), pH 7.4. We then counted cells and diluted them to
2 ⫻ 107/mL in this medium. We immediately harvested an aliquot of cells
by centrifugation as the zero time sample. We monitored the medium pH
regularly and maintained the medium at pH 7.4 with small additions of
NaOH as previously described.14 We took samples at 24-hour intervals and
prepared them for electron microscopy and flow cytometric analysis. In
some experiments, we treated duplicate samples of 2 ⫻ 107 red cells per
milliliter for 5 minutes prior to the in vitro incubation with 0.1% methyl B
cyclodextrin (Sigma) in the incubation medium at 37°C.
In vivo biotin labeling and reinfusion of red blood cells
We labeled red blood cells with biotin in vivo as previously described.15 We
prepared 16 ␮g NHS-Biotin (Calbiochem, La Jolla, CA) per gram of body
weight dissolved in 30 ␮L dimethylacetamide (Calbiochem) and diluted to
0.5 mL in PBS, and injected it through the tail vein of donor mice. At 2
hours after infusion, we collected blood in tubes containing EDTA as an
anticoagulant and then transfused 0.5 mL into each recipient mouse. At 24
and 48 hours after transfusion, we bled the mice and analyzed cells by live
confocal microscopy to detect biotin (with phycoerythrin-streptavidin;
Molecular Probes) and autophagolysosomes (with Lysotracker as described
above). We further analyzed the samples by flow cytometry as described
above. We determined biotin-labeling efficiency by diluting cells and
incubating 1 ⫻ 106 red cells with 15 micrograms of 1 mg/mL
phycoerythrin-streptavidin.
Filipin detection of cholesterol
We preserved blood in vitro in Karnovsky fixative (2.5% paraformaldehyde, 2% glutaraldehyde, 0.2 M cacodylate buffer). The cells were then
incubated for 2 hours in 50 ␮g/mL (from stock solution of 12 mg/mL in
dimethylsulfoxide) of filipin (Sigma) in PBS to label cholesterol and
generate a fluorescent signal. Fluorescence was observed by means of
4⬘6–diamidino–2-phenylindole–2 HCl filters for confocal microscopy and
UV filters for flow cytometry.
Detection of lysosomes and RNA in reticulocytes
Quantitative cholesterol analysis
We visualized lysosomes by washing fresh red blood cells and diluting them
1:1000 in Eagle modified minimum essential medium with low calcium and
without L-glutamine (BioWhittaker, Walkersville, MD) and then incubating
them with Lysotracker (Molecular Probes, Eugene, OR), a pH-specific
marker for lysosomes, or with fluorescein di-(␤-D-galactopyranoside)
(FDG) (Sigma, St Louis, MO), a substrate of ␤-galactosidase that yields a
We prepared erythrocytes for cholesterol quantitation by first centrifuging whole
blood and removing plasma and buffy coat. The cells were then washed 3 times
with PBS and diluted 1:1 in deionized water to cause cell lysis. Total cholesterol
was extracted by adding 100 ␮L the internal standard epicoprostamol (1 mg/mL
in methanol) (EPIC, Sigma Chemicals) either to 100 ␮L red blood cell lysate or
to the positive control, human plasma. Human plasma was used as the positive
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
IMPAIRED ERYTHROPOIESIS IN SR-BI KNOCKOUT MICE
1819
Table 1. Hematological data for wild-type and mutant mice
Genotype
Hematocrit, %
MCV, fL
Reticulocytes, %
MCH concentration, g/dL
MCH, pg
Age 28 to 35 d
WT (n ⫽ 5)
52.9 ⫾ 1.5
57.6 ⫾ 0.5
4.3 ⫾ 0.6
29.2 ⫾ 0.69
15.8 ⫾ 0.6
ApoE⫺/⫺ (n ⫽ 8)
52.5 ⫾ 2.1
62.6 ⫾ 3.6
2.8 ⫾ 1.2
27.3 ⫾ 1.0
16.6 ⫾ 0.5
SR-BI⫺/⫺ (n ⫽ 14)
50.7 ⫾ 2.7
62.5 ⫾ 4.2
11.9 ⫾ 4.0
SR-BI⫺/⫺/apoE⫺/⫺ (n ⫽ 6)
34.2 ⫾ 1.7
84.1 ⫾ 4.1
100
28 ⫾ 1.1
16.1 ⫾ 0.6
17.5 ⫾ 1.3
15.0 ⫾ 0.5
3 mo on normal or cholesterolsupplemented diet; age, 4 mo
WT, normal diet (n ⫽ 5)
54.0 ⫾ 4
50.0 ⫾ 1.5
2.5 ⫾ 0.2
30.8 ⫾ 1.2
15.3 ⫾ 0.3
WT, cholesterol diet (n ⫽ 5)
55.5 ⫾ 0.5
51.1 ⫾ 2
2.4 ⫾ 0.3
29.1 ⫾ 1.9
14.8 ⫾ 0.7
SR-BI⫺/⫺, normal diet (n ⫽ 5)
47.3 ⫾ 3
54.8 ⫾ 3
6.2 ⫾ 1.0
28.4 ⫾ 1.4
15.5 ⫾ 0.5
SR-BI⫺/⫺, cholesterol diet (n ⫽ 7)
39.6 ⫾ 2
63.3 ⫾ 2
18.5 ⫾ 3.0
27 ⫾ 1.0
15.9 ⫾ 0.6
Values are given with SDs. The P values for significantly different measurements are given in the text. Normal mouse reticulocyte counts range from 2% to 6% early in life
and decrease slightly as the mice age.
MCV indicates mean corpuscular volume; MCH, mean corpuscular hemoglobin; WT, wild-type; apoE, apolipoprotein E.
control instead of pure cholesterol to compensate for any extraction effect. The
samples were then saponified by adding 1 mL 10% potassium hydroxide/90%
ethanol (Fisher Scientific, Fair Lawn, NJ) to each sample and incubating for 60
minutes at 60°C. The samples were cooled to room temperature and extracted
with 2 mL hexane (Fisher Scientific) by mixing for 1 minute and then aspirating
off the hexane layer. We repeated this 3 times and then combined the hexane
extracts and evaporated them to dryness under nitrogen at 40°C. Finally, we
resuspended the samples in 100 ␮L pyridine and 100 ␮L N, O-Bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (Alltech Associates, Folsom, CA) and
heated them for 60 minutes at 60°C.16,17 After cooling the extract, we injected 1
␮L into a gas chromatograph mass spectrometer and measured the cholesterol
levels (Hewlett Packard Model 5890/5972 [Atlanta, GA] equipped with a DB-1
column of 30 cm length, 0.25 mm inner diameter, and 0.25 ␮m film thickness
[Alltech Associates]). Unesterified cholesterol was determined as above except
that saponification was omitted.
Figure 1. Morphology of red blood cells from wild-type
and mutant mice. Red blood cell preparations from wildtype, apoE⫺/⫺, SR-BI⫺/⫺, SR-BI⫺/⫺/apoE⫺/⫺, and hbd mice
were examined by means of standard blood-smear WrightGiemsa light microscopy photographed at ⫻ 100 magnification (panels A-E); DIC microscopy photographed at ⫻ 100
magnification (panels F-J) (arrow shows inclusions [panels H,
I] and hemoglobin in target cell [panel J]; transmission
electron microscopy (panels K-P) (panel P is a highermagnification image of an SR-BI⫺/⫺/apoE⫺/⫺ erythrocyte that
shows mitochondria and ribosomes within the membraneenclosed inclusion (arrows).
Results
Mice lacking SR-BI and apoE develop an unusual anemia
SR-BI⫺/⫺/apoE⫺/⫺ mice fed a standard chow diet had low hematocrits and hemoglobin values, which differed significantly from
wild-type controls (Table 1) (P ⬍ .0001). The SR-BI⫺/⫺/apoE⫺/⫺
red blood cells were abnormally large as compared with wild-type
cells (MCV, P ⬍ .0001), but had normal or near-normal cellular
hemoglobin content. All circulating SR-BI⫺/⫺/apoE⫺/⫺ red blood
cells were reticulocytes. Unlike wild-type and apoE⫺/⫺ mice,
whose red blood cells appeared normal in blood smears (Figure 1A,
B), the SR-BI⫺/⫺/apoE⫺/⫺ mice had a nonuniform population of
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1820
HOLM et al
Figure 2. Effects of cholesterol feeding on the morphology of red blood cells
from wild-type and SR-BIⴚ/ⴚ mice. Wild-type (panels A-B) and SR-BI⫺/⫺ (panels
C,D) mice were fed normal, low-cholesterol chow (panels A,C) or 1% cholesterol–
supplemented chow (panels B,D) for 3 months. Then, red blood cells were prepared
and examined by transmission electron microscopy. Bar ⫽ 1 ␮m.
macrocytic red blood cells (Figure 1D) with a targetlike appearance. However, these targetlike cells sometimes appeared to have
multiple intracellular inclusions and were often spiculated. They
were more irregular than classical target forms (eg, the targetlike
erythrocytes from homozygous hbd mice18 in Figure 1E). Spiculated cells could be seen in smears from the SR-BI⫺/⫺ mice fed a
standard chow diet (Figure 1C), but their abnormalities were not as
striking as those from the SR-BI⫺/⫺/apoE⫺/⫺ mice. We also
observed morphologic abnormalities in SR-BI⫺/⫺ and SR-BI⫺/⫺/
apoE⫺/⫺ mice using DIC microscopy. Unlike the normal morphologies of erythrocytes from wild-type and apoE⫺/⫺ mice (Figure
1F,G), many erythrocytes from SR-BI⫺/⫺ mice had irregularities
within their cytoplasms, suggesting the presence of small, membrane-enclosed intracellular inclusions (Figure 1H). Virtually all
the erythroid cells from SR-BI⫺/⫺/apoE⫺/⫺ mice (Figure 1I) had
one or more large, membrane-enclosed intracellular inclusions that
were considerably larger than those seen in SR-BI⫺/⫺ animals.
Their appearance was distinctly different from that of the target
cells of hbd mice (Figure 1J). Furthermore, these cells from
SR-BI⫺/⫺/apoE⫺/⫺ mice stained strongly with both acridine orange
and thiazole orange,19,20 indicating that they retained a high RNA
content, a characteristic of reticulocytes but not mature erythrocytes (Figure 4T and data not shown).
We further characterized the intracellular inclusions using
electron microscopy. Wild-type and apoE⫺/⫺ cells were indistinguishable (Figure 1K,L), exhibiting the biconcave disc morphology
typical of mature red cells. In contrast, some SR-BI⫺/⫺ cells
(approximately 75%, Figure 1M) and all SR-BI⫺/⫺/apoE⫺/⫺ cells
(Figure 1N) were irregularly shaped. We rarely saw mitochondria
and ribosomes in wild-type and apoE⫺/⫺ erythrocytes, but they
were present in approximately 15% of SR-BI⫺/⫺ erythrocytes and
almost all SR-BI⫺/⫺/apoE⫺/⫺ cells. These features are characteristic
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
of immature (stage I) reticulocytes, which are not usually found in
the peripheral circulation. In addition, many of the SR-BI⫺/⫺ cells
had small, membrane-enclosed inclusions that occasionally appeared to contain organelles. All SR-BI⫺/⫺/apoE⫺/⫺ cells had
complex inclusions that resembled those of the SR-BI⫺/⫺ erythrocytes, but were larger. Their contents (mitochondria, ribosomes)
and overall optical density appeared similar to the surrounding
cytoplasm (Figure 1P).
Membrane-enclosed inclusions within erythrocytes increase
with cholesterol feeding and are correlated with cellular
cholesterol content
Because of the possibility that increased hypercholesterolemia
accounted for the increased severity of defects in SR-BI⫺/⫺/
apoE⫺/⫺ mice relative to SR-BI⫺/⫺ mice, we compared the red
blood cells from wild-type and SR-BI⫺/⫺ mice maintained for 3
months on diets consisting either of normal, low-cholesterol chow
or chow supplemented with 1% cholesterol. In wild-type mice, the
high-cholesterol diet did not appear to influence the hematocrit, the
MCV (Table 1; P not significant), or the morphology of the cells
(Figure 2A,B). In contrast, SR-BI⫺/⫺ mice fed cholesterol had
exacerbation of a mild anemia (Table 1) (change in SR-BI⫺/⫺
hematocrit significant; P ⫽ .0007) and marked macrocytosis (Table
1) (change in SR-BI⫺/⫺ MCV significant; P ⫽ .0004). Electron
microscopy showed that SR-BI⫺/⫺ mice fed cholesterol had larger
and more frequent intracellular inclusions than the controls fed
normal chow (Figure 2C,D). However, the cholesterol-feeding–
induced abnormalities in hematocrit, MCV, and erythroid cell
morphology in SR-BI⫺/⫺ mice were not as severe as those seen in
SR-BI⫺/⫺/apoE⫺/⫺ mice.
To determine if the morphological abnormalities of the erythrocytes correlated with their cholesterol compositions, we stained red
blood cells from the different mouse strains with the cholesterolbinding fluorescent dye filipin and measured the filipin staining.
Flow cytometric analysis showed that wild-type erythrocytes and
reticulocytes contained less cholesterol (filipin stain) than erythrocytes from animals with mutations in apoE, SR-BI, or both genes
(Figure 3A; the box in each panel indicates the approximate
distribution of wild-type erythrocytes). Cholesterol levels were
highest in SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes. The filipin staining was
also visualized by confocal microscopy (Figure 3B,C). Although
the plasma membranes of the cells from SR-BI⫺/⫺ and SR-BI⫺/⫺/
apoE⫺/⫺ mice appeared to stain more intensely with filipin than
those of the controls (erythrocytes from normal wild-type mice;
reticulocytes isolated from phlebotomized wild-type mice; and
Figure 3. Quantitation and localization of erythrocyte cholesterol
in wild-type and mutant mice. The cholesterol content of erythrocytes from wild-type, reticulocyte-enriched wild-type, apoE⫺/⫺, SRBI⫺/⫺, and SR-BI⫺/⫺/apoE⫺/⫺ mice was examined by 2 methods. First,
cells were incubated with filipin, to allow fluorescent detection of
cholesterol by flow cytometry (panel A) and confocal microscopy
(panel C). (Note that in panel A the y-axis is logarithmic.) Second,
cholesterol in erythrocyte lysates was measured directly by a biochemical method (panel B). The boxes show the range for 80% of the data
points; the lines within the boxes show the median values; and the
bars show outlying data points (if any). At the bottom of the plot, ⫺
indicates normal mouse chow diet, and ⫹ indicates 3 months on the
high-cholesterol diet.
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BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
erythrocytes from apoE⫺/⫺ mice), the most striking differences
were due to intense staining of intracellular structures in
SR-BI⫺/⫺ and SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes. These intensely
stained intracellular structures resemble the inclusions visualized by other methods (Figure 1) and probably represent the
same vesicles. The levels of unesterified and total (unesterified
plus esterified) cholesterol in the red blood cells, measured
directly with an enzymatic assay (Figure 3B,C), correlated with
the intensity of filipin staining.
The abnormal inclusions are autophagolysosomes
The inclusions in SR-BI⫺/⫺/apoE⫺/⫺ red blood cells had the
appearance of autophagolysosomes, vesicles that result from
sequestration of cytoplasmic contents in a membrane-enclosed
compartment that has low internal pH and contains lysosomal
enzymes.21,22 To explore this possibility, we incubated red blood
cells with Lysotracker, a fluorescent dye that accumulates in low
pH compartments, and with FDG, a substrate for lysosomal
␤-galactosidase. Wild-type and apoE⫺/⫺ erythrocytes (Figure 4A,B)
rarely contained fluorescent inclusions (Figure 4E-F,I-J,M-N). In
contrast, erythrocytes from SR-BI⫺/⫺ and SR-BI⫺/⫺/apoE⫺/⫺ mice
(Figure 4C,D) were stained with these phagolysosomal dyes in
overlapping patterns (Figure 4G,H,K,L,O,P) that were similar to
those of the inclusions described above. Thus, the inclusions are
probably autophagolysosomes.
Figure 4. Fluorescence microscopic and flow cytometric analysis of the
characteristics of red blood cells from wild-type and mutant mice. (A-P) Red
blood cell preparations from wild-type, apoE⫺/⫺, SR-BI⫺/⫺, and SR-BI⫺/⫺/apoE⫺/⫺
mice were simultaneously stained with 2 lysosomal markers: Lysotracker (low-pH
indicator, red) and FDG (lysosomal beta-galactosidase substrate, green), and were
examined by phase contrast (panels A-D) and fluorescence (panels E-P) microscopy.
Panels E-H show Lysotracker staining; panels I-L, FDG staining; and panels M-P,
merged red and green fluorescence images. Yellow indicates spatial overlap of the
signals. (Q-T) Dual-color fluorescence flow cytometric analysis of red blood cells from
the indicated mice stained with thiazole orange to detect RNA (x-axis) and a
phycoerythrin-conjugated monoclonal anti-Trfr antibody to detect cell-surface Trfr
(y-axis). For each cell analyzed, the relative intensities of thiazole orange fluorescence and phycoerythrin fluorescence are indicated by a dot and presented on log
scales. Each panel represents analysis of 10 000 cells. Dots in the lower left quadrant
of the cytogram represent double-negative, mature erythrocytes.
IMPAIRED ERYTHROPOIESIS IN SR-BI KNOCKOUT MICE
1821
Erythroid differentiation is reversibly disrupted in
SR-BIⴚ/ⴚ/apoEⴚ/ⴚ mice
During normal reticulocyte maturation, there is a coordinate
decrease in both cellular RNA content and Trfr expression.23 Both
can be readily measured by means of fluorescent stains and flow
cytometry.12 As expected from the morphologic studies, we could
detect neither RNA nor Trfr in most of the fully differentiated
erythrocytes circulating in wild-type and apoE⫺/⫺ mice (lower left
quadrant of cytograms in Figure 4Q,R). In keeping with the
morphologic studies, SR-BI⫺/⫺ mice had a larger fraction of
immature cells (RNA⫹ and Trfr⫹, upper right quadrant, Figure 4S)
than the wild-type and apoE⫺/⫺ mice, but most of the cells were
double negative. Strikingly, virtually all of the SR-BI⫺/⫺/apoE⫺/⫺
cells retained a high RNA content, and most, but not all, expressed
substantial levels of Trfr (Figure 4T). This suggests that in
SR-BI⫺/⫺/apoE⫺/⫺ mice, some steps in reticulocyte maturation are
completely blocked (normal cytoskeletal remodeling, clearance of
organelles, and RNA) while others can proceed at least partially
(loss of cell-surface Trfr).
Erythrocytes from SR-BI⫺/⫺ animals contained abnormal autophagolysosomes, which were more prominent when the mice were
fed a high-cholesterol diet or when plasma cholesterol levels were
elevated as a consequence of inactivation of the apoE gene
(double-knockout mice). The severity of the morphologic phenotype correlated with cellular cholesterol content. It seemed possible
that disruption in erythroid differentiation might have been due to
loss of SR-BI expression in the reticulocytes themselves (cell
autonomous effects), in other tissues (eg, the liver, which has a
major role in lipoprotein metabolism), or both. Using immunofluorescence methods, we have detected SR-BI protein expression in a
punctate pattern in Trfr⫹ reticulocytes from phlebotomized wildtype mice (data not shown). However, expression of SR-BI in
reticulocytes does not necessarily mean that SR-BI in these cells is
essential for maturation. To address this issue, we examined the
morphologies of erythrocytes isolated from lethally irradiated
apoE⫺/⫺ mice that had subsequently received bone marrow transplantation from apoE⫺/⫺ or SR-BI⫺/⫺/apoE⫺/⫺ donor mice. All
cells in the recipients of bone marrow from apoE⫺/⫺ mice were
SR-BI⫹, while all cells except the bone marrow–derived cells (eg,
erythroid cells) in the recipients of marrow from SR-BI⫺/⫺/apoE⫺/⫺
donors were SR-BI⫹. The apoE⫺/⫺ donor marrow generated
morphologically normal erythrocytes, as expected (data not shown).
The erythrocytes generated from the SR-BI⫺/⫺/apoE⫺/⫺ marrow in
the apoE⫺/⫺ recipient mice did not contain autophagolysosomes
(data not shown). This suggested that accumulation of the autophagolysosomes in the erythrocytes of SR-BI⫺/⫺/apoE⫺/⫺ mice was
not simply a consequence of the absence of SR-BI expression in the
erythroid precursors. These results also suggested that the dyslipidemic environment in the plasma of SR-BI⫺/⫺/apoE⫺/⫺ mice may
have played a critical role in the disruption of reticulocyte
maturation. If that is the case, it might be possible to reverse the
block in development by transferring the abnormal erythrocytes
from SR-BI⫺/⫺/apoE⫺/⫺ mice into normolipidemic wild-type mice.
To examine the role of the extracellular environment in
erythrocyte maturation in vivo, we transfused biotin-labeled erythrocytes from SR-BI⫺/⫺/apoE⫺/⫺ mice into either wild-type or
SR-BI⫺/⫺/apoE⫺/⫺ mice and determined if residence in the circulation of the recipient animal affected the retention of cytoplasmic
autophagolysosomes (Figure 5). We examined a sample of biotinylated donor cells at transfusion and samples of blood from the
recipient animals collected 24 or 48 hours after transfusion by
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1822
HOLM et al
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
Figure 5. Fate of biotinylated erythrocytes from SR-BIⴚ/ⴚ/
apoEⴚ/ⴚ mice transfused into wild-type and SR-BIⴚ/ⴚ/apoEⴚ/ⴚ
recipients. A donor SR-BI⫺/⫺/apoE⫺/⫺ mouse was injected with
biotin to label erythrocytes in vivo. Samples of this blood were
then injected into either wild-type (panels A-C) or SR-BI⫺/⫺/
apoE⫺/⫺ (panels D-F) recipient mice. Samples of blood from these
recipient mice were taken immediately after infusion (panels A,D),
at 24 hours (panels B,E), and at 48 hours (panels C,F), and the
cells were labeled with Lysotracker. Blood cells were analyzed by
confocal microscopy showing biotin in red and Lysotracker in
green. Immediately following transfusion, 9.4% of the cells in the
wild-type recipient were labeled with biotin. This decreased to
9.1% at 24 hours and to 6.0% at 48 hours, for a 48-hour survival
rate of 64%.
staining with fluorescent streptavidin (red signal). We visualized
autophagolysosomes by staining with Lysotracker (green signal).
Prior to transfusion, all biotin-labeled erythrocytes contained
autophagolysosomes (Figure 5A,D). When the labeled SR-BI⫺/⫺/
apoE⫺/⫺ cells were transfused into SR-BI⫺/⫺/apoE⫺/⫺ recipient
mice, circulation for up to 48 hours did not appear to affect the
autophagolysosomes in the cells (Figure 5D-F). In contrast,
autophagolysosomes were efficiently removed from the labeled
cells when they circulated in wild-type mice, with most autophagolysosomes lost by 24 hours (Figure 5A-C). It seems likely that the
loss of autophagolysosomes was a consequence of maturation,
because another marker of immature cells, surface Trfr, was present
on transfused SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes and was substantially
decreased after cells circulated in wild-type recipients. Immediately after transfusion, 82% of the cells with detectable surface
biotin were strongly positive for Trfr, when analyzed by flow
cytometry. By 24 hours, only 66% of the biotin-labeled cells had
detectable Trfr. By 48 hours, only 50% of the biotin-labeled cells
had detectable Trfr, and the amount of Trfr on the Trfr⫹ cells was
significantly less than on the first day. Taken together, the bone
marrow transplantation and biotin-labeling/transfusion experiments indicate that abnormal retention of autophagolysosomes in
SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes is reversible and that these cells
appear to undergo normal maturation when placed in a permissive
environment in vivo.
Expulsion of autophagolysosomes from SR-BIⴚ/ⴚ/apoEⴚ/ⴚ
reticulocytes in vitro
To study the expulsion of autophagolysosomes from SR-BI⫺/⫺/
apoE⫺/⫺ reticulocytes in vitro, we harvested red blood cells from
SR-BI⫺/⫺/apoE⫺/⫺ mice and incubated them in medium containing
normolipidemic fetal calf serum under conditions that support cell
metabolism and cell survival for several days. For positive
controls, we harvested reticulocyte-rich red blood cells from
wild-type mice that had been repeatedly phlebotomized. Expulsion
of autophagolysosomes was assessed by transmission electron
microscopy before (0 hours) and after 72 hours of incubation in
vitro (Figure 6).
At 0 hours, many of the erythrocytes from the phlebotomized
wild-type mice contained small vesicles (Figure 6A, arrowheads).
We interpret this to be the result of marked erythropoietic stress,
due to phlebotomy, that led to the release of immature cells into the
circulation. This observation is consistent with an earlier report
postulating autophagocytosis in patients with severe anemia syndromes.1 At 0 hours, the inclusions in the SR-BI⫺/⫺/apoE⫺/⫺
erythrocytes were larger and more complex than those in the
erythrocytes from the phlebotomized wild-type mice (Figure 6C).
At 72 hours, both wild-type and SR-BI⫺/⫺/apoE⫺/⫺ cells appeared
to be extruding their membrane-enclosed cytoplasmic vesicles
(Figure 6B,E). The cytoplasm of most wild-type cells and many
SR-BI⫺/⫺/apoE⫺/⫺ cells appeared darker and more homogeneous
Figure 6. Effects of in vitro incubation in normolipidemic serum on reticulocytes from phlebotomized wild-type mice and erythrocytes from SR-BIⴚ/ⴚ/apoEⴚ/ⴚ mice.
Erythrocytes from wild-type mice with phlebotomy-induced reticulocytosis (panels A-B) and from SR-BI⫺/⫺/apoE⫺/⫺ mice (panels C,E) were incubated in vitro in medium
containing 20% normolipidemic fetal calf serum for 0 hours (panels A,C) or 72 hours (panels B,E); the cells were then analyzed by transmission electron microscopy. The red
asterisks show membrane-enclosed, cytoplasmic vesicles that appeared to be undergoing expulsion from the SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes (panel E). Arrows denote
cell-free vesicles found in the incubation medium (panel E). Arrowheads show structures in reticulocytes from phlebotomized wild-type mice that appear similar to the
cytoplasmic vesicles seen in SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes (panels A-B). Panels D and F show flow cytometry analysis of RNA content (thiazole-orange staining) versus
forward scatter of the cells (FSC) for SR-BI⫺/⫺/apoE⫺/⫺ samples at 0 hours (panel D) and 72 hours (panel F).
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
than at 0 hours, consistent with maturation. Strikingly, the RNA
content of the SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes decreased over the
course of the incubation (Figure 6D,F). Free phagolysosomal
vesicles could easily be identified in the medium surrounding the
SR-BI⫺/⫺/apoE⫺/⫺ cells (Figure 6E, arrows). Thus, it appears that
incubation in vitro with normolipidemic fetal calf serum was able,
at least in part, to relieve the in vivo block in erythroid differentiation in SR-BI⫺/⫺/apoE⫺/⫺ mice.
We considered the possibility that excess cellular cholesterol
directly interfered with expulsion of the autophagolysosomes. To
investigate this, we briefly treated SR-BI⫺/⫺/apoE⫺/⫺ cells with or
without the cholesterol-sequestering agent methyl cyclodextrin
(MCD, 0.1% solution, 5 minutes)24 prior to incubating them in
vitro as described above (Figure 7). Filipin-staining analysis
showed that the MCD treatment reduced the cholesterol content of
SR-BI⫺/⫺/apoE⫺/⫺ cells to the level observed in erythrocytes from
normal wild-type mice (lower panels). MCD treatment dramatically accelerated the expulsion of autophagolysosomes from the
cells compared with control incubations without MCD treatment
(eg, see Figure 6). After 24 hours of in vitro incubation (Figure 7B),
the intracellular inclusion bodies were substantially smaller than
before incubation (Figure 7A), and most had disappeared after 48
hours of incubation (Figure 7C). At the same time, the cytoplasm of
the SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes became darker and more homogeneous, suggesting that ribosomes are no longer present and that
hemoglobin is more concentrated in the cytoplasm. In addition, the
size and shape of the cells were altered by MCD treatment, as is
apparent from the decrease in FSC (Figure 7, bottom panels). These
results suggest that, by decreasing cellular cholesterol, we have
relieved a block preventing autophagolysosome expulsion. This
supports the possibility that elevated cellular cholesterol is the
primary cause of defective reticulocyte maturation in SR-BI⫺/⫺/
apoE⫺/⫺ mice.
Discussion
Investigation of an unusual anemia in mice with defective lipoprotein metabolism has provided a unique window into late erythroid
Figure 7. Effects of MCD treatment on in vitro vesicle expulsion from erythrocytes from SR-BIⴚ/ⴚ/apoEⴚ/ⴚ mice. Erythrocytes from SR-BI⫺/⫺/apoE⫺/⫺ mice were
preincubated for 5 minutes in medium with (this Figure) or without (not shown;
however, see Figure 6) 0.1% MCD; cells were then incubated in vitro without MCD as
described in Figure 6. Samples were taken before MCD treatment (panel A) and at 24
(panel B) and 48 (panel C) hours of incubation and analyzed by electron microscopy.
At 24 hours (panel B) and 48 hours (panel C), the membrane-enclosed, cytoplasmic
vesicles appear to be undergoing expulsion from the SR-BI⫺/⫺/apoE⫺/⫺ erythrocytes
(red arrows). The plots below each electron micrograph correspond to the photos
above and show a decrease in filipin staining as plotted against FSC. The rectangles
on the 24- and 48-hour plots show the position of the bulk population of cells at 0
hours. Preincubation without MCD (data not shown) gave results similar to those
shown in Figure 6.
IMPAIRED ERYTHROPOIESIS IN SR-BI KNOCKOUT MICE
1823
maturation. Abnormal erythrocyte morphology was observed in
mice lacking the HDL receptor SR-BI. This was exacerbated by
feeding SR-BI⫺/⫺ mice a high-cholesterol diet or, more dramatically, by inactivating the apoE gene along with the SR-BI gene
(SR-BI⫺/⫺/apoE⫺/⫺). These erythrocytes had immature features,
including macrocytosis, irregular shape, high content of RNA and
Trfrs, and large intracellular lysosomelike vesicles that appear to
have arisen by autophagocytosis of organelles, polyribosomes, and
cytoplasm. The size and number of autophagolysosomes were
correlated with the cholesterol content of the erythrocytes. The
autophagolysosomes in SR-BI⫺/⫺/apoE⫺/⫺ cells were expelled
when the mutant erythrocytes were transfused into wild-type
animals, or incubated in vitro with normolipidemic serum. Although SR-BI was expressed in reticulocytes from wild-type mice,
bone marrow transplantation experiments suggested that the accumulation of autophagolysosomes in the SR-BI⫺/⫺ cells was not a
simple cell-autonomous phenomenon but, rather, depended on the
extracellular environment. Indeed, pretreating the cells with the
cholesterol-sequestering agent MCD dramatically accelerated the
in vitro expulsion of the autophagolysosomes. Thus, it appears that
the novel dyslipidemic lipoprotein profile of SR-BI⫺/⫺ mice causes
abnormal cholesterol accumulation in erythrocyte precursors, which
in turn interferes with maturation and results in the accumulation of
autophagolysosomes, RNA, and Trfrs. The appearance of the
erythrocytes from SR-BI⫺/⫺/apoE⫺/⫺ mice suggests a mechanism
by which immature reticulocytes can discard unneeded cellular
structures, reduce cell volume, and complete their maturation.
Do the autophagolysosome-containing, Trfr⫹, and RNA-rich
erythrocytes in SR-BI⫺/⫺/apoE⫺/⫺ mice represent arrested intermediates in the normal red blood cell differentiation pathway, or are
they irrelevant off-pathway, dead-end artifacts induced by the
novel dyslipidemia in SR-BI⫺/⫺ mice? Transfusion experiments
clearly established that the accumulation of autophagolysosomes
and the persistence of RNA and Trfrs in SR-BI⫺/⫺/apoE⫺/⫺
erythrocytes are reversed by circulation in normolipidemic, wildtype animals. Thus, these cells apparently retain the capacity to
progress through the latter stages of erythrocyte development if an
environmental block is released. The observation of autophagosomes in the erythrocytes of diverse human patients with severe
anemia1 and in repeatedly phlebotomized mice (this study) indicates that such structures can be observed independently of SR-BI
deficiency when erythropoiesis is markedly stressed. Although
other explanations are possible, it appears that autophagolysosomes play an evanescent, yet important, role in normal erythroid
differentiation. We propose that they are essential for discarding
cytoplasmic contents and initiating shape, volume, and biochemical changes that ultimately produce organelle-free, biconcave,
mature erythrocytes.
Although our results are perhaps not surprising given the
intriguing role that cholesterol is thought to play in regulated
trafficking and sorting (reviewed in Hoekstra and van IJzendoorn25), the details of the mechanisms by which erythrocytes
expel autophagolysosomes and by which dyslipidemia and excess
cholesterol arrest this expulsion remain to be determined. We do
not yet know if the mechanisms for nuclear expulsion from early
erythrocyte precursors26 and phagolysosomal expulsion from reticulocytes are similar. Nuclei were no longer present in the SR-BI⫺/⫺/
apoE⫺/⫺ erythrocytes. This suggests that nuclear expulsion does
not depend on the loss of autophagolysosomes and is not sensitive
to the environmental factors (eg, dyslipidemia) that lead to
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1824
BLOOD, 1 MARCH 2002 䡠 VOLUME 99, NUMBER 5
HOLM et al
autophagolysosome accumulation in SR-BI⫺/⫺/apoE⫺/⫺ mice. Acceleration of in vitro autophagolysosome expulsion by the cholesterol-sequestering agent MCD may provide a useful tool for the
future analysis of the mechanism of expulsion.
In vivo, reticuloendothelial macrophages of the liver and spleen
play an important role in removing particulate matter (eg, nuclear
remnants, precipitated hemoglobin) from circulating erythroid
cells.27,28 As the erythrocytes pass through narrow vascular spaces,
macrophages are thought to aid in cellular remodeling by actively
removing membrane-enclosed vesicles containing cellular debris.
Although autophagolysosome expulsion from SR-BI⫺/⫺/apoE⫺/⫺
erythrocytes can occur in the absence of phagocytic cells in vitro, it
seems likely that reticuloendothelial macrophages participate in the
removal of residual autophagosomes from circulating immature
reticulocytes in living animals, either subsequent to or concurrently
with their expulsion from cells. Thus, it is possible that insufficient
macrophage-mediated organelle clearance activity or macrophage
dysfunction in SR-BI⫺/⫺/apoE⫺/⫺ mice might contribute to autophagolysosome accumulation in their erythrocytes.
In conclusion, the novel abnormalities of SR-BI⫺/⫺/apoE⫺/⫺
mice have permitted the identification and characterization of
transient intermediates in normal erythroid maturation that were
proposed to exist previously, but were difficult to capture in
morphological studies. The arrest of SR-BI⫺/⫺/apoE⫺/⫺ erythroid
development, apparently dependent upon cholesterol, is reversible
both in vivo and in vitro. Thus, further analysis of these mice may
provide additional insights into the mechanisms underlying erythroid development.
Acknowledgments
We thank Helena Miettinen for critical suggestions, advice, and
help with cholesterol-feeding experiments; Mark Fleming and
Antonio Perez-Atayde for assisting us in the interpretation of
pathology slides; and Mohandas Narla, Ellis Neufeld, David
Clapham, Sam Lux, Joanne Levy, and Robert Levy for helpful
discussions. We thank Jeff Macklis and Lisa Catapano for assisting
us with differential interference contrast microscopy. Howard
Mulhern in the Department of Pathology carried out electron
microscopy at Children’s Hospital, Boston. Michelle Lowe in the
Confocal and Multiphoton Core Facility at the Brigham and
Women’s Hospital performed confocal microscopy. Lynne Montross kindly performed all mouse tail vein injections.
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2002 99: 1817-1824
doi:10.1182/blood.V99.5.1817
Failure of red blood cell maturation in mice with defects in the high-density
lipoprotein receptor SR-BI
Teresa M. Holm, Anne Braun, Bernardo L. Trigatti, Carlo Brugnara, Masa Sakamoto, Monty Krieger and
Nancy C. Andrews
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