1. Healthcare

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Regulated Expression of CD36 During Monocyte-to-Macrophage
Differentiation: Potential Role of CD36 in Foam Cell Formation
By Ho Young Huh, S. Frieda Pearce, Lewis M. Yesner, Joseph L. Schindler, and Roy L. Silverstein
CD36 is an 88-kD integral membrane glycoprotein expressed
on monocytes, platelets, and certain microvascular endothelium serving distinct cellular functions both as an adhesive
receptor for thrombospondin, collagen, and Plasmodiumfalciparuminfected erythrocytes, and as a scavenger receptor
for oxidized low-density lipoprotein and apoptotic neutrophils. In this study, we examined the expression of CD36
during in vitro differentiation of peripheral blood monocytes
into culture-derived macrophages. Steady-state mRNA levels of CD36 showed a transient eightfold increase during
monocyte-to-macrophage differentiation, peaking at the
early macrophage stage (days 3 or 4 in culture), following
a gradual decrease back t o baseline levels by the mature
macrophage stage (days 7 or 8 in culture). lmmunoblotting
with monoclonal antibodies t o CD36 supported this transient, yet significant (8- t o 10-fold) increase in total protein
levels of CD36. The increasedCD36 protein was observed at
the plasma membrane, whereas an intracellular pool of CD36
was also detected from day 2 t o day 6 in culture through
indirect immunofluorescence. A concomitant twofold increase in the cells‘ a b i i i i t o bind ’%thrombospondin at
the early macrophage stage (day 4) verified the functional
competency of the plasma membrane localized CD36, and
supported the presence of an intracellular pool of CD36. The
in vitro differentiated macrophages as well as alveolar macrophages remained responsivet o macrophage colony-stimulating factor (M-CSF), a known transcriptional regulator of
monocyte CD36. The MGSF-induced macrophages resulted
in enhanced foam cell formation, which was inhibitable with
monoclonal antibodies t o CD36. Thus, the transient expression of CD36 during monocyte-to-macrophage dfferentiation, and the ability of M-CSF t o maintain macrophageCD36
at elevated levels, may serve as a critical processin dictating
the functional activity of CD36 during inflammatory responses and atherogenesis.
0 1996 by The American Society of Hematology.
D
plasma membrane receptors critical for monocyte/macrophage function such as FcyR, transferrin receptor, type I
macrophage scavenger receptors, and the mannose receptor
all increase in expression during the monocyte-to-macrophage transition.*-”
CD36 is an 88-kD transmembrane glycoprotein expressed
on monocytes, platelets, and microvascular endothelium. It
functions as an adhesive receptor for thrombospondin-1
(TSP1)i2,’3and ~ o l l a g e n , and
’ ~ mediates cytoadherence of
Plasmodium falciparum-infected erythrocytes to the endothe1i~m.I~
The CD36-TSP1 interaction participates in platelet-tumor cell adhe~ion,’~
and macrophage uptake of aged
ne~trophils.’~.’’
Furthermore, CD36 has also been shown to
function as a scavenger receptor on macrophages for oxidized low-density l i p o p r ~ t e i n , ’suggestive
~ * ~ ~ of a critical role
in macrophage foam cell formation and atherogenesis.
Regulation of CD36 expression and function as a cellular
receptor is complex. We have shown that monocyte adhesion
to tumor necrosis factor (TNF)-activated endothelium increases expression of CD36 on the monocyte cell surface.2”
This adhesion-induced phenomenon appears to be occurring
at the transcriptional level mediated through E-selectin signaling.” Furthermore, soluble mediators macrophage colony-stimulating factor (M-CSF), phorbol myristate acetate
(PMA), and interleukin-4 (E-4)
enhance steady-state mRNA
and protein levels of CD36, whereas lipopolysaccharide
(LPS) and dexamethasone decrease monocyte CD36.” Substrate specificity of CD36 has been proposed to be regulated
in part by posttranslational phosphorylation and dephosphorylation of extracytoplasmic ser/thr residues on CD36,
allowing preferential TSP and collagen binding.”
Earlier studies of monocyte CD36 and TSPl expression
have dealt mainly with leukemic cell lines. For example,
during in vitro differentiation of HL-60 cells to a “macrophagelike” phenotype, TSPl synthesis was suppressed,
whereas a 10-fold increase in TSPl receptor expression was
seemz3However, the identity of this receptor was not addressed. A 10-fold increase in OKM5 antigen, CD36, during
PMA-induced monocytic U937 differentiation has also been
IFFERENTIATION OF human peripheral blood monocytes in vitro has been used extensively as a model
system for investigating macrophage development. Early
morphological descriptions of in vitro monocyte differentiation into culture-derived macrophages and multinucleated
giant cells’.’ have been augmented in recent years with increasing cellular and molecular evidence of correlative alterations in enzymatic activity, secretion of soluble mediators,
and antigen expression. Morphologically, the first few days
in culture are characterized by gradual spreading and flattening, coupled with membrane ruffling and extensions as
the cytoplasm-to-nucleus ratio enhances, typifying a macrophage-like
From day 7, the multinucleated
giant cell population increases steadily and becomes the most
prevalent cell type by day 20.’ During the first week in
culture, as the monocytes acquire macrophage-like morphology, enzymes such as acid phosphatase, B-glucosaminidase,
and macrophage tissue transglutaminase (MTG) have been
shown to increase in their relative activity, whereas lysosomal peroxidase exhibits suppressed a~tivity.4.~~’
Moreover,
From the Program in Cell Biology and Genetics, and the Department of Medicine, the Division of Hematology/Oncology, and the
National Institutes of Health Specialized Center of Research in
Thrombosis, Comell University Medical College, New York, NY.
Submitted May 18, 1995; accepted October 12, 1995.
The first two authors contributed equally to this work.
Supported in part by Grants No. NIH-HL 42540, HL I8828 (National Institutes of Health [NIW Specialized Center of Research in
Thrombosis), and HL 46403 (NIH Program Project in Vascular Cell
Signalling) to R.L.S.
Address reprint requests to Roy L. Silverstein, MD. Division of
Hematology-Oncology, Comell University Medical College, 1300
York Ave. New York, NY 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I996 by The American Society of Hematology.
0006-497I/96/8705-0$3.00/0
2020
Blood, Vol 87, No 5 (March 1). 1996: pp 2020-2028
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MACROPHAGE CD36: ROLE IN FOAM CELL FORMATION
202 1
with PBS and fresh medium added to adherent cells. The cultured
reported.” TSPl expression by human monocytes has been
reported to decrease concomitant with cellular a c t i v a t i ~ n . ~ ~ cells
. ~ ~ were then incubated at 37°C in a humidified 5% CO2 environment
for periods of up to 20 days. Monocyte purity, as determined
In this study, we examine the expression of CD36 and TSPl
by flow cytometq using a monoclonal antibody (MoAb) for CD14,
during in vitro differentiation of peripheral blood monocytes
was greater than 80%. Viability was greater than 98% as assessed
(PBM) into culture-derived macrophages. Our results supby trypan blue exclusion. Alveolar macrophages were obtained from
port the previous data, but further show transient increases
healthy nonsmokers by bronchoalveolar lavage as previously dein steady-state mRNA and protein levels of CD36 accompa~cribed.’~
Ninety percent of the cells isolated through bronchoalnied by corresponding enhancement in the cell’s ability to
veolar lavage were alveolar macrophages confirmed by forward- and
bind TSPl . Although the elevated cell surface expression
side-scatter flow cytometry, whereas greater than 60% were CD14+.
RNase protection assay. RNase protection assays were perof CD36 was transient, in vitro differentiated macrophages
formed as previously
Monocytes were washed, lysed
maintained their responsiveness to transcriptional regulators
in 5 m o m guanidine thiocyanate/O.l m o m EDTA at 2.5 to 5 X
of monocyte CD36 throughout their differentiation pathway.
IO6 cells/mL and hybridization performed directly in 20-pL aliquots
The M-CSF-enhanced CD36 expression, in particular, inof cell lysate for 20 hours at 37°C with antisense 3zP-labeledRNA
creased the macrophages’ ability to take up oxidized lowprobes. RNA probes were labeled to specific activities of 1 to 2
density lipoprotein (ox-LDL) in an in vitro foam cell assay.
x IO9 c p d p g with ”P-UTP. CD36 riboprobes were generated by
Such processes may be critical in maintaining adequate levsubcloning a 792-bp BamHI fragment from the 5’ end of the CD36
els of macrophage CD36 for its adhesive and scavenger
cDNA into pBluescript KS. The resulting plasmid was linearized
functions.
with EcoRI and T7 RNA polymerase used to transcribe an antisense
MATERIALS AND METHODS
Materials. Murine monoclonal anti-CD36 IgG, FA6, was obtained from the Vth International Workshop on Human Leukocyte
Antigens27and 8A6I5 from Dr J. Barnwell (NYU Medical Center,
New York). Polyclonal rabbit-antihuman platelet CD36 IgG was
produced as previously described.28Fluorescein-labeled goat-antimouse IgG was from Kirkegaard & Perry (Gaithersburg, MD).
Horseradish peroxidase-conjugated goat-antirabbit Fab‘2 and the
ECL chemiluminescent peroxidase substrate kit for Western blots
were obtained from Amersham (Arlington Heights, IL). A full-length
CD36 cDNAZ9was obtained from Dr B. Seed (Massachusetts General Hospital, Boston). A 1.1-kb TSP cDNA3’ encoding the amino
terminal 370 amino acids subcloned into pGEM-2 was generously
donated by Dr J. Lawler (Brigham and Women’s Hospital Boston,
MA). A human GAPDH cDNA clone was obtained from the American Type Culture Collection (ATCC; Rockville, MD). Human recombinant M-CSF was generously provided by Genetics Institute
(Boston, MA). Human recombinant IL-4 was from Immunex (Seattle, WA). Restriction enzymes were obtained from Boehringer
Mannheim (Ridgefield, CT). RPMI 1640, Versene, Dulbecco’s phosphate-buffered saline (PBS), fetal bovine serum, and the antibiotics
gentamicin, penicillin, and streptomycin were obtained from GIBCO
(Grand Island, NY). Maximally oxidized human ox-LDL, prepared
by CuS04 reaction with sequential isopycnic ultracentrifugated
LDL,” was obtained from Perimmune (Rockville, MD). Human AB
serum, paraformaldehyde, PMA, oil red 0, dexamethasone, RNase
A and RNase T1, and Escherichia coli LPS were purchased from
Sigma Chemicals (St Louis, MO). Sp6 RNA polymerase, T7 RNA
polymerase, and plasmid pGEM-4Z were from Promega (Madison,
WI). The plasmid pBluescript KS was from Stratagene (La Jolla,
CA). Tissue culture plates were obtained from Costar (Cambridge,
MA). Ficoll and Percoll for monocyte preparation were obtained
from Pbarmacia (Piscataway, NJ). All other reagents were of analytical grade.
Cell isolation and culture. PB mononuclear cells were isolated
from leukocyte-rich fractions diluted 1:l with versene by the FicollPaque technique?’ The mononuclear fraction from the Ficoll gradients was further separated by Percoll gradient centrifugation to obtain
monocytes. The monocytes were washed in PBS and then resuspended in RPMI 1640 containing 0.05% gentamicin and supplemented with 5% heat-inactivated human AB serum. Aliquots of the
cell suspension (5 X IO6 mononuclear cells/mL) were allowed to
adhere to wells of a 24-well tissue culture plate at 37°C in a humidified 5% CO, environment. After 30 minutes the wells were washed
probe of 792 bp. The protected fragment generated by this probe
was 759 bp. The TSPl riboprobe was generated from the pGEM-2/
TSP plasmid linearized with HindIII and transcribed in vitro with
Sp6 RNA polymerase to generate an antisense probe of 1,275 bp.
The protected fragment generated by this probe was 1,200 bp. The
GAPDH riboprobe was generated by subcloning a 744-bp Pst I-Xba
I fragment from the 5‘-most end of the cDNA into pGEM-4Z. The
resulting plasmid was linearized with HindIII and transcribed in
vitro with SP6 RNA polymerase to generate an antisense probe of
554 bp and a protected fragment of the same length. After overnight
hybridization, the samples were digested with RNAse A and RNase
T1 and the protected fragments were analyzed on 5% polyacrylamide
gel electrophoresis in Tris borate buffer. On all gels, radiolabled
probe as well as labeled probe incubated with the RNAses were run
as controls to verify that the protected fragments were of appropriate
size and that the RNase digestion was adequate. Autoradiograms of
the dry gels were assessed by densitometric scanning using a UMAX
(Santa Clara, CA) UC630 flatbed scanner attached to a Macintosh
IIci (Apple Computer, Cupertino, CA) running NIH Image software
(Bethesda, MD) and transcript levels normalized to that obtained
with the control GAPDH riboprobe for each set.
Zmmunoblotting analysis. Monocyte cultures were lysed in 1%
NP-40 in 50 mmom Tris, 5 mmol/L EDTA, and 148 mmom NaCl
at pH 7.4. Total cell lysate was solubilized in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and
proteins (25 pg/lane) resolved through 8% SDS-PAGE. The protein
bands were electrophoretically transferred to nitrocellulose paper,
where the nitrocellulose was then blocked for 2 hours in 20 m o l /
L Tris, 150 mmoVL NaCI, 0.05% Tween 20, pH 7.4 (TBST) with
5% nonfat dry milk. The blocking solution was replaced with murine
monoclonal anti-CD36 IgG FA6 (2 pg/mL) or specific rabbit antiCD36 IgG (20 pg/mL) in TBST for 1 hour, and washed three times
for 10 minutes with the same buffer. Immunoreactive bands were
detected after incubation with horseradish peroxide-conjugated goat
secondary antibodies for 45 minutes followed by addition of ECL
reagent and exposure to photographic film. In studies comparing
intracellular versus cell surface localization, cells were permeabilized by mild detergent lysis with 20 pg/mL digitonin, followed by
low-speed spin (1,500g) to isolate the cytosolic supernatant. The
specificity of the separation procedure was verified through immunoblots to the membrane marker CD3 1.
Indirect immunofluorescence microscopy. Monocytes were
seeded on coverslips placed in 24-or 12-well tissue culture plates
and incubated with soluble mediators or control media for 16 hours
at 37°C. The cells were then placed on ice for I O minutes and then
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HUH ET AL
2022
washed in 4°C PBS for 15 minutes. To detect cell-surface CD36,
cells were prepared by fixing in 2% paraformaldehyde for 30 minutes
on ice. To detect intracellular CD36. cells were permeabilized by
fixation at -20°C in 100% methanol for I O minutes. Cells were then
washed twice in 4°C PBS ( 5 minlwash). The anti-CD36 murine
monoclonal SA6 was added to fixed cells for 30 minutes at 2 pgl
mL. Cells were washed three times for I O minutes each in 4°C PBS
and fluorescein-labeled goat-antimouse IgG (20 pglmL) was added
for 30 minutes. The coverslips were mounted on slides using 15%
polyvinyl alcohol, 65% glycerol in PBS, sealed in the dark, and
photographed with a Nikon epifluorescence microscope (Nikon,
Melville, NY) using Kodak Ektachrome ASA 400 slide film (Eastman Kodak, Rochester, NY).
"'[IJ-TSP binding studies. To quantitate functional CD36 on the
cell surface, "'[I]-TSP binding was used. TSP was purified from
the releasate of thrombin-activated washed platelets by sequential
heparin affinity and anion exchange chromatography and then labeled with Na'"1 using immobilized chloramine T (IODOBEAD;
Pierce Chemical Co. Rockford, IL) as previously described." Binding of "'[I]-TSP to purified PBM was performed as previously described." Experiments were done at 0.1 pmol/L "'[I]-TSP. Nonspecific binding was determined as that not inhibited by EDTA or excess
unlabeled TSP and was generally less than 5% of input. CD36dependent binding was defined as that inhibited by 2 pglmL of the
blocking murine monoclonal anti-CD36 IgG 8A6, and was greater
than 95% of specific binding.
Foam cell assav. Culture-derived macrophages were incubated
in the presence or absence of M-CSF (50 nglmL) for 16 hours. The
cells were subsequently washed, exposed to ox-LDL ( I O pglmL) or
LDL (IO pglmL), while being co-incubated with inhibitory MoAb
to CD36 (8A6.2 pglmL), nonimmune control IgG, or a competitive
inhibitor of the type I scavenger receptor (fucoidan, 200 pglmL),
for 24 hours. After incubation, the macrophage monolayers were
washed, fixed with 2% paraformaldehyde, stained with oil red 0 to
detect intracellular neutral lipids, and counterstained with Wright's
stain.
CD36
TSP
1
0
1
2
3
4
5
(Days in Culture)
6
7
8
Fig 1. Changes in steady-state CD36 and TSPl mRNA levels upon
monocyte differentiation into culture-derived macrophages. Ficolll
Percoll purified human PBMs cultured for up to 8 days were lysed in
5 mol/L guanidine thiocyanate, 0.1 mollL EDTA and hybrididizedwith
'*P-labeled CD36, TSP1, and glyceraldehyde phosphate dehydrogenase (GAPDHIantisense riboprobesat 37°C for 20 hours. The samples
were then digested with RNase T1 and electrophoresed on 5% polyacrylamide gels where autoradiograms were obtained. The first lysate was obtained on the day of the PBM isolation at day 0. Successive lysates where prepared at 24-hour intervals thereafter until day
8 postculture where the cells have fully differentiated into mature
macrophages. Relative levels of steady-state RNA per lane was assessed through densitometric scanning within the linear range of
autoradiographicexposure. Total RNA per lane were normalized by
comparison with total RNA hybrididizedwith a glyceraldehyde phosphate dehydrogenase (GAPDH) riboprobe.
1 2 3 4 5 6 7
-88kDa
Fig 2. Transient increase in CD36 protein levels during monocyteto-macrophagetransition. PBMs were isolated and grown in culture
as described in Fig 1. Well contents were lysed daily with 1% NP40,
resolved by SDS-PAGE, and immunoprobed for CD36 with monoclonal anti-CD36 IgG (FA61. Each lane represents lysate from day 1
to day 7 postculture.
RESULTS
Freshly isolated human PBM were cultured on tissue culture plastic for 8 days where steady-state mRNA and protein
levels of CD36 were measured during in vitro monocyte
differentiation into macrophages. Morphologically, monocytes adhered to the plastic surface by day I , and began to
spread with membrane extensions reaching maximum surface area per cell by day 6 or 7. After the 7 days in culture,
the multinucleated giant cell population increased incrementally from approximately 10%to 90% of total cell population
by day 20. Interested in determining CD36 expression during
the monocyte-to-macrophage lineage, we followed in vitro
differentiating monocytes from day 0 to day 8. PBM initially
showed low levels of steady-state CD36 mRNA until day 1
in culture, as measured by a modified RNase protection assay
(Fig I). Then by day 3 or 4,a 8 t I-fold increase in CD36
mRNA was detected, which gradually decreased to basal
levels by day 7 or 8. To determine if a similar trend was
present for TSPI, a ligand for CD36. steady-state TSP
mRNA was also monitored from day 0 to day 8 during in
vitro monocyte differentiation. Interestingly, TSPl mRNA
was present in highest levels in freshly isolated monocytes
decreasing with time to near-zero levels by day 3 (Fig I),
suggesting asynchronous expression of CD36 and its ligand
TSPl during monocyte-to-macrophage transition.
To correlate the changes in CD36 mRNA levels with
CD36 protein expression, immunoblotting analysis and immunofluorescence microscopy were used. Immunoblots of
cell lysates from in vitro differentiating monocytes were
probed with MoAbs to CD36 (Fig 2). Total protein levels
of CD36 exhibited a transient increase from days 3 through
5 in culture, which gradually decreased to basal levels by
day 7 or 8, similar to the trend of CD36 mRNA changes.
Densitometric tracing of these blots showed an 8-to 10-fold
upregulation of CD36 in day 4 macrophages relative to day
1 or day 7 in culture, which was comparable with the steadystate mRNA changes. To localize the increased CD36 protein, immunofluorescence microscopy with MoAbs to CD36
was performed (Fig 3). Monocytes at day 1 (Fig 3A) showed
rim staining around the plasma membrane consistent with
previously reported surface localization of CD36.'" How-
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MACROPHAGE CD36: ROLE IN FOAM CELL FORMATION
2023
Table 1. Relative Levels of CD36 in the Cell Surface and
Intracellular Compartments During Monocyte-to-Macrophage
Differentiation in the Presence or Absence of M-CSF
Days in Culture
Treatment
Localization
No M-CSF
Intracellular
Cell surface
Total
Intracellular
Cell surface
Total
+M-CSF
Day 1
.18
.82
1.0
1.6
6.8
8.4
Day 4
2 .1 (18%)
.2 (82%)
.3
2 .6 (19%)
2 .9 (81%)
2 1.5
?
?
6.9
2.2
9.1
7.2
7.3
14.5
Day 7
2 .7 (76%)
.4 (24%)
1.1
i .8 (49%)
2 .8 151%)
2 1.6
?
?
.4
1.6
2.2
3.2
3.6
6.8
2 .2 (19%)
.5 (81%)
.7
2 .6 147%)
2 .8 (53%)
2 1.4
?
?
Peripheral blood monocyteslmacrophages were either treated with M-CSF (50
nglmL, 16 hours). or control medium and then permeabilized in 20 pglmL digitonin at 37°C to separate membraneous and cytosolic fractions. These fractions
were analyzed by SDS-PAGE immunoblot with an MoAb to CD36, FA6. Densitometric tracings of the CD36 bands were quantified and expressed in terms of
arbitrary numerical units. The number within parenthesis refers to relative percentage of CD36 within a particular treatment condition. The specificity of the
digitonin separation of plasma membrane versus cytosolic compartments was
verified through immunoblots to a membrane marker. CD31 (data not shown).
The total amount of CD36 was determined through immunoblots of pooled postdigitonin cytosolicend membraneous lysatesas described in Materials and Methods. n = 3.
Fig 3. A transient increase in the intracellular and cell-surface expression of CD36 during monocyte differentiation into macrophages.
PBMs were isolated and grown in culture as described in Fig 1 on
glass coverslips. The cells were fixed daily with methanol at -20°C
t o permeabilize the cell membrane and incubated with anti-CD36
murine monoclonal IgG (8A6). control lgG, or propidium iodide for
nuclear staining. Bound antibody was detected with fluorescein-labeled goat-antimouse F(ab1'2 and observed under 60x and lOOx oil
immersion objectives. (A) Monocytes at day 1 postculture, (B) early
macrophages at day 2, IC1 macrophage at day 4, and (Dl mature
macrophage at day 7 incubated with monoclonal anti-CD36 IgG. (El
Mature macrophage at day 4 postculture incubated with control IgG,
and (F) stained with propidium iodide t o verify presense of cells in
the culture.
ever, at day 2 (Fig 3B), intracellular staining was seen in
addition to the plasma membrane. Interestingly, much of
the intracellular fluorescence within each cell was localized
preferentially toward a leading edge suggestive of polarized
vectorial transport of CD36. At day 4 in culture, when CD36
mRNA and protein were at peak levels, enhanced cell-surface staining for CD36 was observed along membrane extensions and ruffles (Fig 3C), and a distinct pool of CD36
immunofluorescence was observed intracellularly. By day 7,
cell-surface fluorescence decreased back toward basal monocyte levels and the intracellular fluorescence was no longer
detected (Fig 3D). Negative controls were performed using
nonimmune murine IgG (Fig 3E), where the presence of
monocytes was verified by propidium iodide nuclear staining
(Fig 3F).
The relative levels of CD36 within the intracellular versus
plasma membrane pools were quantitated by densitometric
tracings of CD36 immunoblots of digitonin-partitioned
membraneous and cytoplasmic lysates (Table 1). At day 4 of
the monocyte-to-macrophage transition, total CD36 antigen
increased by 8- to IO-fold. Approximately 76% was intracellular, as assessed by digitonin release, compared with less
than 20% in fresh PBM. By day 7, total CD36 returned
toward basal monocyte levels with the relative intracellular
and cell surface percentages close to that of PBM at 19%
and 8 1 %, respectively .To test for the functional competency
of increased surface CD36, '2SI-labeledTSPl binding to in
vitro differentiating PBM was measured at 4°C (Fig 4). Dif-
Day 1
Day 4
Day 7
Fig 4. Increased '%TSPl binding by culture-derived macrophages correlates with the transient increase in CD36 surface expression. PBMs cultured as described in Fig 1 were incubated with '=ITSPl (100 nmol/L) for 30 minutes at 4°C. Bound and free radioactivity
were separated by centrifugation through silicone oil. (31.
Total binding; (E),binding in the presence of 5 mmol/L EDTA; (P),binding in
the presence of antLCD36 IgG (8A61. Data were analyzed through
ANOVA where bound TSPl was expressed as picomoles per milligram of protein. n = 4 and error bars represent standard deviations.
( * P c .05).
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HUH ET AL
2024
ferentiating monocytes at day 4 exhibited a 1.5- to 2-fold
increase in "'I-TSPI binding relative to analogous cultures
of monocyte (day 1) or mature macrophages (day 7). Binding
of TSPl to cell-surface CD36 was specific as it was blocked
by EDTA or inhibitory monoclonal anti-CD36 IgG, 8A6.
Thus, while the upregulated CD36 at the plasma membrane
is functionally competent. a significant amount (76%) of the
increased CD36 appeared to be localized within an intracellular pool at day 4 in culture.
Multiple soluble mediators have been shown to regulate
monocyte CD36, mainly at the transcriptional level." We
wished to investigate whether in vitro differentiated macrophages remained responsive to such regulation of CD36.
Cell cultures were treated with M-CSF, a known upregulator
of monocyte CD36, and monitered for CD36 protein changes
by immunoblotting with MoAbs to CD36 (Fig SA-F). All
cultures at day I , day 4,and at day 7 experienced increased
CD36 total protein levels upon treatment with M-CSF compared to nontreated controls. Interestingly, when mature
macrophages at day 7 were treated with M-CSF and analyzed
by indirect immunofluorescence microscopy, the M-CSFtreated macrophages (Fig SH) revealed an intracellular pool
of CD36 not seen in the nontreated controls (Fig SG). The
relative amounts of CD36 protein at the cell membrane or
within the cytoplasm as quantitated through densitometric
tracings of immunoblots of digitonin-permeablized cells are
summarized in Table 1. The overall effects of M-CSF included a significant increases in the intracellularly localized
CD36 (>S-fold at day I , I .2-fold at day 4,and 8-fold at day
7), as well as enhancement of plasma membrane localized
CD36(>8-fold at day 1, >3-fold at day 4,and >3-fold at day
7). However, the effects of M-CSF were more pronounced at
day 1 and day 7, when basal CD36 levels were relatively
low. In particular, the M-CSF-induced cell-surface and intracellular CD36 at day 7 quantitatively confirmed the immunofluorescence studies of Fig SG-H.
Parallel responses in the steady-state CD36 mRNA levels,
assessed through RNase protection assays, were seen in tissue-derived alveolar macrophages treated with soluble mediators (Fig 6). M-CSF increased steady-state CD36 mRNA
in alveolar macrophages (Fig 6C), whereas LPS and dexamethasone decreased CD36 mRNA levels (Fig 6B and D)
relative to untreated controls (Fig 6A). Thus, culture-derived
macrophages and alveolar macrophages remained responsive
to regulators of monocyte CD36.
CD36 has been shown to function as a scavenger receptor
for ox-LDL on macrophages.'X~"'Therefore, we determined
whether M-CSF would affect macrophage uptake of ox-LDL
in an in vitro foam cell assay (Fig 7). Culture-derived macrophages at day 8 in culture were treated with M-CSF or
control media and then incubated with ox-LDL for 24 hours
in the presence of inhibitors to CD36 and/or the type I macrophage scavenger receptor. Cells were subsequently stained
with oil red 0 to assess intracellular accumulation of neutral
lipids (Fig 7A-H), and the number of oil red 0-positive lipid
droplets per cell were counted. Cells with greater than IO
lipid droplets were scored as a lipid-laden foam cell and
their percentage of the total population determined (Fig 71).
With no ox-LDL exposure, the %day macrophages showed
A B C D -E F
- -
-I
-88kDa
Fig 5. Culture-derivedmacrophages remain responsiveto M-CSF.
PBMs were isolated and treated as described in Fig 1. Cell cultures
at day 1, day 4, and day 7 were lysed in 1% NP40 for immunoblotting
with monoclonal anti-CD36 IgG (FA6). Monocytes at day 1 (A) untreated or (B)treated with M-CSF (50 ng/mL, 16 hours).Mature macrophages at day 4 IC) untreated or (D) treated with M-CSF. Late
macrophages at day 7 (E) untreated or IF) treated with M-CSF. Immunofluorescence microscopy of day 7 (G) untreated or (HI M-CSFtreated macrophages using monoclonal antiCD36 antibody, FA6, as
in Fig 3.
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MACROPHAGE CD36: ROLE IN FOAM CELL FORMATION
A
B
C
2025
D
CD36
GAPDH
m
Fig 6. Alveolar macrophages are responsive to LPS, M-CSF, and
dexamethasone.Alveolar macrophages were isolated by bronchoalveolar lavage and cultured overnight. They were subsequently
treated with (A) media alone, (6) 1 pg/mL LPS, (C) 50 ng/mL M-CSF,
mollL dexamethasonefor 4 hours. The cells were washed,
or (D)
lysed with 5 mollL guanidium thiocyanate, 0.1 mol/L EDTA, and
probed with CD36 and GAPDH riboprobes for RNase protection assay
as in Fig 1.
low levels of oil red 0-positive granules representing the
basal level of intracellular neutral lipids (Fig 7A). Less than
1% of the population were scored as foam cells. Upon oxLDL addition to these macrophages for 24 hours, the overall
neutral lipid content per cell increased (Fig 7B), with the
foam cell percentage increasing to 8% of total cell population. Macrophages that were pretreated with M-CSF for 16
hours, then subsequently incubated with ox-LDL, showed a
dramatic increase in the number of lipid droplets per cell
(Fig 7C), with the foam cell population increasing to 70%.
To verify that the observed phenomenon was specific for
ox-LDL, M-CSF-treated macrophages were incubated with
LDL. These cells possessed low numbers of lipid droplets
(Fig 7D), with a foam cell population of only 2%. When MCSF-treated macrophages were co-incubated with ox-LDL
and inhibitory monoclonal antLCD36 antibody 8A6, a majority of the heavily lipid-laden cell population was lost, as
the foam cell percentage decreased to less than 20% (Fig 7E).
A competitive inhibitor to the type I macrophage scavenger
receptor, fucoidan, had a less dramatic effect in foam cell
number and lipid content as less than 50% of cells remained
as foam cells (Fig 7F). When both 8A6 and fucoidan were
co-incubated with ox-LDL, the two inhibitors had a combinatory effect on foam cell prevention, decreasing the foam
cell percentage to 12% (Fig 7G). Isotype-matched control
IgG had virtually no effect on foam cell percentage (Fig
7H). Thus, the M-CSF-induced CD36 on culture-derived
macrophages was responsible for 65% to 70% of the oxLDL mediated foam cell formation in this in vitro assay.
DISCUSSION
We have shown a transient increase in CD36 expression
during in vitro differentiation of PBMs into culture-derived
macrophages. This phenomenon occurred both at the steadystate mRNA and protein levels, suggestive of transcriptional
regulation of CD36 as the main point of controlling CD36
expression. TSPI, on the other hand, exhibited highest levels
in monocytes, as steady-state TSPl mRNA decreased to negligible levels by the early macrophage stage. This is consistent with previous observations that elicited murine macrophages expressed less TSP than resident
and that PMA “differentiated” HL-60 cells shut off TSP
synthesis.” Such asynchronous expression of CD36 and its
ligand TSPl has also been observed in cytokine-mediated
transcriptional regulation of monocyte CD36.” The high
abundance of TSPl early in the monocyte differentiation
schema could provide a suitable environment for substratemediated monocyte spreading through interactions of TSPl
with CD36 and other receptors. The low levels of TSP expression in differentiated macrophages may render CD36
unoccupied and thus free to interact with its other proposed
ligands, including ox-LDL and apoptotic neutrophils. In this
regard, TSPl has been shown to inhibit CD36-dependent
macrophage uptake of apoptotic neutrophils.I6Furthermore,
monocyte/macrophage CD36 expression has been previously
shown to be controlled at the transcriptional level upon adhesion to TNF activated-endothelium.”’Thus, a variety of cellular stimuli including adhesion, soluble mediators, and differentiation seem to dictate the expression of monocyte/
macrophage CD36, possibly converging at one or more junctions within their individual intracellular signalling pathways.
The transient nature of CD36 expression during the monocyte-to-macrophage transition is analogous to similar trends
observed in other systems of cell differentiation. One example is SCIP, a POU domain transcription factor, which exhibits a transient expression pattern during oligodcndricytc differentiation that may be a critical factor in determining
functionai cell fate.35Of interest, expression of the murine
CD36 gene was recently identified as being dependent on
the B-cell differentiation factor Oct-2, itself a POU octamer
family member.36 Thus, it is reasonable to speculate that
a specific transcription factor, or more likely a battery of
transcriptional factors, may be intimately involved in controlling CD36 transcription during monocyte differentiation.
The recent genomic cloning of the human3’ and mouse CD36
gene (personal communication, July 1995)should aid in such
molecular analysis of the transcriptional mechanisms underlying functional fate determination.
In this article, we also report the presence of an intracellular pool of CD36 in macrophages in addition to its established plasma membrane localization. An intracellular pool
of CD36 was also found in M-CSF- and PMA-treated
PBM,2’ but was not detected in adhesion-induced CD36 expression.2”Thus, it is possible that soluble mediators, including M-CSF, and monocyte-to-macrophage differentiation
may share certain intracellular signaling pathways. The identity and function of the intracellular pool of CD36 is not
clear. CO-immunolocalization studies with intracellular
markers have not shown specific localization to a known
intracellular membrane compartment (personal communication, June 1995). Thus, the intracellular pool may represent
( I ) translated CD36 awaiting transport to the plasma membrane, (2) CD36 that has been initially targeted to the plasma
membrane and subsequently returned cytoplasmically as a
component of receptor cycling, or most likely (3) a combina-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
HUH ET AL
2020
tion of the two,similar to the known behavior of other macrophage cell-surface receptors of lipid moities as in the case
of the LDL-receptor?' Our data showing a marked increase
in CD36-dependent intracellular accumulation of neutral
lipid (Fig 7), despite only a twofold increase in steady-state
surface expression of CD36 (Table l), are consistent with
at least some component of active receptor cycling. Emerging data on the intracellular trafficking of macrophage CD36
should lend insight into the function and mechanism of the
intracellular pool of CD36.
The initial increase and subsequent decrease in CD36 during monocyte differentiation into culture-derived macrophages is unique when compared with the expression patterns of other known scavenger receptors for acetylated and
oxidized-LDL. The type I macrophage scavenger receptor
shows a slower increase in levels upon monocyte-to-macrophage differentiation without a subsequent decrease in ex-
- Ox-LDL
pression." Such data, coupled with differential affinities of
each of the scavenger receptors for alternatively modifiedLDL, particularly acetylated versus oxidized, suggest distinct yet coordinate modes of regulating multiple scavenger
receptor expression on the macrophage cell surface. However, we have also shown that a second signal, such as MCSF or PMA, appears necessary to maintain the elevated
levels of CD36 from the early macrophage to late macrophage stages. Such a model is supported in the context of
inflammatory states, where elevated cytokine levels within
the subendothelial space may be critical for eliciting CD36
expression on the macrophage cell surface, allowing proper
interaction with its proposed ligands. In fact, specific external signals, in the form of soluble mediators, may dictate
the expression and specificity of CD36 for the wide range
of ligands from TSP, collagen, and apoptotic neutrophils
to ox-LDL. The cellular events of atherosclerotic plaque
>lo lipid droplets per cell
+ OX-LDL
MCSF + Ox-LDL
MCSF + LDL
MCSF+ Ox-LDL + @A6
MCSF+Ox-LDL+Fumidan
MCSF+Ox-LDL
MCSF+Ox-LDL+@G
Fig 7. M-CSF-induced CD36 promotes ox-LDL-mediated macrophage foam cell formation. Culture-derived macrophages at day 8
were (A) untreated; or incubated with (B) ox-LDL; IC) M-CSF and oxLDL; (D) M-CSF and LDl; (E) M-CSF, ox-LDL, and CD36 inhibitory
antibody, 8A6; IF) M-CSF, ox-LDL, and fucoidan; IG) M-CSF, ox-LDL
8A6, end fucoidan; or (HIM-CSF, ox-LDL, and control IgG as described
in Materials and Methods. After incubation, the respective cultures
were fixed with 2% paraformaldehvde, stained with oil red 0, and
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
MACROPHAGE CD36: ROLE IN FOAM CELL FORMATION
formation presents a pathologic state where such analysis
may be further extended. The M-CSF-induced macrophages
possessed elevated foam cell number and lipid content,
which was significantly reduced with inhibitory antibodies
to CD36 whereas only partially inhibitable with fucoidan.
Such evidence is suggestive of extensive CD36 involvement
in ox-LDL-mediated macrophage foam cell generation. Elevated levels of M-CSF mRNA and protein have been detected in atherosclerotic lesions of rabbits and humans,39
whereas human M-CSF in the plasma has been shown to
lower plasma cholesterol levels.40Our results suggest that
the increased levels of M-CSF and possibly other cytokines
may maintain CD36 at sufficient levels for effective ox-LDL
binding and uptake, ultimately resulting in enhanced foam
cell formation.
ACKNOWLEDGMENT
We thank Qinghu Zheng for his technical assistance.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1996 87: 2020-2028
Regulated expression of CD36 during monocyte-to-macrophage
differentiation: potential role of CD36 in foam cell formation
HY Huh, SF Pearce, LM Yesner, JL Schindler and RL Silverstein
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