ABCA1 and ABCG1 Protect Against Oxidative
Stress–Induced Macrophage Apoptosis During Efferocytosis
Laurent Yvan-Charvet, Tamara A. Pagler, Tracie A. Seimon, Edward Thorp, Carrie L. Welch,
Joseph L. Witztum, Ira Tabas, Alan R. Tall
Rationale: Antiatherogenic effects of plasma high-density lipoprotein (HDL) include the ability to inhibit apoptosis
of macrophage foam cells. The ATP-binding cassette transporters ABCA1 and ABCG1 have a major role in
promoting cholesterol efflux from macrophages to apolipoprotein A-1 and HDL and are upregulated during the
phagocytosis of apoptotic cells (efferocytosis).
Objective: The goal of this study was to determine the roles of ABCA1 and ABCG1 in preserving the viability of
macrophages during efferocytosis.
Methods and Results: We show that despite similar clearance of apoptotic cells, peritoneal macrophages from
Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ, Abcg1ⴚ/ⴚ, and, to a lesser extent, Abca1ⴚ/ⴚ mice are much more prone to apoptosis during
efferocytosis compared to wild-type cells. Similar findings were observed following incubations with oxidized
phospholipids, and the ability of HDL to protect against oxidized phospholipid-induced apoptosis was markedly
reduced in Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ and Abcg1ⴚ/ⴚ cells. These effects were independent of any role of ABCA1 and
ABCG1 in mediating oxidized phospholipid efflux but were reversed by cyclodextrin-mediated cholesterol efflux.
The apoptotic response observed in Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ macrophages after oxidized phospholipid exposure or
engulfment of apoptotic cells was dependent on an excessive oxidative burst secondary to enhanced assembly of
NADPH oxidase (NOX)2 complexes, leading to sustained Jnk activation which turned on the apoptotic cell death
program. Increased NOX2 assembly required Toll-like receptors 2/4 and MyD88 signaling, which are known to
be enhanced in transporter deficient cells in a lipid raft– dependent fashion.
Conclusions: We identified a new beneficial role of ABCA1, ABCG1 and HDL in dampening the oxidative burst and
preserving viability of macrophages following exposure to oxidized phospholipids and/or apoptotic cells. (Circ
Res. 2010;106:1861-1869.)
Key Words: ABC transporters 䡲 HDL 䡲 cholesterol 䡲 efferocytosis 䡲 oxidative stress
P
apoptotic macrophages in the lung, heart and lesions of
Abcg1⫺/⫺ and Abca1⫺/⫺Abcg1⫺/⫺ mice.9,10,12 The relationship of macrophage apoptosis to lesion development is
complex, and whereas in early lesions efficient clearance of
apoptotic cells by phagocytic macrophages (efferocytosis)
may be associated with decreased lesion development, it is
thought that in advanced lesions impaired efferocytosis leads
to postapoptotic necrosis, inflammation and necrotic core
formation.13–15 In previous studies, we showed substantial
induction of Abca1 and Abcg1 expression in efferocytes
largely attributable to activation of liver X receptor (LXR)
transcription factors and massive efflux of cholesterol to
acceptors in media.16,17 However, even in the absence of
lipoprotein acceptors in media viability of efferocytes was
preserved, reflecting cholesterol esterification and activation
of prosurvival signaling pathways.17 In view of the prominent
accumulation of apoptotic cells in tissues of Abcg1⫺/⫺ and
lasma high-density lipoproteins (HDLs) have a protective
role in atherogenesis, related in part to their ability to
promote cholesterol efflux and suppress vascular inflammation.1–3 The ATP-binding cassette transporters ABCA1 and
ABCG1 promote cholesterol efflux to apolipoprotein
(apo)A-1 and HDL particles, respectively, and have been
shown to decrease macrophage inflammatory and chemokine
gene expression by suppressing signaling via Toll-like receptor (TLR)4/MyD88.4 –7 HDL also has been shown to reduce
macrophage apoptosis in response to oxidized LDL,8 an
effect that in part reflects the ability of ABCG1 to promote
efflux of toxic 7-oxysterols to plasma HDL.8,9
Transplantation of Abca1⫺/⫺Abcg1⫺/⫺ bone marrow into
hypercholesterolemic Ldlr-deficient mice results in markedly
accelerated atherosclerosis and foam cell accumulation in
various tissues including the lung, heart, liver, and intestine.10,11 Moreover, there is evidence of increased numbers of
Original received January 17, 2010; revision received April 19, 2010; accepted April 20, 2010.
From the Division of Molecular Medicine (L.Y.-C., T.A.P., T.A.S., E.T., C.L.W., I.T., A.R.T.), Department of Medicine, Columbia University, New
York; and Department of Medicine (J.L.W.), University of California at San Diego, La Jolla.
Correspondence to Laurent Yvan-Charvet, Division of Molecular Medicine, Department of Medicine, 630 W 168th St, Columbia University, New
York, NY 10032. E-mail ly2159@columbia.edu
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.110.217281
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Non-standard Abbreviations and Acronyms
ABC
apo
ER
HDL
LXR
MAPK
NAC
NOX
ox-PAPC
ROS
siRNA
TLR
ATP-binding cassette transporter
apolipoprotein
endoplasmic reticulum
high-density lipoprotein
liver X receptor
mitogen-activated protein kinase
N-acetylcysteine
NADPH oxidase
oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero3phosphocoline
reactive oxygen species
small interfering RNA
Toll-like receptor
Abca1⫺/⫺Abcg1⫺/⫺ mice, we have directly examined the role
of these transporters in preserving the viability of efferocytes.
Subsequent mechanistic studies have uncovered a novel role
of the transporters in suppressing the oxidative burst mediated by NADPH oxidase (NOX)2 during efferocytosis.
Methods
An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org.
Animals
Abca1⫺/⫺, Abcg1⫺/⫺, and Abca1⫺/⫺Abcg1⫺/⫺ littermates in a
mixed C57BL/6⫻DBA background were as previously described.10
Myd88⫺/⫺ mice on the C57BL/6 background were from Dr. Katherine Fitzgerald (University of Massachusetts). CD36⫺/⫺ mice on the
C57BL/6 background were originally provided by Dr Roy Silverstein (Weill Medical College of Cornell University, New York). All
mice were housed at Columbia University Medical Center according
to animal welfare guidelines. Animals had ad libitum access to both
food and water.
Results
Increased Susceptibility of Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ
Macrophages to Apoptosis After Engulfment of
Apoptotic Cells
Following ingestion of apoptotic Jurkat cells, macrophages
deficient in ABCA1 and/or ABCG1 underwent apoptosis in
the order Abca1⫺/⫺Abcg1⫺/⫺⬎Abcg1⫺/⫺⬎Abca1⫺/⫺, whereas
wild-type (WT) macrophages were resistant to death (Figure
1A). A time course experiment already showed a slight induction of apoptosis in Abca1⫺/⫺Abcg1⫺/⫺ macrophages at 3 hours
following efferocytosis and a dramatic 9-fold increase at 16
hours (Figure 1B). Phagocytosis, as measured by phagocytic
index at 30 minutes after efferocytosis, was unaffected (Figure
1C). However, ingestion of or contact with apoptotic cells by
Abca1⫺/⫺Abcg1⫺/⫺ macrophages appears to be required because addition of conditioned medium from apoptotic Jurkat cell
culture to Abca1⫺/⫺Abcg1⫺/⫺ macrophages did not elicit a
similar apoptotic response (Figure 1D). In terms of the
mechanism we first considered that accumulation of free
cholesterol after ingestion of apoptotic cells may induce
ER-stress with subsequent induction of apoptosis.17 However,
similar to previous observations,17 Abca1⫺/⫺Abcg1⫺/⫺ efferocytes showed increased ACAT activity when compared to
WT, as reflected by enhanced free cholesterol conversion into
cholesteryl ester, thereby preventing induction of CHOP
mRNA and other ER stress markers (Online Figure I). By
contrast, the use of E06 antibody known to recognize oxidized phospholipids and inhibit uptake of apoptotic cells,18
reduced apoptosis in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure
1D). The potential pathophysiological relevance of our
system was illustrated by the finding of regions of enhanced E06 staining surrounding clusters of apoptotic cells
(TUNEL-positive staining) in the lungs of Abcg1⫺/⫺ and
Figure 1. Enhanced susceptibility to apoptosis in Abca1ⴚ/ⴚ, Abcg1ⴚ/ⴚ, and
Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ macrophages after
ingestion of apoptotic Jurkat cells.
Thioglycollate-elicited macrophages from
mice on mixed background C57BL/6⫻DBA
were incubated with apoptotic Jurkat cells
for 30 minutes, extensively washed, and
kept in DMEM 10% FCS for 16 more hours
(A) or the indicated time period (B) before
quantification of apoptosis by Annexin V
staining. In parallel experiments, macrophages were incubated with Cell-Tracker
red prelabeled apoptotic Jurkat cells to
quantify the phagocytic index (PI) (C). Macrophages were incubated for 30 minutes
with cultured media from apoptotic Jurkat
cells or in presence of 0.5 mg/mL E06 antibody competitor during the 30 minutes apoptotic Jurkat cells incubation period (D)
before quantification of apoptosis. Apoptotic
cells were expressed as percentage of total
cells as described in Methods. Results are
means⫾SEM of experiments performed in
triplicate. §P⬍0.05 vs WT; *P⬍0.05 treatment effect.
Yvan-Charvet et al
ABCA1, ABCG1, and Efferocyte Apoptosis
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Figure 2. HDL protects macrophages
from apoptosis induced by ox-PAPC
through modulation of membrane
cholesterol. Macrophages were
exposed to different concentrations of
ox-PAPC (A) or treated for 16hours with
50 ␮g/mL control PAPC or oxidized
PAPC (ox-PAPC) (B). Ox-PAPC–treated
WT, Abcg1⫺/⫺, and Abca1⫺/⫺Abcg1⫺/⫺
macrophages were also incubated simultaneously with different concentrations of
HDL (0 to 100 ␮g/mL) for 16 hours (C
and D). WT and Abca1⫺/⫺Abcg1⫺/⫺
macrophages were pretreated with indicated amount of HDL for 30 minutes
before efferocytosis (E). Pretreatment of
WT and Abca1⫺/⫺Abcg1⫺/⫺ macrophages with 5 mmol/L cyclodextrin (CD)
for 30 minutes before exposure to 50
␮g/mL ox-PAPC or with cyclodextrincholesterol (CD-c, 2.5:1) for 3 hours
before efferocytosis (F). Apoptosis was
quantified at the end of the 16-hour
incubation period and expressed as percentage of total cells as described in
Methods. Results are means⫾SEM.
§P⬍0.05 vs WT; *P⬍0.05 treatment
effect.
Abca1⫺/⫺Abcg1⫺/⫺ bone marrow–transplanted mice compared to controls and Abca1⫺/⫺ mice (Online Figure II).
The findings suggest that HDL and ABCG1 may act to
limit formation of oxidized phospholipids and associated
apoptotic events in the lung.
High-Density Lipoprotein Protects Macrophages
From Apoptosis Induced by Oxidized
Phospholipids in an ABC
Transporter–Dependent Fashion
We recently showed that Abcg1⫺/⫺ macrophages have
enhanced susceptibility to oxidized LDL induced apoptosis, an effect related to a specific role of ABCG1 in
promoting efflux of 7-oxysterols to HDL.8 However,
7-ketocholesterol was undetectable in apoptotic Jurkat
cells and in phagocytic macrophages (data not shown). The
uptake of apoptotic cells by macrophages has many similarities to the uptake of oxidized LDL and oxidized phospholipids are a major bioactive component of both oxidized LDL
and apoptotic cells.19,20 Treatment of macrophages with
oxidized phospholipids (ie, oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3phosphocoline [ox-PAPC]) revealed a dose dependent increase in apoptosis in
Abca1⫺/⫺Abcg1⫺/⫺ macrophages starting as low as 10
␮g/mL ox-PAPC loading (Figure 2A) and at 50 ␮g/mL led to
⫺/⫺
a marked increase in apoptosis in the order Abca1 Abcg1⫺/⫺⬎
Abcg1⫺/⫺⬎ Abca1⫺/⫺⬎WT (Figure 2B), paralleling the effects
of apoptotic Jurkat cells (Figure 1A). We next investigated
the role of HDL on macrophage viability in WT and
Abca1⫺/⫺Abcg1⫺/⫺ macrophages after ox-PAPC exposure.
HDL markedly reduced apoptosis induced by ox-PAPC in
WT macrophages at all concentrations tested and the maximum suppression was reached at 50 ␮g/mL HDL (Figure
2C). The protective effect of HDL was dramatically reduced
in Abcg1⫺/⫺ macrophages (Figure 2C) and virtually abolished in Abca1⫺/⫺Abcg1⫺/⫺ macrophages except at the
highest HDL concentration (Figure 2D). Similar findings
were observed in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure
2F). Because ABCA1 promotes efflux of both cholesterol and
phospholipids from cells,21,22 and a possible role of ABCG1
in phospholipid efflux has also been described,23,24 we first
considered that both transporters might participate in the
efflux of oxidized phospholipids to HDL and thereby also
protect from apoptosis. To test this hypothesis, efflux of
ox-PAPC was determined after loading cells with radiolabeled [14C]-ox-PAPC. The amount of incorporated [14C]oxPAPC is shown in Online Figure III, A. HDL but not apoA-I
showed a slight ability to promote ox-PAPC efflux but
surprisingly, this effect was independent of ABCA1 and
ABCG1 (Online Figure III, B). Thus, the increased suscep-
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June 25, 2010
Figure 3. The enhanced ROS response
in Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ macrophages after
ingestion of apoptotic Jurkat cells or
ox-PAPC loading mediated the apoptotic
response. Thioglycollate-elicited macrophages from WT and Abca1⫺/⫺Abcg1⫺/⫺
mice were incubated with apoptotic Jurkat
cells for 30 minutes, extensively washed and
kept in DMEM 10% FCS for the indicated
time period (A) or treated for 3 hours with 20
␮g/mL ox-PAPC (B) before quantification of
ROS generation by CM-H2DCFDA staining.
Macrophages were incubated in presence of
100 ␮g/mL NAC during exposure to
ox-PAPC (B) or with 100 ␮g/mL NAC or
10 mmol/L glutathione (GSH) during the
course of efferocytosis and apoptosis was
quantified 16 hour later (C). Treatment of
WT and Abca1⫺/⫺Abcg1⫺/⫺ macrophages
with 100 ␮g/mL HDL or pretreatment with
5 mmol/L cyclodextrin (CD) for 30 minutes
before efferocytosis. ROS generation was
quantified 1 hour later (D). Apoptotic cells
and ROS positive cells were expressed as
percentage of total cells as described in
Methods. Results are means⫾SEM of
experiments performed in triplicate. §P⬍0.05
vs WT; *P⬍0.05 treatment effect.
tibility to ox-PAPC-induced apoptosis in Abca1⫺/⫺Abcg1⫺/⫺
macrophages could not be explained by impaired oxidized
phospholipid efflux. Thus, we next considered that effects of
HDL and ABC transporters on macrophage viability could be
mediated by cholesterol efflux. Consistent with this idea,
depletion of membrane cholesterol by cyclodextrin reduced
the percentage of apoptotic cells after ox-PAPC exposure in
both WT and Abca1⫺/⫺Abcg1⫺/⫺ macrophages and completely abolished the apoptosis of Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure 2F). In contrast, the loading of macrophages
with cyclodextrin-cholesterol enhanced the apoptotic response during efferocytosis when both transporters were
missing (Figure 2F).
efferocytes to apoptosis. Two antioxidants, glutathione and
N-acetylcysteine (NAC) showed marked antiapoptotic effects in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure 3C). Similarly, NAC significantly reduced the apoptosis induced by
ox-PAPC in Abca1⫺/⫺Abcg1⫺/⫺ macrophages (Figure 3B).
Remarkably, depletion of membrane cholesterol by either
high concentration of HDL or cyclodextrin clearly reduced
the production of ROS following ingestion of apoptotic
cells in both WT and Abca1⫺/⫺Abcg1⫺/⫺ efferocytes
(Figure 3D).
An Increased Oxidative Burst in
Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ Macrophages After ox-PAPC
Exposure or Engulfment of Apoptotic Cells Is
Responsible for the Enhanced Apoptosis
During bacterial phagocytosis, ROS generation is mediated
by an NADPH oxidase complex, in which gp91phox/NOX2 is
the major catalytic component in macrophages.28 Recent data
have shown that gp91phox/NOX2 may also be activated during
efferocytosis.29 Immunostaining for p47phox clustering (red
staining), one of the active components of the NADPH
oxidase complex known to translocate from a cytosolic pool
to the plasma membrane during active complex formation,
revealed an increase in number of both cluster-positive cells
and p47phox clusters per cell for Abca1⫺/⫺Abcg1⫺/⫺ macrophages compared to controls in the basal state (Figure 4A and
4B). Ox-PAPC increased the number of positive cells and the
number of p47 clusters per cell in WT macrophages and this
was further enhanced in Abca1⫺/⫺Abcg1⫺/⫺ macrophages
(Figure 4A and 4B). As previously shown for bacterial
phagocytosis, 30 minutes after ingestion of apoptotic cells
(green staining), a considerable amount of p47phox staining
was localized in phagolysosomal membranes surrounding the
apoptotic cells (yellow staining: overlap between green apoptotic cells and red p47phox staining) compared to nonphagocytic cells as shown in Figure 4C (left). Performing crosssectional cuts through the phagolysosome indicate more
We recently reported that ABCG1 deficiency induced an exaggerated ROS production in endothelial cells in response to
7-ketocholesterol.25 Oxidized phospholipids are also known to
induce ROS formation,26,27 a mechanism that may play a
crucial role during phagocytosis of bacteria and possibly
apoptotic cells by macrophages.28,29 The ingestion of apoptotic Jurkat cells was associated with a transient increase in ROS
formation in WT macrophages. The increase in ROS was
exaggerated in Abca1⫺/⫺Abcg1⫺/⫺ macrophages (Figure
3A). An enhanced formation of ROS was observed after
exposure to ox-PAPC in DKO macrophages (Figure 3B).
Consistent with this finding, we found that the mRNA
levels of heme oxygenase-1 (HO-1), a marker of oxidative
stress were increased during the course of efferocytosis in
Abca1⫺/⫺Abcg1⫺/⫺ macrophages as well as after ox-PAPC
loading (Online Figure IV). These results led us to investigate if the enhanced oxidative response may be responsible for the increased susceptibility of Abca1⫺/⫺Abcg1⫺/⫺
Increased NOX2 Assembly After ox-PAPC
Exposure or Efferocytosis in
Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ Macrophages
Yvan-Charvet et al
ABCA1, ABCG1, and Efferocyte Apoptosis
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Figure 4. Increased assembly of
NADPH oxidase 2 (NOX2) in Abca1ⴚ/ⴚ
Abcg1ⴚ/ⴚ macrophages after ox-PAPC
exposure or ingestion of apoptotic
Jurkat cells. Confocal analysis of
p47phox stained (red) peritoneal
macrophages from WT and Abca1⫺/⫺
Abcg1⫺/⫺ mice exposed to ox-PAPC for
1 hour (A and B) or to Cell-Tracker
green prelabeled apoptotic Jurkat cells
for 30 minutes (C and D). Arrows indicate p47phox clusters in nonphagocytic
cells (red staining) (A). Representative
images of an ingested apoptotic cell
(green) localized within a phagolysosome
(C left hand panel, in total five different
confocal images were analyzed per genotype). Visualization of the phagolysosome surrounding p47phox staining by
performing a horizontal (indicated by the
green line) and a vertical (indicated by
the red line) cross-cut through the
z-stack (C right hand panels). Inserts on
top and right hand side portray the
flipped image of the phagolysosomes
after the cross-cut. Quantification was
performed as described in the Methods
with the use of ImageJ software (B and
D). Macrophages were incubated in
presence of 10 ␮mol/L NOX2 inhibitor
(DPI) during the course of efferocytosis
(E) or treated with scrambled or NOX2
siRNA before ingestion of apoptotic Jurkat cells (F). At the end of the 16 hour
incubation, apoptosis was determined.
Results are means⫾SEM of an experiment performed in triplicate. §P⬍0.05 vs
WT; *P⬍0.05 treatment effect.
clearly the p47phox lining (red) surrounding the apoptotic cell
(green) (Figure 4C, right). Quantification of the phagolysosomal p47phox staining at 30 minutes after efferocytosis
revealed a 3-fold increased signal in Abca1⫺/⫺Abcg1⫺/⫺
efferocytes compared to WT (Figure 4C and 4D). The use of
NOX2 inhibitor (Figure 4E) and knockdown experiments
(Figure 4F) confirmed that the enhanced apoptotic response
in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes required the activity of
NOX2.
ptotic Jurkat cells (Figure 5A). We also observed a corresponding increase in the cleaved form of caspase 3 in
Abca1⫺/⫺Abcg1⫺/⫺ macrophages (Figure 5A) a marker for
apoptosis.34 This time course of JNK and caspase-3 activation
correlated well with the early induction of apoptosis in
Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure 1B). An inhibitor of
JNK prevented apoptosis of Abca1⫺/⫺Abcg1⫺/⫺ efferocytes
indicating that JNK is necessary in this pathway (Figure 5B).
Sustained JNK Activation After Engulfment of
Apoptotic Cells in Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ Macrophages
Apoptosis of Efferocytes Is Dependent on
TLR4/MyD88 Signaling in
Transporter-Deficient Cells
Apoptosis is thought to represent the outcome of an imbalance between prosurvival and proapoptotic signaling pathways. Neither the prosurvival nuclear factor ␬B or p38
mitogen-activated protein kinase (p38MAPK) pathway,30,31
appeared to be downregulated in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes as reflected by similar protein levels of IKKb and
p38MAPK during the course of efferocytosis (Figure 5A). An
important form of apoptotic signaling induced by ROS
generation involves sustained activation of JNK.32,33 Western
blot analysis of JNK phosphorylation revealed an increase
in both early and late phases of JNK phosphorylation in
Abca1⫺/⫺Abcg1⫺/⫺ macrophages following ingestion of apo-
Myd88 signaling has been shown to play a key role in the
assembly of the NADPH oxidase complex during phagocytosis of Gram-negative bacteria,35 and we have previously
shown that ABC transporter deficient macrophages show increased signaling via TLR4 and TLR2 in a cholesterol lipid raft
dependent fashion.7 Thus, we next determined if increased
signaling via TLR/MyD88 was responsible for increased NOX2
assembly during efferocytosis. Treatment of elicited macrophages with LipidA dramatically increased the number of
p47-immunopositive cells and the number of p47clusters per
cell in Abca1⫺/⫺Abcg1⫺/⫺ macrophages compared to WT
macrophages (Figure 6A and 6B). Although LipidA by itself
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June 25, 2010
line Figure V, A). We next tested whether inhibiting toll-like
receptor signaling prevented the apoptotic response of
Abca1⫺/⫺Abcg1⫺/⫺ efferocytes. Although knockdown of intracellular TLRs by small interfering (si)RNA, including
TLR3, 7 and 8, did not show any beneficial effect, deficiency
of TLR4 and to a lesser extent TLR2 reduced apoptosis in
Abca1⫺/⫺Abcg1⫺/⫺ efferocytes (Figure 6D). We also investigated a possible role of CD36 signaling because it has been
implicated in the recognition of phospholipids present on the
outer leaflet of the plasma membrane of apoptotic cells,19 and
interacts with TLR2 and TLR4.36,37 However, CD36 did not
contribute to apoptosis in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes
when assayed by siRNA knockdown of ABCA1 and ABCG1
in Cd36⫺/⫺ macrophages, siRNA knockdown of CD36 in
Abca1⫺/⫺Abcg1⫺/⫺ efferocytes or by the use of CD36 blocking antibody (Online Figure V, B and C). These findings suggest
that activation of Myd88 signaling through activation of plasma
membrane TLR4, and to a lesser extent TLR2, contributes to the
increased susceptibility of Abca1⫺/⫺Abcg1⫺/⫺ macrophages to
apoptosis following ingestion of apoptotic cells. To confirm this,
we generated Abcg1⫺/⫺Myd88⫺/⫺ mice and performed an efferocytosis assay in elicited macrophages. Lack of MyD88 clearly
abrogated both the apoptosis and the ROS generation observed
in Abcg1⫺/⫺ efferocytes (Figure 6E and 6F).
Discussion
Figure 5. Sustained JNK activation is required for the induction of apoptosis in Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ efferocytes.
Thioglycollate-elicited macrophages from WT and
Abca1⫺/⫺Abcg1⫺/⫺ mice were incubated with apoptotic Jurkat
cells for 30 minutes, extensively washed and kept in DMEM
10% FCS for the indicated time period. Immunoblots of IKKb,
p-p38MAPK, p-JNK, and caspase 3 were performed in a time
course experiment (A). The arrow in the caspase 3 immunoblot
indicates the proactive cleaved form of caspase 3, and the
upper band indicates the uncleaved form. Macrophages were
incubated in presence of 10 ␮mol/L JNK inhibitor (L-JNKI1) during the course of efferocytosis and analyzed for apoptosis 16
hours later (B). Results are means⫾SEM of an experiment performed in triplicate. §P⬍0.05 vs WT.
did not induce significant ROS generation, pretreatment with
LipidA before efferocytosis markedly amplified ROS production (Figure 6C) and enhanced TLR4 signaling led to an
exaggerated apoptotic response during efferocytosis in the
order Abca1⫺/⫺Abcg1⫺/⫺⬎Abcg1⫺/⫺⬎Abca1⫺/⫺⬎WT (On-
Previous studies have shown an increased accumulation of
apoptotic cells in atherosclerotic lesions, lungs and other
tissues in Abcg1⫺/⫺ and Abca1⫺/⫺Abcg1⫺/⫺ mice.9,10,12 In
addition, Abcg1⫺/⫺ macrophages are more susceptible to
apoptosis induced by oxidized LDL or 7-oxysterols, related in
part to a role of ABCG1 in promoting efflux of 7-oxysterols.8
Abca1⫺/⫺Abcg1⫺/⫺ macrophages show increased apoptosis
in response to FC loading (AcLDL⫹ACAT inhibitor), likely
related to the role of the transporters in promoting efflux of
cholesterol from macrophages.10 In this study, we have
uncovered an unexpected role of ABCA1 and ABCG1 in
preserving the viability of macrophages during efferocytosis
and also in mediating a protective role of HDL in defending
macrophages against oxidized phospholipid-induced cell
death. Interestingly, the underlying mechanism is not FCinduced ER stress as we had expected but appears to be
related to a novel role of the ABC transporters and HDL in
dampening the oxidative burst of NOX2 which is activated
during efferocytosis and in response to oxidized phospholipid
uptake. In turn this may be related to the role of the
transporters in diminishing the signaling of TLRs, mediated
by cholesterol efflux and transporter-dependent changes in
membrane lipid organization.4 –7 TLR/MyD88 signaling is
known to induce NOX2 assembly,35 and was shown to have
an essential role in ROS formation and apoptosis in our
system.
In earlier studies, we showed a marked upregulation of
ABCA1 and ABCG1 in efferocytes, mediated mainly by
LXR activation (ABCA1 and ABCG1) and in part by tumor
necrosis factor ␣ signaling via nuclear factor ␬B
(ABCA1).16,38 Consistent with these observations, efferocytes
showed a massive efflux of cholesterol to HDL or apoA-1.17
Even in the absence of cholesterol efflux, there was only a
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ABCA1, ABCG1, and Efferocyte Apoptosis
1867
Figure 6. TLR-dependent Myd88 signaling controls NADPH oxidase
assembly, ROS production, and
the apoptotic response of
Abca1ⴚ/ⴚAbcg1ⴚ/ⴚ efferocytes.
Confocal analysis of peritoneal
macrophages from WT and
Abca1⫺/⫺Abcg1⫺/⫺ mice exposed to
100 ng/mL LipidA for 3 hours (A).
Arrows indicate stained p47phox clusters. Quantification was performed as
described in Methods (B). Peritoneal
macrophages from WT and
Abca1⫺/⫺Abcg1⫺/⫺ mice were exposed
to 100 ng/mL LipidA for 3 hours, following or not by ingestion of apoptotic Jurkat cells and ROS was quantified 3
hours later (C). Macrophages isolated
from WT and Abca1⫺/⫺Abcg1⫺/⫺ mice
were treated with scrambled or different
TLR receptor siRNAs and analyzed for
apoptosis after efferocytosis (D). Macrophages isolated from WT, Abcg1⫺/⫺,
Myd88⫺/⫺, and Abcg1⫺/⫺Myd88⫺/⫺ littermate mice were analyzed for both
apoptosis and ROS generation 16 and 3
hours after efferocytosis, respectively (E
and F). Results are means⫾SEM of
experiments performed in triplicate.
§P⬍0.05 vs WT; *P⬍0.05 treatment
effect.
low level of apoptosis, reflecting compensatory cholesterol
esterification as well as induction of antiapoptotic signaling
pathways via AKT and nuclear factor ␬B.17 The present study
also revealed a compensatory increase in cholesterol esterification in Abca1⫺/⫺Abcg1⫺/⫺ efferocytes, but clearly this
mechanism was insufficient to protect these cells from death
revealing that, although lipoprotein acceptors in media may
not be essential, activity of the 2 main transporters involved
in macrophage cholesterol efflux is crucial to the survival of
macrophages following ingestion of apoptotic cells. Part of
this apparent paradox may reflect the ability of ABCG1 to
promote cholesterol efflux to BSA even in the absence of
cholesterol efflux acceptors,23 as previously used to culture
WT efferocytes.17 More importantly, it likely reflects a
specific ability of ABCA1 and ABCG1 to promote transmembrane sterol redistribution even in the absence of cholesterol acceptors in the medium, as previously suggested.39,40
Consistent with this idea, we previously reported enhanced
lipid raft formation in Abca1⫺/⫺Abcg1⫺/⫺ macrophages in
the absence of lipoprotein acceptors.7
Interestingly, the mechanism underlying the increased plasma
membrane cholesterol-dependent apoptosis in Abca1⫺/⫺Abcg1⫺/⫺
macrophages after ox-PAPC exposure or engulfment of apoptotic cells was found to be an amplified burst of oxidative stress
secondary to increased assembly of NOX2, the major
NADPH oxidase complex present in macrophages.28 Although macrophage ROS generation during bacterial phagocytosis is well known as an important killing mechanism,28
and oxidized phospholipids are also known to promote ROS
generation at least in endothelial cells,26,27 this is the first
report to our knowledge showing that such a mechanism can
also occur in macrophages during efferocytosis. Our studies
are also consistent with important recent observations showing that reconstituted HDL suppresses NOX2 assembly and
activity in neutrophils treated with phorbol ester in a cholesterol lipid raft– dependent fashion,41 and together these studies suggest that this property of reconstituted HDL may be
dependent on activity of the transporters. The new findings
could indicate the importance of the localization of NOX2 in
lipid rafts,42 and are consistent with the previous proposed
role of NOX2 and ROS as signaling molecules in macrophages.43 Recent advances in this area of research have
revealed a close relationship between NOX enzymes and
toll-like receptor signaling during phagocytosis of bacteria.44
Similar to what was observed with phagocytosis of bacteria,35
we found that lack of MyD88 prevented the generation of
ROS during efferocytosis in WT and Abcg1⫺/⫺ cells. We
identified TLR4 as a major component of this regulation in
Abca1⫺/⫺Abcg1⫺/⫺ efferocytes and to a lesser extent TLR2
consistent with our previous findings showing enhanced
1868
Circulation Research
June 25, 2010
TLR2 and TLR4 signaling pathways in Abca1⫺/⫺Abcg1⫺/⫺
macrophages.7 Overall, our data are consistent with a mechanism wherein ABCG1 and to a lesser extent ABCA1
promote membrane sterol redistribution and efflux to HDL,
decreasing lipid raft– dependent assembly of TLR4 and
NOX2 signaling complexes.
Recent studies have shown that in addition to upregulation
of ABCA1 and ABCG1 during efferocytosis, there is induction of the efferocytotic receptor mer-TK by LXRs.38 Together, these studies suggest coordinate functions of LXRs
and the ABC transporters in promoting both efferocytosis and
in preserving viability of phagocytes in the face of the
resultant loading with cholesterol. Defects in these processes
may lead to auto-immune disease,28,38,45 accelerated atherosclerosis with increased accumulation of apoptotic material
and necrotic core formation in advanced plaques,10,13–15 and
here we show evidence as well of a role of these processes in
diseased lungs with increased accumulation of apoptotic cells
and oxidized phospholipids staining with E06 in Abcg1⫺/⫺
and Abca1⫺/⫺Abcg1⫺/⫺ bone marrow–transplanted mice extending a recent report identifying oxidative stress, formation
of oxidized phospholipids and TLR4 signaling as a key
pathway of acute lung injury.46
Sources of Funding
This work was supported by NIH grant HL59541.
Disclosures
None.
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Novelty and Significance
What Is Known?
●
●
●
Expression of Abca1 and Abcg1 is induced in macrophages following
ingestion of apoptotic cells.
The numbers of apoptotic macrophages in lungs and atherosclerotic
lesions are increased in Abcg⫺/⫺ and Abca1⫺/⫺ Abcg1⫺/⫺ mice.
Oxidized LDL induces apoptosis of Abcg1⫺/⫺ and Abca1⫺/⫺Abcg1⫺/⫺
macrophage.
What New Information Does This Article Contribute?
●
●
●
Abcg1⫺/⫺ and Abca1⫺/⫺Abcg1⫺/⫺ macrophages undergo markedly
increased apoptosis following ingestion of apoptotic cells or after
exposure to oxidized phospholipids.
The mechanism involves membrane cholesterol accumulation, increased signaling via TLR4/MyD88 that promotes assembly of
NADPH oxidase (NOX)2 complexes, and excessive ROS formation.
The apoptosis of macrophages following uptake of oxidized phospholipids or apoptotic cells likely leads to a chain reaction of cell
death and inflammation.
The ATP-binding cassette transporters ABCA1 and ABCG1
promote cholesterol efflux to apolipoprotein A-1 and HDL and
likely mediate, in part, the antiatherogenic effect of HDL.
Deficiency of ABCG1 or ABCA1 and ABCG1 in mice leads to
accumulation of apoptotic macrophages in lungs and atherosclerotic lesions. ABCA1 and ABCG1 are highly induced in
macrophages following phagocytosis of apoptotic cells (efferocytosis). Although Abcg1⫺/⫺ and Abca1⫺/⫺Abcg1⫺/⫺ macrophages could ingest apoptotic cells normally, soon afterward
they themselves underwent apoptotic cell death. This was
caused by an excessive oxidative burst attributable to increased
assembly of the NADPH oxidase–NOX2 complex in the plasma
and the endosomal membranes of transporter-deficient macrophages. The findings indicate that HDL and the ABC transporters
normally act to maintain a correct cholesterol content and
distribution in the plasma and endosomal membranes of cholesterol-loaded efferocytes, preventing excessive NOX2 activity.
This represents a new extension of the idea that HDL and the
transporters suppress inflammation during the innate immune
response.