Estrogen and Progesterone Reduce Lipid Accumulation in
Human Monocyte-Derived Macrophages
A Sex-Specific Effect
Jane A. McCrohon, MB, BS, FRACP; Shirley Nakhla, BSc; Wendy Jessup, PhD;
Keith K. Stanley, PhD; David S. Celermajer, MB, BS, PhD, FRACP
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Background—Males have an earlier onset and greater prevalence of clinical atherosclerosis than age-matched females,
which is consistent with an atheroprotective effect of the female sex steroids, estrogen and progesterone. We therefore
examined the effects of estrogen and progesterone on human foam cell formation, a key early event in atherogenesis.
Methods and Results—Monocytes from healthy female and male donors were obtained from white cell concentrates and allowed
to differentiate into macrophages over 10 days. These human monocyte-derived macrophages (MDMs) were exposed to either
control (0.1% vol/vol ethanol) or estrogen or progesterone treatment on days 3 through 10. Lipid loading was achieved on
days 8 through 10 by incubation with acetylated LDL. Lipid from the MDMs was then extracted for analysis of cholesteryl
ester (CE) content. 17b-Estradiol at both physiological (2 nmol/L) and supraphysiological (20 and 200 nmol/L) concentrations
produced a significant reduction in macrophage CE content (8863%, 8862%, and 8564%, respectively; P,0.02 compared
with control). Physiological and supraphysiological levels of progesterone (2, 10, and 200 nmol/L) produced an even more
dramatic reduction in CE content (7469%, 56610%, and 6568%, respectively; P,0.002 compared with control). This effect
could be abrogated by coincubation with the progesterone receptor antagonist RU486. Neither estrogen nor progesterone
produced a reduction in lipid loading in male-donor–derived MDMs. Detailed lipid trafficking studies demonstrated that both
estrogen and progesterone altered macrophage uptake and/or processing of modified LDL.
Conclusions—Physiological levels of estrogen and progesterone are associated with a female-sex–specific reduction in human
macrophage lipid loading, which is consistent with an atheroprotective effect. (Circulation. 1999;100:2319-2325.)
Key Words: cells n atherosclerosis n lipoproteins n lipids
P
remenopausal women have less cardiovascular morbidity
and mortality than similarly aged men.1,2 Animal studies
have supported a potentially antiatherogenic effect of female
sex hormones, with less plaque formation demonstrated in
estrogen-treated cholesterol-fed monkeys3 and rabbits,4 an
effect also observed with combined administration of estrogen and progesterone.3,5 In humans, estrogen therapy is
associated with improved lipoprotein profiles,6,7 vascular
reactivity,8 and cardiovascular outcomes9 in postmenopausal
women in a number of case control and cohort studies.
The effects of progesterone on atherogenesis and event
rates in humans are less well studied. A beneficial effect on
smooth muscle cell proliferation has been suggested10; however, several studies have recently found that the addition of
progesterone to estrogen replacement may attenuate some of
the beneficial effects on lipids,11 fibrinogen,12 and vascular
reactivity.13 The Nurses’ Health Study14 recently documented
a highly significant reduction in coronary event rates with
combined estrogen and progesterone therapy in the setting of
primary prevention; however, the recently reported Heart and
Estrogen-progestin Replacement Study (HERS)15 suggests that
this benefit may not be observed in women with established
coronary disease. Few data exist, however, on the effects of
these hormones on basic atherogenic processes in humans.
Foam cell formation is a key event in the early development of atherosclerosis and is largely due to the uptake of
modified lipoproteins by monocyte-derived macrophages
(MDMs) in the arterial wall.16,17 We therefore examined the
effects of the principal female sex steroids, 17b-estradiol and
progesterone, on macrophage lipid loading and the mechanisms of the observed effects in primary human cells.
Methods
Isolation of Human Monocytes
White cell concentrates (Red Cross Blood Bank) were obtained from
the peripheral blood of healthy human volunteers. All female donors
Received May 18, 1999; revision received July 16, 1999; accepted July 20, 1999.
From the Heart Research Institute (J.A.M., S.N., W.J., K.K.S., D.S.C.) and Department of Cardiology (J.A.M., D.S.C.), Royal Prince Alfred Hospital,
Camperdown, Sydney, Australia, and Department of Medicine (D.S.C.), University of Sydney, Camperdown, Sydney, Australia. Dr Stanley is currently
at The Centre for Immunology, University of NSW and St Vincent’s Hospital, Darlinghurst, Sydney, Australia.
Correspondence to David S. Celermajer, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Rd, Camperdown 2050, Sydney,
Australia. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
2319
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Circulation
December 7, 1999
(n511) were premenopausal (aged 18 to 40 years) with no significant negative medical history. Male donors (n58) were also healthy
and were aged 21 to 58 years. Monocytes were removed within 24
hours of collection by density gradient separation of the white cells
on Lymphoprep (Nycomed Pharma) followed by counterflow centrifugation elutriation at 20°C, as previously described by our
group,18 by use of a Beckman J2-21 M/E centrifuge equipped with a
JE-6B elutriation rotor and a 4.2-mL elutriation chamber (Beckman
Instruments, Inc). The elutriation buffer was HBSS without calcium
or magnesium (Sigma) supplemented with EDTA (0.1 g/L) and 1%
heat-inactivated human serum. The system and tubing were rinsed
with 250 mL each of 70% ethanol, endotoxin-free water, 6%
hydrogen peroxide, endotoxin-free water, and elutriation buffer in
that order before the Lymphoprep-derived mononuclear cell fraction
was loaded at 9 mL/min into the elutriation rotor chamber (2020 rpm
at 20°C). Flow rate was increased by 1 mL/min increments every 10
minutes, and monocytes were typically eluted between 16 and 17
mL/min. Collected fractions were examined by use of a Cytospin
system (Shandon) and Wright’s stain (Diff-Quik, Laboratory Aids).
Monocyte purity of .90% and viability of .95% by trypan blue
exclusion were confirmed by light microscopy.
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Culture of Human MDMs
Monocytes isolated by elutriation were resuspended in phenol
red–free RPMI (Life Technologies), plated onto 24-mm-diameter
tissue culture wells, and allowed to adhere for $1.5 hours at 37°C
under 5% CO2 in air. The media were then removed and the adherent
monocytes washed twice gently with PBS before the addition of
phenol red–free RPMI containing 10% postmenopausal female
human serum, penicillin G (50 U/mL), and streptomycin (50
mg/mL). Media changes occurred every 2 to 3 days, and the various
treatments were added from days 3 through 10 with each media
change. Treatment conditions included 17b-estradiol (2, 20, and 200
nmol/L; Sigma), progesterone (2, 10, and 200 nmol/L; Fluka), RU
486 (100 nmol/L; Sigma), and ethanol (0.1% vol/vol) as control
(used to dissolve the hormones). Each experiment used at least
triplicate cultures for each condition. In addition, experiments were
performed to compare the effects of the above hormones in maleand female-donor MDMs propagated in either male serum or
postmenopausal female serum to ensure that any effects observed
were not merely nonspecific perturbations due to removal of the cells
from their usual hormonal environment (for example, androgen
withdrawal in the male MDMs).
Macrophage differentiation occurred over the 10-day period, and
lipid loading was achieved on days 8 through 10 during a 48-hour
incubation with 50 mg/mL acetylated LDL (AcLDL) in phenol
red–free RPMI containing 10% (vol/vol) lipoprotein-deficient human serum (d.1.25). Cell viability for each treatment condition was
established by trypan blue exclusion, with viability levels of 90% to
94%.
Monocyte to macrophage differentiation was similar in all treatment groups as assessed by cell-surface expression of CD16 (FcgRIII) (Sigma) by use of an ELISA technique involving a second
antibody of sheep anti-mouse antibody/horseradish peroxidase conjugate (Amersham International) and ABTS substrate (Kirkegaard
and Perry Laboratories).19 At 4, 7, 10, and 12 days, CD16 expression
rose under control conditions from extremely low levels of
0.0960.02 on day 3 (OD at 405 nm) to 0.17660.02, 0.33860.04,
and 0.36060.02 at days 7, 10, and 12, respectively (n53). There was
no significant difference in CD16 antigen expression in these control
cells and those exposed to estrogen 2 nmol/L or progesterone 10
nmol/L during days 3 through 12 of differentiation (P$0.3). Similarly, nonspecific binding (assessed by use of isotype mouse IgG)
was not significantly different between conditions for each time
point.
Preparation of LDL
LDL (1.05.d.1.02 g/mL) was isolated from plasma of healthy,
normolipidemic fasting subjects by 2-step centrifugation at 10°C
with a Beckman L8-M centrifuge and Ti70 rotor at 50 000 rpm
(242 000g) for 24 hours. The LDL was dialyzed 4 times against 1 L
of deoxygenated PBS (calcium and magnesium free; Flow Laboratories) containing 0.1 mg/mL chloramphenicol (Boehringer Mannheim) and 1.0 mg/mL EDTA. The LDL was stored in the dark at 4°C
and used within 2 weeks.
Acetylation of LDL
LDL was acetylated at 4°C by a modification of a previous method20
with 6 mL of acetic anhydride per 1 mg of LDL protein. After
acetylation, LDL was again dialyzed 4 times in 1 L of PBS
containing chloramphenicol (0.1 g/L) and EDTA (1 mg/mL) over 16
hours to remove excess saturated sodium acetate and acetic anhydride, filtered (0.45 mmol/L), and stored in the dark at 4°C to be used
within 1 week. Adequate acetylation of LDL was confirmed before
use by observation of a relative electrophoretic mobility of .2.5 for
AcLDL compared with native LDL on 1% Universal agarose gels
(Ciba-Corning) in Tris-barbitone buffer (pH 8.6) at 90 V for 45
minutes.
Iodination of AcLDL
AcLDL was iodinated with 125I (carrier-free sodium iodide, activity
200 MBq) by the iodine monochloride method.21 The 125I-labeled
AcLDL was passed through a PD-10 column (Sephadex G-25M,
Pharmacia), to remove unreacted iodide and then dialyzed 4 times
against 1 L of PBS/EDTA/chloramphenicol, filtered (0.45 mmol/L),
and stored at 4°C to be used within 1 week. Specific activity was
assessed before use and ranged between 250 and 750 counts/min per
1 ng of LDL protein.
Preparation of Macrophage Cell Extracts
After the human MDMs were washed 3 times with ice-cold PBS,
cells were lysed with 0.6 mL of cold 0.2 mol/L NaOH at 4°C for 15
minutes. From the lysate, 0.2 mL was used for cell protein estimation, and the remaining 0.4 mL was added to 0.6 mL of ice-cold PBS
and immediately extracted into methanol (2.5 mL) and hexane (5
mL) in the presence of 20 mmol/L butylated hydroxytoluene (Sigma)
and 2 mmol/L EDTA. Samples were stored after extraction at 280°C
until analysis for free cholesterol (FC) and cholesteryl ester (CE) was
performed, usually within 7 days.
Analysis of Cholesterol and CEs
Cholesterol and CEs were separated by reverse-phase high-performance liquid chromatography at room temperature on a C-18 column
(Supelco) as described previously.22 CEs were analyzed with an
eluent of acetonitrile/isopropanol (30:70 vol/vol), whereas FC values
were assessed with acetonitrile/isopropanol/water (44:54:2 vol/vol/
vol), with detection at 210 nm absorbance for both parameters
(Activon UV-200 absorbance detector).
Cholesterol and CEs were quantified by the derivation of standard
curves by use of commercially available standards (Sigma). The
curves expressed a linear relation between the chromatographic peak
areas and the mass of the standard, which enabled us to quantify
individual cholesterol compounds in nanomoles per milligram of cell
protein.
Protein Estimation
All protein estimations were performed by the bicinchoninic acid
method (Sigma) with BSA used as a standard. Samples were
incubated for 60 minutes at 60°C before measurement of absorbance
at 562 nm.
I125-Labeled AcLDL Trafficking Experiments
For all of the following lipoprotein trafficking experiments, MDMs
were treated as described previously. Instead of AcLDL loading on
day 8, cells were treated with 125I-labeled AcLDL to assess lipidhandling mechanisms.
Cell-Surface Lipoprotein Binding at 4°C
After 8 days of propagation, cells were washed twice with PBS and
cooled to 4°C for 20 minutes, and cell-surface– bound AcLDL was
determined after a 4-hour incubation at 4°C with 125I-AcLDL (1, 2,
McCrohon et al
Female Hormones Reduce Foam Cell Formation
2321
5, 8, 10, 20, 30, and 40 mg/mL) with and without a 30-fold excess of
unlabeled AcLDL to assess specific and nonspecific binding. Each
condition was performed in triplicate cultures. After incubation,
macrophages were washed 5 times with ice-cold PBS containing 2
mg/mL BSA, then washed 3 times with PBS as described by
Goldstein et al.23 Cells were lysed with 0.2 mol/L NaOH, and lysates
were assessed for protein content and cell-associated (cell surface)
radioactivity. Binding parameters were analyzed by the LIGAND
computer program.24
Incubation at 37°C Followed by Surface Binding at 4°C
Macrophages were incubated at 37°C for 4 hours with 20 mg/mL
unlabeled AcLDL and washed as described previously at 4°C, and
the media were replaced with RPMI (containing 2 mg/mL BSA and
20 mg/mL 125I-AcLDL) for an additional 4 hours at 4°C to reassess
surface binding–site availability after metabolism of AcLDL at
37°C.
Retroendocytosis Studies
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Previous studies in the THP-1 cell line have shown that estrogen
action in reducing macrophage lipoprotein metabolism is independent of lysosomal degradation of AcLDL.25 We therefore explored
the possibility that estrogen and progesterone might affect the
membrane traffic of AcLDL in human macrophages. A role for sex
steroid hormone modulation of cellular lipoprotein-receptor pathways has been demonstrated in the estrogen-mediated increase in
hepatocyte LDL transcytosis via the asialoglycoprotein receptor.26 In
macrophages, internalized lipoprotein-scavenger receptor complex
dissociates in the acidic environment of the endosome, with the
receptor recycling to the cell surface and the majority of the AcLDL
proceeding to lysosomal degradation.27 A shift in the balance
between degradation and retroendocytosis of intact AcLDL would
contribute to a reduction in net uptake of modified lipoprotein. We
examined rates of retroendocytosis by internalizing labeled AcLDL
4 times between 15 minutes and 2 hours and then measuring the
release of intact AcLDL to the extracellular media over 60 minutes.
At 8 days, cells were incubated with 20 mg/mL 125I-AcLDL at
37°C for either 15, 30, or 120 minutes. At each time point, the
respective wells were rapidly cooled to 4°C to inhibit additional
intracellular transport and washed as described previously28 with the
exception of an acid wash (0.2 mol/L acetic acid, 0.5 mol/L NaCl,
pH 2.4) to remove surface-bound 125I-AcLDL, followed by the final
3 PBS washes. By removing $80% of the specific surface-bound
AcLDL during acid washing29 before the chase incubation, these
studies ensured that the intact AcLDL in the TCA-treated media was
derived largely from inside the cell rather than from dissociation of
labeled ligand from surface receptors. These experiments were also
performed in the presence of 30 times excess cold AcLDL to account
for nonspecific binding.
The chase incubation involved the addition of fresh RPMI
(containing 2 mg/mL BSA and 100 mg/mL of unlabeled AcLDL) at
37°C for 1 hour. The media were then collected and precipitated with
TCA, and the radioactivity of the pellet was measured (intact AcLDL
released into the media from inside cells). In addition, a separate
chase of only 15 minutes was performed on the cells that received a
2-hour pulse, to preferentially measure retroendocytosis from organelles close to the surface (eg, early endosomes). Viability after
each trafficking experiment was assessed by the trypan blue exclusion method and confirmed to be .90%.
Statistical Analysis
Results are expressed as mean6SEM of $3 wells per condition per
donor, with n511 for female-donor MDMs and n58 for male-donor
MDMs. Because of individual donor variability, results for each
donor have been expressed as a percentage of control values, and
individual experimental results (in percent of control values) have
been pooled to give the final results for male and female-donor cells
under control and hormone-treated conditions. Statistical significance was analyzed by a 1-way ANOVA and post hoc pairwise
testing between conditions. Statistical significance was inferred at a
2-sided P value ,0.05.
Figure 1. A, 17b-estradiol (E) treatment produced a significant
reduction in human macrophage CE content compared with
controls. This effect was independent of the classic estrogen
receptor (ie, effect not abrogated by estrogen receptor antagonist ICI 182780 [ICI] or reproduced by estrogen receptor agonist
diethylstilbestrol [DES]). Results are expressed as percentage of
control values and are from 11 female donors. *P50.02 control
vs E2nM. There was no significant difference between physiological (E2nM) and supraphysiological levels (E20nM, E200nM)
of estradiol. B, Progesterone (P) treatment produced marked
reduction in CE content. Progesterone effect could be abrogated by coincubation with progesterone receptor antagonist
RU 486. Results are expressed as percentage of control values
and are from 11 female donors. *P50.002 control vs P2nM.
There was no significant difference between physiological (P2nM
and P10nM) and supraphysiological (P200nM) progesterone
doses.
Results
Effects of Estrogen and Progesterone on
Macrophage Lipid Loading
17b-Estradiol produced a significant reduction in macrophage CE accumulation in premenopausal female MDMs
(n511) (Figure 1A). This occurred at both physiological
concentrations and supraphysiological concentrations of 17bestradiol (8863%, 8862%, and 8564% for 2, 20, and 200
nmol/L estrogen, respectively; P,0.02 for each concentration compared with control but no significant differences
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December 7, 1999
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between concentrations). This effect could not be reversed by
coincubation with the estrogen receptor antagonist ICI
182780 (shown in Figure 1A). Furthermore, MDMs exposed
to the potent nonsteroidal estrogen receptor agonist diethylstilbestrol did not show a significant reduction in CE content
(9564%; P50.1 compared with controls), which further
suggests that the effect of 17b-estradiol occurred independently of the classic estrogen receptor known to be present in
these cells.
Physiological levels of progesterone produced an even
more dramatic reduction in CE accumulation (7469%,
56610%, and 6568% for 2, 10, and 200 nmol/L progesterone, respectively; P,0.002 for each concentration compared
with control but no significant difference between concentrations) (Figure 1A). This reduction in cellular CE content with
progesterone could be abrogated by coincubation with the
progesterone receptor antagonist RU 486 (11268% and
91613% for 2 nmol/L progesterone per 100 nmol/L RU 486
and 10 nmol/L progesterone per 100 nmol/L RU 486,
respectively; P.0.5 compared with controls) (Figure 1). RU
486 treatment alone was not significantly different from
controls (9368%, P50.1).
Intracellular FC levels were also reduced in the estrogentreated female-donor MDMs (8664%, 8863%, and 8566%
for 2, 20, and 200 nmol/L estrogen, respectively; P,0.02 for
each concentration compared with controls) but not in
progesterone-treated MDMs (10868%, 10568%, and
117611% for 2, 10, and 200 nmol/L progesterone, respectively; P.0.1 compared with controls).
By contrast, CE accumulation in MDMs from male donors
was not decreased by either 17b-estradiol or progesterone
(for example, 10569% and 11568% for estrogen 200
nmol/L and progesterone 10 nmol/L, respectively; P.0.05
compared with controls). Similarly, FC levels in male-donor
MDMs were not significantly influenced by either female
hormone (data not shown).
These sex-specific effects of estrogen and progesterone
were also observed when the male- and female-donor MDMs
were propagated in phenol red–free RPMI supplemented with
10% male serum (as opposed to 10% postmenopausal female
serum). There was no significant effect of 17b-estradiol or
progesterone on lipid loading of male-donor MDMs compared with controls (data not shown), but there was a
persistent reduction in lipid loading in female-donor MDMs
exposed to female hormones in the presence of male or
postmenopausal female serum in the growth media. For
example, cellular CE and FC values (expressed as nmol/mg
cell protein) for MDMs from the same female donor, maintained in RPMI containing 10% postmenopausal female
serum compared with 10% male serum, were not significantly
different from each other for each of the treatment conditions
(control 277612 versus 30163, P50.1; estrogen 200 nmol/L
23369 versus 264612, P50.1; progesterone 10 nmol/L
221618 versus 245615, P50.4).
Estrogen and Progesterone Action on Macrophage
Uptake and Processing of Modified LDL
To understand the effects of estrogen and progesterone on
AcLDL loading, we conducted binding and intracellular
Figure 2. There was no significant difference in surface binding
at 4°C between conditions at any concentration of AcLDL
(P.0.1). These data are representative of 3 equivalent
experiments.
processing studies of radiolabeled AcLDL in premenopausal
female-donor macrophages, in which there were significant
hormonal effects on intracellular lipid loading.
Surface Binding Studies
Surface binding studies performed at 4°C over a range of
concentrations for radiolabeled AcLDL with or without a
30-fold excess of unlabeled ligand showed no significant
difference in binding between control cells and cells treated
with estrogen (Figure 2A) or progesterone (Figure 2B). When
specific and nonspecific binding kinetics were analyzed by
the LIGAND computer program,24 scavenger receptor affinity and binding-site numbers were not significantly different
between treatment groups (Kd values 1.031028, 9.731029,
and 9.631029 mol/L for control, estrogen 2 nmol/L, and
progesterone 10 nmol/L, respectively, P.0.9; receptor site
concentrations 8.8310211, 7.1310211, and 7.1310211 mol/L
for control, estrogen 2 nmol/L, and progesterone 10 nmol/L,
respectively, P.0.4).
Because of the previously reported temperature-sensitive
nature of the scavenger receptor ligand-binding domain,30
binding at 4°C may not be representative of in vivo binding.
We therefore incubated the MDMs with unlabeled AcLDL
for 4 hours at 37°C and then rapidly cooled the cells to 4°C
before challenging them with a single concentration of
McCrohon et al
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Figure 3. Surface receptor occupancy for modified LDL at 37°C
was significantly lower for both 17b-estradiol (E) and progesterone (P) vs controls as assessed by subsequent surface binding
of I125-AcLDL at 4°C. These results were representative of 3
equivalent experiments. *P,0.05; **P,0.001.
radioactive ligand and excess AcLDL. In this protocol,
binding represents the steady state, with unoccupied receptor
sites remaining at the surface during uptake at 37°C. Compared with controls, estrogen- and progesterone-treated macrophages were able to bind a significantly greater proportion
of labeled AcLDL (11465% for estrogen 2 nmol/L and
161615% for progesterone 10 nmol/L versus 10064% control, P,0.05 and 0.001, respectively) (Figure 3). This suggests that at physiological temperatures, estrogen and progesterone reduce scavenger receptor occupancy and therefore
subsequent internalization of ligand-receptor complexes.
Retroendocytosis Studies
Progesterone significantly increased the amount of intact
AcLDL that had undergone retroendocytosis under all conditions tested (P,0.008) (Figure 4). Furthermore, the fractions of intact ligand that had undergone retroendocytosis
after 15 minutes of labeling (Figure 4, A), 30 minutes of
labeling (B), and a 2-hour labeling period (C) were similar.
Because early endosomes would be filled by the 15-minute
time point, this suggests that retroendocytosis predominantly
occurs from the early endosomal compartment. Release of
AcLDL from the cells was also rapid, with the majority
occurring during the first 15 minutes of the 60-minute chase
(Figure 4, D). This observation is also consistent with the
recycling time of ligands from an early endosomal compartment. Retroendocytosis in estrogen-treated macrophages was
not significantly different from control cells.
Discussion
In this study, we found that physiological concentrations of
both 17b-estradiol and progesterone decreased CE formation
in human MDMs. This effect was sex specific, being observed in female- but not male-donor cells. The estrogen
effect was not mediated via the classic estrogen receptor and
was due predominantly to an effect on scavenger receptor
occupancy and therefore lipoprotein uptake. The progesterone effect, in contrast, was receptor mediated and due to
Female Hormones Reduce Foam Cell Formation
2323
Figure 4. Retroendocytosis of undegraded AcLDL was significantly greater in progesterone-treated human MDMs than in
control and estrogen-treated cells (*P,0.008). Fraction of undegraded ligand that was retroendocytosed was similar for early
and late time points, implying a predominant effect on early
stages of intracellular lipid trafficking. These data are pooled
from 3 separate experiments, with results expressed as a percentage of control.
changes in both lipoprotein uptake and intracellular processing, in particular to increased retroendocytosis of modified
LDL. Both estrogen and progesterone receptors are known to
exist in macrophages.
Previous studies have suggested other potentially antiatherogenic effects of estrogen, including beneficial effects
on the lipid profile,6,7 coagulation factors,12 cell adhesion
molecule expression,31,32 arterial plaque size, and cellular
proliferation.33 In an immortalized cell line that does not
express estrogen receptors, supraphysiological doses of 17bestradiol have been shown to decrease lipid uptake by
macrophages25; however, the effects of physiological levels
of estrogen and its antagonists on foam cell formation and the
mechanism of lipoprotein uptake in female and male primary
human macrophages have not been studied previously. Our
observation of an uptake-mediated, receptor-independent reduction in macrophage lipid loading in female cells only is
consistent with other recently published work33,34 that has
also noted sex-specific effects of sex steroid hormones on
atherosclerotic processes.
Few previous studies have examined the effect of progesterone on basic atherogenic processes. The mechanism for the
observed benefit of progesterone on MDM lipid loading in
the present study most likely relates to both reduced modified
LDL internalization at 37°C and greater retroendocytosis of
undegraded lipoprotein to the surface. Time-course experiments (Figure 4) suggested that retroendocytosis must occur
from early endosomal compartments of the cell, because it is
close to its maximal value after 15 minutes, which is
approximately twice the t1/2 (8 minutes) of passage through
this compartment in Chinese hamster ovary cells.27 The
precise effects on the endosome remain uncertain. Altered
endosomal acidification affecting receptor-ligand dissociation is one plausible explanation, although previously reported effects with estrogen in hepatocytes were small.35
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December 7, 1999
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Modulation of receptor affinity and sorting are other possible
methods of sex steroid action.
Previous studies36 in the mouse cell line J774 have suggested that progesterone inhibits FC translocation from the
plasma membrane, leading to decreased CE accumulation in
foam cell macrophages. This mechanism was not observed in
the present study, in which decreased intracellular CE levels
in progesterone-treated MDMs were not accompanied by
significant increases in FC concentration. A reduction in
acyl-CoA:cholesterol acyltransferase (ACAT) activity may
also lead to reduced cellular CE content; however, progesterone has been shown to be only a weak inhibitor of ACAT
activity in previous studies in mouse peritoneal
macrophages.37
Competitive ligand-receptor– binding experiments at 4°C
did not reveal altered scavenger receptor binding kinetics
between treatment groups; however, our data showing less
ligand-receptor occupancy at 37°C with estrogen and progesterone treatment could be attributed to a change in ligandreceptor affinity at 37°C. Hence, 4°C binding data may not be
representative of in vivo hormone effects, consistent with the
previously described temperature-sensitive nature of the
scavenger receptor ligand-binding domain. 30,38 The
progesterone- and estrogen-related increase in radiolabeled
AcLDL surface binding at 4°C after a 37°C incubation could
be due to (1) reduced cell-surface scavenger receptor occupancy with modified LDL at 37°C or (2) an increase in
cell-surface scavenger receptor numbers. The latter explanation is unlikely, because our data showed no treatment
differences in cell-surface binding site number in MDMs
exposed to hormones for 6 days at 37°C until just before the
4°C binding experiments. A plausible explanation is that
estrogen and progesterone decrease receptor affinity for
AcLDL at 37°C, leading to an overall decrease in ligand
binding and internalization at this temperature. We have
attempted to examine receptor-ligand affinity at 37°C indirectly using receptor occupancy as a marker because of the
inadequacy of Scatchard binding kinetics at this temperature
and the limitations imposed by inhibitors of internalization
such as N-ethylmaleimide, whose broad-specificity alkylating
potential could also modify sulfhydryl groups in the scavenger receptor, potentially confounding interpretation of binding data.
Sex-specific reduction in lipid loading under the influence
of the female sex steroids, estrogen and progesterone, occurs
in the presence of either male or postmenopausal female
serum. This suggests that the sex differences are unlikely to
be related to nonspecific perturbations in the cell environment
but rather to specific cellular characteristics. One plausible
explanation would be a difference in sex steroid hormone
receptor levels or hormonally responsive cellular pathways
between the sexes; this possibility requires additional study.
Foam cell formation is intimately involved in early atherogenesis39,40 and has been studied extensively in mouse and
more recently in immortalized human cell lines.41,42 The
methods used to purify primary monocytes from healthy
donors have enabled the study of macrophage lipoprotein
uptake with primary human cells from both males and
females. This might be expected to reflect the in vivo
situation more closely than could previously be accomplished. The presence of sex steroid receptors in these
primary cells43– 45 has been demonstrated recently and is
important in an examination of sex steroid modulation of
lipoprotein metabolism. The use of AcLDL is well established as a standard form of modified lipoprotein in foam cell
experiments, because it is specific for the scavenger receptor
pathway for lipoprotein uptake.46 The probable importance of
this pathway in contributing to atherosclerosis has been
confirmed in several animal studies.47
In summary, the present study has shown that physiological levels of estrogen and progesterone are associated with
reduced foam cell formation in a primary human cell culture
system. Our data demonstrate that sex steroid hormones
might influence macrophage lipoprotein metabolism by
changing ligand-receptor interactions and membrane traffic.
These results are consistent with a role for both estrogen and
progesterone in the atheroprotection conferred by female
premenopausal status and the observed sex difference in
atherosclerosis development.
Acknowledgments
Dr McCrohon is supported by Sydney University and the Department of Cardiology, Royal Prince Alfred Hospital. Dr Jessup is
supported by the NHMRC. Dr Celermajer is supported by the
Medical Foundation, University of Sydney. We thank Dr David
Handelsman and Dr Paul Williams from the Department of Endocrinology, University of Sydney, for their technical advice
and assistance.
References
1. Wingard DL, Suarez L, Barrett-Connor E. The sex differential in mortality from all causes and ischemic heart disease. Am J Epidemiol. 1983;
117:165–172.
2. Lerner DJ, Kannel WM. Patterns of coronary heart disease morbidity and
mortality in the sexes: a 26-year follow-up of the Framingham Study. Am
Heart J. 1986;111:383–390.
3. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shicely CA,
Adams MR. Estrogen and progesterone replacement therapy reduces low
density lipoprotein accumulation in the coronary arteries of surgically
postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:
1995–2002.
4. Haarbo J, Nielsen LB, Stender S, Christiansen C. Aortic permeability to
LDL during estrogen therapy: a study in normocholesterolemic rabbits.
Arterioscler Thromb. 1994;14:243–247.
5. Haarbo J, Leth-Espensen P, Stender S, Christiansen C. Estrogen monotherapy and combined estrogen-progestogen replacement therapy
attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest. 1991;87:1274 –1279.
6. Nabulsi AA, Folsom AR, White A, Patsch W, Heiss G, Wu KK, Szklo M.
Association of hormone-replacement therapy with various cardiovascular
risk factors in postmenopausal women. N Engl J Med. 1993;328:
1069 –1075.
7. Sacks FM, Walsh BW. The effects of reproductive hormones on serum
lipoproteins: unresolved issues in biology and clinical practice. Ann N Y
Acad Sci. 1990;592:272–285.
8. Lieberman EH, Gerhard MD, Uehata A, Walsh BW, Selwyn AP, Ganz P,
Yeung AC, Creager MA. Estrogen improves endothelium-dependent,
flow-mediated dilation in post-menopausal women. Ann Intern Med.
1994;121:936 –941.
9. Bush TL, Barrett-Connor E, Cowan LD, Criqui MH, Wallace RB,
Suchindran CM, Tyroler HA, Rifkind BM. Cardiovascular mortality and
non-contraceptive use of estrogen in women: results from the Lipid
Research Clinics Program Follow-up Study. Circulation. 1987;75:
1102–1109.
10. Lee WS, Harder JA, Yoshizumi M, Lee ME, Haber E. Progesterone
inhibits arterial smooth muscle cell proliferation. Nat Med. 1997;9:
1005–1008.
McCrohon et al
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
11. The Writing Group for the PEPI Trial. The Postmenopausal Estrogen/
Progestin Interventions (PEPI) trial. JAMA. 1995;273:199 –208.
12. Gebara OCE, Mittleman MA, Sutherland P, Lipinska I, Matheny T, Xu P,
Welty FK, Wilson PWF, Levy D, Muller JEM, Tofler GH. Association
between increased estrogen status and increased fibrinolytic potential in
the Framingham Offspring Study. Circulation. 1995;91:1952–1958.
13. Sorensen KE, Dorup I, Hermann AP, Mosekilde L. Combined hormone
replacement therapy does not protect women against the age-related
decline in endothelium-dependent vasomotor function. Circulation. 1998;
97:1234 –1238.
14. Grodstein F, Stampfer MJ, Colditz GA, Willett WC, Manson JA, Joffe M,
Rosner B, Fuchs C, Hankinson SE, Hunter DJ, Hennekens CH, Speizer
FE. Postmenopausal hormone therapy and mortality. N Engl J Med.
1997;336:1769 –1775.
15. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B,
Vittinghoff E, for the Heart and Estrogen/progestin Replacement Study
(HERS) Research Group. Randomized trial of estrogen plus progestin for
secondary prevention of coronary heart disease in postmenopausal
women. JAMA. 1998;280:605– 613.
16. Masuda J, Ross R. Atherogenesis during low level hypercholesterolemia
in the non-human primate, I: fatty streak formation. Arteriosclerosis.
1990;10:164 –177.
17. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witzum
JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins and contain oxidationspecific lipid-protein adducts. J Clin Invest. 1991;87:90 –99.
18. Garner B, Dean RT, Jessup W. Human macrophage-mediated oxidation
of low-density lipoprotein is delayed and independent of superoxide
production. Biochem J. 1994;301:421– 428.
19. Andreesen R, Brugger W, Scheibenbogen C, Kreutz M, Leser HG, Rehm
A, Löhr GW. Surface phenotype analysis of human monocyte to macrophage maturation. J Leukoc Biol. 1990;47:490 – 497.
20. Brown MS, Goldstein JL, Krieger M, Ho YK, Anderson RGW.
Reversible accumulation of cholesteryl esters in macrophages incubated
with acetylated lipoproteins. J Cell Biol. 1979;82:597– 613.
21. Contreras MA, Bale WF, Spar IL. Iodine monochloride (ICl) iodination
techniques. Methods Enzymol. 1983;92:277–292.
22. Kritharides L, Jessup W, Dean RT. A method for staging the degree of
LDL oxidation by the separation of cholesterol and cholesteryl ester
oxidation products using HPLC. Anal Biochem. 1993;213:79 – 89.
23. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of
low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:
241–260.
24. Munson J. A User’s Guide to LIGAND: Data Analysis and Curve-Fitting
for Ligand-Binding Experiments. Bethesda, Md: National Institutes of
Health; 1993.
25. Sulistiyani, St Clair RW. Effect of 17b-estradiol on metabolism of acetylated low-density lipoprotein by THP-1 macrophages in culture. Arterioscler Thromb Vasc Biol. 1997;17:1691–1700.
26. Burgess JW, Stanley KK. 17b-Ethynylestradiol increases transcytosis of
asialoglycoproteins in rat liver. J Biol Chem. 1994;269:3482–3488.
27. Dunn KW, Maxfield FR. Delivery of ligands from sorting endosomes to
late endosomes occurs by a maturation of sorting endosomes. J Cell Biol.
1992;117:301–310.
28. Warren RA, Green FA, Enns CA. Saturation of the endocytic pathway for
the transferrin receptor does not affect the endocytosis of the epidermal
growth factor receptor. J Biol Chem. 1997;272:2116 –2121.
29. Davis CG, Goldstein JL, Südhof TC, Anderson RGW, Russell DW,
Brown MS. Acid-dependent ligand dissociation and recycling of LDL
receptor mediated by growth factor homology region. Nature. 1987;326:
760 –765.
30. Zhang H, Yang Y, Steinbrecher UP. Structural requirements for the
binding of modified proteins to the scavenger receptor of macrophages.
J Biol Chem. 1993;268:5535–5542.
Female Hormones Reduce Foam Cell Formation
2325
31. Caulin-Glaser T, Watson CA, Pardi R, Bender JR. Effects of 17bestradiol on cytokine-induced endothelial cell adhesion molecule
expression. J Clin Invest. 1996;98:36 – 42.
32. Caulin-Glaser T, Farrell W, Pfau SE, Zaret B, Bunger K, Setaro JF,
Brennan JJ, Bender JR, Cleman MW, Cabin HS, Remetz MS. Modulation
of circulating cell adhesion molecules in postmenopausal women with
coronary artery disease. J Am Coll Cardiol. 1998;31:1555–1560.
33. Hanke H, Hanke S, Finking G, Muhic-Lohrer A, Mück AO, Schmahl FW,
Haasis R, Hombach V. Different effects of estrogen and progesterone on
experimental atherosclerosis in female versus male rabbits: quantification
of cellular proliferation by bromodeoxyuridine. Circulation. 1996;94:
175–181.
34. Bruck B, Brehme U, Gugel N, Hanke S, Finking G, Lutz C, Benda N,
Schmahl FW, Haasis R, Hanke H. Gender-specific differences in the
effects of testosterone and estrogen on the development of atherosclerosis
in rabbits. Arterioscler Thromb Vasc Biol. 1997;17:2192–2199.
35. Van Dyke RW, Root KV. Ethinyl estradiol decreases acidification of rat
liver endocytic vesicles. Hepatology. 1993;18:604 – 613.
36. Mazzone T, Krishna M, Lange Y. Progesterone blocks intracellular translocation of free cholesterol derived from cholesterol ester in macrophages. J Lipid Res. 1995;36:544 –551.
37. Lange Y. Cholesterol movement from plasma membrane to rough endoplasmic reticulum. J Biol Chem. 1994;269:3411–3414.
38. Doi T, Higashino K, Kurihara Y, Wada Y, Miyazaki T, Nakamura H,
Uesugi S, Imanishi T, Kawabe Y, Itakura H, Yazaki Y, Matsumoto A,
Kodama T. Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors.
J Biol Chem. 1993;268:2126 –2133.
39. Gerrity RG. The role of the monocyte in atherogenesis, I: transition of
blood-borne monocytes into foam cells in fatty lesions. Am J Pathol.
1981;103:181–190.
40. Aquel NM, Ball RY, Waldmann H, Mitchinson MJ. Monocytic origin of
foam cells in atherosclerotic plaques. Atherosclerosis. 1984;53:265–271.
41. Tsuchiya S, Kobayashi Y, Goto Y, Okomura H, Nakai S, Kono T, Taka
K. Induction of maturation in cultured human monocytic leukaemia cell
lines by a phorbol diester. Cancer Res. 1982;42:1530 –1536.
42. Auwerx J. The human leukaemia cell line, THP-1: a multifaceted model
for the study of monocyte-macrophage differentiation. Experientia. 1991;
47:22–31.
43. Danel L, Souweine G, Monier JC, Saez S. Specific estrogen binding sites
in human lymphoid cells and thymic cells. J Steroid Biochem. 1983;18:
559 –563.
44. Cutolo M, Accardo S, Villagio B, Barone A, Sulli A, Coviello D,
Carrabio C, Felli L, Miceli D, Farrugio R, Carruba G, Castagnetta L.
Androgen and estrogen receptors are present in primary cultures of
human synovial macrophages. J Clin Endocrinol Metab. 1996;81:
820 – 827.
45. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Müller KH,
Sharkey AM, Smith SK. Vascular endothelial growth factor is produced
by peritoneal fluid macrophages in endometriosis and is regulated by
ovarian steroids. J Clin Invest. 1996;98:482– 489.
46. Kamps JA, Kruijit J, Kuiper J, van Berkel TJC. Characterization of the
interaction of acetylated LDL and oxidatively modified LDL with human
liver parenchyma and Kupffer cells in culture. Arterioscler Thromb.
1992;12:1079 –1087.
47. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K,
Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y,
Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T,
Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y,
Horiuchi S, Takahashi K, Kruijit JK, van Berkel TJC, Steinbrecher UP,
Ishibashi S, Maeda N, Gordon S, Kodama T. A role for scavenger
receptors in atherosclerosis and susceptibility to infection. Nature. 1997;
386:292–296.
Estrogen and Progesterone Reduce Lipid Accumulation in Human Monocyte-Derived
Macrophages: A Sex-Specific Effect
Jane A. McCrohon, Shirley Nakhla, Wendy Jessup, Keith K. Stanley and David S. Celermajer
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Circulation. 1999;100:2319-2325
doi: 10.1161/01.CIR.100.23.2319
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