Autoimmunity, May 2011; 44(3): 1–10

q Informa UK, Ltd.

ISSN 0891-6934 print/1607-842X online
DOI: 10.3109/08916934.2010.530626

Atherosclerosis development in SLE patients is not determined by

monocytes ability to bind/endocytose Ox-LDL

LINA M. YASSIN1, JULIÁN LONDOÑO2,3, GUILLERMO MONTOYA2,3,

JUAN B. DE SANCTIS4, MAURICIO ROJAS1, LUIS A. RAMÍREZ5, LUIS F. GARCÍA1, &
GLORIA VÁSQUEZ1,5
1
Grupo de Inmunologı́a Celular e Inmunogenética, Facultad de Medicina, Universidad de Antioquia, Medellı́n, Colombia,
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2
Grupo de Investigación en Sustancias Bioactivas, Instituto de Quı́mica Farmacéutica, Universidad de Antioquia, Medellı́n,
Colombia, 3Grupo de Inmunomodulación, Facultad de Medicina, Universidad de Antioquia, Medellı́n, Colombia, 4Instituto de
Imunologı́a, Universidad Central de Venezuela, Caracas, Venezuela, and 5Grupo de Reumatologı́a, Facultad de Medicina,
Universidad de Antioquia, Medellı́n, Colombia

(Submitted 6 April 2010; revised 30 September 2010; accepted 6 October 2010)

Abstract
For personal use only.

Patients with systemic lupus erythematosus (SLE) have a high risk of developing cardiovascular disease; however, the
mechanisms involved in the early onset of atherosclerosis in these patients are not clear. Scavenger receptors, CD36 and
CD163 are expressed by mononuclear phagocytes and participate in the binding and uptake of oxidized low-density
lipoproteins (Ox-LDL), contributing to foam-cells formation and atherosclerosis development. The aim of the present study
was to evaluate CD36þ and CD163þ expression and Ox-LDL removal by monocytes from SLE and atherosclerotic patients,
compared to similar age-range healthy controls. Healthy controls, SLE, and atherosclerotic patients were evaluated for carotid
intima media thickness (CIMT), lipid profile, and native LDL (N-LDL) and Ox-LDL binding/endocytosis. SLE patients
presented decreased high-density lipoproteins (HDL) and increased Triglyceride levels, and half of the SLE patients
had increased CIMT, compared to their healthy controls (HCSLE). The number of CD14þCD163þ cells was increased
in atherosclerosis healthy controls (HCAtheros) compared to HCSLE, but there were no differences between SLE or
atherosclerotic patients and their respective healthy controls. Clearance assays revealed a similar capacity to bind/endocytose
Ox-LDL by monocytes from SLE patients and HCSLE, and an increased binding and endocytosis of Ox-LDL by monocytes
from atherosclerotic patients, compared to HCAtheros. The decreased CD36 and CD163 expression observed in
atherosclerotic and SLE patients, respectively, suggest that these inflammatory conditions modulate these receptors
differentially. The increased CIMTobserved in SLE patients cannot be explained by Ox-LDL binding/endocytosis, which was
comparable to their controls.

Keywords: Atherosclerosis, SLE, scavenger receptors, OxLDL, endocytosis

Introduction [1,3], important for recruiting monocytes [4], and

Atherosclerosis is nowadays considered a chronic
inflammatory disease occurring in the arterial walls
[1,2]. The process is initiated when plasma levels of
very low-density lipoproteins (VLDL) and LDL rise,
diffuse into the artery wall to an extent that exceeds
the capacity for elimination, and are retained in the
extracellular matrix activating the endothelium [1].
The endothelial activation is characterized by an
increased expression of ICAM-1 and V-CAM-1,
production of the monocyte chemotactic protein-1
detaching of endothelial cells generating gaps between
them [2]. Increased intercellular space and activation
of the endothelium allows for the accumulation of
LDL, and the adherence and migration of lympho-
cytes and monocytes to the subendothelium and
intima [1].
LDL are oxidized by reactive molecular species
generated by the endothelial cells and activated
monocytes/macrophages [5]. Ox-LDL can be recog-
nized by scavenger receptors (SR), expressed on the

Correspondence: G. Vásquez, Grupo de Reumatologı́a, Facultad de Medicina, Universidad de Antioquia, Calle 64 51D-154, Bloque 6,
Medellı́n, Colombia. Tel: 0574-2106453. Fax: 0574-2106450. E-mail: glomavas@gmail.com
 2 L. M. Yassin et al.
recruited monocytes and macrophages [6]. Mono-
cytes differentiate into macrophages in the presence of
macrophage colony stimulating factor produced by
endothelial cells (EC) and smooth muscle cells [7].
Macrophages up-regulate the SR expression, increas-
ing Ox-LDL uptake [6] and leading to the delivery of
Ox-LDL into the lysosomes, where they are hydro-
lyzed to protein and cholesteryl ester.
Cholesterol is then released into the cytoplasm,
where it accumulates and suffers re-esterification,
transforming the macrophages into foam cells, the
hallmark of the atherosclerotic plaque [5,8]. The
interaction between EC, monocytes/macrophages,
and T cells within the arterial walls, results in the
activation and production of inflammatory cytokines
such as IL-1b, TNF-a, and IFN-g, increasing tissue
damage and promoting the formation of atherosclero-
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tic plaques [1,2].
Systemic lupus erythematosus (SLE) is also a
systemic inflammatory disease, in which, among other
alterations, there is strong evidence of early develop-
ment of atherosclerosis and its consequences, like
myocardial infarction [9– 13]. Evidence of the link
between SLE and atherosclerosis was provided by a
mouse model of SLE-atherosclerosis (gld.apoE 2/2 ).
For personal use only.
These mice showed accelerated atherosclerosis
induced by the autoimmune phenotype, which was
associated with a decreased ability to remove
apoptotic cells [14]. In humans, comparisons between
SLE patients and healthy controls with similar
demographic characteristics and risk factors, showed
that the prevalence of atherosclerosis was higher in
SLE patients [10].
Also, there is a statistically significant increase of
coronary heart disease and stroke in SLE patients that
cannot be fully explained by the traditional Framing-
ham risk factors [15]. Thus, the inflammatory
phenomenon occurring in SLE patients may trigger
the development of atherosclerosis [10].
In this respect, SRs such as CD36 and CD163,
besides their capacity to bind Ox-LDL, also bind
apoptotic cells [6], which are the main targets of the
autoimmune response in SLE [16]. We previously
observed a decreased apoptotic cells removal in SLE
patients associated with cellular activation regardless
of CD36 [17].
To further explore possible mechanisms for athero-
sclerosis development in SLE, the present study
compared the carotid intima media thickness
(CIMT), the lipid profile and the expression of
CD36 and CD163 in SLE patients, healthy SLE
controls, atherosclerotic patients and healthy athero-
sclerosis controls, as well as the capacity of their
monocytes to bind/endocytose Ox-LDL or native
LDL (N-LDL). Even though more than half of SLE
patients had increased CIMT, these patients did not
show differences in the number of CD36þ and
CD163þ monocytes, nor in Ox-LDL binding and
endocytosis, compared to HCSLE.
However, HCAtheros showed increased number of
circulating CD163þ monocytes, as compared to
HCSLE, but no differences were observed between
patients and their respective healthy controls. The
expression of CD163 was decreased in SLE patients
monocytes compared to HCSLE, meanwhile mono-
cytes from atherosclerotic patients presented
decreased expression of CD36 compared to HCAtheros.
Also, Ox-LDL binding and endocytosis was increased
in atherosclerotic patients as compared to HCAtheros.
Patients and methods
Reagents
RPMI-1640, PBS, and fetal calf serum (FCS) were
purchased from GIBCO-BRL (Grand Island, NY,
USA); penicillin, streptomycin, Ficoll-Hypaque,
pooled human serum AB (PHS), and Limulus
Amebocyte Lysate assay kits (LAL) from Biowhittaker
(Walkersville, MD, USA); trypan blue, sodium azide,
bovine serum albumin, and 1,10 dioctadecyl-3,3,30 ,30 -
tetramethylindocarbocyanine (DiI) from SIGMA (St
Louis, MO, USA) and Invitrogen (Carlsbad, CA,
USA). Dimethyl sulfoxide (DMSO) was purchased
from MERCK (Darmstadt, Germany). Anti-CD14-
FITC (clone M5E2), anti-CD36-PE (clone CB38),
anti-CD163-PE (clone GHI/61), anti-HLA-DR-Cy
(clone TÜ36) and isotype control antibodies IgG2ak-
FITC (clone G155-178), IgG1k-PE (MOPC-21),
IgMk-PE (clone G155-178), and IgG2bk-PE-Cy5
(clone 27-35) were purchased from BD Bioscience
(San José, CA, USA). Anti-CD36-FITC (clone
FA6.152) was obtained from Beckman-Coulter (Brea,
CA, USA) and anti-CD163-FITC (clone ED2) from
AbD Serotec (Oxford, UK). CALTAG Cal-Lyse
Lysing Solution was obtained from Caltag Laboratories
Inc. (Burlingame, CA, USA). Quantification Kit-Rapid
was purchased from Fluka Chemika AG (Buchs,
Switzerland) and the kit to evaluate the lipid profile
(Architect/Aeroset system) was obtained from Abbott
Diagnostics (Chicago, IL, USA). All media were
negative for LPS as evaluated by LAL assay.
Patients and controls
Thirty-eight SLE patients diagnosed according to the
American College of Rheumatology Criteria (ACR)
[18] and 21 patients with a previous vascular event
explained by atherosclerosis and no autoimmune
associated disease were recruited at the Hospital
Universitario San Vicente de Paul, Medellı́n, Colom-
bia. Two groups of healthy controls were also
recruited: 29 healthy controls with similar age-range
to SLE patients (HCSLE) from medical and laboratory
personnel of the Universidad de Antioquia, and 10

healthy controls with similar age-range to athero-
sclerotic patients (HCAtheros) at the outpatient clinic of
the Universidad Hospital Caracas, Venezuela. Patients
with SLE were classified according to the systemic
lupus erythematosus disease activity index (SLEDAI)
[19]. Healthy controls were included if they had
normal blood pressure, were nonsmokers, and showed
normal results on a routine physical examination. All
patients and controls signed an informed consent
form previously approved by the Ethics Committee of
the Instituto de Inmunologı́a, Universidad Central
de Venezuela and by the Ethics Committee of the
Instituto de Investigaciones Médicas, Facultad de
Medicina, Universidad de Antioquia.
Clinical parameters
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The presence of atherosclerosis was established by
measuring the CIMT. Seven HCSLE, 10 HCAtheros,
16 SLE patients, and 9 atherosclerotic patients were
evaluated for carotid duplex (Aloka Prosound 3500
Vascular echography) in the vascular service of
Hospital Universitario San Vicente de Paul, Medellı́n.
The serum total cholesterol (TC), triglycerides
(TGC), high-density lipoproteins (HDL), and LDL
levels were measured using the spectrophotometric
For personal use only.
technique at the Hospital Universitario San Vicente de
Paul clinical laboratory and the Cardiology outpatient
clinic of the Hospital Universitario de Caracas in 14
atherosclerotic patients, 21 SLE patients, 5 HCSLE,
and 10 HCAtheros.
LDL isolation, oxidation, and labeling
Fifty milliliters of sodium citrate-anticoagulated blood
were obtained from healthy nonsmoking normolipe-
mic donors (age 20 – 25) and centrifuged at 400g for
Figure 1. LDL isolation, oxidation, and labeling. (A) SDS-PAGE of lipoprotein fractions CM/VLDL (second lane), LDL fraction (third
lane), HDL fraction (fourth lane), and whole plasma (fifth lane). The first lane shows the molecular weight ladder. (B) Agarose gel showing the
electrophoretic mobility differences between N-LDL and Ox-LDL. (C) Flow cytometry LDL-DiI labeling efficiency; nonlabeled Ox-LDL
(shaded histogram), DiI-labeled Ox-LDL (opened histogram).
Ox-LDL binding/endocytosis in SLE 3
20 min. LDL were purified in a discontinuous density
gradient according to the method of Wilson et al. [20].
Human plasma (3.2 ml) from healthy donors was
centrifuged at 105,000g (Beckman XL-100) at 58C for
12 h in the presence of 1.6 ml NaCl (1006 g/ml) to
obtain the mixture of chylomicrons and VLDL.
The remaining plasma was mixed with 1.6 ml of
KBr (1.182 g/ml) and centrifuged at the same
conditions for 18 h, in order to obtain the LDL
fraction at the upper phase and the HDL fraction in
the lower phase. Purity of these fractions was
determined by SDS-PAGE electrophoresis, as com-
pared to total plasma (Figure 1A). Proteins in the
LDL fraction were quantified by Bradford using a
commercial kit, recovering 350 – 400 mg/ml of protein.
To obtain Ox-LDL, the purified LDL fraction was
incubated with an oxidizing solution of CuSO4
(100 mM) for 12 h at 378C [21], and the reaction
stopped by transferring the LDL solution to 48C.
Oxidation of the lipid fraction was quantitatively
determined by the thiobarbituric acid reactive
substances method [22], with minor modifications,
using a solution of thiobarbituric acid (0.67%),
trichloroacetic acid (15%), and HCl (0.1N). Changes
in the net charge of the protein fraction (Apo B 100) of
LDL due to oxidation, were evaluated by agarose
(0.8%) gel electrophoresis in barbital buffer (0.05 M,
pH 8.6, 100 V) for 2 h and stained with sudan black
(0.1% in ethanol) for 10 h (Figure 1B).
LDL fluorescent labeling with DiI was performed as
previously described [23,24], with minor modifications.
Ox-LDL and N-LDL were incubated with 300 mg
DiI/mg of protein for 12 h at 378C in the dark. Labeled
Ox-LDL (Ox-LDL-DiI) and N-LDL (N-LDL-DiI)
were centrifuged for 10 min at 400g at 48C. Fluor-
escence efficiency was evaluated by flow-cytometry
(Figure 1C) and labeled LDL stored at 48C until use.
 4 L. M. Yassin et al.

Evaluation of CD36 and CD163 expression by flow of Ox-LDL-DiI (0.2, 0.5, 1.0, 10 mg/ml) or with
cytometry 10 mg/ml of N-LDL-DiI, in a final volume of 1 ml of
complete medium (RPMI-1640 plus 10% FCS,
One hundred microliters of EDTA-anticoagulated
penicilline-100 U/ml, Streptomycin-100 mg/ml)
blood from patients and controls were stained with
(CM) for 1 h at 378C. Thereafter, the cells were
anti-CD14-FITC (15 ml) and anti-HLA-DR-Cy
centrifuged at 400g for 5 min and incubated with
(10 ml), plus anti-CD163-PE (10 ml), anti-CD163-
washing buffer for 10 min at RT and washed again.
FITC (5 ml), anti-CD36-PE (6 ml), or anti-CD36-
Cells were stained with 5 ml of anti-CD14-FITC for
FITC (5 ml), or with their respective isotype control
30 min at 48C and evaluated by flow cytometry.
antibodies and incubated for 20 min in the dark at
Binding/endocytosis of Ox-LDL-DiI and N-LDL-
room temperature (RT). Then, 100 ml of Caltag
DiI was analyzed in the CD14þ gate. Two parameters
buffer were added to each tube, incubated for 10 min
were evaluated: binding/endocytosis (total) of LDL
under the same conditions, followed by addition of
and endocytosis, in the presence of 0.4% trypan blue
1 ml of distilled water and incubated for 15 min.
[25]. Binding of LDL was calculated (total – LDL
Thereafter, the cells were placed in washing buffer
endocytosed).
(PBS, 0.1% NaN3, 2% PHS) and evaluated by flow-
cytometry (Coulter Epics XLe FlowCytometer,
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Coulter International Corporation, Hialeah, FL, Statistical analysis

USA) and analyzed using the software Windows
Unpaired t-test was used for comparisons of CD36
Multiple Document Interface 2.8, WinMDI (Scripts
and CD163, CIMT, lipid profile parameters, and
Research Institute, La Jolla, CA, USA). Leukocytes
N-LDL binding/endocytosis evaluation. Two-way
were counted in hemocytometer with 0.3% acetic acid.
ANOVA with Bonferroni post-test was used for Ox-
LDL binding/endocytosis assays. Pearson correlation
Peripheral blood mononuclear cells (PBMC) isolation was used to correlate the number of cells expressing
CD36 and CD163 with the SLEDAI score. The data
PBMCs from patients and controls were isolated by
was analyzed with GraphPad Prism version 5.0
For personal use only.

centrifugation on Ficoll-Hypaque gradient, placed in

freezing medium (90% heat inactivated FCS: 10%
DMSO) and frozen at 2 1968C until use for
endocytosis assays.
Ox-LDL and N-LDL binding/endocytosis assays
Five hundred thousand (5 £ 105) PBMCs from
healthy controls, atherosclerotic patients, and SLE
patients were incubated with different concentrations
(GraphPad Software, San Diego, CA, USA).
Results
Clinical and demographic characteristics of studied subjects
The mean age for HCSLE, SLE patients, HCAtheros,
and atherosclerotic patients were 28 ^ 8, 34 ^ 12,
65 ^ 5, and 61 ^ 9, respectively (Table I). SLE
patients were under different immunosuppression

Table I. Clinical characteristics of patients and controls.

HCSLE SLE HCAtheros Atherosclerosis

Number 29 38 10 21
Age 28 ^ 8* 34 ^ 12 65 ^ 5 61 ^ 9
Treatment – PDN, CLQ, – Captopril,
Azathioprine, Metoprolol, Warfarin,
Cyclophosphamide, ASA and statins
Mycophenolate mofetil
CIMT 0.66 ^ 0.17 0.87 ^ 0.29 0.73 ^ 0.13 1.15 ^ 0.26†
n¼7 n ¼ 16 n ¼ 10 n¼9
TC levels (mg/dl) 191 ^ 24‡ 218 ^ 63 163 ^ 22 214 ^ 69{
n¼5 n ¼ 21 n ¼ 10 n ¼ 14
TGC levels (mg/dl) 86 ^ 12 248 ^ 177 87 ^ 9 181 ^ 130§
n¼5 n ¼ 20 n ¼ 10 n ¼ 13
LDL levels (mg/dl) 112 ^ 22 117 ^ 25 95 ^ 9 129 ^ 59
n¼5 n ¼ 13 n ¼ 10 n ¼ 12
HDL levels (mg/dl) 61 ^ 6 43 ^ 17k 52 ^ 11 51 ^ 17
n¼5 n ¼ 18 n ¼ 10 n ¼ 13

*Data show the mean ^ SD. HC, Healthy controls; HCSLE, Healthy controls with similar age-range to SLE patients; HCAtheros, Healthy
controls with similar age-range to atherosclerosis patients; PDN, Prednisolone; CQL, chloroquine; ASA, Acetylsalicylic acid; CIMT, Carotid
Intima Media Thickness; TC, Total Cholesterol; mg/dl, milligrams/deciliter; TGC, Triglycerides; LDL, Low Density Lipoproteins; and
HDL, High Density Lipoproteins. Results were analyzed by unpaired t-test; † ( p , 0.001) compared to HCAtheros; ‡ ( p , 0.05) compared to
HCAtheros; { ( p , 0.05) compared to HCAtheros; § ( p , 0.05) compared to HCAtheros; k ( p , 0.05) compared to HCSLE.

schedules with prednisone, chloroquine, cyclopho-
sphamide, mycophenolate mofetil, and azathioprine,
and their SLEDAI score was 12 ^ 9. Atherosclerotic
patients were also under different treatments, with
captopril, metoprolol, warfarin, acetylsalicylic acid
(ASA) and statins (Table I).
Among the clinical parameters, CIMT was found
increased in all nine atherosclerotic patients tested
(1.15 mm ^ 0.26) compared to 10 HCAtheros evalu-
ated (0.73 mm ^ 0.13, p , 0.05). The 16 SLE
patients presented a CIMT of 0.87 mm ^ 0.29,
which did not reach statistical significant difference
when compared with 7 HCSLE evaluated; however,
since normal values of CIMT range from 0.36 to
0.9 mm and CIMT higher than 0.9 mm is associated
with a high prevalence of cardiovascular disease [26],
it is noteworthy that 9/16 of the SLE patients (56%)
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evaluated presented a CIMT higher than 0.9 mm.
TC levels were increased in all 14 atherosclerotic
patients (214 ^ 69 mg/dl) compared to 10 HCAtheros
tested (163 ^ 22 mg/dl p , 0.05). LDL levels were
similar between the patients and their respective
healthy controls. TGC levels showed a nonsignificant
trend to increased values in the 20 SLE patients
(248 ^ 177 mg/dl) compared to 5 HCSLE evaluated
(86 ^ 12 mg/dl). HDL levels were decreased in all 18
For personal use only.
SLE patients (43 ^ 17 mg/dl) as compared to the 5
HCSLE (61 ^ 6 mg/dl, p , 0.05) studied.
LDL isolation, oxidation, and labeling
The fractions obtained from plasma of healthy
donors by ultracentrifugation, in the presence of
salts, as described in patients and methods, were
compared with whole plasma by SDS-PAGE
(Figure 1A). The LDL fraction showed mainly one
band of high molecular weight (. 170 kDa) that may
correspond to the protein ApoB100, according to the
molecular weight described for apolipoproteins [5].
Several bands were observed in the fractions that
according to the isolation process should contain
CM/VLDL and HDL. Since oxidation increases the
negative net charge of LDL [27], the oxidation and
purity of Ox-LDL was verified by agarose gel
electrophoresis, confirming that the Ox-LDL band
migrated to the cathode, compared with the N-LDL
band (Figure 1B). DiI labeling efficiency, as
confirmed by flow cytometry, showed that 80 – 90%
of LDL were labeled, compared with nonlabeled
LDL (Figure 1C).
CD14þCD163þ cells are increased in HCAtheros compared
to HCSLE, and CD36 and CD163 expression are
differentially altered in atherosclerotic and SLE patients
Since CD36 and CD163 are involved in Ox-LDL and
apoptotic cells removal [6], the number (Figure 2A,B)
and the expression (Figure 2C,D) of these receptors
Ox-LDL binding/endocytosis in SLE 5
were compared in monocytes from patients and
controls. There was no difference in the number of
circulating CD14þCD36þ cells/ml between SLE
(153.1 ^ 205.5) or atherosclerotic patients (251 ^
157.6) and their respective controls (161.3 ^ 211.7
and 219.6 ^ 74.2) nor between HCSLE and HCAtheros
(Figure 2A). However, the number of CD14þCD163þ
cells was increased in HCAtheros (377.1 ^ 71.75)
compared to HCSLE (235.4 ^ 164.7, p , 0.05), but
no differences were observed between SLE patients
(283.6 ^ 248.2) and HCSLE or between atherosclero-
tic patients (289.2 ^ 126.8) and HCAtheros
(Figure 2B).
Comparison of CD36 expression among the groups
studied showed that CD36 MFI on CD14þ cells was
decreased in atherosclerotic patients (22.0 ^ 28)
compared with HCAtheros (43 ^ 23.3, p , 0.05)
(Figure 2C). Contrariwise, CD163 MFI was found
decreased in SLE patients (32 ^ 18.1) compared to
HCSLE (67.5 ^ 17.8, p , 0.0001) (Figure 2D), but
there were no differences between HCSLE and
HCAtheros neither for CD36 MFI (Figure 2C) nor
for CD163 MFI (Figure 2D).
Of note, in SLE patients the number of
CD14þCD36þ or CD163þ cells did not correlate
with the disease activity, as measured by SLEDAI
(data not shown). These results suggest that the
differences observed in the number of circulating
CD14þCD163þ cells may be due to the age difference
between SLE patients/HCSLE and atherosclerotic
patients/HCAtheros, rather than due to SLE or
atherosclerosis activity.
SLE patients and HCSLE have similar Ox-LDL
binding/endocytosis
Binding/endocytosis of Ox-LDL are important pro-
cesses for atherosclerosis development [2], thus
PBMCs were incubated with N-LDL-DiI and Ox-
LDL-DiI, and analyzed by flow cytometry. Figure 3
shows the CD14þ cells not incubated with Ox-LDL
(Figure 3A), total binding/endocytosis (Figure 3B)
and endocytosis of DiI-labeled Ox-LDL (Figure 3C)
performed in a representative healthy control. Binding
and endocytosis of Ox-LDL at concentrations ranging
from 0.2 to 10 mg/ml, showed a dose-dependent
increase in patients and controls (Figure 4).
Monocytes from SLE patients, compared to
HCSLE, showed no differences in Ox-LDL binding
to CD14þ cells (15.7 ^ 13.5 vs. 11.5 ^ 11) at
10 mg/ml. At 0.2 mg/ml atherosclerotic patients pre-
sented increased Ox-LDL binding (4.99 ^ 4.94)
compared to HCAtheros (1.55 ^ 0.75, p , 0.05)
(Figure 4A). Ox-LDL endocytosis was increased in
atherosclerotic patients compared to HCAtheros at
1 mg/ml (20.66 ^ 14.71 vs. 7.78 ^ 1.69, p , 0.05)
and at 10 mg/ml (52.2 ^ 21.56 vs. 11.71 ^ 3.89,
p , 0.01). HCAtheros presented a decreased Ox-LDL
 6 L. M. Yassin et al.

A B
1200 1200 *

CD14+CD163+ cells/µl
CD14+CD36+ cells/µl
1000 1000

Number of
Number of
800 800
600 600
400 400
200 200
0 0

E s is E s is
SL E ro os SL
SL
E he
ro
ros
HC SL C At
he
cle
r HC C At cle
H s H s
ro ro
he he
At At

C D ***
*

CD163 MFI in CD14+ cells

CD36 MFI in CD14+ cells

100 100
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80 80

60 60

40 40

20 20

0 0

SL
E E ro
s sis SL
E E ro
s sis
HC SL At
he
le ro HC SL At
he le ro
HC ro
sc HC ro
sc
he he
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At At

Figure 2. Number and expression of CD36 and CD163 on monocytes from healthy controls, atherosclerotic patients, and SLE patients.
(A) Number of CD14þCD36þ and (B) CD14þCD163þ cells per microliter. (C) CD36 and (D) CD163 MFI in CD14þ cells from HCSLE
(n ¼ 29), SLE patients (n ¼ 38), HCAtheros (n ¼ 10), and atherosclerotic patients (n ¼ 21), evaluated by flow cytometry. Results show
individual values and means ^ SD. *p , 0.05, ***p , 0.001. Results were analyzed by unpaired t-test.

endocytosis (11.71 ^ 3.4) compared to HC SLE The percentage of bound N-LDL to CD14þ cells
(40.72 ^ 25.22, p , 0.001) at 10 mg/ml, suggesting was decreased in atherosclerotic patients (8.03 ^ 4.68)
that the age decreases the capacity of CD14þ cells to and in HCSLE (8.08 ^ 8.38, p , 0.05) compared to
endocytose Ox-LDL (Figure 4B). HCAtheros (15.51 ^ 4.46, p , 0.01), but no differences

A B C
104 104 104
2.64% 24.3% 19.6%

103 103 103

Ox-LDL-Dil

102 102 102

101 101 101

104
0
10 100 100
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

CD14-FITC

Figure 3. Dot plot of Ox-LDL binding/endocytosis by monocytes from a healthy control donor. (A) Representative dot plot from a healthy
control showing CD14þ cells not exposed to Ox-LDL, (B) total Ox-LDL-DiI (10 mg/ml) binding/endocytosis, and (C) Ox-LDL-DiI
endocytosis (with trypan blue) by CD14þ cells. Monocytes were gated based on forward and side scatter light parameters. Ox-LDL-DiI
fluorescence is shown on the Y-axis and CD14-FITCþ on the X-axis.
 Ox-LDL binding/endocytosis in SLE 7

A B
40 80

Ox-LDL endocyt. by CD14+ cells

Ox-LDL binding to CD14+ cells
*** HCSLE
SLE
30 60
HCAtheros.
Atherosclerosis
20 40 *

10 * 20

0 0

0.2 0.5 1.0 10 0.2 0.5 1.0 10

Figure 4. Binding/endocytosis of Ox-LDL by monocytes from patients and controls. (A) Percentage of CD14þ cells from HCSLE, SLE
patients, HCAtheros, and atherosclerotic patients that have bound and (B) endocytosed Ox-LDL-DiI (mg/ml), evaluated by flow cytometry as
described in materials and methods. Results show means ^ SD and were analyzed by two-way ANOVA with Bonferroni post-test. *p , 0.05,
Autoimmunity Downloaded from informahealthcare.com by 190.71.172.154 on 02/12/11

***p , 0.001.

were observed between HCSLE and SLE patients to be determinant for atherosclerosis, since a similar
(9.64 ^ 6.08). N-LDL endocytosis by CD14þ cells number of CD36þ and CD163þ monocytes was
was not different between the groups (data not shown). observed in SLE patients, compared to HCSLE and in
In addition, there was no difference in Ox-LDL atherosclerotic patients compared to HCAtheros.
binding/endocytosis between SLE patients with high The decreased CD36 and CD163 MFI observed,
and low CIMT (data not shown). respectively, in atherosclerotic and SLE patients could
be explained by Statins treatment [34] in athero-
sclerotic patients and by the inflammatory state [35]
For personal use only.

Discussion present in SLE patients. Furthermore, CD36 and

The risk of cardiovascular disease is very high in SLE; CD163 expression differences could suggest that the
however, the exact nature of the underlying vascular role of these receptors in atherosclerosis development
pathology is not clear [28]. According to the Framing- is different in atherosclerotic and SLE patients.
ham’s study, dyslipidemia with low levels of HDL and Even though there were not differences in the number
high levels of LDL is a risk factor for developing of CD14þCD36þ and CD14þCD163þ cells between
atherosclerosis [29,30]. Our SLE patients presented HCAtheros and atherosclerotic patients, HCAtheros pre-
decreased HDL levels and a trend toward increased sented an increased number of CD163 in monocytes,
TGC levels compared to HCSLE. This pattern is similar compared to HCSLE, suggesting that aging, besides
to the reported ‘lupus pattern’ of dyslipoproteinemia, other undetermined factors, may influence the number
defined by elevated levels of VLDL and TGC, and low of circulating CD163þ monocytes.
HDL levels [31], which was associated with athero- Aging has been previously reported to be
sclerosis presented in SLE patients [10]. accompanied by increased C reactive protein, serum
Increased CIMT is a marker for atherosclerosis that amyloid-A protein, and alpha-1-acid glycoprotein
correlates with established coronary heart disease [32]. [36], suggesting an increased pro-inflammatory state
Because CIMT was used as criterion for early diagnosis in elderly subjects [37]. Additionally, systemic inflam-
of atherosclerosis, we measured CIMT in patients and matory stimuli such as a coronary artery bypass graft
controls. In adults, CIMT normal values range from surgery with cardiopulmonary bypass, was reported to
0.36 to 0.9 mm and values higher than 0.9 mm are increase CD163 expression on monocytes [38].
associated with high cardiovascular disease prevalence Nevertheless, the role of CD163 and CD36 in
[26]. As expected, the highest CIMTs were observed in atherosclerosis development is not clear yet, since
atherosclerotic patients. In SLE patients CIMT was not there are conflicting findings about their positive or
statistically increased, but 56% of SLE patients negative contribution to atherosclerosis [39 – 44].
presented CIMT .0.9 mm, even though they were However, there is evidence suggesting that SR class
much younger than the atherosclerotic patients, A family members, including CD163 [6], are
supporting the previous reports of early atherosclerosis implicated in the pathological deposition of cholesterol
development in SLE patients [33]. on arterial walls during atherogenesis, as a result of
Ox-LDL removal by SRs is a well-established event receptor-mediated uptake of oxidized LDL [8,45,46].
in lipid deposition, foam cells formation, and Ox-LDL binding and endocytosis by monocytes
atherosclerosis development [5,6], the latter being were increased in atherosclerotic patients compared to
increased in SLE [10]. However, the presence of these HCAtheros; but N-LDL binding was decreased in
receptors on the monocyte membrane does not seem atherosclerotic patients compared to their controls.
 8 L. M. Yassin et al.
However, the differences in Ox-LDL binding and
endocytosis observed between atherosclerotic patients
and HCAtheros cannot be explained by the number of
CD36þ nor CD163þ monocytes, since they were not
different between them.
These findings suggest that SR expressed on
monocytes from atherosclerotic patients may present
functional alterations that could lead to increased
Ox-LDL removal or that other SR, such as MARCO
or LOX-1, were also contributing to atherosclerosis
development.
In SLE patients, binding and endocytosis of Ox-
LDL by monocytes were not different to HCSLE,
suggesting that the early atherosclerosis development
reported in SLE patients [2,10] is not related to
alterations in Ox-LDL removal. Besides, the similar
N-LDL uptake observed between SLE patients and
Autoimmunity Downloaded from informahealthcare.com by 190.71.172.154 on 02/12/11
HCSLE is not in agreement with the previously
reported decreased LDL internalization by leukocytes
from SLE patients. However, these differences could
be explained by the different experimental conditions
used for the N-LDL uptake assays [47].
Ox-LDL binding/endocytosis by SR, besides parti-
cipating in foam cells formation and atherosclerosis
development, may lead to an inflammatory response
through the activation of MAPKs and NFkB,
For personal use only.
resulting in the production of the inflammatory
cytokines IL-1b and TNF-a [48 –50], which are
major players in atherosclerosis development [51].
In conclusion, our results support that aging
increases the number of circulating CD163þ mono-
cytes, but does not seem to determine an increase in
Ox-LDL removal capacity. Even though SLE patients
presented decreased HDL levels and increased TGC
levels, Ox-LDL binding/endocytosis was similar to
their controls and hence cannot explain the increased
CIMT observed in a high percentage of SLE patients.
Thus, it would be possible to suggest that the
mechanisms involved in atherosclerosis development
are different in the presence of a concomitant
autoimmune disease, such as SLE, but further studies
are needed to explore this possibility.
Acknowledgements
Thanks to the Hospital San Vicente de Paul for
allowing the development of the project, to the Flow
Cytometry Unit at the SIU (Sede de Investigación
Universitaria), Universidad de Antioquia, and to Libia
M. Rodrı́guez for her helpful input concerning the
statistical analysis. The authors also thank the patients
and the healthy volunteers.
Declaration of interest: This work was supported by
Colciencias, Grant 11150416348 and “Proyecto de
Sostenibilidad”, Universidad de Antioquia. LMY was
beneficiary of a doctoral fellowship from Colciencias
and received partial funding from “Proyecto de
Sostenibilidad” Universidad de Antioquia. Authors’
salaries were paid either by Colciencias (LMY, JL and
GM), the Universidad de Antioquia (MR, LAR, LFG
and GV) or by Universidad Central de Venezuela
(JBS). None of the authors has any potential financial
conflict of interest related to this manuscript. The
authors alone are responsible for the content and
writing of the paper.
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