January 2011
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CD11c/CD18 Expression Is Upregulated on Blood
Monocytes During Hypertriglyceridemia and Enhances
Adhesion to Vascular Cell Adhesion Molecule-1
AQ: 1
R. Michael Gower, Huaizhu Wu, Greg A. Foster, Sridevi Devaraj, Ishwarlal Jialal,
Christie M. Ballantyne, Anne A. Knowlton, Scott I. Simon
P1-foo
Objective—Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and
emigration across inflamed endothelium. Involvement of !2-integrin CD11c/CD18 in atherogenesis was recently shown
in dyslipidemic mice, which motivates our study of its inflammatory function during hypertriglyceridemia in humans.
Methods and Results—Flow cytometry of blood from healthy subjects fed a standardized high-fat meal revealed that at
3.5 hours postprandial, monocyte CD11c surface expression was elevated, and the extent of upregulation correlated with
blood triglycerides. Monocytes from postprandial blood exhibited an increased light scatter profile, which correlated
with elevated CD11c expression and uptake of lipid particles. Purified monocytes internalized triglyceride-rich
lipoproteins isolated from postprandial blood through low-density lipoprotein–receptor–related protein-1, and this also
elicited CD11c upregulation. Laboratory-on-a-chip analysis of whole blood showed that monocyte arrest on a vascular
cell adhesion molecule-1 (VCAM-1) substrate under shear flow was elevated at 3.5 hours and correlated with blood
triglyceride and CD11c expression. At 7 hours postprandial, blood triglycerides decreased and monocyte CD11c
expression and arrest on VCAM-1 returned to fasting levels.
Conclusion—During hypertriglyceridemia, monocytes internalize lipids, upregulate CD11c, and increase adhesion to
VCAM-1. These data suggest that analysis of monocyte inflammation may provide an additional framework for
evaluating individual susceptibility to cardiovascular disease. (Arterioscler Thromb Vasc Biol. 2011;31:00-00.)
Key Words: adhesion molecules ! atherosclerosis ! leukocytes
N
eointimal thickening due to monocyte recruitment and
lipid deposition in arteries begins during childhood,1,2
but complications due to atherosclerosis do not manifest until
later in life. Prevention requires early identification of individuals at risk for cardiovascular disease. Conventional risk
factors, including smoking, diabetes, hypertension, or dyslipidemia, can identify those at increased risk; however, many
individuals with cardiovascular events have only 1 or none of
these factors.3 Thus, there is a need for personalized tests of
susceptibility to detect disease and guide therapy.
In animal models, hypercholesterolemia induces vascular
cell adhesion molecule-1 (VCAM-1) expression on aortic
endothelium, and its induction precedes accumulation of
macrophages and T lymphocytes at sites of lesion formation.4,5 The !1-integrin very late antigen-4 (VLA-4) is the
primary monocyte receptor that binds to VCAM-1 and
supports rolling,6 which on activation mediates cell arrest.7
VLA-4/VCAM-1 interactions are important for the progres-
sion of atherosclerosis, as hypercholesterolemic mice that
express VCAM-1 lacking one of the VLA-4 binding domains
exhibit reduced lesion area.8 Recent evidence suggests the
!2-integrin CD11c/CD18 also recognizes VCAM-1 as an
adhesive ligand during monocyte recruitment in shear flow.
Antibody blocking experiments on inflamed human aortic
endothelium revealed that CD11c and VLA-4 cooperate to
support arrest and transendothelial migration.9 Furthermore,
our recent study showed that monocytes isolated from mice
deficient in CD11c exhibited lower arrest efficiency and an
inability to adhesion strengthen on recombinant VCAM-1
under shear flow.10
Monocyte CD11b/CD18 is elevated during postprandial
lipemia and following exposure to triglyceride-rich lipoproteins (TGRL)11,12; however, the effect on CD11c/CD18 expression is unknown. It is also unclear how native TGRL
modulates monocyte upregulation of these proteins. Lowdensity lipoprotein (LDL)-receptor related protein-1 (LRP-1)
Received on: May 25, 2010; final version accepted on: October 15, 2010.
From the Department of Biomedical Engineering (R.M.G., G.A.F., S.I.S.), Laboratory for Atherosclerosis and Metabolic Research (S.D., I.J.),
Molecular and Cellular Cardiology, Cardiovascular Division, Department of Medicine (A.A.K.), and Department of Medical Pharmacology (A.A.K.),
University of California, Davis, Calif; Section of Atherosclerosis and Vascular Medicine, Department of Medicine (H.W., C.M.B.) and Section of
Leukocyte Biology, Department of Pediatrics (H.W., C.M.B.), Baylor College of Medicine, Houston, Texas; Center for Cardiovascular Disease
Prevention, Methodist DeBakey Heart and Vascular Center, Houston, Texas (H.W., C.M.B).
Correspondence to Scott I. Simon, PhD, Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Dr, Davis,
CA 95616. E-mail [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
DOI: 10.1161/ATVBAHA.110.215434
1
<zjs;Original Article> • <zjss;> • <zdoi;10.1161/ATVBAHA.110.215434>
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2
Arterioscler Thromb Vasc Biol
January 2011
mediates TGRL clearance from the blood in the liver and
TGRL uptake into macrophages.13,14 We demonstrate that
blood monocytes express LRP-1 and it is involved in binding
TGRL, which leads to upregulation of monocyte CD11c in
vitro. Thus, we hypothesized that during periods of hypertriglyceridemia, CD11c may be upregulated on circulating
monocytes and contribute to enhancing arrest on VCAM-1.
We report that in healthy subjects, monocyte uptake of
circulating lipid and upregulation of cell surface CD11c
correlates with blood triglyceride following a high-fat meal.
Applying a laboratory-on-a-chip assay of monocyte adhesion
to VCAM-1 under shear flow revealed that arrest correlated
with postprandial triglycerides, was dependent on CD11c,
and returned to fasting levels as the triglyceride peak subsided. As atherosclerosis is a chronic disease exacerbated by
monocyte recruitment to nascent lesions where VCAM-1 is
elevated,15 these data may provide an additional framework
for evaluating susceptibility to atherosclerosis.
Methods
An expanded Methods section is available online at
http://atvb.ahajournals.org.
Subjects
Forty healthy subjects (24 female) were recruited from the general
population on the basis of an approved institutional review board
protocol at the University of California, Davis. Clinical characteristics of all participants are listed in the online Supplemental Table I.
Study Meal and Design
The meal consisted of 1230 calories, 40% of which were from fat.
Additional nutritional information is provided in the supplemental
materials. Venous blood was obtained after a 10-hour overnight fast,
and then the meal was administered. Participants returned 3.5 hours
postprandial, a period that coincides with the average spike in
triglycerides after ingestion of a high-fat meal,12 and again at 7 hours
for a final blood draw.
Analytic Methods
Lipid and glucose measurements were conducted at the University of
California, Davis, Medical Center Clinical Laboratory using the
Beckman Coulter UniCel DxC 800 Synchron Clinical System. LDL
cholesterol was calculated by the Friedewald equation. Cytokines
were analyzed in fasting and postprandial serum samples by Milliplex XMAP technology. Multiplexed cytokines were analyzed on the
Bioplex 200 system.
Antibodies
Commercially available antibodies are listed in the supplemental
data. CD11c monoclonal antibody 496K, which induces a lowaffinity conformation and inhibits ligand binding, was obtained from
ICOS Corp (Bothell, Wash).16
Flow Cytometry
Immediately following venipuncture, whole blood was cooled to
4°C. After labeling, unbound antibody was removed, and red blood
cells were lysed. Data were acquired on a FACScan cytometer
(Becton Dickinson, San Jose, Calif). Monocytes were identified by
CD14 fluorescence above 99% of isotype control and side scatter
profile as shown in Supplemental Figure I. Fluorescence activated
cell sorting and Oil Red O staining are described in the supplemental
data.
In Vitro Exposure of Monocytes to TGRL
Mononuclear cell (MNC) and TGRL isolations are described in the
supplemental data.
Freshly isolated MNCs from fasting subjects were incubated with
Alexa Fluor 488 –labeled TGRL at 100 "g of apolipoprotein B
(apoB)/mL at 37°C for 30 minutes and then cooled to 4°C. To
remove surface-bound lipoproteins, MNCs were washed in Hanks’
balanced salt solution containing 5 mmol/L EDTA (pH 6.0). For
experiments monitoring cell surface receptors with fluorescently
conjugated antibodies, unlabeled TGRL was used. In blocking
experiments, MNCs were incubated with 50 "g/mL of the LRP-1
antagonist receptor-associated protein (RAP)17 before incubation
with TGRL. Confocal microscopy is described in the supplemental
data.
Whole-Blood Adhesion Assay
Design and assembly of the microfluidic device and the whole blood
adhesion assay are described in the supplemental data.
Monocyte adhesion to VCAM-1 in whole blood has been described previously.18 In this study, we have adapted those protocols
to our custom microfluidic device. Briefly, diluted whole blood was
withdrawn through a microfluidic chamber sealed to a glass coverslip derivatized with VCAM-1. Following the assay, arrested cells
were fixed with methanol and stained using Wright Stain (Fisher
Scientific, Pittsburgh, Pa). A total differential count was conducted
along the flow channel. Monocytes were identified by morphology,
including cell diameter, large cytoplasm to nucleus ratio, and fine
reticular chromatin.
Statistics
Multiple groups were compared using 1-way ANOVA with the
Tukey post test. Fasting and postprandial measurements were compared with a paired Student t test. All other comparisons were made
with an unpaired Student t test. All analysis was carried out using
GraphPad Prism, version 5.0c, for Mac.
Results
Blood Triglycerides and Monocyte Inflammation
Are Elevated Postprandial
Blood triglyceride concentration increased an average of 85%
from fasting levels 3.5 hours postprandial, a period that
coincides with the peak in triglycerides after ingestion of a
high-fat meal.12 Glucose and apoB100 remained unchanged
at this time point, but there were significant decreases in total
cholesterol, LDL, and high-density lipoprotein (HDL) cholesterol (Supplemental Table II).
Surface receptors were detected by flow cytometry of
antibody-labeled whole blood samples to define a baseline for
monocyte inflammation and avoid activation that occurs
during isolation.19 Following the peak in blood triglycerides
at 3.5 hours, monocytes exhibited a significant increase in
cell surface expression of CD14, CD11b, and CD11c and a
decrease in CD62L (Figure 1). In contrast, VLA-4 expression
was not increased (data not shown). Granulocytes did not
exhibit a significant increase in any measured surface antigens (Supplemental Figure II).
Monocyte markers of inflammation were increased postprandially, and we hypothesized that cytokines levels may
also be increased and associated with the observed activation.
Tumor necrosis factor-#, interferon- $, interleukin-1 !,
interleukin-6, and interleukin-8 were all significantly increased after the meal, whereas interleukin-10, a potent
antiinflammatory cytokine,20 remained unchanged. It is noteworthy that the relative increase in cytokines did not correlate
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Gower et al
CD11c Is Upregulated on Postprandial Monocytes
3
Table 2. Correlations Between the Postprandial Increase in
Monocyte CD11c and Blood Lipids
Pearson r
95% CI
P Value
r2
Fasting
Figure 1. Postprandial changes in monocyte surface receptors.
A, Monocyte surface CD11c, CD14, CD11b, and CD62L in fasting (light bars) and 3.5 hours postprandial (dark bars) blood for
40 subjects. Expression is displayed as median fluorescence
intensity (MFI). Significance was measured by the paired Student t test. *P"0.001.
T1
with the change in monocyte surface CD11c or triglyceride
level in blood. Endotoxin was not a factor in the inflammatory response because levels detected in serum were low
(4 IU/mL) and remained unchanged by the meal (Table 1).
Increase in Monocyte CD11c Correlates With
Blood Lipid Levels
T2
We have reported that CD11c expression is increased on
monocytes in dyslipidemic mice,10 and we were interested in
whether CD11c correlated with blood lipid levels in our
human cohort. Relative increases in CD11c correlated the
strongest with the absolute value of a subject’s fasting and
postprandial triglycerides (Table 2) but not the percentage
increase in triglycerides (Supplemental Figure IIIA). Percentage increase in CD11c also correlated with apoB100, nonHDL cholesterol, total cholesterol, and fasting LDL but not
HDL (Table 2).
Inflammatory Response Following Triglyceride
Increase Subsides by 7 Hours
The extent of monocyte CD11c increase correlated with
postprandial triglycerides at 3.5 hours, and we hypothesized
that its expression might decrease as triglycerides reverted
back to fasting levels. To address this hypothesis, we recalled
6 subjects selected for high postprandial triglycerides
Table 1.
Triglyceride
0.65
0.42 to 0.81
"0.0001
0.43
Apolipoprotein B100
0.51
0.22 to 0.72
0.001
0.26
Total cholesterol:HDL
ratio
0.46
0.17 to 0.68
0.004
0.21
Non-HDL cholesterol
0.46
0.16 to 0.68
0.004
0.21
Total cholesterol
0.45
0.16 to 0.68
0.004
0.21
LDL cholesterol
0.42
0.12 to 0.66
0.008
0.18
HDL cholesterol
#0.22
#0.50 to 0.11
0.194
0.05
Triglyceride
0.62
0.37 to 0.78
"0.0001
0.38
Non-HDL cholesterol
0.54
0.27 to 0.74
0.0004
0.29
Apolipoprotein B100
0.45
0.15 to 0.67
0.005
0.20
Total cholesterol:HDL
ratio
0.44
0.14 to 0.67
0.005
0.20
Postprandial
Total cholesterol
0.44
0.14 to 0.67
0.005
0.20
HDL cholesterol
#0.27
#0.54 to 0.06
0.106
0.07
LDL cholesterol
0.23
#0.12 to 0.53
0.197
0.05
Correlations between the postprandial increase in monocyte CD11c expression from fasting levels (as measured by flow cytometry) and blood lipids. Data
are from 40 subjects (24 female). Data are partitioned on fasting and
postprandial values and listed in descending order based on strength of
correlation.
(410!116 mg/dL, mean!SD) and robust monocyte CD11c
increase. Venous blood was obtained after a 10-hour overnight fast, and then the meal was administered. Participants
returned 3.5 and 7 hours following ingestion for postprandial
blood draws. Clinical characteristics and serum biochemistry
at 0, 3.5, and 7 hours postprandial are displayed in Supplemental Tables III and IV. For these subjects, triglyceride
concentration at 7 hours showed a trend to decrease but was
not significantly different from 3.5 hours (Figure 2A). However, the elevation in CD11c and CD11b expression on
monocytes was significantly decreased at 7 hours compared
with the level at 3.5 hours (Figure 2B).
Serum Cytokine Levels
Sensitivity
(Lower Limit)
Fasting
(0 Hours)
Postprandial
(3.5 Hours)
TNF-#
0.05
4.7!0.5
7.6!1.1***
IFN-$
0.29
4.5!0.9
7.9!1.3**
IL-1!
0.06
1.1!0.3
2.1!0.7*
IL-6
0.10
8.9!2.8
12.8!3.4**
IL-8
0.11
5.8!0.9
IL-10
0.15
10.4!2.2
Endotoxin
0.10
4.2!0.3
8.7!1.8*
12.9!2.7
4.3!0.2*
Data are displayed as mean!SE from 40 subjects (24 female). Endotoxin
units are IU/mL. All other units are pg/mL. Significance was measured by the
paired Student t test. TNF indicates tumor necrosis factor; IFN, interferon; IL,
interleukin.
*P"0.05, **P"0.01, ***P"0.001.
Figure 2. Inflammatory response following increase in triglyceride subsides by 7 hours. Six subjects were selected for high
postprandial triglycerides (410!116 mg/dL; mean!SD) to study
the duration of monocyte activation. A, Subject serum triglyceride concentration at 0, 3.5, and 7 hours postprandial. Significance tested by repeated-measures ANOVA with the Tukey
post test. B, Percentage change in monocyte surface CD11c,
CD14, and CD11b from fasting values at 3.5 and 7 hours postprandial. Significance was measured by the Student t test.
*P"0.05.
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Arterioscler Thromb Vasc Biol
January 2011
Figure 3. Monocytes exhibit
lipid droplets and elevated
CD11c postprandial. A, Representative scatter plots of
monocytes from blood at 0,
3.5, and 7 hours postprandial
for 6 subjects with high postprandial triglycerides (410!116
mg/dL; mean!SD). B, Percentage of monocytes in each
quadrant (Q) at each time
point. C, CD11c expression on
monocytes in each quadrant
(Q) at each time point. Significance was tested by repeatedmeasures ANOVA with the
Tukey post test. *P"0.05. NS
indicates not significant. At
each time point, the CD11c
expression in Q3$Q4 was significantly higher than CD11c
expression in Q1$Q2 as
tested by the paired Student t
test. D and E, Representative
images of monocytes sorted
from fasting and 3.5 hours
postprandial blood stained
with Oil Red O (neutral lipid)
and hematoxylin (nucleus).
F3
Monocytes Take Up Lipid Particles in
Hypertriglyceridemic Blood
Monocytes Internalize TGRL and Upregulate
CD11c In Vitro
We have previously reported that monocytes in the blood of
hypercholesterolemic mice fed a high-fat diet internalized
lipids, and this correlated with elevated light scatter and cell
surface CD11c as detected by flow cytometry and confirmed
by histology.10 Here, we report that monocyte light scatter
significantly increased in the blood of subjects at 3.5 hours
postprandial (Figure 3A and 3B). Monocytes with elevated
light scatter profiles expressed significantly more CD11c than
cells with low light scatter (Figure 3C). By 7 hours, these
trends reversed, as indicated by a decrease in the scatter
distribution and CD11c expression of monocytes in blood.
Inspection of peripheral blood films confirmed a 1-fold
increase in the percentage of vacuolated monocytes at the
3.5-hour time point (Supplemental Figure IVA and IVB). To
establish whether these vacuoles consisted of neutral lipids,
monocytes from whole blood were sorted on the basis of
CD14 expression and scatter profile. Monocytes from 3.5
hours postprandial blood exhibited punctate Oil Red O–positive droplets, which were not detected in fasting blood
(Figure 3D and 3E and Supplemental Figure IVC and IVD).
We hypothesized that the lipid droplets observed in monocytes were due to uptake of TGRL in the blood. In support of
this, we found that monocytes expressed LRP-1 (Supplemental Figure VA), an LDL-family receptor that mediates macrophage uptake of TGRL.13,14 To determine its potential role,
TGRL was isolated from postprandial blood, labeled with
Alexa Fluor 488, and incubated with MNCs from fasting
healthy subjects. Internalization was visualized by confocal
immunofluorescence microscopy and quantified by flow
cytometry. The LRP-1 antagonist RAP17 inhibited TGRL
uptake by 30%, whereas TGRL endocytosis was abrogated at
10°C (Figure 4A and 4B). We also found that LRP-1 antibody
binding was inhibited in the presence of TGRL, suggesting
that binding of lipoprotein particles resulted in receptor
internalization (Supplemental Figure VB).
Additional experiments were performed to examine the
capacity of isolated TGRL to induce CD11c upregulation on
monocytes. A 30-minute incubation with TGRL induced a
40% increase in CD11c expression, a level similar to that
observed ex vivo, and this increase was inhibited by RAP
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CD11c Is Upregulated on Postprandial Monocytes
5
Figure 5. Monocyte arrest on VCAM-1 increases postprandially
and is dependent on CD11c. A, Correlation of monocyte arrest
on recombinant VCAM-1 with postprandial triglycerides for 17
subjects. Pearson r&0.7, P"0.01, R2&0.5. B, Dependence of
monocyte arrest on CD11c and VLA-4 at 0, 3.5, and 7 hours
postprandial for 6 subjects with high postprandial triglycerides
(410!116 mg/dL; mean!SD). Significance tested by repeatedmeasures ANOVA with the Tukey post test. *P"0.05 versus IgG
at 3.5 hours.
Figure 4. Internalization of TGRL in vitro increases monocyte
surface CD11c. A, Confocal images of MNCs incubated with
Alexa Fluor 488 –labeled TGRL (100 "g apoB/mL) alone and in
the presence of the LRP-1 antagonist RAP (50 "g/mL) at 37°C
or 10°C. Surface-bound lipoproteins were removed by washing
cells with 5 mmol/L EDTA. White arrows, monocytes; green,
TGRL. Monocytes were identified by morphology. Scale bar:
20 "m. B, Monocyte internalization of TGRL labeled with Alexa
Fluor 488 as measured by flow cytometry. Monocytes were
identified by scatter profile. Conditions are the same as A. Significance was tested by 1-way ANOVA with the Tukey post test.
C, Monocyte surface CD11c following incubation with TGRL
(100 "g apoB/mL) or TGRL$RAP (50 "g/mL) measured by flow
cytometry. Monocytes were identified by CD14 expression. Data
from 4 to 6 independent experiments. Significance was tested
by the Student t test.
(Figure 4C). These data indicate that monocytes in part use
LRP-1 to bind TGRL, which induces upregulation in CD11c
expression. Because this assay was performed in an MNC
fraction (to avoid activation of monocytes during isolation),
we cannot discount a paracrine effect from lymphocytes.
However, monocyte lipoprotein uptake and CD11c expression increase was inhibited by RAP, suggesting that CD11c
upregulation was a direct consequence of this interaction.
Monocyte Adhesion to VCAM-1 Under Shear
Stress Correlates With Postprandial Triglycerides
and Depends on CD11c
To assess the consequences of elevated postprandial triglycerides on monocyte function, we measured monocyte adhesion to recombinant VCAM-1 in diluted whole blood under
defined shear stress at fasting, 3.5 hours postprandial, and 7
hours postprandial using a laboratory-on-a-chip microfluidic
flow channel (Supplemental Figure VIA to VIC). Monocytes
in diluted and sheared whole blood recruited avidly to the
VCAM-1 substrate (Supplemental Figure IVD) and were
rarely observed rolling, but they quickly transitioned to firm
arrest, in agreement with previous work.18 A direct correlation was observed between a subject’s increase in monocyte
arrest from fasting and his or her peak in postprandial
triglycerides at 3.5 hours (Figure 5A).
Previously, we have reported that cooperative binding of
CD11c and VLA-4 to VCAM-1 contributes to the conversion
of monocyte rolling to arrest.9,10 To investigate the contribution of CD11c to the enhanced monocyte adhesion, we
measured monocyte arrest in the presence of CD11c blocking
antibody 496K, VLA-4 blocking antibody HP2.1, or IgG
control for 6 subjects selected for high postprandial triglycerides and CD11c expression (Figure 5B). Monocyte arrest
was significantly elevated at 3.5 hours but decreased to
fasting levels by 7 hours. Blood monocyte counts in our
subjects ranged from 2%105 to 10%105/mL and remained
constant at 3.5 and 7 hours after the meal, consistent with
previous studies.12 This confirms that increased monocyte
arrest was due to postprandial changes in monocyte function,
not simply changes in blood concentration. Inhibition of
CD11c with antibody significantly decreased monocyte adhesion at 3.5 hours postprandial but not at fasting or 7 hours
postprandial. Consistent with previous observations,18 blocking VLA-4 function with HP2.1 significantly blocked adhesion at all time points, demonstrating that monocyte arrest
was dependent on binding to VCAM-1. Moreover, the contribution of CD11c to adhesion correlated with the time point
of its peak in expression.
Discussion
We quantified the acute inflammatory response of circulating
monocytes following an increase in blood triglycerides,
which was characterized by elevated expression of CD11c, an
integrin receptor recently shown to regulate adhesion to
VCAM-1 on inflamed endothelium.9 This was accompanied
by an increase in CD11b expression and a reduction in
CD62L, suggesting monocyte activation. Monocyte activation was found to subside by 7 hours postprandial, a time
when blood triglyceride was declining. Monocytes in post-
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Arterioscler Thromb Vasc Biol
January 2011
prandial blood exhibited cytoplasmic lipid droplets, suggesting that lipid uptake in the circulation induces cell surface
expression of CD11c. Increase in monocyte arrest on
VCAM-1 also correlated with postprandial triglycerides, and
this was dependent on integrin cooperativity between VLA-4
and CD11c. These data are consistent with a recent report
showing that apoE#/# mice fed a high-fat diet have increased
CD11c expression on blood monocytes, which take up lipid
and accumulate in atherosclerotic lesions.10 In that study,
genetic deletion of CD11c in crossbred apoE#/#/CD11c#/#
mice significantly diminished the efficiency of monocyte
arrest on VCAM-1 in shear flow and reduced atherosclerosis.
These data in human and mouse models implicate CD11c as
a functional integrin that participates in recruitment on
VCAM-1 during periods of elevated blood lipids, such as the
postprandial phase, a period when incidents linked to coronary disease are prevalent.21
Our data set assessed a cohort of healthy subjects selected
without regard to age or gender. The most striking change in
serum biochemistry following the meal was an 85% increase
in triglycerides. Epidemiological evidence links elevated
nonfasting triglyceride with increased risk of atherosclerosis
and associated cardiovascular events.22,23 A causal relationship between triglyceride and monocyte CD11c levels was
recently demonstrated in humans with metabolic syndrome
and obese mice.24 In both cases, high triglyceride levels
correlated with elevated expression of CD11c on blood
monocytes that decreased following diet-induced weight loss.
Here, we observed in an acute study design that postprandial
hypertriglyceridemia transiently increased monocyte CD11c
and primed them for adhesion to VCAM-1. Our studies
provide a plausible link between the epidemiological data in
humans and mouse models that reveal increased monocyte
recruitment to nascent arterial lesions under conditions in
which VCAM-1 is elevated.4,8 For example, in dyslipidemic
mice, atherosclerotic lesion size is increased in proportion to
the number of accumulated monocytes.25 The importance of
CD11c in this pathogenesis was highlighted by our recent
study showing that apoE#/#/CD11c#/# mice have smaller
aortic lesions because of decreased macrophage content.10
A novel finding of this study was the close correlation
between postprandial triglyceride concentration, CD11c expression, and monocyte adhesion under shear flow. Increase
in CD11c and monocyte adhesion did not correlate with the
relative increase in triglycerides following the meal but rather
with the absolute concentration in the blood. Stable monocyte
arrest was dependent on VLA-4 and CD11c to recognize
distinct epitopes on VCAM-1.9 Furthermore, VLA-4 was
dependent on high-affinity CD11c that supported increased
levels of monocyte arrest because the blocking antibody
496K acts allosterically on CD11c by stabilizing a low
affinity conformation.16 These data are consistent with our
previous observation of cooperativity between CD11c and
VLA-4 in binding VCAM-1 on TGRL-primed and inflamed
human aortic endothelium.9 A potential mechanism by which
upregulation of CD11c expression potentiates VLA-4 function may involve cross-talk in signaling such as that previously demonstrated between !1 and !2.26 Thus, the clinical
relevance of elevated CD11c on monocytes in the postpran-
dial circulation is their enhanced potential to home to lesions
where endothelial VCAM-1 expression is elevated.15
Our study and others demonstrate that monocytes internalize
lipids during postprandial lipemia and after exposure to
TGRL.11,12,27 How blood monocytes internalize unmodified
lipoprotein particles is unclear. Recent studies using cell lines
and mice indicate that macrophages uptake native lipoproteins
through LRP-1, apoB48 receptor, CD36, and pinocytosis.13,14,28 –31 We show that freshly isolated monocytes internalized TGRL through LRP-1 (and possibly other LDL-family
receptors), resulting in upregulation of CD11c. Interestingly,
several in vivo studies with LDL receptor#/# or apoE#/# mice
indicated that macrophage-specific LRP-1 deficiency was associated with increased atherosclerotic lesions.29,32 Although this
suggests an atheroprotective role for LRP-1, our data and those
of others demonstrate an essential role of LRP-1 in macrophage
uptake of remnant lipoproteins and highlight the complexity of
LRP-1 in atherogenesis.13,14 In this regard, we are actively
studying how binding or internalization of lipoproteins upregulates CD11c expression. A plausible pathway may involve
signaling following ligand binding to LRP-1, which induces
activation of PLC and a rise in intracellular IP3 and Ca2$ levels
that in turn activate protein kinase C.33,34 Because protein kinase
C is important for cell degranulation through its modulation of
proteins involved in late exocytosis,35,36 a mechanism contributing to monocyte activation could be that LRP-1 binding TGRL
triggers activation of protein kinase C and release of CD11c
stored in secretory vesicles.19
In summary, we show an increase in monocyte inflammatory response in a population of subjects with high circulating
triglycerides. Monocytes were found to internalize lipids and
upregulate cell surface CD11c as early as 3.5 hours after a
high-fat meal, a period coincident with peak blood triglycerides. The inflammatory response was transient, subsiding by
7 hours. Using a dynamic blood film in a laboratory-on-achip format revealed that monocyte arrest on VCAM-1 was
dependent on CD11c and correlated with a subject’s postprandial triglycerides. Thus this assay may prove useful in
identifying high-risk individuals early in disease progression.
Given that the initial presentation of coronary disease for
many patients is sudden death or myocardial infarction, these
data warrant longitudinal studies to determine whether monocyte CD11c expression correlates with the development of
more severe atherosclerotic disease.
AQ:6 –7
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Acknowledgments
The authors thank Dr Donald Staunton of CisThera Inc. for development of antibodies and technical advice and Nadine Raymond for
invaluable assistance in clinical coordination.
Sources of Funding
This work was supported by National Institutes of Health Grants
HL086350 (to R.M.G.), HL082689 (to S.I.S.), K24-AT00596 (to I.J.),
HL077281 (to A.A.K.), HL079071 (to A.A.K.), DK078847 (to
C.M.B.), and R01-HL098839 (to H.W.); an American Heart Association award (to H.W.); and a Howard Hughes Medical Institution Med
into Grad Fellowship, University of California, Davis (to R.M.G.).
Disclosures
None.
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balt2/zhq-atv/zhq-atv/zhq00110/zhq9222-10z xppws S!1 10/26/10 7:37 4/Color Figure(s): F3-4 Art: 215434 Input-elr
Gower et al
References
1. Stary HC. Evolution and progression of atherosclerotic lesions in
coronary arteries of children and young adults. Arteriosclerosis. 1989;9:
I19 –I32.
2. Strong JP, Malcom GT, McMahan CA, Tracy RE, Newman III WP,
Herderick EE, Cornhill JF, for the Pathobiological Determinants of Atherosclerosis in Youth Research Group. Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention
from the Pathobiological Determinants of Atherosclerosis in Youth
Study. J Am Med Assoc. 1999;281:727–735.
3. Khot UN, Khot MB, Bajzer CT, Sapp SK, Ohman EM, Brener SJ, Ellis
SG, Lincoff AM, Topol EJ. Prevalence of conventional risk factors in
patients with coronary heart disease. J Am Med Assoc. 2003;290:
898 –904.
4. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear
leukocyte adhesion molecule during atherogenesis. Science. 1991;251:
788 –791.
5. Sakai A, Kume N, Nishi E, Tanoue K, Miyasaka M, Kita T. P-selectin
and vascular cell adhesion molecule-1 are focally expressed in aortas of
hypercholesterolemic rabbits before intimal accumulation of macrophages and T lymphocytes. Arterioscler Thromb Vasc Biol. 1997;17:
310 –316.
6. Alon R, Kassner PD, Carr MW, Finger EB, Hemler ME, Springer TA.
The integrin VLA-4 supports tethering and rolling in flow on VCAM-1.
J Cell Biol. 1995;128:1243–1253.
7. Chen C, Mobley JL, Dwir O, Shimron F, Grabovsky V, Lobb RR,
Shimizu Y, Alon R. High affinity very late antigen-4 subsets expressed on
T cells are mandatory for spontaneous adhesion strengthening but not for
rolling on VCAM-1 in shear flow. J Immunol. 1999;162:1084 –1095.
8. Cybulsky MI, Iiyama K, Li H, Zhu S, Chen M, Iiyama M, Davis V,
Gutierrez-ramos JC, Connelly PW, Milstone DS. A major role for
VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest. 2001;
107:1255–1262.
9. Sadhu C, Ting HJ, Lipsky B, Hensley K, Garcia-Martinez LF, Simon SI,
Staunton DE. CD11c/CD18: novel ligands and a role in delayed-type
hypersensitivity. J Leukoc Biol. 2007;81:1395–1403.
10. Wu H, Gower RM, Wang H, Perrard X-YD, Ma R, Bullard DC, Burns
AR, Paul A, Smith CW, Simon SI, Ballantyne CM. Functional role of
CD11c$ monocytes in atherogenesis associated with hypercholesterolemia. Circulation. 2009;119:2708 –2717.
11. Alipour A, van Oostrom AJHHM, Izraeljan A, Verseyden C, Collins JM,
Frayn KN, Plokker TWM, Elte JWF, Castro Cabezas M. Leukocyte
activation by triglyceride-rich lipoproteins. Arterioscler Thromb Vasc
Biol. 2008;28:792–797.
12. van Oostrom AJ, Rabelink TJ, Verseyden C, Sijmonsma TP, Plokker
HW, De Jaegere PP, Cabezas MC. Activation of leukocytes by postprandial lipemia in healthy volunteers. Atherosclerosis. 2004;177:
175–182.
13. Fujioka Y, Cooper AD, Fong LG. Multiple processes are involved in
the uptake of chylomicron remnants by mouse peritoneal macrophages. J Lipid Res. 1998;39:2339 –2349.
14. Kuchenhoff A, Harrach-Ruprecht B, Robenek H. Interaction of apo
E-containing lipoproteins with the LDL receptor-related protein LRP.
Am J Physiol. 1997;272:C369 –C382.
15. O’Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE. Neovascular
expression of E-selectin, intercellular adhesion molecule-1, and vascular
cell adhesion molecule-1 in human atherosclerosis and their relation to
intimal leukocyte content. Circulation. 1996;93:672– 682.
16. Sadhu C, Hendrickson L, Dick KO, Potter TG, Staunton DE. Novel tools
for functional analysis of CD11c: activation-specific, activationindependent, and activating antibodies. J. Immunoassay Immunochem.
2008;29:42–57.
17. Bu G. Receptor-associated protein: a specialized chaperone and antagonist for members of the LDL receptor gene family. Curr Opin Lipidol.
1998;9:149 –155.
CD11c Is Upregulated on Postprandial Monocytes
7
18. Reinhardt PH, Kubes P. Differential leukocyte recruitment from whole
blood via endothelial adhesion molecules under shear conditions. Blood.
1998;92:4691– 4699.
19. Miller LJ, Bainton DF, Borregaard N, Springer TA. Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface. J Clin Invest. 1987;
80:535–544.
20. Kleemann R, Zadelaar S, Kooistra T. Cytokines and atherosclerosis: a
comprehensive review of studies in mice. Cardiovasc Res. 2008;79:
360 –376.
21. Gertler MM, Driskell MM, Bland EF, Garn SM, Lerman J, Levine SA,
Sprague HB, White PD. Clinical aspects of coronary heart disease; an
analysis of 100 cases in patients 23 to 40 years of age with myocardial
infarction. J Am Med Assoc. 1951;146:1291–1295.
22. Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. Fasting
compared with nonfasting triglycerides and risk of cardiovascular events
in women. J Am Med Assoc. 2007;298:309 –316.
23. Boquist S, Ruotolo G, Tang R, Bjorkegren J, Bond MG, de Faire U,
Karpe F, Hamsten A. Alimentary lipemia, postprandial triglyceride-rich
lipoproteins, and common carotid intima-media thickness in healthy,
middle-aged men. Circulation. 1999;100:723–728.
24. Wu H, Perrard XD, Wang Q, Perrard JL, Polsani VR, Jones PH, Smith
CW, Ballantyne CM. CD11c expression in adipose tissue and blood and
its role in diet-induced obesity. Arterioscler Thromb Vasc Biol. 2009.
25. Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P,
Weissleder R. Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc Natl Acad Sci U S A.
2006;103:10340 –10345.
26. van den Berg JM, Mul FP, Schippers E, Weening JJ, Roos D, Kuijpers
TW. !1 integrin activation on human neutrophils promotes !2 integrinmediated adhesion to fibronectin. Eur J Immunol. 2001;31:276 –284.
27. den Hartigh LJ, Connolly-Rohrbach JE, Fore S, Huser TR, Rutledge JC.
Fatty acids from very low-density lipoprotein lipolysis products induce
lipid droplet accumulation in human monocytes. J Immunol. 2010;184:
3927–3936.
28. Gianturco SH, Ramprasad MP, Song R, Li R, Brown ML, Bradley WA.
Apolipoprotein B-48 or its apolipoprotein B-100 equivalent mediates the
binding of triglyceride-rich lipoproteins to their unique human monocytemacrophage receptor. Arterioscler Thromb Vasc Biol. 1998;18:968 –976.
29. Overton CD, Yancey PG, Major AS, Linton MF, Fazio S. Deletion of
macrophage LDL receptor-related protein increases atherogenesis in the
mouse. Circ Res. 2007;100:670 – 677.
30. Calvo D, Gomez-Coronado D, Suarez Y, Lasuncion MA, Vega MA.
Human CD36 is a high affinity receptor for the native lipoproteins HDL,
LDL, and VLDL. J Lipid Res. 1998;39:777–788.
31. Kruth HS, Huang W, Ishii I, Zhang WY. Macrophage foam cell formation
with native low density lipoprotein. J Biol Chem. 2002;277:34573–34580.
32. Hu L, Boesten LS, May P, Herz J, Bovenschen N, Huisman MV, Berbee
JF, Havekes LM, van Vlijmen BJ, Tamsma JT. Macrophage low-density
lipoprotein receptor-related protein deficiency enhances atherosclerosis in
ApoE/LDLR double knockout mice. Arterioscler Thromb Vasc Biol.
2006;26:2710 –2715.
33. Misra UK, Gawdi G, Pizzo SV. Ligation of low-density lipoprotein
receptor-related protein with antibodies elevates intracellular calcium and
inositol 1,4,5-trisphosphate in macrophages. Arch Biochem Biophys.
1999;372:238 –247.
34. Misra UK, Chu CT, Gawdi G, Pizzo SV. The relationship between low
density lipoprotein-related protein/#2-macroglobulin (#2M) receptors
and the newly described #2M signaling receptor. J Biol Chem. 1994;269:
18303–18306.
35. Chung SH, Polgar J, Reed GL. Protein kinase C phosphorylation of
syntaxin 4 in thrombin-activated human platelets. J Biol Chem. 2000;
275:25286 –25291.
36. Fujita Y, Sasaki T, Fukui K, Kotani H, Kimura T, Hata Y, Sudhof TC,
Scheller RH, Takai Y. Phosphorylation of Munc-18/n-Sec1/rbSec1 by
protein kinase C: its implication in regulating the interaction of Munc18/n-Sec1/rbSec1 with syntaxin. J Biol Chem. 1996;271:7265–7268.
JOBNAME: AUTHOR QUERIES PAGE: 1 SESS: 3 OUTPUT: Tue Oct 26 07:39:25 2010
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