CD11c/CD18 expression is upregulated on blood monocytes during hypertriglyceridemia and enhances adhesion to VCAM-1

Abstract

Objective

Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and emigration across inflamed endothelium. Involvement of β2-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 LRP-1, and this also elicited CD11c upregulation. Lab-on-a-chip analysis of whole blood showed that monocyte arrest on a 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.

Conclusions

During hypertriglyceridemia, monocytes internalize lipid, upregulate CD11c, and increase adhesion to VCAM-1. These data suggest that analysis of monocyte inflammation may provide additional framework for evaluating individual susceptibility to cardiovascular disease.

Neointimal 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 one 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 β₁-integrin very late antigen-4 (VLA-4) is the primary monocyte receptor that binds to VCAM-1 and supports rolling,⁶ which upon activation mediates cell arrest.⁷ VLA-4/VCAM-1 interactions are important for the progression 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 β₂-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 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. LDL-receptor related protein-1 (LRP-1) 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 enhance arrest on VCAM-1.

We report in healthy subjects that monocyte uptake of circulating lipid and upregulation of cell surface CD11c correlates with blood triglyceride following a high fat meal. Applying a lab-on-a-chip assay of monocyte adhesion to VCAM-1 under shear flow revealed that arrest correlated with postprandial triglyceride, 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 additional framework for evaluating susceptibility to atherosclerosis.

Methods

An expanded Methods section is available online.

Subjects

Forty healthy subjects (24 female) were recruited from the general population based on an approved institutional review board protocol at the University of California, Davis. Clinical characteristics for all participants are listed in Online Table 1.

Study meal and design

The meal consisted of 1230 calories, 40% of which were from fat. Additional nutritional information is provided in the supplement. 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.

Analytical Methods

Lipid and glucose measurements were conducted at the UC 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 online data supplement. CD11c monoclonal antibody 496K, which induces a low affinity conformation and inhibits ligand binding, was obtained from ICOS corporation, (Bothell, WA).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 was acquired on a BD FACScan cytometer (San Jose, CA). 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 is described in the supplement.

In vitro exposure of monocytes to triglyceride rich lipoproteins

Mononuclear cell (MNC) and triglyceride rich lipoprotein (TGRL) isolations are described in the online data supplement.

Freshly isolated MNCs from fasting subjects were incubated with AlexaFluor488-labeled TGRL at 100µg apoB/mL at 37°C for 30min and then cooled to 4°C. To remove surface bound lipoproteins, MNCs were washed in HBSS containing 5mM 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 50µg/mL of LRP-1 antagonist, Receptor Associated Protein (RAP),¹⁷ before incubation with TGRL. Confocal microscopy is described in the online supplement.

Whole Blood Adhesion Assay

Design and assembly of the microfluidic device and the whole blood adhesion assay are described in the online data supplement.

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 one-way ANOVA with 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 Graph Pad Prism version 5.0c for Mac.

Results

Blood triglycerides and monocyte inflammation are elevated postprandial

Blood triglyceride concentration increased an average of 85 percent 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 apolipoprotein B100 remained unchanged at this time point, but there were significant decreases in total, LDL, and HDL cholesterol (Online Table 2).

Surface receptors were detected by flow cytometry of antibody-labeled whole blood samples in order 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).

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) for 40 subjects. Expression is displayed as median fluorescence intensity (MFI). Significance measured by student-paired t-test. *P<0.001

Monocyte markers of inflammation were increased postprandial and we hypothesized that cytokines levels may also be increased and associated with the observed activation. TNF-α, IFN-γ, IL1-β, IL-6 and IL-8 were all significantly increased after the meal, whereas IL-10, a potent anti-inflammatory cytokine,²⁰ remained unchanged. It is noteworthy that the relative increase in cytokines did not correlate with the change in monocyte surface CD11c or triglyceride level in blood. Endotoxin was not a factor in the inflammatory response since levels detected in serum were low (4 IU/mL) and remained unchanged by the meal (Table 1).

Table 1.

Serum cytokine levels

	Sensitivity (Lower Limit)	Fasting (0hr)	Postprandial (3.5hr)
TNF-α	0.05	4.7±0.5	7.6±1.1***
IFN-γ	0.29	4.5±0.9	7.9±1.3**
IL-1β	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	8.7±1.8*
IL-10	0.15	10.4±2.2	12.9±2.7
endotoxin	0.10	4.2±0.3	4.3±0.2

Data are displayed as mean ± standard error from 40 subjects (24 female). Endotoxin units are IU/mL. All other units are pg/mL. Significance measured by student-paired t-test.

^*P<0.05,

^**P<0.01,

^***P<0.001

Increase in monocyte CD11c correlates with blood lipid levels

We have reported that CD11c expression is increased on monocytes in dyslipidemic mice,¹⁰ and we were interested if CD11c correlated with blood lipid levels in our human cohort. Relative increases in CD11c correlated strongest with the absolute value of a subject’s fasting and postprandial triglyceride (Table 2), but not the percent increase in triglyceride (Supplemental Figure IIIA). Percent increase in CD11c also correlated with apolipoprotein B100, non-HDL cholesterol, total cholesterol and fasting LDL, but not HDL (Table 2).

Table 2.

Correlations Between the Postprandial Increase in Monocyte CD11c and Blood Lipids

	Pearson r	95% CI	P value	r2
Fasting
Triglyceride	0.65	0.42 – 0.81	< 0.0001	0.43
Apolipoprotein B100	0.51	0.22– 0.72	0.001	0.26
Total cholesterol:HDL ratio	0.46	0.17 – 0.68	0.004	0.21
Non-HDL cholesterol	0.46	0.16 – 0.68	0.004	0.21
Total cholesterol	0.45	0.16 – 0.68	0.004	0.21
LDL cholesterol	0.42	0.12 – 0.66	0.008	0.18
HDL cholesterol	−0.22	−0.50 – 0.11	0.194	0.05
Postprandial
Triglyceride	0.62	0.37 – 0.78	< 0.0001	0.38
Non-HDL cholesterol	0.54	0.27 – 0.74	0.0004	0.29
Apolipoprotein B100	0.45	0.15 – 0.67	0.005	0.20
Total cholesterol:HDL ratio	0.44	0.14 – 0.67	0.005	0.20
Total cholesterol	0.44	0.14 – 0.67	0.005	0.20
HDL cholesterol	−0.27	−0.54 – 0.06	0.106	0.07
LDL cholesterol	0.23	−0.12 – 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 from 40 subjects (24 female). Data is partitioned on fasting and postprandial values and listed in descending order based on strength of correlation. Abbreviations: CI, confidence interval; HDL, high density lipoprotein; LDL, low density lipoprotein.

Inflammatory response following triglyceride increase subsides by seven 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 six subjects selected for high postprandial triglyceride (410±116mg/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 Online Tables 3 and 4. 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 significantly decreased at 7 hours compared to the 3.5 hours (Figure 2B).

Figure 2. Inflammatory response following increase in triglyceride subsides by seven hours.

Six subjects were selected for high postprandial triglyceride (410±116mg/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 Tukey post-test. B, Percent change in monocyte surface CD11c, CD14, and CD11b from fasting values at 3.5 and 7 hours postprandial. Significance measured by student t-test. * P<0.05.

Monocytes take up lipid particles in hypertriglyceridemic blood

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,B). 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 percent of vacuolated monocytes at the 3.5 hour time point (Supplemental Figure IV A,B). To establish whether these vacuoles consisted of neutral lipids, monocytes from whole blood were sorted based on CD14 expression and scatter profile. Monocytes from 3.5 hour postprandial blood exhibited punctate Oil Red-O positive droplets, which were not detected in fasting blood (Figure 3 D,E and Supplemental Figure IV C,D).

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 triglyceride (410±116mg/dL; mean±SD). B, Percentage of monocytes in each quadrant at each time point. C, CD11c expression on monocytes in each quadrant at each time point. Significance tested by repeated measures ANOVA with Tukey post test. * P<0.05; NS=not significant. At each time point the CD11c expression in Q3+Q4 is significantly higher than CD11c expression in Q1+Q2 as tested by student-paired t-test. D,E Representative images of monocytes sorted from fasting and 3.5hr postprandial blood stained with Oil Red O (neutral lipid) and hematoxylin (nucleus).

Monocytes internalize triglyceride-rich lipoprotein and upregulate CD11c in vitro

We hypothesized that the lipid droplets observed in monocytes were due to uptake of triglyceride-rich lipoproteins (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 Alexafluor488, and incubated with mononuclear cells from fasting healthy subjects. Internalization was visualized by confocal immunofluorescence microscopy and quantified by flow cytometry. LRP-1 antagonist, Receptor Associated Protein (RAP),¹⁷ inhibited TGRL uptake by 30%, while TGRL endocytosis was abrogated at 10°C (Figure 4A,B). 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).

Figure 4. Internalization of TGRL in vitro increases monocyte surface CD11c.

A, Confocal images of mononuclear cells incubated with alexafluor488 labeled TGRL (100 µg apoB/mL) alone and in the presence of LRP-1 antagonist RAP (50µg/mL) at 37°C or 10°C. Surface bound lipoproteins were removed by washing cells with 5mM EDTA. White arrows: monocytes; Green: TGRL. Monocytes identified by morphology. Scale bar: 20 µm. B, Monocyte internalization of TGRL labeled with alexafluor488 as measured by flow cytometry. Monocytes identified by scatter profile. Conditions are the same as panel A. Significance tested by one-way ANOVA with Tukey post test. C, Monocyte surface CD11c following incubation with TGRL (100µg apoB/mL) or TGRL + RAP (50µg/mL) measured by flow cytometry. Monocyte identified by CD14 expression. Data from 4–6 independent experiments. Significance tested by student t-test. TGRL: Triglyceride Rich Lipoprotein. RAP: Receptor Associated Protein.

Additional experiments were performed to examine the capacity of isolated TGRL to induce CD11c upregulation on monocytes. Thirty-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 (Figure 4C). These data indicate that monocytes in part utilize LRP-1 to bind TGRL, which induces upregulation in CD11c expression. Since this assay was performed in a mononuclear cell 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 triglyceride and depends of CD11c

To assess the consequences of elevated postprandial triglyceride on monocyte function we measured monocyte adhesion to recombinant VCAM-1 in diluted whole blood under defined shear stress at fasting, 3.5 hours, and 7 hours postprandial using a lab-on-a-chip microfluidic flow channel (Supplemental Figure VI A–C). Monocytes in diluted and sheared whole blood recruited avidly to the VCAM-1 substrate (Supplemental Figure IVD) and were rarely observed rolling, but 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 their peak in postprandial triglycerides at 3.5 hours (Figure 5A).

Figure 5. Monocyte arrest on VCAM-1 increases postprandial 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 six subjects with high postprandial triglycerides (410±116mg/dL; mean±SD). Significance tested by repeated measures ANOVA with Tukey post test. *P<0.05 versus IgG at 3.5 hrs.

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 six subjects selected for high postprandial triglyceride 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 2–10 × 10⁵/mL and remained constant at 3.5 and 7 hours of 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, but not at fasting or 7 hours postprandial. Consistent with previous observations¹⁸ 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 postprandial 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 ApoE^−/− mice fed a high fat diet increased CD11c expression on blood monocytes, which took up lipid and accumulated 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 dataset 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 non-fasting 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 increased in proportion to the number of accumulated monocytes.²⁵ The importance of CD11c in this pathogenesis was highlighted by our recent study showing apoE^−/−/CD11c^−/− mice have smaller aortic lesions due to 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 triglyceride 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 since 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 previously demonstrated between β₁- and β₂.²⁶ Thus the clinical relevance of elevated CD11c on monocytes in the postprandial 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 LDLR^−/− and/or apoE^−/− mice indicated that macrophage-specific LRP-1 deficiency was associated with increased atherosclerotic lesions.^{29, 32} While this suggests an atheroprotective role for LRP-1, our data and 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 and/or internalization of lipoproteins upregulate 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 PKC.^{33, 34} Since PKC 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 PKC 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. Employing a dynamic blood film in a lab-on-a-chip format revealed that monocyte arrest on VCAM-1 was dependent on CD11c and correlated with a subject’s postprandial triglyceride. 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 if monocyte CD11c expression correlates with the development of more severe atherosclerotic disease.