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

Abstract

Objective: Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and emigration across inflamed endothelium. Involvement of β(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.

Conclusions: 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.

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

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.

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).

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.

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.