Recruitment of foamy monocytes to inflamed endothelium expressing VCAM-1 contributes to the development of plaque during atherogenesis. Foamy CD11c+ monocytes arise in the circulation during the onset of hypercholesterolemia and recruit to nascent plaque, but the mechanism of CD11c/CD18 and very late Ag-4 (VLA-4) activation and cooperation in shear-resistant cell arrest on VCAM-1 are ill defined. Within 1 wk of the onset of a Western high-fat diet (WD) in apolipoprotein E–deficient mice, an inflammatory subset of foamy monocytes emerged that made up one fourth of the circulating population. These cells expressed ∼3-fold more CD11c/CD18 and 50% higher chemokine receptors than nonfoamy monocytes. Recruitment from blood to a VCAM-1 substrate under shear stress was assessed ex vivo using a unique artery-on-a-chip microfluidic assay. It revealed that foamy monocytes from mice on a WD increased their adhesiveness over 5 wk, rising to twice that of mice on a normal diet or CD11c−/− mice fed a WD. Shear-resistant capture of foamy human or mouse monocytes was initiated by high-affinity CD11c, which directly activated VLA-4 adhesion via phosphorylated spleen tyrosine kinase and paxillin within focal adhesion complexes. Lipid uptake and activation of CD11c are early and critical events in signaling VLA-4 adhesive function on foamy monocytes competent to recruit to VCAM-1 on inflamed arterial endothelium.

Monocyte recruitment from the circulation to inflamed endothelium expressing VCAM-1 is an obligate step during the formation of atherosclerotic lesions and represents a potential target for drug-based therapies to ameliorate plaque formation (1, 2). However, precise identification of the inflammatory monocytes that contribute to atherogenesis remains elusive. Mouse models of hypercholesterolemia, such as apolipoprotein E–deficient (apoE−/−) mice, are commonly used to study atherogenesis (35). Fed a Western high-fat diet (WD), these mice circulate high levels of lipoproteins at concentrations that closely match those in hypertriglyceridemic humans (6). With the onset of hyperlipidemia, monocytes are mobilized from bone marrow and splenic reservoirs to the circulation, whereupon a fraction accumulate lipid and traffic to inflamed endothelium expressing VCAM-1 (1, 4, 5). A critical step in atherogenesis is the early recruitment of inflammatory monocytes and their differentiation into foamy macrophages in nascent plaque; however, the mechanism through which lipid uptake initiates monocyte activation and integrin-mediated shear-resistant arrest is ill defined (5).

Two major classes of monocytes increase their numbers in the circulation during hypercholesterolemia and recruit to inflamed endothelium: classical or inflammatory monocytes are characterized as Ly6ChighCCR2highCX3CR1low in mice and CD14++CD16 in humans. These subsets are thought to be the precursors of inflammatory foamy macrophages in established aortic lesions (4). A second subset, denoted as patrolling or anti-inflammatory monocytes that express Ly6ClowCCR2lowCX3CR1high in mice and CD14+CD16+ in humans, recruit to inflamed endothelium early in the process of lesion formation, give rise to CD11c+ foamy macrophages, extravasate at the endothelial interface, and accumulate in organs (7, 8). The propensity for the trafficking and emigration of specific monocyte subsets is dictated by their relative expression of CCR2, CCR5, and CX3CR1, which signal integrin activation and guide cell migration to arterial sites of nascent plaque formation (7, 8). A direct comparison of inflammatory activation in the context of lipid-mediated integrin-dependent recruitment on VCAM-1 between mouse and human monocyte subsets is lacking.

An early event in inflammation associated with hyperlipidemia is membrane upregulation of β2-integrin CD11c/CD18 on circulating monocytes. In contrast, β1-integrin very late Ag-4 (VLA-4) expression levels remain constant on circulating monocytes (3, 5, 9, 10). Durable bond formation on VCAM-1 involves an upshift in VLA-4 affinity and subsequent receptor coalescence within focal clusters that promote high-avidity binding and shear-resistant adhesion on inflamed endothelium (1013). High-affinity CD11c can also recognize VCAM-1 on mouse and human endothelium and, in the high-affinity conformation, signals the activation of VLA-4, thereby enhancing recruitment of monocytes and exacerbating plaque formation (3, 10, 14). In fact, the relative expression of CD11c on foamy monocytes increases with cardiovascular risk in subjects with metabolic syndrome, and receptor number increases as a function of the severity of myocardial injury in ischemic patients (10). Its functional role in atherogenesis was recently demonstrated by genetic deletion in apoE−/−/CD11c−/− double-knockout mice, which were protected from compartmentalization of monocytes in adipose tissue and in atherosclerotic plaque (3, 15). Thus, the dynamics of CD11c expression during the onset of hypertriglyceridemia and its role in the activation of VLA-4 may provide a means of predicting their participation in atherogenesis.

In the current study, we measured the emergence of foamy monocytes within 1 wk of the onset of hypercholesterolemia in mice and used an ex vivo model to produce triglyceride-rich lipoprotein (TGRL)-loaded foamy monocytes from humans fed a high-fat meal to better delve into the mechanism by which CD11c activates VLA-4 during inflammation. Our experimental strategy was 2-fold: phenotypic changes were tracked in monocytes sampled from the circulation of apoE−/− and CD11c−/−/apoE−/− mice fed a normal versus a high-fat diet for 5 wk. We assessed ex vivo the molecular pathway associated with CD11c- and VLA-4–dependent capture of monocytes on VCAM-1 using a unique artery-on-a-chip (A-chip) microfluidic assay. Our data reveal that, in hypertriglyceridemic mice, a subset of lipid-laden monocytes expressing Ly6ClowCD11chigh emerge in the circulation and exhibit an enhanced capacity to activate VLA-4. This subset is functionally similar to human CD14++CD16+ (Mon2) monocytes that take up TGRL and capture on inflamed aortic endothelium (16). Conversion of CD11c from low to high affinity was sufficient to activate VLA-4 within focal clusters that redistribute to lipid rafts where spleen tyrosine kinase (Syk) and paxillin stabilize integrin-mediated monocyte arrest. These data reveal the earliest phenotypic changes in monocyte activation during hyperlipidemia and indicate that CD11c expression and signaling on foamy monocytes provides a biomarker and potential target for intervention of atherogenesis.

Genetic deletion of CD11c in C57BL/6 mice and breeding with apoE−/− strain to generate CD11c−/−/apoE−/− mice was achieved as described previously (3). Both apoE−/− and CD11c−/−/apoE−/− mice were backcrossed for 12 generations onto a C57BL/6 wild-type strain. Male mice were fed a normal chow diet (ND; PicoLab Rodent Chow 5010; LabDiet, St. Louis, MO) until 8 wk of age and then switched to a WD (21% fat [w/w], 0.15% cholesterol [w/w]; Dyets, Bethlehem, PA) and maintained on it for 1–5 wk. This was shown to induce hypercholesterolemia in mice lacking the apoE gene. The α4-(Y991A) C57BL6 strain was maintained on an ND and used at 10–12 wk of age.

At predetermined time points, mice were sacrificed using CO2 asphyxiation, and blood was obtained via cardiac puncture using a 3-ml syringe (BD, San Diego, CA) and 30 gauge × 1” needle (BD, San Diego, CA) and injected into Vacutainer collection tubes. For experiments studying primary human monocytes, subjects were recruited from the general population near Davis, CA. Blood was obtained by venipuncture into Vacutainer collection tubes and maintained at room temperature. For flow cytometry and blood differential counts, blood was drawn into tubes containing K2EDTA. For laboratory-on-a-chip adhesion assays, blood was drawn into tubes containing sodium heparin. Blood differential counts were determined using a COULTER AcT diff Hematology Analyzer (Beckman-Coulter).

The following Abs were purchased from BioLegend (San Diego, CA) and used for flow cytometry and immunofluorescence microscopy: anti-mouse CD11c (Alexa Fluor [AF]488; clone N418), anti-mouse CD115 (PE conjugated; clone AFS98), anti-mouse CD49d (PE conjugated or unconjugated; clone R-12), anti-mouse Ly6C (PerCP/Cy5.5 conjugated or AF647 conjugated; clone HK1.4), anti-human CD14 (AF488 conjugated or PE conjugated; clone M5E2), anti-human CD16 (PE/Cy5 conjugated; clone 3G8), anti-human CD11c (PE conjugated or unconjugated; clone BU15), and anti-human Syk (unconjugated; clone 4D10.2). The following Abs were purchased from Abcam (Cambridge, MA) and used for immunoprecipitation, Western blot, and immunofluorescence microscopy: anti-human paxillin (unconjugated; clone 5H11), anti-human CD11c (unconjugated; clone MM0422-3J16), and anti-human CD49d (clone EPR1355Y and clone HP2.1). The following secondary Abs were purchased from Life Technologies (Grand Island, NY): goat anti-mouse IgG (AF488 conjugated or HRP conjugated) and goat anti-rabbit IgG (AF555 conjugated or HRP conjugated). CD11c allosteric agonist 496B, which activates CD11c-promoting ligand binding, and CD11c allosteric antagonist 496K, which inactivates CD11c-inhibiting ligand binding, were generously provided by Eli Lilly Corp. (17). For flow cytometry, the stain indices for each Ab were calculated using the formula:

SI=XposXneg2SDneg

where Xpos is the median fluorescence intensity (MFI) of positive stained population, Xneg is the MFI of a population that does not express the target receptor, and SDneg is the SD of the negative population. For function-blocking studies, Abs were used at 10 μg/ml. The paxillin inhibitor (6-B345TTQ; Sigma Aldrich) and p-Syk inhibitor (622387-85-3; EMD Millipore) were used at a concentration of 50 μM and 50 nM, respectively. Inhibitors were incubated with cells for 10 min at 37°C.

Mouse whole blood obtained via cardiac puncture was drawn and immediately cooled to 4°C. After labeling with fluorescent Abs for 30 min, RBCs were removed with RBC Lysis buffer (BioLegend). Data were acquired on a BD FACScan cytometer within 2 h of cardiac puncture. Cells were characterized as having positive expression of membrane adhesion molecules (CD11c, CD49d) and chemokine receptors (CCR2, CX3CR1, CCR5) based on a significant increase in mean fluorescence intensity over fluorescence-minus-one controls. Foamy monocytes were defined as CD115+ cells that had a side scatter (SSC) value > 2 SD above the mean SSC of monocytes from ND-fed mice. This criterion for defining foamy monocytes was assessed using the lipid dye BODIPY to stain intracellular lipid. More than 95% of SSChigh monocytes stained positive for BODIPY.

Isolation of TGRL from hypertriglyceridemic subjects was described previously (10). Blood samples for lipoprotein isolation were collected into BD Vacutainer plasma separator tubes containing lithium heparin 3.5 h after consumption of the high-fat meal. They were spun at room temperature for 10 min at 1300 × g to remove cellular particulate. Following centrifugation, plasma was transferred to ultracentrifuge tubes (Beckman-Coulter) and centrifuged at 280,000 × g at 14°C for 18 h. TRGLs were obtained by aspirating the top layer of plasma (ρ < 1.0063 g/ml). The isolate’s apolipoprotein B (apoB) content was measured with a total human apoB ELISA (ALerCHEK).

Human mononuclear cells were obtained as described previously (9). Heparinized whole blood was diluted 1:1 with HBSS without Ca2+ and Mg2+, layered over mononuclear cell separation medium Lymphoprep (ρ = 1.077 g/ml; Axis-Shield, Oslo, Norway), and spun at 800 × g for 25 min at 25°C. The mononuclear band was isolated with a 16 gauge × 2.5” safety catheter and spun down at 200 × g for 10 min. Mononuclear cells were washed twice in HBSS without Ca2+ and Mg2+ and reconstituted in HBSS containing 1 mM Ca2+ and 1 mM Mg2+.

We prepared 25-mm-diameter No. 1.5 glass coverslips in a 1:1 solution of H2SO4 and H2O2, rinsed them with water, and dipped them in a solution of acetone (Sigma-Aldrich) and 4% (v/v) 3-aminopropyltriethoxysilane (Pierce). Goat F(ab′)2 anti-human IgG Fc (100 μg/ml; Pierce) was incubated on the glass for 1 h at room temperature. Coverslips were incubated with 5 μg/ml mouse recombinant VCAM-1–Fc chimera (R&D Systems), followed by 0.1% human serum albumin (HSA) to block nonspecific binding of leukocytes. This method of linking VCAM-1 results in a density ∼ 2000 molecules/μm2.

The device was designed to allow the use of four independent flow channels with dimensions of 60 μm × 2 mm × 8 mm (h × w × l). An interconnected spider web pattern surrounding the channels allowed the device to be vacuumed sealed over a glass coverslip. The microfluidic networks were designed in AutoCAD (Autodesk) and printed as a photomask by CAD/Art services. A master mold for the device was created by coating a 200-mm-diameter silicon wafer with SU-8 50 photoresist (MicroChem) to a height of 60 μm and then exposing the wafer to UV light through the photomask containing the microfluidic channel pattern, as described previously (18). Microfluidic chambers were fabricated by pouring polydimethylsiloxane (PDMS) Sylgard 184 prepolymer (Dow Corning) over the master silicon wafers. Reservoir and vacuum access holes were punched directly into the PDMS chambers using a needle.

Heparinized mouse whole blood was added to the A-chip chamber reservoir and withdrawn through a single channel using a syringe pump (Kent Scientific) at a flow rate resulting in a physiological shear stress of 2 dyn/cm2 at the glass fluid interface (a wall shear stress corresponding with athero-prone regions in the arterial vasculature) (19). Arrested monocytes were fixed with 1× Lyse/Fix Buffer (BioLegend), and the coverslip was removed from the PDMS chamber and incubated with HBSS containing 1% HSA for 30 min at room temperature. Coverslips were washed twice with 1× Dulbecco’s PBS (DPBS) and stained with PE anti-CD115 and BODIPY.

Mouse blood was perfused through the microfluidic chamber for 4 min over a substrate of recombinant mouse VCAM-1. HBSS was allowed to flow through for 4 min to wash unbound cells, and 1× Lyse/Fix Buffer was perfused through for 2 min to fix arrested cells. The glass coverslip was removed from the chamber and placed in a dish containing HBSS + 1% HSA for 30 min at room temperature. For experiments imaging colocalization of VLA-4 with CD11c (or CD45), cells were stained with 5 μg/ml PE anti-CD49d and 5 μg/ml AF488 anti-CD11c (or 5 μg/ml AF488 anti-CD45) for 30 min and then washed twice with DPBS. Coverslips were mounted in Prolong Gold Antifade Reagent with DAPI (Life Technologies) and imaged on a Nikon Eclipse TE2000-S microscope, coupled to a Hamamatsu ORCA-ER CCD camera, using a Nikon Apo TIRF 60× oil objective (N.A. = 1.49). Fluorophores were excited with a 2-mW HeNe 488-nm laser and an adjustable (1–20 mW) 552-nm OBIS laser diode (Coherent). For colocalization analysis, monocytes were identified using DAPI nuclear stain to visualize the kidney bean–shaped morphology specific to monocytes. ImageJ v1.46 was used to calculate the Pearson correlation to compare the overlap of CD49d (red)- and CD11c (or CD45) (green)-labeled receptors.

Fluorescent beads coated with ligand were used to probe VLA-4 receptor function on cells in suspension, as described previously (20). Carboxylated fluorescent latex beads (diameter = 2 μm; Life Technologies) were washed three times in DPBS free of Ca or Mg ions and incubated in 10 μg/ml recombinant human fibronectin (Fn) for 1 h at 37°C. Beads were washed twice in DBPS, dispersed by sonication, and counted before use. Monocytes (5 × 105) were mixed with 1 × 107 Fn-coated beads in 500 μl HBSS with Ca and Mg ions in a mixing tube containing a small magnetic stir bar. The sample was placed in a specially designed mixing chamber to maintain the sample at 37°C and stir suspensions at 500 rpm with a magnetic bar, corresponding to between 1 and 2 dyn/cm2, which is equivalent to the hydrodynamic shear stress in the microfluidic channels (21). The mixing chamber was positioned just upstream of the sample-injection nozzle of a FACScan (Becton Dickinson). Monocytes and discrete beads were discriminated based on forward scatter and SSC parameters, and a live gate was placed over the monocyte population to limit detection to monocytes and monocyte-bead events. A total of 10,000 events was collected at a sampling rate of 10 ms over the course of the 10-min total collection time. The mean number of beads/monocyte (NB) was calculated by summing the product of the fraction of monocytes (MONb) with the number of beads bound (b) and dividing by the total number of monocytes with and without (MON0) Fn beads:

NB=b=16MONb×b/(MON0+b=16MONb)

With this technique, the maximum number of beads/cell that could be resolved was six at each time point measured. Monocytes that bound more beads were summed at 6.

Monocytes maintained in HBSS + Ca/Mg + 0.1% HSA were stimulated with 496B (10 μg/ml), TGRL (apoB, 100 μg/ml), MCP-1 (30 nM), or Mn2+ (1 mM) in the presence of 4 nM LDV-FITC with continuous mixing in a customized mixing device that was described previously. Binding of LDV-FITC to monocytes was monitored constantly using flow cytometry; it reached maximum binding by 10 min of incubation.

Human mononuclear cells were isolated from whole blood and suspended in HBSS with Ca and Mg ions. We measured CD11c on lymphocytes in the mononuclear cell preparation and confirmed that lymphocytes did not express CD11c (data not shown). Mononuclear cells were incubated for 15 min at 37°C with allosteric Abs against CD11c that activate (496B), inactivate (496K), or bind CD11c but do not alter its conformation (BU15). Separately, mononuclear cells were incubated with TGRL (100 μg/ml apoB) or intralipid for 30 min at 37°C and washed in HBSS containing 2 mM EDTA to remove surface-bound lipoproteins. Mononuclear cells were lysed with IP Lysis/Wash Buffer (Pierce), and protein was measured using a bicinchoninic acid assay (Pierce). Samples were diluted to 700 μg/ml protein using IP Lysis/Wash Buffer and incubated with anti-CD11c (clone MM0422-3J16) for 4 h at 4°C. Protein A/G agarose resin from the Pierce Classic IP Kit was used to pull down CD11c, and the resin was washed four times with IP Lysis/Wash Buffer. Equal concentrations of protein sample were subjected to gel electrophoresis, and protein was transferred to a polyvinylidene membrane. The membranes were blocked with 5% milk for 1 h, and primary Abs were used to probe for CD49d, paxillin, Syk, and p-Syk. An HRP-conjugated secondary Ab was used to stain the primary Abs, and membranes were developed using ECL (SuperSignal West Pico Chemiluminescent Substrate; Pierce). Data from blots were collected using a ChemiDoc MP system, and the protein density in each band was calculated using ImageJ v1.46.

Data are presented as mean ± SEM, unless otherwise specified. Multiple groups were compared using one-way ANOVA with the Tukey posttest. All analyses were carried out using GraphPad Prism v6.0d for Mac (GraphPad Software, La Jolla, CA).

All protocols and procedures involving apoE−/−, CD11c−/−/apoE−/−, and α4-(Y991A) mice were approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Whole-blood monocytes and TRGLs were obtained from human subjects in accordance with an institutional review board–approved protocol at the University of California, Davis. Written consent was obtained from human subjects prior to enrollment in the study.

Hypercholesterolemia promotes monocyte uptake of circulating lipid and recruitment to arterial sites of nascent plaque development within weeks of initiating a WD in apoE−/− mice (5, 22). This motivated assessment of the dynamics of emergence of foamy monocytes in the circulation of WD- versus ND-fed apoE−/− mice over a 5-wk study interval. Blood monocytes were identified by flow cytometry based on positive expression of CD115 and were characterized as foamy based on elevated SSC and BODIPY fluorescence intensity above that on nonfoamy controls (Fig. 1A). Foamy monocytes increased in the circulation most rapidly within the first week of the initiation of a WD and plateaued at ∼30% of total monocytes at 5 wk (Fig. 1B). Control mice fed a nonfat diet exhibited no detectable change in lipid content of circulating monocytes or neutrophils or lymphocytes after 1 wk, indicating that lipid uptake in the blood is specific to monocytes and the effect of a WD (Supplemental Fig. 1A, 1B). The number of circulating monocytes that were foamy remained equivalent between apoE−/− and CD11c−/−/apoE−/− mice over 5 wk of the WD (Fig. 1B, Supplemental Fig. 1C). Discrimination of monocytes based on Ly6C expression revealed that the vast majority of foamy monocytes in blood of mice fed a WD were Ly6Clow (∼80%), which outnumbered Ly6Chigh by ∼4:1; this ratio did not change significantly over 5 wk, regardless of CD11c expression (Fig. 1C). We conclude that, during the early onset of hypercholesterolemia in apoE−/− mice, a subset of foamy Ly6Clow monocytes emerges, and CD11c−/− monocytes maintain the ability to take up lipid in circulation.

FIGURE 1.

Foamy monocytes are predominantly Ly6Clow and CD11chigh and are prevalent in blood after 1 wk of a high-fat diet in apoE−/− mice, independent of CD11c expression. (A) Monocytes were identified based upon CD115 positivity. Monocytes that had taken up lipid and became foamy were discriminated by SSC intensity. BODIPY fluorescence (open graph) was measured on both foamy (SSC high) and nonfoamy (SSC low) populations and compared with unstained control (filled graph). (B) Percentage of foamy monocytes in apoE−/− mice consuming an ND (gray line) or a WD (solid black line) and in CD11c−/−/apoE−/− mice consuming a WD (dashed black line) (n = 4–5 mice/time point). ###,***p < 0.005, repeated-measures ANOVA. (C) Proportion of foamy monocytes that are Ly6Clow or Ly6Chigh. ***p < 0.005 denotes significance comparing percentage of foamy Ly6Clow to percentage of foamy Ly6Chigh using a paired Student t test. (D) CD11c expression measured on foamy (solid black line) and nonfoamy (dashed black line) monocytes from apoE−/− mice fed a WD and nonfoamy monocytes from apoE−/− fed an ND (gray line) (n = 4–5 mice/time point). Dotted line represents fluorescence staining of CD11c on CD11c−/− monocytes. ****p < 0.05, foamy versus nonfoamy from WD mice, one-way ANOVA with Tukey posttest. (E) VLA-4 expression measured on monocytes from apoE−/− and CD11c−/−/apoE−/− fed an ND or WD for 1 wk (n = 3 mice/group).

FIGURE 1.

Foamy monocytes are predominantly Ly6Clow and CD11chigh and are prevalent in blood after 1 wk of a high-fat diet in apoE−/− mice, independent of CD11c expression. (A) Monocytes were identified based upon CD115 positivity. Monocytes that had taken up lipid and became foamy were discriminated by SSC intensity. BODIPY fluorescence (open graph) was measured on both foamy (SSC high) and nonfoamy (SSC low) populations and compared with unstained control (filled graph). (B) Percentage of foamy monocytes in apoE−/− mice consuming an ND (gray line) or a WD (solid black line) and in CD11c−/−/apoE−/− mice consuming a WD (dashed black line) (n = 4–5 mice/time point). ###,***p < 0.005, repeated-measures ANOVA. (C) Proportion of foamy monocytes that are Ly6Clow or Ly6Chigh. ***p < 0.005 denotes significance comparing percentage of foamy Ly6Clow to percentage of foamy Ly6Chigh using a paired Student t test. (D) CD11c expression measured on foamy (solid black line) and nonfoamy (dashed black line) monocytes from apoE−/− mice fed a WD and nonfoamy monocytes from apoE−/− fed an ND (gray line) (n = 4–5 mice/time point). Dotted line represents fluorescence staining of CD11c on CD11c−/− monocytes. ****p < 0.05, foamy versus nonfoamy from WD mice, one-way ANOVA with Tukey posttest. (E) VLA-4 expression measured on monocytes from apoE−/− and CD11c−/−/apoE−/− fed an ND or WD for 1 wk (n = 3 mice/group).

Close modal

It was reported that CD11c−/−/apoE−/− mice develop smaller atherosclerotic lesions after 12 wk on a WD and that monocytes from these mice exhibit a reduced capacity to arrest on VCAM-1 under hydrodynamic shear stress (3). Furthermore, CD11c expression on circulating monocytes increases with the extent of hypertriglyceridemia in humans and hypercholesterolemia in mice (3, 10, 15). This suggests that CD11c upregulation occurs as a consequence of metabolic and inflammatory signaling, but the dynamics of this process has not been examined. CD11c expression increased 3-fold after 1 wk on foamy Ly6Clow monocytes, whereas no such increase was detected on nonfoamy monocytes (Fig. 1D). Lipid uptake also elicited CD11c upregulation on Ly6Chigh monocytes, but receptor numbers were 5-fold lower compared with Ly6Clow monocytes (Supplemental Fig. 2A, 2B). Elevated expression of CD11c on all foamy monocytes was sustained out to 5 wk, whereas VLA-4 was expressed at equivalent levels and remained constant on all subsets of circulating monocytes, regardless of lipid uptake (Fig. 1E). Moreover, no significant change in CD11c or VLA-4 expression on circulating neutrophils was detected (Supplemental Fig. 2D, 2E). Thus, CD11c expression increased most rapidly early in the course of diet-induced hypercholesterolemia, mirroring the dynamics of the emergence of foamy Ly6Clow monocytes in the circulation of apoE−/− mice.

Mice deficient in the MCP-1 receptor (CCR2), fractalkine receptor (CX3CR1), or RANTES receptor (CCR5) exhibit impaired monocyte recruitment and development of mature plaque (23). This observation prompted measurement of CCR2, CX3CR1, and CCR5 chemokine receptors and adhesion function of blood monocytes following chemokine stimulation (Supplemental Fig. 2C). No significant differences in chemokine receptor expression were detected on nonfoamy monocytes in blood from WD-fed (Fig. 2A) and ND-fed mice (data not shown). In contrast, foamy monocytes from apoE−/− mice fed a WD for 1 wk upregulated CCR2 and CX3CR1 (28 and 70% increase in MFI) compared with nonfoamy monocytes, respectively. In contrast, no significant change in CCR5 on foamy or nonfoamy monocytes was detected. Monocyte adhesive capacity was assessed ex vivo by shearing cell suspensions with an excess of fluorescence beads coated with the VLA-4 ligand Fn and measuring the kinetics of capture by flow cytometry (Fig. 2B) (24). VLA-4 adhesion was low on nonfoamy monocytes, which did not increase bead capture from baseline following the onset of shear mixing. Foamy monocytes exhibited ∼2-fold greater binding avidity compared with nonfoamy monocytes, which was abrogated by the addition of anti–VLA-4 (Fig. 2B). Bead capture increased ∼2-fold above baseline upon stimulation with MCP-1 (30 nM) or fractalkine (10 nM) (Fig. 2C). In contrast, there was no significant change in VLA-4 avidity following the addition of RANTES, which was consistent with the low expression level and lack of increase in CCR5 on foamy and nonfoamy monocytes. These data indicate that foamy monocytes in the blood of hypercholesterolemic mice have an increased baseline capacity to adhere via VLA-4 and are primed for a heightened response to chemokine activation by MCP-1 and fractalkine within 1 wk of the initiation of a WD.

FIGURE 2.

Foamy monocytes adopt an inflammatory phenotype associated with increased chemokine receptors, VLA-4 adhesion, and colocalization with CD11c within lipid rafts. (A) Receptor expression of CCR2, CX3CR1, and CCR5 measured on foamy and nonfoamy monocytes from apoE−/− mice fed a high-fat diet for 1 wk (n = 4–5 mice). *p < 0.05, foamy versus nonfoamy monocytes, paired Student t test. (B) A real-time readout of VLA-4 avidity based upon binding of Fn beads was performed by flow cytometry, as described in 2Materials and Methods. Capture of Fn beads sheared with foamy versus nonfoamy monocytes over 10 min in the presence of 30 nM MCP-1 or anti-VLA-4 blocking Ab (n = 3–4 separate experiments). Significance was determined comparing foamy versus nonfoamy monocytes using multiple Student t test. *p < 0.05 and **p < 0.01 denote the time points at which Fn bead binding to foamy monocyte and foamy monocytes + MCP-1, respectively, is significantly greater than nonfoamy monocytes. (C) Percentage increase in VLA-4 adhesion over control (0.01% DMSO) following addition of MCP-1 (30 nM), fractalkine (10 nM), or RANTES (15 nM) (n = 3 separate experiments). Significance was determined comparing foamy versus nonfoamy monocytes using a paired Student t test. *p < 0.05, **p < 0.01. (D) Monocytes from apoE−/− mice were labeled with Abs against CD11c, VLA-4, and lipid rafts (CTxB). Confocal photomicrographs are representative of 30 monocytes observed from a mouse fed an ND or a high-fat diet (n = 3 mice/group). Scale bar, 10 μm. (E) Integrin coalescence in membrane lipid rafts was determined using Pearson correlation. Significance was determined comparing ND- versus WD-fed mice using a Student t test. *p < 0.05, **p < 0.01.

FIGURE 2.

Foamy monocytes adopt an inflammatory phenotype associated with increased chemokine receptors, VLA-4 adhesion, and colocalization with CD11c within lipid rafts. (A) Receptor expression of CCR2, CX3CR1, and CCR5 measured on foamy and nonfoamy monocytes from apoE−/− mice fed a high-fat diet for 1 wk (n = 4–5 mice). *p < 0.05, foamy versus nonfoamy monocytes, paired Student t test. (B) A real-time readout of VLA-4 avidity based upon binding of Fn beads was performed by flow cytometry, as described in 2Materials and Methods. Capture of Fn beads sheared with foamy versus nonfoamy monocytes over 10 min in the presence of 30 nM MCP-1 or anti-VLA-4 blocking Ab (n = 3–4 separate experiments). Significance was determined comparing foamy versus nonfoamy monocytes using multiple Student t test. *p < 0.05 and **p < 0.01 denote the time points at which Fn bead binding to foamy monocyte and foamy monocytes + MCP-1, respectively, is significantly greater than nonfoamy monocytes. (C) Percentage increase in VLA-4 adhesion over control (0.01% DMSO) following addition of MCP-1 (30 nM), fractalkine (10 nM), or RANTES (15 nM) (n = 3 separate experiments). Significance was determined comparing foamy versus nonfoamy monocytes using a paired Student t test. *p < 0.05, **p < 0.01. (D) Monocytes from apoE−/− mice were labeled with Abs against CD11c, VLA-4, and lipid rafts (CTxB). Confocal photomicrographs are representative of 30 monocytes observed from a mouse fed an ND or a high-fat diet (n = 3 mice/group). Scale bar, 10 μm. (E) Integrin coalescence in membrane lipid rafts was determined using Pearson correlation. Significance was determined comparing ND- versus WD-fed mice using a Student t test. *p < 0.05, **p < 0.01.

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To examine the mechanism underlying integrin-mediated bead capture, we labeled and imaged lipid rafts on the membrane of foamy monocytes in suspension, because they are known to play an important role in the coalescence and clustering of activated integrins and facilitate stable shear-resistant adhesion (2527). Monocytes were isolated from mouse blood and labeled with fluorescent Abs against VLA-4, CD11c, and fluorescent cholera toxin subunit B (CTxB), which preferentially binds to the ganglioside GM1 protein expressed in lipid rafts. Although CD11c and VLA-4 were diffusely expressed, these integrins were twice as likely to colocalize on the plasma membrane in general and ∼5-fold more likely to coalesce within lipid raft domains on monocytes from WD-fed versus ND-fed controls (Fig. 2D, 2E). These data indicate that the increase in VLA-4 adhesive function on freshly isolated foamy monocytes in suspension correlates with CD11c upregulation and integrin colocalization within lipid rafts.

Monocytes in circulation take up lipid and are primed for recruitment on inflamed endothelium, predominantly via VLA-4 binding to VCAM-1 (28, 29). We used an A-chip microfluidic device to quantify ex vivo the propensity of foamy monocytes isolated from hyperlipidemic humans and mice to capture on a VCAM-1 substrate under shear (10). Human aortic endothelial cells (HAECs) stimulated with the inflammatory cytokine TNF-α provide a proatherogenic model to study integrin-mediated recruitment of monocytes (28, 30). Mononuclear cells were isolated from hypertriglyceridemic subjects in the postprandial state and subsequently sheared over a substrate of inflamed HAECs in the A-chip microchannels (Supplemental Fig. 3A, 3B). The prevalence of foamy monocyte capture was assessed by immunofluorescence labeling of recruited cells with the lipid partitioning dye BODIPY (Supplemental Fig. 3A). Foamy monocytes sheared over inflamed HAECs were captured twice as efficiently as nonfoamy monocytes, and adhesion was 30% dependent on CD11c and 70% dependent on VLA-4 (Supplemental Fig. 3B). To directly compare human and mouse monocyte capture on VCAM-1, we next examined monocyte recruitment in a reduced system in which the glass substrate was coated with rVCAM-1–IgG. Annealing VCAM-1 to the substrate at a site density equivalent to that on inflamed HAECs (data not shown), we detected a 2:1 recruitment efficiency of foamy/nonfoamy monocytes, similar to capture on the HAEC monolayer. Further, monocyte capture was abrogated with an anti-VLA-4–blocking Ab, yet the efficiency of capture remained partially dependent on CD11c (Supplemental Fig. 3B). This reduced A-chip system provided an ideal means of examining ex vivo the dynamics of integrin-mediated capture of circulating monocytes on VCAM-1 over the 5-wk course of a WD versus an ND in apoE−/− mice. Whole-blood samples were collected and sheared over rVCAM-1–IgG in the A-chip device at a wall shear stress of 2 dyn/cm2; captured monocytes were fixed and stained with H&E for identification based on characteristic morphology and expression of CD115 (Supplemental Fig. 3C, 3D). Following 1 wk of a WD, the capture efficiency of monocytes, defined as the number arrested normalized to the whole-blood count, increased >2-fold from a baseline of ∼3 arrested up to 10/1000 infused monocytes (Fig. 3A). Over the ensuing 4 wk, monocytes from mice fed a WD increased by ∼4-fold over baseline. In comparison, blood monocytes from CD11c−/−/apoE−/− mice exhibited only an ∼2-fold increase, recruiting with an efficiency equivalent to that of mice fed an ND. By comparison, the recruitment of neutrophils in blood on VCAM-1 arrested with a 100-fold lower efficiency than foamy monocytes (Supplemental Fig. 3E). These data indicate that monocytes from hypercholesterolemic mice markedly increase their ability to recruit to VCAM-1 within 1 wk of initiation of a WD. Moreover, the steady increase in the efficiency of monocyte recruitment observed with up to 5 wk of a WD required CD11c expression.

FIGURE 3.

Foamy monocytes recruit on a VCAM-1 substrate under shear stress in a lipid raft- and VLA-4–dependent manner. (A) Whole blood was sheared ex vivo on rVCAM-1 in a laboratory chip microfluidic device. Recruitment efficiency of monocytes plotted for apoE−/− versus CD11c−/−/apoE−/− mice (n = 4–5/time point) fed ND or WD for 1–5 wk. **p < 0.01, ***p < 0.005, WD versus ND mice, one-way ANOVA with Tukey posttest. (B) Focal adhesions enriched in integrins and lipid rafts support monocyte arrest on VCAM-1. Ab-tagged CD11c and VLA-4 are seen within the contact region. Images are representative of 30–50 arrested cells/channel. Scale bar, 10 μm. (C) Integrin enrichment within the region of membrane contact on VCAM-1, as detected by TIRF microscopy for arrested monocytes from WD (n = 5) versus ND (n = 5) mice and disruption of lipid rafts after MβCD treatment. Significance was determined using one-way ANOVA followed by a Tukey posttest. **p < 0.01, ***p < 0.005. (D) VLA-4 coalescence with CD11c within focal adhesions quantified using Pearson coefficient. Significance was determined using one-way ANOVA followed by a Tukey posttest. **p < 0.01, ***p < 0.005. (E) Following arrest, foamy monocytes were identified using CD115 (red), and lipid droplets were stained with BODIPY (green). Images are representative of 100–150 arrested cells/channel. Scale bar, 10 μm. (F) Capture efficiency of foamy versus nonfoamy monocytes identified on chip following shearing of blood isolated from apoE−/− or CD11c−/−/apoE−/− mice on ND and 1-wk WD (n = 5 separate experiments). Significance was determined comparing nonfoamy to foamy monocytes in apoE−/− mice after one wk WD using a paired Student t test. *p < 0.05, ***p < 0.005.

FIGURE 3.

Foamy monocytes recruit on a VCAM-1 substrate under shear stress in a lipid raft- and VLA-4–dependent manner. (A) Whole blood was sheared ex vivo on rVCAM-1 in a laboratory chip microfluidic device. Recruitment efficiency of monocytes plotted for apoE−/− versus CD11c−/−/apoE−/− mice (n = 4–5/time point) fed ND or WD for 1–5 wk. **p < 0.01, ***p < 0.005, WD versus ND mice, one-way ANOVA with Tukey posttest. (B) Focal adhesions enriched in integrins and lipid rafts support monocyte arrest on VCAM-1. Ab-tagged CD11c and VLA-4 are seen within the contact region. Images are representative of 30–50 arrested cells/channel. Scale bar, 10 μm. (C) Integrin enrichment within the region of membrane contact on VCAM-1, as detected by TIRF microscopy for arrested monocytes from WD (n = 5) versus ND (n = 5) mice and disruption of lipid rafts after MβCD treatment. Significance was determined using one-way ANOVA followed by a Tukey posttest. **p < 0.01, ***p < 0.005. (D) VLA-4 coalescence with CD11c within focal adhesions quantified using Pearson coefficient. Significance was determined using one-way ANOVA followed by a Tukey posttest. **p < 0.01, ***p < 0.005. (E) Following arrest, foamy monocytes were identified using CD115 (red), and lipid droplets were stained with BODIPY (green). Images are representative of 100–150 arrested cells/channel. Scale bar, 10 μm. (F) Capture efficiency of foamy versus nonfoamy monocytes identified on chip following shearing of blood isolated from apoE−/− or CD11c−/−/apoE−/− mice on ND and 1-wk WD (n = 5 separate experiments). Significance was determined comparing nonfoamy to foamy monocytes in apoE−/− mice after one wk WD using a paired Student t test. *p < 0.05, ***p < 0.005.

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To assess the distribution of CD11c and VLA-4 receptors within the plane of adhesive contact of monocytes recruited on the VCAM-1 substrate, we used total internal reflection fluorescence (TIRF) microscopy, which restricts the detection of fluorescent reporters to within ∼100 nm of the glass substrate where integrin/VCAM-1 bonds reside (Fig. 3B). The number of CD11c and VLA-4 receptors in contact with VCAM-1 was ∼1-fold greater on arrested monocytes from WD mice versus ND mice (Fig. 3C). Moreover, these integrins were twice as likely to coalesce in focal clusters on monocytes from blood of WD mice versus ND mice, such that 80% of VLA-4 was spatially colocated with CD11c following arrest (Fig. 3D). Treatment of monocytes with the lipid raft inhibitor methyl-β-cyclodextrin (MβCD) reduced integrin coalescence within the region of contact with VCAM-1 to the level observed with monocytes from ND mice (Fig. 3C, 3D). This phenomenon was integrin specific, because coalescence between VLA-4 and CD45, a highly expressed membrane receptor on monocytes, remained at the baseline level of 20%, which was equivalent between monocytes from ND and WD mice (Supplemental Fig. 4A–C).

The prevalence of foamy monocytes captured on VCAM-1 was assessed on the A-chip by immunofluorescence labeling of membrane CD115 and intracellular lipid with BODIPY (Fig. 3E) (10). Recruitment efficiency was equivalent between nonfoamy monocytes obtained from ND and WD mice (data not shown). In contrast, foamy monocytes from apoE−/− mice fed a WD recruit ∼3-fold more efficiently than nonfoamy monocytes, and this increase was attributed to CD11c, because recruitment efficiency was equivalent between foamy and nonfoamy monocytes in blood from apoE−/−/CD11c−/− mice (Fig. 3F). Inhibition of lipid rafts with MβCD reduced recruitment of foamy monocytes to a level similar to nonfoamy monocytes. In all cases, blocking VLA-4 binding to VCAM-1 by pretreatment with Ab abrogated recruitment. These data indicate that hypercholesterolemia amplifies the adhesive capacity of monocytes to capture on VCAM-1 in a manner dependent on spatial colocalization of CD11c and VLA-4 into bond clusters that coalesce within lipid raft microdomains. This integrin cooperativity was a function of lipid uptake to form foamy monocytes in the circulation, regardless of subset, because the relative ratio of foamy Ly6Clow/Ly6Chigh that arrested on VCAM-1 was maintained at 4:1, reflecting their respective numbers in the circulation of apoE−/− mice (data not shown).

We next addressed the mechanism by which monocyte uptake of lipoprotein in suspension elicits CD11c activation and VLA-4–dependent adhesion under shear stress. Mononuclear cells were isolated from blood collected from ND-fed mice and incubated with TGRL. This TGRL was derived from human plasma and consisted of very low-density lipoprotein and chylomicron lipid particles (28), previously reported to elicit a proatherogenic response based on the TGRL particles' capacity to upregulate VCAM-1 on HAECs and induce activation of VLA-4 adhesion following uptake by human monocytes (9, 10). Mouse monocytes became foamy following in vitro exposure to TGRL, as confirmed by an increase in SSC and BODIPY staining, similar to that observed ex vivo in circulating monocytes from apoE−/− mice fed a WD (Fig. 4A). Monocyte activation following TGRL uptake was assessed based on upregulation of CD11c expression, an upshift in VLA-4 affinity, and VLA-4–dependent capture of Fn beads. VLA-4 affinity was detected as increased binding of the small fluorescently labeled peptide LDV-FITC to monocytes incubated with TGRL.

FIGURE 4.

Monocyte uptake of TGRL elicits increased CD11c expression and VLA-4 adhesion in lipid rafts dependent on paxillin binding and lipid rafts. Mouse mononuclear cells were isolated from blood from apoE−/− mice maintained on an ND. Cells were incubated with human TGRL (100 μg/ml apoB) for 30 min at 37°C. (A) FACS plots of foamy and nonfoamy monocytes following uptake of TGRL. Monocytes containing lipid stained positive for BODIPY. Prior to incubation with TGRL, mouse mononuclear cells were treated with 0.01% DMSO (vehicle control), 10 nM MβCD, or paxillin inhibitor 6-B345TTQ (50 μM). (B) CD11c expression on foamy versus nonfoamy monocytes. Significance was determined using paired Student t test. *p < 0.05, **p < 0.01. (C) VLA-4 affinity measured by LDV-FITC (30 nM) binding to foamy versus nonfoamy monocytes at the time of maximum adhesion (∼10 min). Significance was determined using a Student t test. **p < 0.01, ***p < 0.005. (D) VLA-4 adhesion measured by Fn bead binding in sheared monocyte suspensions (n = 4 separate experiments using TGRL isolated from four hypertriglyceridemic human subjects). **p < 0.01, ***p < 0.005, Student t test. Mouse mononuclear cells were isolated from blood from α4-(Y991A) or wild-type mice and incubated with human TGRL at 100 μg/ml apoB for 30 min at 37°C. (E) CD11c expression on foamy versus nonfoamy monocytes. VLA-4 affinity (F) and VLA-4 adhesion (G) on foamy versus nonfoamy monocytes measured at the time of maximum adhesion (n = 5 separate experiments fed TGRL isolated from hypertriglyceridemic human subjects). For (E)–(G), **p < 0.01, Student t test.

FIGURE 4.

Monocyte uptake of TGRL elicits increased CD11c expression and VLA-4 adhesion in lipid rafts dependent on paxillin binding and lipid rafts. Mouse mononuclear cells were isolated from blood from apoE−/− mice maintained on an ND. Cells were incubated with human TGRL (100 μg/ml apoB) for 30 min at 37°C. (A) FACS plots of foamy and nonfoamy monocytes following uptake of TGRL. Monocytes containing lipid stained positive for BODIPY. Prior to incubation with TGRL, mouse mononuclear cells were treated with 0.01% DMSO (vehicle control), 10 nM MβCD, or paxillin inhibitor 6-B345TTQ (50 μM). (B) CD11c expression on foamy versus nonfoamy monocytes. Significance was determined using paired Student t test. *p < 0.05, **p < 0.01. (C) VLA-4 affinity measured by LDV-FITC (30 nM) binding to foamy versus nonfoamy monocytes at the time of maximum adhesion (∼10 min). Significance was determined using a Student t test. **p < 0.01, ***p < 0.005. (D) VLA-4 adhesion measured by Fn bead binding in sheared monocyte suspensions (n = 4 separate experiments using TGRL isolated from four hypertriglyceridemic human subjects). **p < 0.01, ***p < 0.005, Student t test. Mouse mononuclear cells were isolated from blood from α4-(Y991A) or wild-type mice and incubated with human TGRL at 100 μg/ml apoB for 30 min at 37°C. (E) CD11c expression on foamy versus nonfoamy monocytes. VLA-4 affinity (F) and VLA-4 adhesion (G) on foamy versus nonfoamy monocytes measured at the time of maximum adhesion (n = 5 separate experiments fed TGRL isolated from hypertriglyceridemic human subjects). For (E)–(G), **p < 0.01, Student t test.

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VLA-4 affinity and adhesion increased ∼1-fold, and this correlated with an ∼7-fold upregulation of CD11c on foamy monocytes compared with nonfoamy monocytes in the same suspensions (Fig. 4B–D). Pretreatment with the lipid raft inhibitor MβCD abrogated the increase in VLA-4 affinity and avidity, but it did not affect the upregulation of CD11c, indicating that uptake of TGRL and CD11c upregulation were independent of lipid raft formation shown to be necessary for downstream VLA-4 activation.

Paxillin is a cytoplasmic adapter protein that binds to the α4 subunit of VLA-4 and is necessary for monocyte adhesion to β1 integrin ligands under shear (31). Pretreatment with the paxillin antagonist 6-B345TTQ prevents its interaction with the cytoplasmic domain of α4 and is reported to reduce monocyte recruitment during inflammation (32). Treatment with this antagonist did not diminish uptake of TGRL and subsequent upregulation of CD11c, but it abrogated the increase in VLA-4 affinity and concomitant Fn bead capture (Fig. 4B–D). To confirm that it was paxillin association with the α4 cytoplasmic domain that stabilized high-affinity VLA-4, we measured changes in VLA-4 affinity and Fn bead binding on monocytes from α4-(Y991A) mice, which lack the intracellular domain that supports paxillin binding to VLA-4. Uptake of TGRL by monocytes isolated from α4-(Y991A) mice induced foaminess and activated the increased expression of CD11c to a similar level as in control wild-type C57BL6 mice (Fig. 4E). However, activation of VLA-4 affinity and avidity in foamy monocytes from α4-(Y991A) mice was prevented (Fig. 4F, 4G). These data indicate that paxillin binding at the Y991A domain is a critical step in TGRL-mediated activation of VLA-4 to bind ligand. We conclude that monocyte uptake of lipid elicits upregulation of CD11c, which, in turn, activates VLA-4 affinity and avidity preferentially on foamy monocytes in a lipid raft– and paxillin-dependent manner.

Previous studies in T cells revealed that CCR2 ligation by MCP-1 signals activation of VLA-4 to form focal adhesions with VCAM-1 through cytosolic association with p-Syk and paxillin (33, 34). We hypothesized that a similar mechanism governs activation of foamy monocyte–induced via outside-in signaling by high-affinity CD11c. Outside-in signaling of VLA-4 was compared with inside-out signaling via MCP-1 binding to CCR2 by measuring the level of LDV-FITC binding and the kinetics of Fn bead capture by human monocytes in suspension. The high-affinity conformation of CD11c was detected with the Ab reporter mAb 3.9. Outside-in signaling was induced by the binding of 496B, a mAb that recognizes the I-domain allosteric site of CD11c, upshifting the receptor to a high-affinity conformation (10, 17). Titrating the activation of high-affinity CD11c by addition of TGRL, MCP-1, or 496B elicited a proportional increase in VLA-4 capture of Fn beads and binding of LDV-FITC (Fig. 5A, 5B). Allosteric activation of CD11c with 496B elicited the steepest increase in VLA-4 adhesion to Fn beads, such that an equivalent amount of bead capture required activation of half the number of high-affinity CD11c stimulated by MCP-1. Outside-in or inside-out signaling of VLA-4 activation was dependent on paxillin and Syk phosphorylation, as shown by pretreatment with the respective inhibitors that lowered Fn bead binding to the baseline of untreated controls (Fig. 5B, 5C). In contrast, activation of high-affinity CD11c by all stimuli was unaffected by inhibition of paxillin and p-Syk (Fig. 5D). Thus, human and mouse monocytes exhibit a similar functional response of VLA-4 that correlates with the extent of outside-in signaling via high-affinity CD11c.

FIGURE 5.

High-affinity CD11c activates VLA-4 adhesion within a macromolecular complex initiated by p-Syk and dependent upon paxillin. (A) Human mononuclear cells isolated from blood were treated with anti-CD11c agonist 496B (0.5, 1, 2, 5, 10, 20 μg/ml), MCP-1 (0.1, 1, 5, 10, 30, 100 nM). or TGRL (apoB: 25, 50, 100 μg/ml). VLA-4 adhesion plotted versus high-affinity CD11c expression detected by anti-CD11c mAb 3.9. VLA-4 binding to LDV-FITC at the time of maximum adhesion (B), VLA-4 adhesion (C), and high-affinity CD11c (D) on mononuclear cells pretreated with 0.01% DMSO, 50 μM paxillin inhibitor, or 3 μM p-Syk inhibitor for 15 min and then incubated with anti-CD11c Ab BU15 (control; 10 μg/ml), anti-CD11c agonist 496B (10 μg/ml), TGRL (100 μg/ml apoB), MCP-1 (30 nM), or Mn2+ (1 mM). For (B)–(D), significance comparing CD11c agonist, TGRL, MCP-1, and Mn2+ to untreated control was determined using a one-way ANOVA with Tukey posttest. Significance comparing the effect of inhibitors on the stimulated group was determined using a one-way ANOVA with Tukey posttest. **p < 0.01, ***p < 0.005. (E) CD11c association with VLA-4 on human monocytes that were isolated and suspended for 15 min at 37°C with control anti-CD11c BU15 and activated with TGRL (100 μg/ml apoB) or allosteric anti-CD11c agonist 496B (10 μg/ml). Western blot detection of CD11c, VLA-4, paxillin, p-Syk, and p-Ser988 on monocytes that were lysed and immunoprecipitated by anti-CD11c coupled to protein-A/G agarose resin. (FI) Protein content/band was normalized to CD11c density, and the fold change for VLA-4 and paxillin following activation with anti-CD11c agonist 496B or TGRL was compared with that obtained by treatment with the control anti-CD11c BU15. p-Syk was normalized to total Syk protein for each condition to establish the p-Syk/Syk ratio, and its fold change was compared with the control anti-CD11c BU15 (n = 3 separate experiments). *p < 0.05 versus untreated sample, multiple Mann–Whitney tests.

FIGURE 5.

High-affinity CD11c activates VLA-4 adhesion within a macromolecular complex initiated by p-Syk and dependent upon paxillin. (A) Human mononuclear cells isolated from blood were treated with anti-CD11c agonist 496B (0.5, 1, 2, 5, 10, 20 μg/ml), MCP-1 (0.1, 1, 5, 10, 30, 100 nM). or TGRL (apoB: 25, 50, 100 μg/ml). VLA-4 adhesion plotted versus high-affinity CD11c expression detected by anti-CD11c mAb 3.9. VLA-4 binding to LDV-FITC at the time of maximum adhesion (B), VLA-4 adhesion (C), and high-affinity CD11c (D) on mononuclear cells pretreated with 0.01% DMSO, 50 μM paxillin inhibitor, or 3 μM p-Syk inhibitor for 15 min and then incubated with anti-CD11c Ab BU15 (control; 10 μg/ml), anti-CD11c agonist 496B (10 μg/ml), TGRL (100 μg/ml apoB), MCP-1 (30 nM), or Mn2+ (1 mM). For (B)–(D), significance comparing CD11c agonist, TGRL, MCP-1, and Mn2+ to untreated control was determined using a one-way ANOVA with Tukey posttest. Significance comparing the effect of inhibitors on the stimulated group was determined using a one-way ANOVA with Tukey posttest. **p < 0.01, ***p < 0.005. (E) CD11c association with VLA-4 on human monocytes that were isolated and suspended for 15 min at 37°C with control anti-CD11c BU15 and activated with TGRL (100 μg/ml apoB) or allosteric anti-CD11c agonist 496B (10 μg/ml). Western blot detection of CD11c, VLA-4, paxillin, p-Syk, and p-Ser988 on monocytes that were lysed and immunoprecipitated by anti-CD11c coupled to protein-A/G agarose resin. (FI) Protein content/band was normalized to CD11c density, and the fold change for VLA-4 and paxillin following activation with anti-CD11c agonist 496B or TGRL was compared with that obtained by treatment with the control anti-CD11c BU15. p-Syk was normalized to total Syk protein for each condition to establish the p-Syk/Syk ratio, and its fold change was compared with the control anti-CD11c BU15 (n = 3 separate experiments). *p < 0.05 versus untreated sample, multiple Mann–Whitney tests.

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To quantify the association between CD11c and VLA-4 integrins in response to monocyte activation in suspension, immunoprecipitation and Western blot analysis were used following allosteric activation of CD11c with 496B or uptake of TGRL. Because phosphorylation of Syk and recruitment of paxillin to the α4-cytoplasmic domain are required for stabilizing high-affinity VLA-4 integrins at sites of focal adhesion, we probed the complex pulled down by immunoprecipitation of CD11c with Abs specific to these cytosolic molecules (31, 35, 36). In response to activation with TGRL or 496B, there was an ∼3-fold increase in p-Syk, paxillin, and VLA-4 (Fig. 5E–I). Paxillin binding to the α4-cytoplasmic domain and a conformational shift of VLA-4 to high affinity requires dephosphorylation of α4 at the serine-988 region (Ser988) (37). To assess the phosphorylation state of Ser988 following activation of CD11c, we probed the immunoprecipitated complex using an Ab specific to p-Ser988. Activation of CD11c with 496B or TGRL resulted in a concomitant decrease in p-Ser988 to the baseline level detected in vehicle controls (Fig. 5E, 5I). Thus, activated CD11c induces the dephosphorylation of Ser988 that provides a substrate for the assembly of paxillin and p-Syk to bind and stabilize high-affinity VLA-4 at focal sites of adhesion.

To probe the sequence of events involved in the dephosphorylation of Ser988 and the association of p-Syk and paxillin with the integrin complex, monocytes were pretreated with p-Syk or paxillin inhibitors prior to activation of CD11c. Treatment with paxillin inhibitor reduced the amount of VLA-4 pulled down with CD11c to the baseline and prevented dephosphorylation of Ser988 at the α4-cytoplasmic domain. However, paxillin inhibition did not affect the binding of p-Syk to activated CD11c (data not shown). Treatment with the p-Syk inhibitor reduced both paxillin and VLA-4 association with CD11c and suppressed dephosphorylation of Ser988 (Fig. 5F–I). This result suggests that phosphorylation of Syk by high-affinity CD11c precedes its association with paxillin and VLA-4. Collectively, these data indicate a sequence that is initiated by lipid uptake and an upshift in CD11c affinity that triggers the association with VLA-4, contemporaneous with activation of Syk, recruitment of paxillin, and dephosphorylation of Ser988 within a macromolecular membrane complex that supports stable bond formation with Fn or VCAM-1.

To further examine how induction of the high-affinity conformation of CD11c facilitates VLA-4–mediated adhesion within lipid rafts, we examined, in real-time, the kinetics of Fn bead capture and imaged integrin coalescence within the region of adhesive contact in fixed and stained samples. Human monocytes in suspension were stimulated with either MCP-1 or TGRL, and CD11c was imaged with the nonblocking control Ab BU15, or locked into low affinity by pretreatment with the allosteric inhibitor Ab 496K, or treated with MβCD to inhibit lipid raft formation (Fig. 6A–C). A similar rate of VLA-4–mediated capture of Fn beads was induced following stimulation with TGRL or MCP-1. However, locking CD11c in a low-affinity conformation with 496K or inhibition of lipid raft formation blocked to baseline the Fn bead capture activated by TGRL uptake. In contrast, stimulation with MCP-1 elicited bead capture that was independent of high-affinity CD11c or recruitment of VLA-4 into lipid rafts (Fig. 6B, 6C). Confocal imaging of the region of adhesive contact revealed the coalescence of high-affinity CD11c with VLA-4 within lipid rafts (e.g., capture frequency: 1 in 5 monocytes with beads) (Fig. 6D). The few monocytes with bound Fn beads (e.g., capture frequency: <1 in 10 monocytes with beads) following TGRL activation in the presence of 496K or MβCD inhibitors exhibited fewer focal clusters of VLA-4 within diffuse lipid rafts. MCP-1 activation resulted in uniformly distributed VLA-4, which did not necessarily redistribute to lipid raft domains. We conclude that uptake of lipoprotein and formation of foamy monocytes induce the activation of CD11c and its coalescence within lipid rafts, which, in turn, precipitates the focal clustering of high-avidity VLA-4 competent for shear-resistant ligand binding (Fig. 7).

FIGURE 6.

High-affinity CD11c elicits VLA-4 coalescence in lipid rafts to activate adhesion under shear stress. Human mononuclear cells isolated from blood were pretreated with anti-CD11c Abs BU15 (A) or 496K (B) or MβCD (C), and VLA-4 bead adhesion was measured for 10 min to establish a baseline for each treatment. Activation was elicited by addition of TGRL (100 μg/ml apoB) or MCP-1 (30 nM), and kinetics of VLA-4 capture of Fn beads at ∼1 dyn/cm2 was measured in real-time by FACS. (D) Confocal fluorescence imaging of captured Fn beads by monocytes in shear. Bright-field images and images showing Abs recognizing high-affinity CD11c (green), total VLA-4 (red), and lipid rafts–CTxB (cyan). Scale bars, 5 μm.

FIGURE 6.

High-affinity CD11c elicits VLA-4 coalescence in lipid rafts to activate adhesion under shear stress. Human mononuclear cells isolated from blood were pretreated with anti-CD11c Abs BU15 (A) or 496K (B) or MβCD (C), and VLA-4 bead adhesion was measured for 10 min to establish a baseline for each treatment. Activation was elicited by addition of TGRL (100 μg/ml apoB) or MCP-1 (30 nM), and kinetics of VLA-4 capture of Fn beads at ∼1 dyn/cm2 was measured in real-time by FACS. (D) Confocal fluorescence imaging of captured Fn beads by monocytes in shear. Bright-field images and images showing Abs recognizing high-affinity CD11c (green), total VLA-4 (red), and lipid rafts–CTxB (cyan). Scale bars, 5 μm.

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FIGURE 7.

Signaling events following monocyte uptake of lipid and activation of CD11c. (A) Monocytes take up lipid in the blood via low-density lipoprotein receptor related protein-1, which results in an upshift in affinity of CD11c via a PLC-dependent mechanism. (B) Activation of CD11c in response to TGRL uptake and PLC signaling results in coupling with ITAMs and phosphorylation of Syk within lipid rafts. (C) p-Syk is associated with clustering of paxillin and VLA-4 with CD11c within focal adhesions. (D) Monocyte capture on VCAM-1 is mediated by high-avidity focal adhesion domains within lipid rafts containing activated CD11c and VLA-4.

FIGURE 7.

Signaling events following monocyte uptake of lipid and activation of CD11c. (A) Monocytes take up lipid in the blood via low-density lipoprotein receptor related protein-1, which results in an upshift in affinity of CD11c via a PLC-dependent mechanism. (B) Activation of CD11c in response to TGRL uptake and PLC signaling results in coupling with ITAMs and phosphorylation of Syk within lipid rafts. (C) p-Syk is associated with clustering of paxillin and VLA-4 with CD11c within focal adhesions. (D) Monocyte capture on VCAM-1 is mediated by high-avidity focal adhesion domains within lipid rafts containing activated CD11c and VLA-4.

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Identification of the inflammatory subset of circulating monocytes that recruit to nascent plaque during atherogenesis has been elusive. We report that Ly6ClowCD11chigh constitute the major subset (>80%) of foamy monocytes that emerge in the circulation within 1 wk of the onset of hypercholesterolemia. This subset adopted a proinflammatory phenotype primed for enhanced VLA-4 adhesion in response to signaling via chemokine receptors (CCR2, CX3CR1) and uptake of lipids. Shear-resistant monocyte arrest on VCAM-1 resulted from a redistribution of high-affinity CD11c and VLA-4 into focal adhesions that were stabilized by paxillin within lipid raft–enriched membrane microdomains. Monocyte uptake of native lipoprotein high in triglycerides was sufficient to activate CD11c from a low- to a high-affinity state, which induced phosphorylation of Syk in a process that precipitated its physical coupling with VLA-4 and paxillin. These data reveal a novel mechanism by which human and mouse monocytes activate VLA-4–dependent recruitment on VCAM-1 that is distinct from chemokine signaling and regulated by the relative number of high-affinity CD11c expressed on an inflammatory subset of foamy monocytes.

Of the two major monocyte subsets that circulate in murine blood, Ly6Chigh are typically associated with a proinflammatory phenotype (38, 39), whereas Ly6Clow are often referred to as patrolling cells that monitor vascular integrity and function in angiogenesis (40). However, Ly6Clow also recruit to sites of plaque formation, and their numbers in circulation correlate with lesion size in apoE−/− mice, suggesting that they also contribute to atherogenesis (23). Supporting this is the recent study of Robbins et al. (5), who reported using an apoE−/− model that foamy macrophages in nascent plaque are primarily derived from monocyte subsets that recruit over the initial 4 wk of hypercholesterolemia. We observed Ly6Clow and Ly6Chigh foamy monocytes in the circulation of apoE−/− mice within 1 wk of a WD, and their relative numbers were maintained at an ∼4:1 ratio throughout the 5 wk of observation. Importantly, both subsets of foamy monocytes increased their capacity to activate VLA-4 adhesion in a manner dependent upon the relative expression of CD11c. Using the identical mouse model, Xu et al. (41) reported that proliferating monocytes from the circulation of apoE−/− mice express CD11c and are present at sites of nascent plaque formation as early as 2 wk following the onset of hypercholesterolemia. These foamy monocytes in the plaque were shown to produce inflammatory cytokines, such as TNF-α and IL-1β. Likewise, we observed ex vivo that monocytes from the circulation, when incubated with TGRL and captured on VCAM-1, produced TNF-α and IL-1β (Supplemental Fig. 4D). Thus, foamy monocytes (both Ly6Chigh and Ly6Clow) upregulate CD11c and increase their recruitment efficiency to VCAM-1 during the early stage of plaque formation.

CD11c expression is upregulated on the plasma membrane from a storage pool in cytosolic granules within minutes of lipid uptake (42). Neither CD11c expression nor membrane upregulation was required for uptake and internalization of lipid, as supported by the fact that equivalent numbers of foamy monocytes were detected in blood samples from apoE−/− and CD11c−/−/apoE−/− mice fed a WD. In this regard, Xu et al. (41) also found that CD11c was upregulated on foamy monocytes within hours of a bolus injection and uptake of isolated cholesterol ester-rich very low-density lipoproteins into the circulation. Similarly, we show that exposure to proatherogenic TGRL also induces the formation of foamy monocytes, suggesting that lipoprotein uptake is the chief source of this mode of activation. Emergence of foamy monocytes and an increase in their capacity to recruit to VCAM-1 under shear stress rose most dramatically within the first week of a WD, at which time they adhered twice as efficiently as those from mice on an ND. The capacity for lipid uptake to activate CD11c and increase VLA-4 affinity and avidity on foamy monocytes was consistent between mouse and human models. Human Mon2 (CD14++CD16+) isolated postprandial from hypertriglyceridemic human subjects or those activated ex vivo following TGRL uptake upregulated CD11c affinity that directly correlated with the extent of VLA-4–mediated adhesion (10). The mechanism for CD11c-mediated activation of VLA-4 adhesion to ligand is likely dependent on both an increase in its affinity for ligand and redistribution into focal clusters, given that the overall number of VLA-4 receptors on activated monocytes remains constant (43). Examination of integrin distribution beneath arrested monocytes revealed twice as many VLA-4 and CD11c receptors colocalized in focal clusters within lipid raft microdomains adherent to VCAM-1, a process required for enhanced recruitment efficiency.

Cooperativity between β1 and β2 integrins appears to involve a cis-interaction between high-affinity CD11c and VLA-4. This activation occurs downstream of Syk phosphorylation, which triggers the dephosphorylation of Ser988 on the α4 subunit, which leads to subsequent recruitment of paxillin (37, 44) and stabilization of high-affinity VLA-4 (34, 45). Outside-in signaling was achieved using the CD11c-activating Ab 496B to allosterically stabilize a high-affinity conformation and VLA-4–mediated activation, which was functionally equivalent to monocyte uptake of TGRL. This approach established a causal link between the upshift in CD11c affinity state and an increase in VLA-4 affinity and adhesive capacity. A putative mechanism for VLA-4 activation via high-affinity CD11c may involve its redistribution in membrane rafts proximal to Src-homology 2 domains on SLP-76 or CrkL, adaptor proteins that were shown to trigger activation following interaction with integrin cytodomains (4648). A hierarchy in the level of VLA-4 activation was detected, with signaling via the allosteric agonist 496B most potent and TGRL and MCP-1 less potent. These data suggest that conversion of CD11c to high affinity is a direct regulator of VLA-4 adhesion and implicate p-Syk activation of paxillin as a downstream regulator in the signaling pathway. This conclusion is supported by previous reports that phosphorylation of Syk at the cytodomain of CD18 activates binding of paxillin (49), which serves as a substrate in stabilizing α4β1 into a high-affinity conformation (31, 32). With regard to the sequence, we found that inhibiting paxillin binding or phosphorylation of Syk had no effect on the conversion of CD11c to a high-affinity conformation but abrogated the downstream dephosphorylation of Ser988 on the α4 cytosolic subunit that is required for activation of VLA-4. Using the allosteric antagonist Ab 496K to maintain CD11c in a low-affinity conformation, we found that the upshift in CD11c affinity was critical for TGRL, but not MCP-1, activation of VLA-4 adhesion. A similar level of inhibition was observed by depleting lipid rafts with MβCD, which interfered with VLA-4 and CD11c colocalization and consequently diminished the efficiency of monocyte Fn bead capture and arrest on VCAM-1. We conclude that lipid uptake–induced high-affinity CD11c functions as a direct regulator of VLA-4 avidity through activation of p-Syk and dephosphorylation of Ser988 on the α4 subunit, both of which are associated with focal adhesion formation stabilized by paxillin within lipid rafts at sites of shear-resistant adhesion on VCAM-1.

A question that remains unanswered is what is the precise mechanism by which lipoprotein uptake by monocytes confers activation of CD11c? We recently reported that uptake of TGRL via low-density lipoprotein receptor related protein-1 mediates signaling through phospholipase C (PLC) (9, 10). In the current study, we show that this pathway is necessary for CD11c activation, as depicted in Fig. 7A. Further, we propose that local release of intracellular calcium occurs downstream of lipid uptake, which is known to precipitate PIP3 and small-GTPases, including Rap1, which can activate CD11c at its cytodomain (11, 50). Uptake of oxidized low-density lipoprotein and recognition by TLR4 signals through phosphorylation of Syk Src-homology 2 domains, which, in turn, can activate ITAMs (51). The mechanism by which CD11c becomes active and recruited within lipid rafts may occur in a manner similar to ITAM-mediated outside-in activation of the β2 integrin LFA-1 (52). Upon redistribution with lipid rafts, active CD11c may associate with constitutively available signaling molecules, such as ITAMs, that facilitate phosphorylation and signaling via Syk, as depicted in Fig. 7B (53). We showed that phosphorylation of Syk by high-affinity CD11c triggered the dephosphorylation of Ser988, which provided a substrate for binding of paxillin to VLA-4 in a focal adhesion complex within lipid rafts (Fig. 7C). In this manner, high-affinity CD11c coupled in the membrane to p-Syk could associate with paxillin to initiate conversion of VLA-4 to high-affinity where they coalesce into focal clusters that facilitate bond formation with VCAM-1 (Fig. 7D).

In conclusion, our data reveal that within a week of diet-induced hypercholesterolemia, a subset of monocytes takes up lipid and adopts an inflammatory phenotype associated with increased CD11c expression and shear-resistant capture on VCAM-1. Human Mon2 inflammatory monocytes also activated VLA-4–mediated adhesion in response to uptake of TGRL, which elicited an upshift in CD11c affinity state that amplified monocyte recruitment to VCAM-1 under shear stress. We conclude that CD11c expression and activation may serve as a reliable biomarker of the proatherogenic state of monocytes in circulation, as well as a potential target for inhibiting their early recruitment into nascent plaque.

We thank Britta Drier and Keith Bailey for assistance with optimizing immunoprecipitation and Western blots. We also thank Mark Ginsberg and Marina Slepak for providing the α4-(Y991A) mice. We appreciate the generous provision of mAbs by Don Staunton of CysThera and by Eli Lilly Corp.

This work was supported by National Institutes of Health Grants HL082689 and A047294 (to S.I.S.) and HL098839 (to H.W.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

A-chip

artery on a chip

AF

Alexa Fluor

apoB

apolipoprotein B

CTxB

cholera toxin subunit B

DPBS

Dulbecco’s PBS

Fn

fibronectin

HAEC

human aortic endothelial cell

HSA

human serum albumin

MβCD

methyl-β-cyclodextrin

MFI

median fluorescence intensity

ND

normal chow diet

PDMS

polydimethylsiloxane

PLC

phospholipase C

SSC

side scatter

Syk

spleen tyrosine kinase

TIRF

total internal reflection fluorescence

TRGL

triglyceride-rich lipoprotein

VLA-4

very late Ag-4

WD

Western high-fat diet.

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The authors have no financial conflicts of interest.

Supplementary data