Novel Tools for Functional Analysis of CD11c: Activation‐Specific, Activation‐Independent, and Activating Antibodies

Sadhu, Chanchal; Hendrickson, Lee E.; Dick, Ken O.; Potter, Tamara G.; Staunton, Donald E.

doi:10.1080/15321810701735062

Cited by 11 publications

(9 citation statements)

References 31 publications

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“…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.…”

Section: Discussionsupporting

confidence: 90%

CD11c/CD18 Expression Is Upregulated on Blood Monocytes During Hypertriglyceridemia and Enhances Adhesion to Vascular Cell Adhesion Molecule-1

Gower

Foster

et al. 2011

ATVB

144

141

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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.

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Section: Discussionsupporting

confidence: 90%

CD11c/CD18 Expression Is Upregulated on Blood Monocytes During Hypertriglyceridemia and Enhances Adhesion to Vascular Cell Adhesion Molecule-1

Gower

Foster

et al. 2011

ATVB

144

141

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“…Outside-in signaling was induced by the binding of 496B, a monoclonal antibody that recognizes the I-domain allosteric site of CD11c upshifting the receptor to a high affinity conformation. (10, 36) 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 (Figure 5A and 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.…”

Section: Resultsmentioning

confidence: 98%

“…The following secondary antibodies were purchased from Life Technologies (Green 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 antibody were calculated using the formula:…”

Section: Methodsmentioning

confidence: 99%

CD11c/CD18 Signals Very Late Antigen-4 Activation To Initiate Foamy Monocyte Recruitment during the Onset of Hypercholesterolemia

Foster

Chidambaram

et al. 2015

The Journal of Immunology

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Recruitment of foamy monocytes to inflamed endothelium expressing vascular cell adhesion molecule-1 (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 antigen-4 (VLA-4) activation and cooperation in shear resistant cell arrest on VCAM-1 is ill defined. Within one week of the onset of a Western high-fat diet (WD) in apoE-/- mice, an inflammatory subset of foamy monocytes emerged comprising ¼ of the circulating population. These cells expressed ∼3-fold more CD11c/CD18 and 50% higher chemokine receptors than non-foamy monocytes. Recruitment from blood to a VCAM-1 substrate under shear stress was assessed ex-vivo using a unique artery-chip microfluidic assay. It revealed that foamy monocytes from mice on a WD increased their adhesiveness over 5 weeks, rising to twice that of mice on a normal diet (ND) or CD11c-/- 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.

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“…We evaluated the integrin specificity during arrest by addition of function-blocking antibodies before shearing blood over a VCAM-1 substrate. Pretreatment of blood samples with antibody 496K, which binds to an allosteric site on the metal-ion-dependent adhesion site (MIDAS) domain and inactivates high-affinity CD11c binding function (18), reduced the arrest fraction of Mon2 for high-risk and MI subjects to levels equivalent to that of low-risk subjects (Fig. 5E).…”

Section: Cd11c Expression and Vla-4 Affinity Are Increased On Mon2 Fomentioning

confidence: 99%

“…To determine whether CD11c affinity state influences integrin binding to VCAM-1 and the efficiency of monocyte recruitment in shear, blood was pretreated with allosteric antibodies that stabilize the inactivated (496K) and activated (496B) ligandbinding conformation of CD11c (18). Fresh blood obtained from fasting subjects was treated with allosteric or isotype control antibody and subsequently sheared on VCAM-1 and compared for VLA-4 receptor number and focal clustering on captured monocytes (Fig.…”

Section: Monocyte Capture On Vcam-1 Is Regulated By Activation Of Cd11cmentioning

confidence: 99%

On-chip phenotypic analysis of inflammatory monocytes in atherogenesis and myocardial infarction

Foster¹,

Gower²,

Stanhope

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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, and each may play distinct roles during atherogenesis and myocardial infarction. We studied a range of subjects that included otherwise healthy patients with elevated serum triglyceride levels to patients presenting with acute myocardial infarction. Our objective was to correlate an individual's risk with the activation state of each monocyte subset as a function of changes in adhesion receptor expression using flow cytometric quantitation of integrins and L-selectin membrane expression. A microfluidic-based laboratory-on-a-chip was developed to quantify the adhesion efficiency of monocytes sheared in whole blood on vascular cell adhesion molecule-1, while characterizing adhesion receptor expression and topography on captured monocytes. CD14 ++ CD16+ monocytes adhered with sevenfold higher efficiency than other subsets, and in patients with myocardial infarction the capture efficiency of this subset was double that for healthy subjects. In patients with hypertriglyceridemia, this increase in monocyte adhesion was attributable to CD14 ++ CD16+ uptake of triglyceride-rich lipoproteins and subsequent signaling via a Phospholipase C-dependent mechanism to increase CD11c expression, very late antigen-4 function, and integrin coclustering within focal adhesive sites on vascular cell adhesion molecule-1. In summary, we introduce a unique laboratory-on-a-chip method for quantifying the activation state of monocyte subsets. These experiments reveal that CD11c/CD18 is an inducible integrin whose expression correlates with a monocyte inflammatory state in subjects at risk for atherogenesis and in patients with myocardial infarction.B2-integrin | cell arrest | shear flow

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Novel Tools for Functional Analysis of CD11c: Activation‐Specific, Activation‐Independent, and Activating Antibodies

Cited by 11 publications

References 31 publications

CD11c/CD18 Expression Is Upregulated on Blood Monocytes During Hypertriglyceridemia and Enhances Adhesion to Vascular Cell Adhesion Molecule-1

CD11c/CD18 Expression Is Upregulated on Blood Monocytes During Hypertriglyceridemia and Enhances Adhesion to Vascular Cell Adhesion Molecule-1

CD11c/CD18 Signals Very Late Antigen-4 Activation To Initiate Foamy Monocyte Recruitment during the Onset of Hypercholesterolemia

On-chip phenotypic analysis of inflammatory monocytes in atherogenesis and myocardial infarction

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