Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Conversion of iMo to an inflammatory phenotype is associated with nuclear translocation of NF-κB and production of IL-1β that requires ADAM17 proteolytic activity. (A) NF-κB cytosolic to nuclear translocation (n = 6/group). (B) Intracellular IL-1β production in brefeldin A–treated iMo at 2 h of shear and treatment with allosteric affinity stabilizing anti-CD11c or IgG control that binds the common epitope of CD11c (n = 6 per group) (two-way ANOVA with Tukey posttest, *p < 0.05 from control within each Ab treatment, #p < 0.05 between CAD and NSTEMI within Ab treatments, $p < 0.05 from the CD11c IgG treatment for each patient cohort). (C) Combined expression of nuclear NF-κB (n = 4; CAD; n = 2, NSTEMI) and (D) cytosolic IL-1β (n = 3 CAD, n = 2 NSTEMI) after treatment with ADAM17 inhibitor TMI-1 or vehicle in the presence of the IgG control to CD11c or the low affinity promoting Ab after 2 h of shear-resistant arrest to VCAM-1 at 2 dynes/cm2. Two-way ANOVA with Tukey posttest, *p < 0.05 from the control treatment compared with that of the ADAM17 inhibitor.

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Translocation Assay, Activity Assay, Shear, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Monocyte recruitment to inflamed endothelium under shear stress is β1- and β2-integrin–dependent on binding to VCAM-1. (A) Monocytes were perfused into the A-Chip onto TNF-α–stimulated HAEC in the presence of blocking Abs to CD11c, CD11b, VLA-4, or a nonspecific isotype IgG (Control) and TEM of adherent cells (white arrows) assessed by phase-contrast imaging after 10 min of shear at 2 dynes/cm2. Images display a time course of monocyte arrest and transmigration (phase dark) over 2 min. Scale bar, 10 μm. Monocyte subsets were determined based upon on-chip analysis of relative fluorescence intensity of Abs to CD14, CD16, and CCR2, which enabled the assessment of the number of arrested per field of view (FOV) at original magnification ×40. (B) Quantification of arrested iMo (filled bars) and the number of those arrested cells that have transmigrated (open bars) over 10 min of constant shear (2 dynes/cm2). Data are represented as mean arrested or transmigrated cells per FOV ± SD (n = 4–5 per group) (two-way ANOVA with Tukey posttest *p < 0.05 between isotype control within each cohort TEM color, #p < 0.05 compared with healthy, $p < 0.05 between CAD and NSTEMI cohorts for each Ab blocking treatment).(C) Monocyte subset arrest frequency on rVCAM-1 on the A-Chip. Data are represented as mean ± SD monocyte subset fraction of total arrested measured on the day of percutaneous coronary intervention or venous blood draw from healthy (n = 6), CAD (n = 8), and NSTEMI (n = 20) analyzed via two-way ANOVA with Tukey posttest. *p < 0.05 between healthy age-matched subject groups.

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Shear, Binding Assay, Blocking Assay, Control, Imaging, Transmigration Assay, Fluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Recruitment of iMo on VCAM-1 is regulated by the expression level and affinity state of CD11c, which discriminates cardiac patients from healthy controls. (A) Integrin dependence of iMo arrest following treatment with a pan–anti-CD11c mAb to a nonblocking common epitope (IgG), an allosteric anti-CD11c low affinity–inducing mAb, and anti–VLA-4 functional blocking mAb (healthy, n = 5; CAD, n = 5; NSTEMI, n = 21). Data are represented as mean ± SD iMo arrest number (two-way ANOVA with Tukey posttest, *p < 0.05 between patient groups, #p < 0.05 from the anti-CD11c IgG control). Arrest fraction of iMo/10,000 monocytes infused into the A-Chip plotted against an individual patient’s mean receptor number for (B) CD11c (n = 6, healthy; n = 8, CAD; n = 20, NSTEMI), (C) VLA-4 (n = 6, healthy; n = 10, CAD; n = 7, NSTEMI), (D) CD11b (n = 6, healthy; n = 10, CAD; n = 7, NSTEMI), and (E) CX3CR1 (n = 6, healthy; n = 8, CAD; n = 7, NSTEMI) receptor expression measured on whole blood monocytes via FACS analysis with the resulting Pearson correlation. (F) Principle component plot of integrin and chemokine receptor expression from six random individuals from each of the healthy, CAD, and NSTEMI groups, in which each data point represents a unique individual. The plot represents a two-dimensional space that contains 80.7% of the original expression data, in which the x-axis or dimension 1 (Dim1) represents 52.6% of the original expression data, and the y-axis given by dimension 2 (Dim2) represents 28.1% of the original input of expression data. (G) Heatmap of the integrin and chemokine receptor parameters used in the principal component clustering analysis. Relative weighting of the correlations depicted by a gradient in which blue is the minimum, white (0) is the median, and red indicates the maximum.

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Expressing, Functional Assay, Blocking Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: iMo form focal adhesions through activated CD11c and diffusion of VLA-4 that precedes conversion to a CD16− phenotype postarrest on VCAM-1. (A) TIRF images of iMo CD11c (green) and VLA-4 (red) expression at time of attachment to human rVCAM-1 (T = 0) and at 60 min postarrest at 2 dynes/cm2 shear stress for healthy, CAD, and NSTEMI (scale bar, 10 μm). Quantification of healthy (black, n = 7), CAD (blue, n = 7), and NSTEMI (orange, n = 5) iMo (B) CD11c and (C) CD49d (VLA-4) membrane receptor MFI per cell area at time of arrest to establish baseline fluorescence/cell area (bar graphs) and dynamically over time using live TIRF. Data are represented as percentage change from baseline fluorescence at the time of attachment for CD11c or VLA-4 receptors (one-way ANOVA with Tukey posttest, *p < 0.05 comparing the maximal percentage change to the baseline at T = 0. Scale bar, 10 μm). (D) Representative images of iMo from healthy, CAD, and NSTEMI patients depict CD16 expression (red) and CD14 (green) at t = 0 min (time of arrest) and 60 min postarrest. (E) CD16 receptor expression on adherent iMo assessed from the time of capture to 60 min postadhesion from healthy (n = 6), CAD (n = 7), and NSTEMI (n = 5) patients under constant flow or under static conditions (translucent points and lines). iMo conversion was classified as expressing fewer than 10,000 receptors, which was equivalent to background nonspecific fluorescence intensity. Data are represented as mean CD16 receptor number at time of arrest to VCAM-1 (one-way ANOVA with Tukey posttest, *p < 0.05 comparing the peak increase/decrease the baseline at T = 0. Scale bar, 10 μm). (F) Fraction of iMo that have converted relative to baseline CD16 receptor expression from healthy (n = 6), CAD (n = 7), and NSTEMI (n = 7) patients at 75 min under constant shear and treatment with CD11c allosteric or a pan-CD11c Ab control. Data are represented as mean CD16 expression/time ± SD (two-way ANOVA with Tukey posttest, #p < 0.05 compared with healthy controls, *p < 0.05 compared with the CD11c IgG treatment for each healthy age-matched subject cohort, $p < 0.05 between CAD and NSTEMI). Scale bar, 10 μm.

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Diffusion-based Assay, Expressing, Shear, Membrane, Fluorescence, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: An allosteric shift in CD11c affinity following iMo arrest on VCAM-1 induces membrane coalescence with CD16 and subsequent cleavage by ADAM17. (A) Schematic depicting the coalescence and (B) immunofluorescent images of CD11c (green), ADAM17 (purple), and CD16 (red) within focal adhesive contact on VCAM-1 at the time point of capture (scale bar, 10 μm). Associated Pearson correlation of the coalescence of membrane (C) CD11c–CD16 and (D) CD11c–ADAM17 receptors under static conditions from healthy, CAD, and NSTEMI iMo (n = 4–5). (E) Schematic representation of high affinity CD11c coclustered with CD16. This results in outside-in signaling associated with phosphorylation of Syk and association with ADAM17. (F) Western blot of coimmunoprecipitation of CD11c with ADAM17 and CD16 and associated signaling component p-Syk following 15 min of shear stress or static conditions on arrested monocytes from a healthy donor using allosteric affinity modulating mAbs to CD11c or a pan anti-CD11c control IgG (n = 7). (G) CD16, (H) p-Syk/Total Syk, and (I) ADAM17 band density normalized to CD11c protein expression quantified by densitometry (n = 7). Data are represented as mean band intensity normalized to CD11c ± SD (Student t test, *p < 0.05 between static and shear conditions) (n = 7). (J) Schematic representation of phenotypic conversion dependent upon ADAM17-mediated cleavage of CD16 and an allosteric shift to low affinity CD11c at focal sites of VLA-4–mediated adhesion. (K) ADAM17-dependent cleavage of CD16 on iMo from CAD and NSTEMI when treated with ADAM17 inhibitor (TMI-1 at 12 nM) (translucent lines) or DMSO vehicle control (filled lines) over 75 min at 2 dynes/cm2 postarrest to VCAM-1. * represents a significant difference between vehicle and TMI-1 at the given time point that is sustained after 15 min of shear. One-way ANOVA with Tukey posttest, *p < 0.05. (L) iMo conversion frequency after 75 min of shear postarrest to VCAM-1 in the presence of ADAM17 inhibitor or vehicle control. Data are represented as mean conversion ± SD (Student t test. *p < 0.05 between treated and vehicle control).

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Membrane, Adhesive, Phospho-proteomics, Western Blot, Shear, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: An Allosteric shift in CD11c affinity activates a pro-atherogenic state in arrested intermediate monocytes

doi: 10.4049/jimmunol.2000485

Figure Lengend Snippet: Integrin expression and iMo arrest as a function of measures of myocardial injury and incidence of MI in cardiac patients. Bivariate plots of iMo average integrin receptor expression (CD11c [black]; CD11b [blue]; VLA-4 [red]) from each NSTEMI patient versus corresponding serum measures of (A) CK-MB, (B) LVEF%, (C) and peak troponin T (CD11c: n = 33; CD11b: n = 30; VLA-4: n = 31), with resulting Pearson correlation coefficient. (D) Number of iMo arrested under shear stress in the A-Chip from patients experiencing a primary NSTEMI (n = 9) versus those returning to the clinic for a repeat MI within 3 y (n = 4). Student t test ***p < 0.001.

Article Snippet: Blocking Abs to CD11c (3.9) and VLA-4 (HP2/1) were purchased from BioLegend and GeneTex, respectively.

Techniques: Expressing, Shear