Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) expression is induced with macrophage inflammatory polarization. Murine bone marrow–derived macrophages were differentiated in culture and stimulated with IL-4 or interferon-γ (IFNγ)/lipopolysaccharide (LPS) to resemble tissue resident or inflammatory macrophages, respectively. A: Macrophage polarization into resident and inflammatory phenotype was confirmed by transcriptional analyses (quantitative RT-PCR) of the relevant genes. Data were normalized to unstimulated (naive) macrophages. B–E: Flow cytometry (B), Western blot (C), quantitative RT-PCR (D) and immunofluorescence staining (E) analyses show an induction of ICAM-1 expression in inflammatory (MϕIFNγ/LPS) but not in resident (MϕIL-4) or in undifferentiated and unstimulated macrophages. F and G: Differentiated macrophages were stimulated (for 24 hours) as indicated and ICAM-1 expression was assessed (representative flow diagram, F) and quantified (G) using flow cytometry analyses. n = 3 to 5 independent experiments. ∗∗P < 0.01 versus naive macrophages; ††P < 0.01. Scale bars = 20 μm. Arg1, arginase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; MFI, mean fluorescence intensity; mRNA, messenger RNA; TGF-β1, transforming growth factor β1; TNFα, tumor necrosis factor α; Unstim, unstimulated.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Flow Cytometry, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Inflammatory macrophages in murine and human colon tissue express intercellular adhesion molecule-1 (ICAM-1). A: Epithelial injury/colitis was induced by introduction of dextran sulfate sodium (DSS) 3% (w/v) to drinking water for 7 days in CX3CR1-GFP macrophage reporter mice. Macrophages were isolated from the lamina propria of distal colons and ICAM-1 expression by CX3CR1/CD45/CD64-positive Ly6Chi inflammatory (P1) versus Ly6Clow resident (P2) macrophages was analyzed by flow cytometry. B–D: Nonactive (noninflamed) control and active inflammatory bowel disease (IBD) colon tissue sections were stained by immunofluorescence for pan macrophage marker CD68 and ICAM-1. B: The number of CD68-positive macrophages per high-power field was quantified. C: The percentage of macrophages that were positive for both CD68 and ICAM-1 were quantified. D: Representative images show an increased number of CD86 and ICAM-1 expressing macrophages (yellow cells) in IBD tissue. n = 4 independent experiments, with at least 10 fields quantified for human sections. ∗∗P < 0.01. Scale bars = 20 μm.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Isolation, Expressing, Flow Cytometry, Staining, Immunofluorescence, Marker

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) contributes to efferocytosis by inflammatory macrophages ex vivo. Apoptotic cell (AC) engulfment assays were performed with bone marrow (BM)-derived macrophages undifferentiated or differentiated as indicated. A: Jurkats or murine BM-derived polymorphonuclear leukocytes (PMNs) were labeled fluorescently, irradiated to induce apoptosis, and incubated with undifferentiated macrophages or with MϕIL-4 and MϕIFNγ/LPS. B: Efferocytotic ability was examined in MϕIFNγ/LPS isolated from wild-type (WT) versus ICAM-1 knockout (KO) mice. C: Representative images depict impaired engulfment of Jurkat (JK) cells by ICAM-1 KO MϕIFNγ/LPS. D: Quantitative RT-PCR analyses were performed to determine inflammatory polarization of ICAM-1 KO MϕIFNγ/LPS. Data shown as KO relative to WT expression for each gene. n = 4 independent experiments for each Jurkats and PMNs. ∗P < 0.05, P < 0.01. Scale bars = 20 μm. IFNγ, interferon-γ; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mRNA, messenger RNA; TNFα, tumor necrosis factor α.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Ex Vivo, Derivative Assay, Labeling, Irradiation, Incubation, Isolation, Knock-Out, Quantitative RT-PCR, Expressing

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) facilitates apoptotic cell clearance in vivo. Apoptotic cell (AC) engulfment by peritoneal macrophages was examined. Mice were treated with thioglycollate to induce Mϕ recruitment to the peritoneal cavity. Mice were then injected with fluorescently tagged intestinal epithelial cells (IECs), which were isolated from E-cadherin–cyan fluorescence protein (CFP) mice and irradiated to induce apoptosis. A and B: Wild-type (WT) and ICAM-1 knockout (KO) Mϕs were isolated by lavage, and engulfment of IEC-CFP (acquisition of CFP signal) by MHCII+/CD64+ peritoneal macrophages was quantified. C: ICAM-1 expression was confirmed in WT but not in KO macrophages. N = 3 independent experiments with 2 to 3 mice for each experimental condition. ∗∗P < 0.01.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: In Vivo, Injection, Isolation, Fluorescence, Irradiation, Knock-Out, Expressing

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) promotes efferocytosis by mediating macrophage–apoptotic cell (AC) binding. Engulfment of apoptotic (A) Jurkat (JK) cells and (B) polymorphonuclear leukocytes (PMNs) by wild-type bone marrow–derived MϕIFNγ/LPS or (C) intestinal epithelial cells (IECs) by thioglycollate-recruited peritoneal MHCII+/CD64+ macrophages, with or without the addition of function blocking ICAM-1 or control IgG antibodies (Abs) was examined. Inhibition of ICAM-1 adhesive interactions impairs efferocytosis for all conditions. n = 3 independent experiments for each condition. ∗P < 0.05, ∗∗P < 0.01. IFNγ, interferon-γ; LPS, lipopolysaccharide.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Binding Assay, Derivative Assay, Blocking Assay, Inhibition

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) facilitates efferocytosis in human THP-1 macrophages. A–E: THP-1 macrophages were differentiated toward inflammatory phenotype by PMA treatment (THP-1PMA) with and without further stimulation with interferon-γ (IFNγ)/lipopolysaccharide (LPS). A and B: Messenger RNA (mRNA) transcript (A) and protein analyses (B) shows a significant induction in ICAM-1 expression. C: Knockdown of ICAM-1 protein was confirmed by Western blot. D: Apoptotic cell (AC) (Jurkats) engulfment assays were performed with/without siRNA-mediated down-regulation of ICAM-1 expression, showing impaired engulfment in the absence of functional ICAM-1. E: Engulfment assays were performed similarly with/without the addition of function blocking ICAM-1 or control IgG antibodies (Abs). F: AC engulfment assays were performed using primary human monocyte–derived macrophages activated with IFNγ/LPS treatment, with/without the addition of function blocking ICAM-1 or control IgG Abs. Inhibition of ICAM-1 adhesive interactions impaired THP-1 and primary macrophage efferocytosis. n = 4 independent experiments. ∗P < 0.05, ∗∗P < 0.01. Diff, differentiated; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Si-ICAM-1, small interfering-ICAM-1; Si-Scr, small-interfering scrample; Undiff, undifferentiated.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Expressing, Western Blot, Functional Assay, Blocking Assay, Derivative Assay, Inhibition

Journal: The American Journal of Pathology

Article Title: Intercellular Adhesion Molecule 1 Functions as an Efferocytosis Receptor in Inflammatory Macrophages

doi: 10.1016/j.ajpath.2019.12.006

Figure Lengend Snippet: Intercellular adhesion molecule-1 (ICAM-1) redistributes to the engulfment site to mediate apoptotic cell (AC) attachment. AC [Jurkat (JK)] engulfment assays by wild-type MϕIFNγ/LPS were performed. A: In the process of engulfment, MϕIFNγ/LPS were fixed and fluorescently stained for surface ICAM-1 (no permeabilization). Confocal microscopy was used to determine ICAM-1 localization with respect to engulfed ACs. Representative images show ICAM-1 clustering at the site of engulfed AC (white arrows). B: MϕIFNγ/LPS were treated with Jasplakinolide (Jas) to block the ability of macrophages to engulf ACs. C: AC attachment to ϕIFNγ/LPS was quantified with/without the addition of function blocking ICAM-1 or control IgG antibodies (Abs). D: Representative images show a significant reduction in AC attachment to ϕIFNγ/LPS with ICAM-1 inhibition. n = 3 independent experiments for each condition. ∗P < 0.05, ∗∗∗P < 0.001. Scale bars = 20 μm (A and D). IFNγ, interferon-γ; LPS, lipopolysaccharide.

Article Snippet: An anti-mouse ICAM-1 (YN1/1.7.4) purified or conjugated to fluorescein isothiocyanate, anti–CD64-PE, and fluorescein isothiocyanate–conjugated IgG control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA), anti-human ICAM-1 (15.2) was purchased from Serotec (Raleigh, NC), major histocompitibility complex II (MHCII; M5/114.15.2) was purchased from BioLegend, horseradish-peroxidase–conjugated anti-mouse and anti-rabbit IgGs were purchased from Jackson Immunoresearch (West Grove, PA), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (FF26A/F9) was purchased from BioLegend, human anti–glyceraldehyde-3-phosphate dehydrogenase was purchased from Sigma-Aldrich, and anti-mouse–Alexa 488 was purchased from Life Technologies (Waltham, MA).

Techniques: Staining, Confocal Microscopy, Blocking Assay, Inhibition