rat  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Expression and function of CXCL16 in cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and effect of CXCL16 neutralizing antibody on neutrophil and mononuclear leukocyte adhesion. HUAEC were stimulated with 1% CSE, INF-γ, or TNF-α (20 ng/ml) for 1, 4, or 24 h. Relative quantification of mRNA levels for CXCL16 and GAPDH after (A) 1 h and (B) 4 h ( n = 5–8 independent experiments). Columns show fold increase in CXCL16 mRNA expression relative to control GAPDH. Values are expressed as mean ± SEM of the 2 −ΔΔCt values. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (C) Protein expression was determined by flow cytometry. Results are expressed as mean of fluorescence intensity (MFI) ( n = 7–8 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (D) Following a similar protocol, CXCL16 was visualized in non-permeabilized HUAEC by immunofluorescence (green). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) ( n = 4–5 independent experiments). (E,F) Endothelial cells were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, human neutrophils (E) or mononuclear cells (F) (1 × 10 6 cells/ml) incubated with or without EDTA were perfused over the monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte accumulation quantified ( n = 5–7 independent experiments). Values are expressed as the mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to the stimulus MOPC-21-treated group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Quantitative Proteomics, Control, Flow Cytometry, Fluorescence, Immunofluorescence, Incubation

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Inhibition of cigarette smoke extract (CSE)-induced CXCL16 expression in arterial endothelial cells. (A) CXCL16 expression was determined by flow cytometry in human umbilical arterial endothelial cells preincubated or not with apocynin (30 µM) or allopurinol (100 µM) for 1 h and then stimulated with 1% CSE for 24 h ( n = 7 independent experiments). Results are expressed as mean of fluorescence intensity (MFI). Values are expressed as the mean ± SEM. * P < 0.05 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group. Endothelial cells were transfected with (B) Nox2, (C) Nox4, or (D) Nox5 siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 5–11 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group in control siRNA transfected cells.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Transfection, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Cigarette smoke extract (CSE)-induced CXCL16 overexpression is decreased by RhoA, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB inhibition in human umbilical arterial endothelial cells (HUAEC). (A) CXCL16 expression was determined by flow cytometey in endothelial cells preincubated or not with a RhoA inhibitor (C3 transferase, 2 µg/ml) for 4 h and then stimulated with 1% CSE for 24 h. Results are expressed as mean of fluorescence intensity (MFI) ( n = 5 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 relative to values in the medium group; + P < 0.05 relative to 1% CSE group. (B) HUAEC were transfected with RhoA siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to their respective group in control siRNA-transfected cells. (C) HUAEC were stimulated with 1% CSE for 24 h. Some cells were pretreated with PD098059 (20 µM), SB202130 (20 µM), or MOL-294 (2.5 µM) for 1 h before CSE stimulation. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 4–7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to values in the 1% CSE group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Over Expression, Inhibition, Expressing, Fluorescence, Transfection, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Percentage of circulating platelets expressing PAC-1, P-selectin, CXCL16, and CXCR6 from active and not active smoking chronic obstructive pulmonary disease (COPD) patients and aged-matched controls by flow cytometry. Platelets were stained with conjugated antibodies against (A) CD41 and PAC-1, (B) CD41 and P-selectin, (C) CD41 and CXCL16, and (D) CD41 and CXCR6. Results are expressed as percentage of positive cells ( n = 15 aged-matched controls, n = 17 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the control group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Leukocyte recruitment by cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and CXCL16 plasma levels from whole blood of active and not active smokers patients with chronic obstructive pulmonary disease (COPD) and aged-matched controls. HUAEC were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, whole blood from patients with COPD active or not active smokers and healthy aged-matched controls incubated (A) without, or (B) with EDTA, was perfused over endothelial monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte adhesion quantified ( n = 13 aged-matched controls, n = 14 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as the mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 or ++ P < 0.01 relative to 1% CSE group; Δ P < 0.05 or ΔΔ P < 0.01 relative to the values in the aged-matched control group. (C) CXCL16 plasmatic levels were measured by ELISA ( n = 17 aged-matched controls, n = 17 active smokers COPD patients, n = 18 ex-smokers COPD patients). Values are expressed as the mean ± SEM.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Clinical Proteomics, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Effect of cigarette smoke (CS) exposure in CXCR6-expressing and CXCRC6 knockout mice. Heterozygous (CXCR6 −/+ ) and homozygous (CXCR6 −/– ) mice were exposed or not to CS for 3 days and responses were examined 16 h later. (A) Leukocyte–arteriolar endothelium interactions was measured by intravital microscopy. Results are expressed as mean ± SEM ( n = 5–8 animals per group). * P < 0.05 or ** P < 0.01 relative to non-exposed animals; + P < 0.05 relative to CXCR6 −/+ mice. (B) Relative quantification of CXCL16 and β-actin mRNA was determined by RT-PCR. Columns show fold increase in expression of CXCL16 mRNA relative to control GAPDH values ( n = 5 independent experiments). Values are represented as mean ± SEM of the 2 −ΔΔCt values. ** P < 0.01 relative to non-exposed animals. (C) Cremaster muscle was fixed for CXCL16 and endothelium (CD31) staining. CXCL16 expression is shown in green (stained with an Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody) and vessel endothelium (red) was stained with a PE-conjugated anti-mouse CD31 monoclonal antibody. Overlapping expression of CXCL16 and CD31 is shown in yellow. Results are representative of five to six animals per group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Knock-Out, Intravital Microscopy, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Staining

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Expression and function of CXCL16 in cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and effect of CXCL16 neutralizing antibody on neutrophil and mononuclear leukocyte adhesion. HUAEC were stimulated with 1% CSE, INF-γ, or TNF-α (20 ng/ml) for 1, 4, or 24 h. Relative quantification of mRNA levels for CXCL16 and GAPDH after (A) 1 h and (B) 4 h ( n = 5–8 independent experiments). Columns show fold increase in CXCL16 mRNA expression relative to control GAPDH. Values are expressed as mean ± SEM of the 2 −ΔΔCt values. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (C) Protein expression was determined by flow cytometry. Results are expressed as mean of fluorescence intensity (MFI) ( n = 7–8 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (D) Following a similar protocol, CXCL16 was visualized in non-permeabilized HUAEC by immunofluorescence (green). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) ( n = 4–5 independent experiments). (E,F) Endothelial cells were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, human neutrophils (E) or mononuclear cells (F) (1 × 10 6 cells/ml) incubated with or without EDTA were perfused over the monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte accumulation quantified ( n = 5–7 independent experiments). Values are expressed as the mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to the stimulus MOPC-21-treated group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Quantitative Proteomics, Control, Flow Cytometry, Fluorescence, Immunofluorescence, Incubation

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Inhibition of cigarette smoke extract (CSE)-induced CXCL16 expression in arterial endothelial cells. (A) CXCL16 expression was determined by flow cytometry in human umbilical arterial endothelial cells preincubated or not with apocynin (30 µM) or allopurinol (100 µM) for 1 h and then stimulated with 1% CSE for 24 h ( n = 7 independent experiments). Results are expressed as mean of fluorescence intensity (MFI). Values are expressed as the mean ± SEM. * P < 0.05 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group. Endothelial cells were transfected with (B) Nox2, (C) Nox4, or (D) Nox5 siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 5–11 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group in control siRNA transfected cells.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Transfection, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Cigarette smoke extract (CSE)-induced CXCL16 overexpression is decreased by RhoA, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB inhibition in human umbilical arterial endothelial cells (HUAEC). (A) CXCL16 expression was determined by flow cytometey in endothelial cells preincubated or not with a RhoA inhibitor (C3 transferase, 2 µg/ml) for 4 h and then stimulated with 1% CSE for 24 h. Results are expressed as mean of fluorescence intensity (MFI) ( n = 5 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 relative to values in the medium group; + P < 0.05 relative to 1% CSE group. (B) HUAEC were transfected with RhoA siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to their respective group in control siRNA-transfected cells. (C) HUAEC were stimulated with 1% CSE for 24 h. Some cells were pretreated with PD098059 (20 µM), SB202130 (20 µM), or MOL-294 (2.5 µM) for 1 h before CSE stimulation. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 4–7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to values in the 1% CSE group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Over Expression, Inhibition, Expressing, Fluorescence, Transfection, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Percentage of circulating platelets expressing PAC-1, P-selectin, CXCL16, and CXCR6 from active and not active smoking chronic obstructive pulmonary disease (COPD) patients and aged-matched controls by flow cytometry. Platelets were stained with conjugated antibodies against (A) CD41 and PAC-1, (B) CD41 and P-selectin, (C) CD41 and CXCL16, and (D) CD41 and CXCR6. Results are expressed as percentage of positive cells ( n = 15 aged-matched controls, n = 17 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the control group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Leukocyte recruitment by cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and CXCL16 plasma levels from whole blood of active and not active smokers patients with chronic obstructive pulmonary disease (COPD) and aged-matched controls. HUAEC were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, whole blood from patients with COPD active or not active smokers and healthy aged-matched controls incubated (A) without, or (B) with EDTA, was perfused over endothelial monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte adhesion quantified ( n = 13 aged-matched controls, n = 14 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as the mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 or ++ P < 0.01 relative to 1% CSE group; Δ P < 0.05 or ΔΔ P < 0.01 relative to the values in the aged-matched control group. (C) CXCL16 plasmatic levels were measured by ELISA ( n = 17 aged-matched controls, n = 17 active smokers COPD patients, n = 18 ex-smokers COPD patients). Values are expressed as the mean ± SEM.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Clinical Proteomics, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Effect of cigarette smoke (CS) exposure in CXCR6-expressing and CXCRC6 knockout mice. Heterozygous (CXCR6 −/+ ) and homozygous (CXCR6 −/– ) mice were exposed or not to CS for 3 days and responses were examined 16 h later. (A) Leukocyte–arteriolar endothelium interactions was measured by intravital microscopy. Results are expressed as mean ± SEM ( n = 5–8 animals per group). * P < 0.05 or ** P < 0.01 relative to non-exposed animals; + P < 0.05 relative to CXCR6 −/+ mice. (B) Relative quantification of CXCL16 and β-actin mRNA was determined by RT-PCR. Columns show fold increase in expression of CXCL16 mRNA relative to control GAPDH values ( n = 5 independent experiments). Values are represented as mean ± SEM of the 2 −ΔΔCt values. ** P < 0.01 relative to non-exposed animals. (C) Cremaster muscle was fixed for CXCL16 and endothelium (CD31) staining. CXCL16 expression is shown in green (stained with an Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody) and vessel endothelium (red) was stained with a PE-conjugated anti-mouse CD31 monoclonal antibody. Overlapping expression of CXCL16 and CD31 is shown in yellow. Results are representative of five to six animals per group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Knock-Out, Intravital Microscopy, Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Staining

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Mo were cultured with or without the stimuli listed on the x-axis (concentrations as in Figure 1). Cell culture supernatants were collected at indicated time points and analyzed for the presence of CXCL16 by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± SEM from 3 independent experiments using 3 different donors with each condition tested in triplicate.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Mo were cultured with or without the indicated stimuli. Summary data are shown for the percent of CXCL16+ADAM10+ cells as a function of cell stimulus. Data are from 3 independent experiments using 3 different donors and are presented as the mean ± SEM.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Cell Culture

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Static adhesion of Mϕ to CASMCs, which were cultured with 25 µg/ml oxLDL for 24 h. The adhesion assay was performed as described in Materials and Methods. A, Prior to the adhesion assay, Mo or Mϕ were blocked for 45 min at 37°C with the following agents: 5 µg/ml isotype control rat IgG2b or CX3CR1 mAb or 4 µg/ml rat IgG2a or CXCL16 mAb. B, Mo were nucleofected with 150 nM of the indicated CXCL16-specific (CXCL16) or negative control (-Cmed) sRNAi and then cultured for 24 h in the presence or absence of atherogenic lipids. Data in A are from 3 independent experiments using Mo from 3 different donors with each condition tested in duplicate. Results in B are from 4 independent experiments using 4 different donors, each condition tested in triplicate. Data are expressed as the mean ± SEM.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Cell Culture, Cell Adhesion Assay, Control, Negative Control

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Uptake of Dil-oxLDL was performed as detailed in Materials and Methods. Cell-associated fluorescence was expressed as nanograms of Dil-oxLDL per milligram of total cellular protein. A, Prior to Dil-oxLDL internalization assay, Mϕ were incubated for 45 min at 37°C with 4 µg/ml of isotype control or mAbs recognizing CD36, SR-A, CXCL16 or CD68. B, Mo were nucleofected with 150 nM of the indicated CXCL16-specific (CXCL16) or negative control (-Cmed) sRNAi and then cultured for 24 h with or without 50 µg/ml oxLDL, 10 µg/ml 9-HODE or 10 µg/ml 13-HODE. Values shown are the mean (±SEM) of n=4 donors for each condition.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Fluorescence, Incubation, Control, Negative Control, Cell Culture

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Uptake of Dil-HDL was performed as described in Materials and Methods. Cell-associated fluorescence was expressed as nanograms of Dil-HDL per milligram of total cellular protein. A, Mϕ, which were differentiated in presence of 50 µg/ml oxLDL for 24 h, were incubated with 4 µg/ml rat IgG2a or CXCL16 mAb before Dil-HDL internalization assay. B, Mo were nucleofected with 150 nM of CXCL16-specific (CXCL16) or negative control (-Cmed) sRNAi and then cultured for 24 h with or without 50 µg/ml oxLDL, 10 µg/ml 9-HODE or 10 µg/ml 13-HODE. Data represent the mean ± SEM from 4 donors. *p < 0.05 and **p < 0.01 vs the corresponding unblocked control value in A.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Fluorescence, Incubation, Negative Control, Cell Culture, Control

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Mo were nucleofected with 150 nM of CXCL16-specific (CXCL16) or negative control (-Cmed) sRNAi and then cultured for 24 h with 50 µg/ml acLDL in the presence of radioactive cholesterol. Cells were equilibrated, washed and efflux was stimulated by incubating Mϕ in media containing lipid-poor acceptors for 6, 12 or 24 h. A, Macrophage 3H-Cholesterol efflux to human HDL. B, The release of 3H-Cholesterol to purified human ApoA-1. Results represent the mean ± SEM and are from 3 independent experiments using 3 different donors with each condition tested in triplicate. *p < 0.05, comparing the indicated value to the corresponding negative control sRNAi (-Cmed) value for the same time point.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Negative Control, Cell Culture, Purification

Journal:

Article Title: Atherogenic Lipids Induce HDL Uptake and Cholesterol Efflux in Human Macrophages by Upregulating Transmembrane Chemokine CXCL16, without Engaging CXCL16-dependent Cell Adhesion

doi: 10.4049/jimmunol.0804112

Figure Lengend Snippet: Mo were nucleofected with 150 nM of CXCL16-specific (CXCL16) or negative control (-Cmed) sRNAi and then cultured for 12, 24 or 48 h with or without 50 µg/ml oxLDL. Cells were harvested at indicated times, RNA isolated as described in Materials and Methods, and accumulation of mRNA for ABCA1 (A), ABCG1(B) and ApoE(C) was examined. Results represent the mean ± SEM and are from 5 independent experiments with each condition tested in triplicate. Donors were the same in A–C. *p < 0.05 and **p < 0.01 vs the corresponding negative control sRNAi (-Cmed) value for the same time point.

Article Snippet: Monoclonal antibodies (mAbs) included: rat anti-human CX3CR1 (Caltag-MedSystems Ltd, Buckingham, UK), rat anti-human CXCL16 (R&D Systems Europe Ltd, Abingdon, UK), mouse anti-human CD14, CD36, CD68 (BD Biosciences, Oxford, UK) and SR-A (CosmoBio, Tokyo, Japan).

Techniques: Negative Control, Cell Culture, Isolation