Journal: Nature Communications
Article Title: EGFL7 reduces CNS inflammation in mouse
Figure Lengend Snippet: T lymphocytes express ligand αvβ3 integrin and adhere to EGFL7. a Colocalization of CD4+ cells (green) and EGFL7 (red) in perivascular ECM area in MS brain lesions (upper panel = frontal, lower panel = temporal). Collagen IV = white. Nuclei (DAPI) = blue. Representative of n = 4 MS donors, ≥ 3 lesions/donor. b Expression of αvβ3 integrin on human CD14 monocytes ex vivo (left), memory CD4 T lymphocytes ex vivo (center), and in vitro activated memory CD4 T lymphocytes (right), as assessed by flow cytometry. Representative of n = 6 donors. c Expression of αvβ3 integrin on human CD4 T lymphocytes ex vivo and upon activation. Percentage and mean fluorescence intensity (MFI). n = 6 donors. d Expression of αvβ3 integrin on human CD4 T lymphocytes ex vivo from healthy controls, untreated MS or treated MS. Flow cytometry analysis. 1 dot = 1 donor. e , f Expression of αvβ3 integrin by murine CD4 T lymphocytes ( e ) ex vivo and on day 1–5 in vitro upon activation, and ( f ) in naïve and MOG 35–55 -immunized (EAE) mice at different time points of EAE. n = 4–5 mice/condition, 1 dot = 1 mouse. g – k Adhesion assay. g Representative microscopy images of CFSE-labeled activated human CD4 T lymphocytes adhering to rhEGFL7 or BSA 1% after 24 h. Scale bar = 100 μm. Amount of human ( h ) and murine ( j ) activated CFSE-labeled CD4 T cells adhering to coated rh or rmEGFL7, ICAM-1 or fibronectin, normalized to BSA 1%, according to fluorescence intensity as assessed by plate-reader. n = 6 human donors and n = 8 mice. Amount of human ( i ) and murine ( k ) cells adhering to coated EGFL7, ICAM-1 or fibronectin, following pre-treatment with rhEGFL7 ( i ) or rmEGFL7 ( k ); anti-αvβ3 integrin neutralizing antibody ( i ) or RGD-peptide ( k ); or relevant control (rat serum, isotype or RAD-peptide). n = 7 human donors and n = 8 mice. Statistical analysis performed by Wilcoxon matched-pairs signed rank test ( c ) and one-way ANOVA followed by Tukey’s multiple comparison test ( d – k ). * p
Article Snippet: The following primary antibodies were used: mouse anti-human collagen IV (Abcam; 1:200) or goat anti-human collagen IV (Millipore; 1:80), mouse anti-human CD31 (BD Biosciences; 1:200), rabbit anti-human EGFL7 (clone 57; 1:100), mouse anti-human CD4 (BD Biosciences; 1:80), rat anti-mouse CD31 (BD Biosciences; 1:500), rat anti-mouse laminin-alpha4 (RnD systems; 1:500), donkey anti-mouse IgG-AF488 (Invitrogen; 1:500), rabbit anti-mouse collagen IV (BioRad; 1:100), rat anti-mouse CD4 (BD Biosciences AF647-labeled; 1:200), rabbit anti-mouse fibrinogen (Innovative Research; 1:1500), rabbit anti-mouse CD146 (Abcam; 1:200), rat anti-mouse CD106 (eBioscience; 1:100) and rat anti-mouse CD54 (eBioscience; 1:100).
Techniques: Mass Spectrometry, Expressing, Ex Vivo, In Vitro, Flow Cytometry, Cytometry, Activation Assay, Fluorescence, Mouse Assay, Cell Adhesion Assay, Microscopy, Labeling