Fig. 4: High-affinity LFA-1 and Mac-1 clustering were distinctly localized on neutrophils during transendothelial migration upon stimulation with N-formylmethionyl-leucyl-phenylalanine. | Experimental & Molecular Medicine

Fig. 4: High-affinity LFA-1 and Mac-1 clustering were distinctly localized on neutrophils during transendothelial migration upon stimulation with N-formylmethionyl-leucyl-phenylalanine.

From: LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) distinctly regulate neutrophil extravasation through hotspots I and II

Fig. 4

a CD18 expression (left panel), CD11a expression (middle panel), and the CD18-to-CD11a ratio (right panel) were visualized at two different time points during transendothelial migration of a neutrophil in LFA-1 FRET (CD11a-mYFP/CD18-mCFP) mice. Red, endothelial cellular border imaged with an Alexa 594-stained anti-CD31 antibody. Two-photon intravital two-dimensional imaging of LFA-1 FRET mice was performed. The cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) ratio is visualized using the rainbow scale. The dotted arrow shows the direction of transendothelial migration of the neutrophil. Scale bar, 10 μm. See Video 8. The images are representative of at least five independent videos. b CD18 expression (left panel), CD11b expression (middle panel), and the CD18-to-CD11b ratio (right panel) were visualized at two different time points during transendothelial migration of a neutrophil in Mac-1 FRET (CD11b-mYFP/CD18-mCFP) mice. Red, endothelial cellular border imaged with an Alexa-594 stained anti-CD31 antibody. Two-photon intravital two-dimensional imaging of Mac-1 FRET mice was performed. The CFP/YFP ratio is visualized using the rainbow scale. The dotted arrow shows the direction of transendothelial migration of the neutrophil. Scale bar, 10 μm. See Video 9. The images are representative of at least five independent videos. c, d The affinity and clustering of LFA-1 and Mac-1 were quantified based on CFP, YFP, and the CFP-to-YFP ratio during two-photon intravital imaging of both LFA-1 FRET and Mac-1 FRET mice. *P < 0.0001. A.U. arbitrary units. e The intensities of CD18 and CD11a in LFA-1 mice and CD18 and CD11b in Mac-1 FRET mice were, respectively, quantified along a line from the trailing edge to the leading edge of the transendothelial migratory neutrophils. The images are representative of analyses of at least five independent neutrophils