Dynamic regulation of LFA-1 and Mac-1 during neutrophil extravasation in live mice (CAM1P.232)

Abstract LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are key integrins that regulate leukocyte adhesion and migration in inflamed tissues. Despite considerable advances in our understanding of leukocyte migration, real-time regulation of leukocyte integrins in vivo is not well characterized. To this end, we generated knock-in mice in which CD11a, CD11b, and CD18 were fused with mYFP and mCFP, respectively, to generate the CD11a-mYFP, CD11b-mYFP, and CD18-mCFP mice. We then crossed these mice to generate LFA-1 FRET (CD11a-mYFP/CD18-mCFP) and Mac-1 FRET (CD11b-mYFP/CD18-mCFP) mice. Using FRET assay with multiphoton intravital microscopy (MP-IVM), we now can directly measure LFA-1 and Mac-1 activation during neutrophil extravasation in inflamed tissues. Neutrophils showed distinct patterns of distribution of active integrins and their clustering at different steps in the multistep extravasation cascade. Experiments with blocking antibodies and knock-out mice confirmed that LFA-1 and Mac-1 are regulated by distinct activation kinetics in vivo. Our data suggest that these knock-in mice will be useful to visualize the subcellular distribution and activation of LFA-1 and Mac-1 in real-time during leukocyte migration in intact tissues.