Analysis of integrin (CD11b/CD18) movement during neutrophil adhesion and migration on endothelial cells

Little is known of the distribution of cell surface molecules during the adhesion and migration of leucocytes on endothelial cells. We have used confocal microscopy and a Fab fragment of a non-inhibitory monoclonal antibody recognizing the integrin CD11b/CD18 (Mac-1) to study the movement of this adhesion molecule over time. We found that during the initial stage of neutrophil contact with TNF-α activated human umbilical vein endothelial cells (HUVEC), there is a rapid accumulation of Mac-1 at the contact area between the two cell types. As the neutrophil spreads, Mac-1 redistributes away from this initial contact area. During neutrophil migration on HUVEC, Mac-1 was redistributed to the leading edge of the migrating cell, suggesting that the existing cell surface pool of adhesion molecules is dynamic and can be recruited to the leading front as the cell changes direction. As neutrophils migrate on HUVEC, Mac-1-dense macroaggregates are rapidly formed and broken down at the contact plane between the two cells. The confocal microscope, coupled with the use of non-inhibitory antibodies labelled with photostable fluorophores, is a useful tool for the study of the movement of cell surface molecules over time.