Kinetics of LFA-1 binding to ICAM-1 studied in a cell-free system

Neutrophil capture on inflamed endothelium is controlled by dynamic regulation of the integrin CD11a/CD18 (LFA-1). Small molecules, antibodies, and certain divalent cations binding to specific epitopes on the integrin are able to stabilize either a closed (low affinity) or open (high affinity) state. To determine the relationship between LFA-1 conformation and affinity for ICAM-1 we assembled a cell-free system consisting of CD11a/CD18 heterodimer adhered to latex microspheres. The kinetics of dimeric ICAM-1 binding to the LFA-1 on the microspheres was measured via flow cytometry and a real time conformational shift into a lower affinity state was observed by addition of a small molecule inhibitor.