Abstract
The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is central to the regulation of adhesion in leukocytes. In this report, we investigated the mechanisms by which phorbol myristate acetate (PMA) promotes LFA-1-dependent cell adhesion. The adhesion of PMA-stimulated cells to immobilized ICAM-1 was quantified in direct force measurements acquired by atomic force microscopy (AFM). Enhanced adhesion of PMA-stimulated cells to immobilized ICAM-1 stemmed from an increase in the number of LFA-1/ICAM1 complexes formed between the two opposing surfaces on contact, rather than by affinity modulation of LFA-1. Single molecule force measurements revealed that the force spectrum of the LFA-1/ICAM-1 complex formed by PMA-stimulated cells is identical to the force spectrum of the complex formed by resting cells. Thus, PMA stimulation does not modify the mechanical strength of the individual LFA-1/ICAM-1 interaction. Instead, the enhanced cell adhesion of PMA-stimulated cells appeared to be a complex process that is correlated with changes in the mechanical properties of the cell. We estimated that changes in the elasticity of the cell gave rise to a greater than 10-fold increase in cell adhesion. We investigated this further by examining the contribution of LFA-1 receptor redistribution on the cell surface to cell adhesion. Using cell cross-linking that retarded receptor movement on the cell surface, we found a 20% reduction in the energy of de-adhesion. Finally, we examined alternative methods of cell stimulation that relied on increases in [Ca2+]i. The use of thapsigargin and ionomycin revealed similar increases in the energy of de-adhesion as stimulation of cells with PMA.