Interactions between leukocyte function-associated antigen-1 (LFA-1) with its cognate ligand, intercellular adhesion molecule-1 (ICAM-1) play a crucial role in leukocyte adhesion. Because the cell and its adhesive components are subject to external perturbation from the surrounding flow of blood, it is important to understand the binding properties of the LFA-1/ICAM-1 interaction in both steady state and in the presence of an external pulling force. Here we report on atomic force microscopy (AFM) measurements of the unbinding of LFA-1 from ICAM-1. The single molecule measurements revealed the energy landscape corresponding to the dissociation of the LFA-1/ICAM-1 complex and provided the basis for defining the energetic determinants of the complex at equilibrium and under the influence of an external force. The AFM force measurements were performed in an experimental system consisting of an LFA-1-expressing T cell hybridoma, 3A9, attached to the end of the AFM cantilever and an apposing surface expressing ICAM-1. In measurements covering three orders of magnitude change in force loading rate, the LFA-1/ICAM-1 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier, respectively. The addition of Mg(2+), a cofactor that stabilizes the LFA-1/ICAM-1 interaction, elevated the unbinding force of the complex in the slow loading regime. In contrast, the presence of EDTA suppressed the inner barrier of the LFA-1/ICAM-1 complex. These results suggest that the equilibrium dissociation constant of the LFA-1/ICAM-1 interaction is regulated by the energetics of the outer activation barrier of the complex, while the ability of the complex to resist a pulling force is determined by the divalent cation-dependent inner activation barrier.

Single-molecule measurements of the interaction of leukocyte function-associated antigen-1 (LFA-1), expressed on Jurkat T cells, with intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2) were conducted using atomic force microscopy (AFM). The force spectra (i.e., unbinding force versus loading rate) of both the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions were acquired at a loading rate range covering 3 orders of magnitude (50-60,000 pN/s) and revealed a fast loading regime and a slow loading regime. This indicates that the dissociation of both complexes involves overcoming a steep inner and a wide outer activation barrier. LFA-1 binding to ICAM-1 and ICAM-2 was strengthened in the slow loading regime by the addition of Mg(2+). Differences in the dynamic strength of the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions can be attributed to the presence of wider barriers in the ICAM-2 complex, making it more responsive to a pulling force than the ICAM-1 complex.

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–ICAM-1 complexes formed between the two apposing 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 appears to be a complex process that correlates with changes in the mechanical properties of the cell. We estimate that changes in the elasticity of the cell gave rise to a more than 10-fold increase in cell adhesion.

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple-bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study.

The recruitment of T lymphocytes to lymphoid organs or sites of inflammation is a crucial step in adaptive immunity. These processes require endothelial activation and expression of adhesion molecules, including E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, the complete characterization of the adhesion strength and dynamics between lymphocytes and endothelial cells has been hampered by the lack of sensitive quantitative techniques. Here we report on the application of atomic force microscopy to characterize the interaction between individual pairs of living T lymphocytes (i.e., Jurkat cells) and human umbilical vein endothelial cells (HUVECs). The detachment of individual cell-cell conjugates was a complex process involving several step-like rupture events and the viscoelastic deformation of cells on the scale of several microns. Adhesion between Jurkat cells and activated endothelial cells increased with compression force and contact time, with the most dramatic changes occurring within the first half second of contact. After 0.25 sec of contact, E-selectin, ICAM-1, and VCAM-1 contributed to 18%, 39%, and 41% of total adhesion strength, respectively, suggesting that ICAM-1 and VCAM-1 contributed more than the selectins in supporting cell attachment.