Atomic force microscopy measurement of leukocyte-endothelial interaction

1 Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101; 2 Department of Medicine, The University of Tokyo, Tokyo 113-8655, Japan; 3 Department of Pediatrics, State University of New York, Stony Brook 11794; and 4 Departments of Medicine and Pharmacology, New York Medical College, Valhalla, New York 10595 Submitted 28 May 2003 ; accepted in final form 10 September 2003 Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40–100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor- -activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against 1 -integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti- V 3 . Interestingly, adhesion was also inhibited in a dose-dependent manner (IC 50 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti- 1 and anti- V 3 antibodies. In conclusion, these data demonstrate the role played by 1 -integrins in leukocyte-endothelial adhesion and transmigration and the role played by V 3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes. integrins; cell-cell adhesion; arginine-glycine-aspartic acid peptide Address for reprint requests and other correspondence: V. T. Moy, Dept. of Physiology and Biophysics, Univ. of Miami School of Medicine, Miami, FL 33101-6430 (E-mail: vmoy{at}miami.edu ).