A surrogate method for assessment of beta(2)-integrin-dependent adhesion of human eosinophils to ICAM-1

We have developed and validated an inexpensive and equivalent method for me asuring eosinophil adhesion by beta(2)-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene . The number of adherent eosinophils on BSA or ICAM-1 coated microplates wa s quantified by residual eosinophil peroxidase activity. Non-stimulated eos inophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimul ation, either BSA or ICAM-1 caused comparable and concentration-dependent a dhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 1 5 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA o r ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fM LP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for beta(2)-integrin; neither alpha-CD 49d mAb directed against the alpha(4)-chain or alpha-CD29 directed against the common beta(1)-chain of VLA-4 blocked adhesion to BSA or ICAM-1 control s. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinosit ol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated prote in kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinop hil adhesion to either BSA or ICAM-1 comparably. These results indicate tha t BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to beta(2)-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable fo r assessment of signal transduction pathways mediating adhesion. (C) 2000 E lsevier Science B.V. All rights reserved.