Journal of Immunological Methods 240 (2000) 157–164 www.elsevier.nl / locate / jim A surrogate method for assessment of b -integrin-dependent 2 adhesion of human eosinophils to ICAM-1 Xiangdong Zhu, Ramnath Subbaraman, Hiroyuki Sano, Benjamin Jacobs, * ˜ Akiko Sano, Evan Boetticher, Nilda M. Munoz, Alan R. Leff Section of Pulmonary and Critical Care Medicine, Department of Medicine and Department of Pharmacological and Physiological Sciences, Pediatrics, Anesthesia and Critical Care, and Committees on Clinical Pharmacology and Cell Physiology, Division of Biological Sciences, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA Received 29 December 1999; received in revised form 4 April 2000; accepted 5 April 2000 Abstract We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by b -integrin 2 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 was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 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 or 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, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for b -integrin; neither a-CD49d mAb directed 2 against the a -chain or a-CD29 directed against the common b -chain of VLA-4 blocked adhesion to BSA or ICAM-1 4 1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to b -integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to 2 those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.  2000 Elsevier Science B.V. All rights reserved. Keywords: Molecular adhesion; Adhesion assay; Inflammatory cells 1. Introduction Abbreviations: BSA, bovine serum albumin; EPO, eosinophil peroxidase; fMLP, formyl-met-leu-phe; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; Eosinophil adhesion by surface integrin to im- MEK, mitogen activated protein kinase kinase; PAF, platelet- munoglobulin supergene, ICAM-1, is an essential activating factor; PI-3 kinase, phosphatidylinositol 3-kinase step for cell migration in allergic inflammation as in *Corresponding author. Tel.: 11-773-702-1859; fax: 11-773- asthma (Wegner et al., 1990; Leff et al., 1991; 702-9181. E-mail address: aleff@medicine.bsd.uchicago.edu (A.R. Leff) Wardlaw et al., 1994). The most widely used method 0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759(00)00192-7