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 measuring 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 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 beta(2)-integrin; neither alpha-CD49d 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 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 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 for assessment of signal transduction pathways mediating adhesion.
Zhu
X
X
Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, IL 60637, USA.
Subbaraman
R
R
Sano
H
H
Jacobs
B
B
Sano
A
A
Boetticher
E
E
Muñoz
N M
NM
Leff
A R
AR
D000730:Androstadienes / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D018821:CD18 Antigens / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000187:drug effects / Q000276:immunology*; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D006969:Hypersensitivity, Immediate; D016023:Integrins / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D015848:Interleukin-5 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D012710:Serum Albumin, Bovine; D000077191:Wortmannin
D000730:Androstadienes; D002070:Butadienes; D018821:CD18 Antigens; D016023:Integrins; D015848:Interleukin-5; D009570:Nitriles; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor; D047428:Protein Kinase Inhibitors; C113580:U 0126; D018799:Intercellular Adhesion Molecule-1; D012710:Serum Albumin, Bovine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019833:Genistein; D000077191:Wortmannin