Measurement of neutrophil and eosinophil adhesion to E-selectin, VCAM-1, and ICAM-1 by the use of tranfected fibroblast cell lines

Abstract

A method which enables the specific measurement of neutrophil and eosinophil adhesion to the endothelial cell adherence receptors E-selectin, VCAM-1 and ICAM-1 has been developed. The method is based on continuous cultures of cell lines of transfected hamster kidney fibroblasts (BHK-21), that selectively express each of the endothelial cell adhesions molecules. Isolated granulocytes are added to the cultured adherent fibroblasts at a ratio of 20:1 and the cells are coincubated for 60 min at 37°C. After removal of the nonadherent granulocytes the amount of adherent granulocytes could be measured by addition of detergent and a peroxidase substrate. Selective measurement of neutrophil and eosinophil adhesion was accomplished by addition of detergent to the adherent cells, collection of extracts followed by measurement of the concentration of an eosinophil (eosinophil cationic protein) and a neutrophil (myeloperoxidase) granule protein, respectively, in the extracts. At basal conditions neutrophils and eosinophils showed significant adhesion to E-selectin and eosinophils a low degree of adhesion to VCAM-1. Significant adhesion of neutrophils and eosinophils to ICAM-1 and of eosinophils to VCAM-1 was selectively induced by addition of manganese ions (Mn 2+) at a concentration of 0.5 mmol/1. Neutrophils demonstrated a significantly higher adhesion to E-selectin than eosinophils, while eosinophil adhesion to ICAM-1 was significantly higher than that of neutrophils. In conclusion, a method to compare the adhesive capacity of neutrophil and eosinophil granulocytes towards specific endothelial cell adhesion molecules has been developed.