AUGMENTATION OF STIMULATED EOSINOPHIL DEGRANULATION BY VLA-4 (CD49D)-MEDIATED ADHESION TO FIBRONECTIN

We examined the effect of VLA-4-mediated adhesion to purified fibronectin (FN) on the stimulated release of the granular protein, eosinophil peroxidase (EPO), in human peripheral blood eosinophils. In initial studies, optimal time-course and concentration-effect relationships were determined; eosinophil adhesion to FN-coated styrene plates was maximal in wells coated with 10 mu g/ml FN after incubation in the wells for 60 min (17,097 +/- 3,670 adherent eosinophils/well versus 6,789 +/- 925 adherent eosinophils/well in control wells; P < 0.005). Treatment of eosinophils with 10(-8) to 10(-6) M formylmethionylleucyl-phenylalanine (FMLP) + 5 mu g/ml cytochalasin B (CYTB) caused a concentration-dependent increase in EPO release, which was augmented by preincubation of eosinophils for 120 min in FN-coated (10 mu g/ml) styrene wells versus eosinophils preincubated in control wells. At 10(-6) M FMLP + CYTB, initial adhesion to FN for 120 min caused an increase in the secretion of EPO from 367 +/- 26 to 485 +/- 25 ng/10(6) eosinophils (P = 0.0001). Treatment of eosinophils during incubation in FN-coated wells with the anti-VLA-4 monoclonal antibody HP2/1 attenuated stimulated EPO secretion caused by 10(-6) M FMLP + CYTB from 497 +/- 40 to 285 +/- 26 ng/10(6) eosinophils (P < 0.02). Similarly, treatment with HP2/1 caused a decrease in eosinophil adhesion to FN-coated styrene from 12,693 +/- 1,866 to 6,206 +/- 852 adherent cells/FN-coated well (P < 0.001). Treatment with the anti-CD18 monoclonal antibody, R15.7, and the nonbinding control monoclonal antibody, 3G8, at the same concentration as for HP2/1 had no effect on EPO secretion caused by FMLP + CYTB or eosinophil adhesion to FN-coated styrene. We demonstrate that the integrin adhesion molecule VLA-4 selectively mediates adhesion of isolated human eosinophils to purified FN in vitro and that the VLA-4-mediated eosinophil adhesion to FN also augments stimulated eosinophil degranulation.