ACTIVATION-DEPENDENT AND INDEPENDENT VLA-4 BINDING-SITES ON VASCULAR CELL-ADHESION MOLECULE-1

Vascular cell adhesion molecule-1 (VCAM) is a cytokine-inducible membe r of the immunoglobulin superfamily which binds to the integrin VLA-4. VCAM is expressed predominantly on the vascular endothelium where it is involved in the recruitment of mononuclear cells and lymphocytes to sites of inflammation. Two forms of VCAM containing six and seven Ig domains (VCAM-6d; VCAM-7d) are generated by alternative splicing but t he physiological significance of this is unknown. We have utilised VCA M deletion mutants, VCAM-transfected cell lines and monoclonal antibod ies to assess the functional importance of the individual VCAM domains . We have identified two binding sites on VCAM-7d located in domains 1 and 4 that are involved in the adhesion of the U937 human myelomonocy tic cell line. Adhesion to domain 1 is temperature-independent, inhibi ted by the anti-VCAM mAbs 4B2 or IE10, and insensitive to PMA activati on. In contrast, adhesion to domain 4 is temperature sensitive, unaffe cted by mAbs 4B2 or IE10 and augmented by PMA. Adhesion to both domain s can be totally inhibited by the anti-VLA4 mAb, 2B4. The anti-VCAM mA b 4B2 inhibits adhesion of U937 cells to stably transfected VCAM 7d-CH O cells at 4 degrees C, but, at 37 degrees C the effect of 4B2 on adhe sion is modest with incubation times of less than 60 minutes duration. With longer incubation times, its effectiveness gradually increases, so that by 2 hours >75% of the response can be blocked. Co-incubation with PMA prevents this time-dependent enhancement of 4B2 efficacy but has no significant effect on the inhibitory activity of the anti-VLA-4 mAb 2B4. These data can be explained by postulating a two stage ligan d-receptor interaction that involves activation-induced changes in the avidity of VLA-4 for domain 4 of VCAM.