VASCULAR CELL-ADHESION MOLECULE (VCAM)-IG FUSION PROTEIN DEFINES DISTINCT AFFINITY STATES OF THE VERY LATE ANTIGEN-4 (VLA-4) RECEPTOR

The Very Late Antigen-4 receptor (VLA-4) (alpha(4) beta(1)) is constit utively expressed on leukocytes and plays a role in cell trafficking, activation and development through its interaction with two alternativ e ligands, Vascular Cell Adhesion Molecule (VCAM-1) and fibronectin (F N). VLA-4-dependent cell adhesion is augmented by various stimuli, suc h as divalent cations, certain beta(1)-specific monoclonal antibodies (mAbs) and cell activation. However, the steps of the adhesive process which they affect are currently undefined. In order to investigate wh ether or not these stimuli affect the primary step, VLA-4/ligand bindi ng, we employed a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a s oluble ligand for VLA-4. Using this soluble ligand, we have directly d emonstrated that the VLA-4 receptor can exist in at least three differ ent affinity states on the cell surface. Two distinct high affinity st ates are induced on normal peripheral blood T cells, one by the anti-b eta 1 mAb TS2/16, and one of 15-20 fold higher affinity by the divalen t cation Mn2+. Interestingly, activation through the T cell receptor ( TcR), through CD31 or by the Macrophage Inflammatory Protein-lp chemok ine (MIP-1 beta) do not detectably increase VLA-4 affinity although th ey do augment VLA-4 dependent cell adhesion in vitro. Thus, VCAM-Ig bi nding defines high affinity VLA-4 receptors, revealing unique effects of the TS2/16 mAb and Mn2+ cations in vitro, and distinguishes VLA-4/V CAM interactions from subsequent steps in cell adhesion.