Lymphocyte adhesion through very late antigen 4: evidence for a novel binding site in the alternatively spliced domain of vascular cell adhesion molecule 1 and an additional alpha 4 integrin counter-receptor on stimulated endothelium

Recent studies demonstrate that alternative splicing of mRNA from a single gene can produce two forms of vascular cell adhesion molecule 1 (VCAM-1): a six-immunoglobulin (Ig) domain form (VCAM-6D) and a seven-Ig domain form (VCAM-7D). Using a COS cell transient expression assay, we investigated whether VCAM-6D and VCAM-7D differ functionally in adhesion to the integrin VLA-4 (CD49d/CD29) on lymphoid cells. Binding of lymphoid cell lines and peripheral blood lymphocytes was completely blocked by VLA-4 monoclonal antibody (mAb) and one VCAM-1 mAb (4B9) to both VCAM-6D and VCAM-7D, whereas one VCAM-1 mAb (E1/6) completely blocked binding to VCAM-6D but only partially inhibited binding to VCAM-7D. We conclude that there is one VLA-4 binding site in the six Ig domains shared between VCAM-6D and VCAM-7D, and that the alternatively spliced domain 4 present in VCAM-7D provides a second VLA-4 binding site that is blocked by 4B9 but not the E1/6 mAb. We compared the inhibitory effects of anti-VCAM-1 and anti-VLA-4 mAbs on lymphoid cell adhesion to cultured human umbilical vein endothelial cells (HUVEC). The anti-VCAM-1 mAb 4B9 blocked the binding of PBL and lymphoid tumor cells to stimulated HUVEC better than the anti-VCAM-1 mAb E1/6. Because VCAM-7D is the predominant form of VCAM-1 expressed by stimulated endothelial cells, this difference in VCAM-1 mAb inhibition is attributed to lymphoid cell binding to VCAM-7D on stimulated HUVEC. Although the anti-VLA-4 mAb and anti-VCAM-1 mAb 4B9 equally inhibited PBL binding to stimulated HUVEC, mAb 4B9 inhibited the binding of two lymphoid cell lines significantly less than anti-VLA-4 mAb. Combination of 4B9 mAb with function-blocking antiserum to human fibronectin, a second known ligand for VLA-4, also failed to inhibit as much as anti-VLA-4 mAb. These findings suggest that adhesion of lymphoid cell lines through VLA-4 or other alpha 4 integrins may involve inducible counter-receptor(s) on endothelium distinct from either VCAM-1 or fibronectin. Time course experiments indicate that the fraction of alpha 4 integrin-dependent binding that can be blocked by anti-VCAM-1 mAb E1/6 rises and peaks within 2 h of tumor necrosis factor (TNF) stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)