Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes.
The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.
Pulido
R
R
Sección de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.
Elices
M J
MJ
Campanero
M R
MR
Osborn
L
L
Schiffer
S
S
García-Pardo
A
A
Lobb
R
R
Hemler
M E
ME
Sánchez-Madrid
F
F
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D002448:Cell Adhesion; D002460:Cell Line; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D039001:Integrin alpha Chains; D019012:Integrin beta1; D016023:Integrins / Q000276:immunology*; D016029:Receptors, Very Late Antigen / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C063493:CDw49d; D000939:Epitopes; D039001:Integrin alpha Chains; D019012:Integrin beta1; D016023:Integrins; D016029:Receptors, Very Late Antigen