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(Journal of Leukocyte Biology. 2000;68:641-649.)
© 2000 by Society for Leukocyte Biology

{alpha}Mß2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to {alpha}M I domain that is divalent cation-sensitive

Randal P. Orchekowski*, Janet Plescia{dagger}, Dario C. Altieri{dagger} and Mary Lynn Bajt*

* Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan; and
{dagger} Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut

The ß2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the {alpha}M subunit, which directly induces adhesion of {alpha}Mß2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the {alpha}M I domain. The {alpha}M I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of {alpha}Mß2 ligand-binding affinity. Thus, these studies have identified a novel {alpha}M I domain activation epitope of {alpha}Mß2 and support the idea that the I domain modulates the activational state of the ß2 integrins.

Key Words: adhesion • active conformation • inflammatory immune response




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