title: AlphaMbeta2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to alphaM I domain that is divalent cation-sensitive.
abstract: The beta2 (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 alphaM subunit, which directly induces adhesion of alphaMbeta2-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 alphaM I domain. The alphaM I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of alphaMbeta2 ligand-binding affinity. Thus, these studies have identified a novel alphaM I domain activation epitope of alphaMbeta2 and support the idea that the I domain modulates the activational state of the beta2 integrins.
authors.affiliation: Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan, USA.
authors.forename: R P
authors.initials: RP
authors.lastname: Orchekowski
authors.forename: J
authors.initials: J
authors.lastname: Plescia
authors.forename: D C
authors.initials: DC
authors.lastname: Altieri
authors.forename: M L
authors.initials: ML
authors.lastname: Bajt
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism; D019556:COS Cells / Q000166:cytology / Q000378:metabolism; D002413:Cations, Divalent; D002448:Cell Adhesion / Q000502:physiology; D003179:Complement C3b / Q000378:metabolism; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D000939:Epitopes; D016177:Macrophage-1 Antigen; D018799:Intercellular Adhesion Molecule-1; D008345:Manganese; D003179:Complement C3b; D005340:Fibrinogen; D008274:Magnesium