title: Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1).
abstract: Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.
authors.affiliation: Department of Immunology, Genentech, Inc., South San Francisco, California 94080.
authors.forename: P W
authors.initials: PW
authors.lastname: Berman
authors.forename: G R
authors.initials: GR
authors.lastname: Nakamura
authors.forename: L
authors.initials: L
authors.lastname: Riddle
authors.forename: H
authors.initials: H
authors.lastname: Chiu
authors.forename: K
authors.initials: K
authors.lastname: Fisher
authors.forename: M
authors.initials: M
authors.lastname: Champe
authors.forename: A M
authors.initials: AM
authors.lastname: Gray
authors.forename: P
authors.initials: P
authors.lastname: Ward
authors.forename: S
authors.initials: S
authors.lastname: Fong
mesh_terms: D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D016177:Macrophage-1 Antigen / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002241:Carbohydrates; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins