Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1).
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.
Berman
P W
PW
Department of Immunology, Genentech, Inc., South San Francisco, California 94080.
Nakamura
G R
GR
Riddle
L
L
Chiu
H
H
Fisher
K
K
Champe
M
M
Gray
A M
AM
Ward
P
P
Fong
S
S
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
D002241:Carbohydrates; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins