Human phagocytes have multiple lipid A-binding sites.
Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.
Golenbock
D T
DT
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021.
Hampton
R Y
RY
Raetz
C R
CR
Wright
S D
SD
D000943:Antigens, Differentiation / Q000502:physiology; D001665:Binding Sites; D018845:CD11 Antigens; D018821:CD18 Antigens; D016264:Dextran Sulfate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008050:Lipid A / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010586:Phagocytes / Q000378:metabolism*; D016028:Receptors, Leukocyte-Adhesion / Q000502:physiology
D000943:Antigens, Differentiation; D018845:CD11 Antigens; D018821:CD18 Antigens; D008050:Lipid A; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016028:Receptors, Leukocyte-Adhesion; D016264:Dextran Sulfate