Detection and biochemical characteristics of the receptor for complexes of soluble CD14 and bacterial lipopolysaccharide.
Soluble CD14 (sCD14) has been found to bind LPS and mediate LPS activation of several cell types. It has been postulated that sCD14-LPS complexes induce cell responses by interacting with a cell surface structure, which, in turn, triggers cell activation. There has been no biochemical evidence, however, for a direct interaction of sCD14 with a cell surface structure, and the putative receptor has not been identified. To rigorously test this hypothesis, we studied the interaction of human rsCD14 with cells in the absence of serum and in the presence and the absence of LPS. We found 1) there was specific and saturable binding of 125I-sCD14, indicative of a typical receptor-ligand interaction, to several cell types, including endothelial cells, epithelial cells, astrocytes, and human monocytes; 2) specific binding to all the cell types and IL-6 induction in membrane-bound CD14 (mCD14)-negative cells occurred only when both sCD14 and LPS were present; 3) competitive displacement experiments of 125I-sCD14 binding to astrocytes and Scatchard plots revealed a binding of high affinity (Kd = 3.3 +/- 0.4 nM) and approximately 25,000 single class binding sites/cell; 4) the steady state for the association of 125I-sCD14 was obtained after 180-200 min; 5) chemical cross-linking experiments revealed the association of sCD14 with a binding structure of approximately 216 kDa; 6) binding of 125I-sCD14 to CD14-expressing cell transfectants was about 50% lower than that to nontransfected cells. Maximal binding, however, was recovered after removing mCD14, suggesting that the sCD14-LPS receptor may also interact with mCD14. These results provide direct biochemical evidence for the existence of a cell surface signal-mediating binding structure for LPS-bearing sCD14 and suggest that this structure may represent the signaling unit of the postulated multimeric LPS receptor in mCD14-bearing cells.
Vita
N
N
SANOFI Research, Labège Center, France.
Lefort
S
S
Sozzani
P
P
Reeb
R
R
Richards
S
S
Borysiewicz
L K
LK
Ferrara
P
P
Labéta
M O
MO
D000818:Animals; D001254:Astrocytoma; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003432:Cross-Linking Reagents; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011971:Receptors, Immunologic / Q000032:analysis* / Q000737:chemistry*; D012995:Solubility; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011971:Receptors, Immunologic