SumlnaryCD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14, rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14. R cent work has described several serum and cell surface proteins that are necessary for responses of leukocytes to low concentrations of bacterial LPS (endotoxin) (1). LPSbinding protein (LBP),I an acute phase reactant, binds LPS (2) and greatly enhances the sensitivity of cells to LPS (3). Normal serum and plasma also enhance responses to LPS, and a multicomponent factor termed septin has been proposed to serve this function (4). LBP (5) and septin (4) each bind to LPS-coated particles and promote the interaction of these particles with CD14 (6, 7), a glycosylphosphatidylinositol (GPI)-anchored protein of monocytes, macrophages, and PMN (8, 9, 10). CD14 is necessary for serum-or LBPmediated responses of cells to LPS, such as the production 1 Abbreviations used in this paper: CHO, Chinese hamster ovary; ELPS, sheep erythrocytes coated with LPS; ELPS-LBP, ELPS opsonized with LBP; GPI, glycosylphosphatidylinositol; HAP, Dulbecco's PBS with 0.5 U/ml aprotinin, 0.05% human serum albumin, 3 mM D-glucose; HUVEC, human umbilical vein endothelial cells; LBP, LPS-binding protein; NHP, normal human plasma; PD-EDTA, Dulbecco's PBS lacking Ca 2+ and Mg 2+ with 1 mM EDTA; Ra, strain R60; Re, strain R595; rLBP, recombinant LBP; rsCD14, recombinant soluble CD14. of TNF by monocytes (6) and an increase in the adhesive properties of 132-integrins on PMN (7).Cells that do not express CD14, such as endothelial cells, also respond to low concentrations of LPS in the presence of serum. We have shown that these resp...

SummaryWe have recently shown that lipopolysaccharide (LPS)-binding protein (LBP) is a lipid transfer protein that catalyzes two distinct reactions: movement of bacterial LPS (endotoxin) from LPS micelles to soluble CD14 (sCD14) and movement of LPS from micelles to reconstituted high density lipoprotein (R-HDL) particles. Here we show that LBP facilitates a third lipid transfer reaction: movement of LPS from LPS-sCD14 complexes to R-HDL particles. This action of LBP is catalytic, with one molecule of LBP enabling the movement of multiple LPS molecules into R-HDL. LBP-catalyzed movement of LPS from LPS-sCD14 complexes to R-HDL neutralizes the capacity of LPS to stimulate polymorphonuclear leukocytes. Our findings show that LPS may be transferred to R-HDL either by the direct action of LBP or by a two-step reaction in which LPS is first transferred to sCD14 and subsequently to R-HDL We have observed that the two-step pathway of LPS transfer to R-HDL is strongly favored over direct transfer. Neutralization of LPS by LBP and R-HDL was accelerated more than 30-fold by addition of sCD14. Several observations suggest that sCD14 accelerates this reaction by serving as a shuttle for LPS: addition of LBP and sCD14 to LPS micelles resulted in LPS-sCD14 complexes that could diffuse through a 100-kD cutoff filter; LPS-sCD14 complexes appeared transiently during movement of LPS to R-HDL facilitated by purified LBP; and sCD14 could facilitate transfer of LPS to R-HDL without becoming part of the final LPS-R-HDL complex. Complexes of LPS and sCD14 were formed transiently when LPS was incubated in plasma, suggesting that these complexes may play a role as intermediates in the neutralization of LPS under physiological conditions. These findings detail a new activity for sCD14 and suggest a novel mechanism for lipid transfer by LBP.

Lipopolysaccharide binding protein (LBP) is a plasma protein known to facilitate the diffusion of bacterial LPS (endotoxin). LBP catalyzes movement of LPS monomers from LPS aggregates to HDL particles, to phospholipid bilayers, and to a binding site on a second plasma protein, soluble CD14 (sCD14). sCD14 can hasten transfer by receiving an LPS monomer from an LPS aggregate, and then surrendering it to an HDL particle, thus acting as a soluble "shuttle" for an insoluble lipid. Here we show that LBP and sCD14 shuttle not only LPS, but also phospholipids. Phosphatidylinositol (PI), phosphatidylcholine, and a fluorescently labeled derivative of phosphatidylethanolamine (R-PE) are each transferred by LBP from membranes to HDL particles. The transfer could be observed using recombinant LBP and sCD14 or whole human plasma, and the plasma-mediated transfer of PI could be blocked by anti-LBP and partially inhibited by anti-CD14. sCD14 appears to act as a soluble shuttle for phospholipids since direct binding of PI and R-PE to sCD14 was observed and because addition of sCD14 accelerated transfer of these lipids. These studies define a new function for LBP and sCD14 and describe a novel mechanism for the transfer of phospholipids in blood. In further studies, we show evidence suggesting that LBP transfers LPS and phospholipids by reciprocal exchange: LBP-catalyzed binding of R-PE to LPS • sCD14 complexes was accompanied by the exit of LPS from sCD14, and LBP-catalyzed binding of R-PE to sCD14 was accelerated by prior binding of LPS to sCD14. Binding of one lipid is thus functionally coupled with the release of a second.

Phospholipid transfer protein (PLTP) and lipopolysaccharide-binding protein (LBP) are lipid transfer proteins found in human plasma. PLTP shares 24% sequence similarity with LBP. PLTP mediates the transfer and exchange of phospholipids between lipoprotein particles, whereas LBP transfers bacterial lipopolysaccharide (LPS) either to lipoprotein particles or to CD14, a soluble and cell-surface receptor for LPS. We asked whether PLTP could interact with LPS and mediate the transfer of LPS to lipoproteins or to CD14. PLTP was able to bind and neutralize LPS: incubation of LPS with purified recombinant PLTP (rPLTP) resulted in the inhibition of the ability of LPS to stimulate adhesive responses of neutrophils, and addition of rPLTP to blood inhibited cytokine production in response to LPS. Transfer of LPS by rPLTP was examined using fluorescence dequenching experiments and native gel electrophoresis. The results suggested that rPLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to reconstituted HDL particles, but it did not transfer LPS to CD14. Consonant with these findings, rPLTP did not mediate CD14-dependent adhesive responses of neutrophils to LPS. These results suggest that while PLTP and LBP both bind and transfer LPS, PLTP is unable to transfer LPS to CD14 and thus does not mediate responses of cells to LPS. Lipopolysaccharide (LPS 1; endotoxin) is a membrane lipid of Gram-negative bacteria that acts as a potent inflammatory stimulus in humans and other mammals (1). Recent work has suggested that a plasma protein called LPS-binding protein (LBP) is important in trafficking LPS in blood. LBP can transfer LPS to lipoprotein particles, resulting in the functional neutralization of LPS (2). LBP also mediates functional responses of cells to LPS (3) by facilitating the transfer of LPS to CD14 (4), a glycoprotein found both as a soluble monomer in the blood (soluble CD14, sCD14) and as a glycosylphosphatidylinositol-linked membrane protein (mCD14) on monocytes, macrophages, and neutrophils.LBP has sequence similarity to two other plasma lipid transfer proteins, phospholipid transfer protein (PLTP, 24% amino acid identity) (5) and cholesteryl ester transfer protein (CETP, 23% amino acid identity) (3). LBP, PLTP, and CETP are found in plasma associated with HDL particles (2, 6, 7). CETP facilitates the transfer of cholesteryl esters, triglycerides, and phospholipids between lipoproteins (7). PLTP mediates the exchange and transfer of phospholipids between lipoprotein particles (6). PLTP also mediates high density lipoprotein (HDL) conversion, the transformation of HDL into smaller and larger particles (8 -10). Through these activities, PLTP may regulate HDL level and composition and thereby affect cholesterol metabolism (6). Because our previous studies indicated that the transfer of LPS may be important in modulating inflammatory responses to LPS, and because LBP and PLTP share sequence similarity as well as related functions, we investigated the ability of PLTP to interact with LP...