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...

Gram-negative bacterial lipopolysaccharide (LPS)1 (endotoxin) is one of the most potent and ubiquitous of the known bacterial signal molecules. Animals have sensitive mechanisms for recognizing the presence of LPS in tissues. Monocytes, macrophages, and neutrophils, which express the LPS binding receptor, CD14 (1, 2), and its signal transducer, Toll-like receptor-4 (3), are particularly sensitive to LPS (4). They respond by producing inflammatory mediators that amplify and diversify the LPS signal, triggering host defenses that wall off and destroy invading bacteria. For reasons that are not entirely clear, however, the response to LPS can also be harmful, resulting in severe sepsis, septic shock, and even death. Mechanisms that regulate responses to LPS are therefore likely to be very important for the host.HDL is the most abundant of the lipoproteins in human plasma and interstitial fluids. It can remove both phospholipids and unesterified cholesterol from cells (reviewed in Ref. 5), and HDL-mediated cellular cholesterol efflux and the subsequent delivery of HDL-cholesterol to the liver (reverse cholesterol transport) are thought to help protect animals from atherosclerosis. This study addresses another facet of the HDL particle: its ability to bind LPS and neutralize its biological activity. There is abundant evidence that HDL and other plasma lipoproteins play an important role in controlling host responses to LPS. Numerous studies have shown that complexes of LPS with HDL and other lipoproteins have little or no stimulatory activity, either in vitro or in vivo (6 -9), and there is now strong evidence that HDL and other lipoproteins can neutralize endotoxin in vivo (10 -17).Two plasma lipid transfer proteins, LPS-binding protein (LBP) (18,19) and phospholipid transfer protein (PLTP) (20), promote the binding of purified LPS to lipoproteins, whereas only LBP can facilitate LPS binding to CD14 on cell membranes (mCD14) or soluble CD14 (sCD14) in plasma (1,21,22). sCD14 can rapidly transfer LPS to mCD14 on cells (23), and it also facilitates the activation of cells that do not express mCD14 (24). Although sCD14 can also transfer LPS to HDL (19), it has been said to contribute very little to the movement of LPS to lipoproteins in whole plasma (25).In this study, we sought to determine whether HDL and plasma LPS transfer proteins could remove LPS from host cells. We found that HDL facilitates the release of cell-bound LPS, that sCD14 enhances this release, and that the removal of LPS from the cells attenuates proinflammatory responses. This new pathway of LPS traffic to lipoproteins may be important for regulating host responses to Gram-negative bacteria. These findings also raise the possibility that efflux of microbial ligands from macrophages to HDL contributes to the antiinflammatory potency of HDL in processes such as atherosclerosis. EXPERIMENTAL PROCEDURESCells and Reagents-Human monocytic THP-1 cells were cultured as described previously (26). CD14 expression was induced either by culturing the cells in 1...

Phospholipid transfer protein (PLTP)1 plays important and diverse roles in lipoprotein metabolism (1, 2). Plasma PLTP transfers phospholipids between lipoproteins and remodels high density lipoprotein (HDL) to generate lipid-poor particles (3-7), and hepatic PLTP facilitates the production of triglyceride-rich apoB-containing particles in mice (8, 9). PLTP is expressed ubiquitously in human and mouse tissues (5, 10, 11), suggesting that it may locally modulate lipid metabolism in peripheral tissues. Little is known, however, about the interactions of PLTP with peripheral cells and its effects on cellular lipid metabolism.One possible function of peripheral PLTP is to transport lipids from cells to lipoproteins. Cholesterol efflux from cells occurs by at least two distinct mechanisms mediated by different components in HDLs (12, 13). HDL phospholipids sequester cholesterol that desorbs from the plasma membrane, and lipidpoor HDL apolipoproteins remove both cholesterol and phospholipids by an active transport process. A cell surface receptor called scavenger receptor B1 facilitates cholesterol transport from cells to HDL phospholipids (13), whereas a sterol-inducible membrane transporter called ATP-binding cassette transporter A1 (ABCA1) mediates the removal of cholesterol and phospholipids by lipid-poor apolipoproteins (14). It is possible that PLTP enhances one or both of these lipid efflux pathways in peripheral tissues.We reported previously that PLTP enhanced cholesterol and phospholipid efflux from cholesterol-loaded human fibroblasts in the presence of HDL particles (15). In contrast, PLTP had no effect on lipid efflux from fibroblasts isolated from a patient with Tangier disease, an HDL deficiency syndrome discovered later to be caused by mutations in ABCA1 (16 -19). These findings suggested that PLTP enhanced lipid efflux only by ABCA1-dependent mechanisms. The discovery of ABCA1 has allowed us to address in more detail the role of ABCA1 in this PLTP-enhanced lipid efflux. Our current findings confirm that ABCA1 expression is required for PLTP lipid efflux activity and that this probably involves direct interactions of PLTP with ABCA1. These studies suggest a model whereby PLTP mimics apolipoproteins in removing cellular lipids by the ABCA1 pathway, except that PLTP requires the presence of lipoprotein acceptor particles for optimum lipid transport activity. EXPERIMENTAL PROCEDURESLipoproteins, ApoA-I, and Phospholipid Vesicles-LDL and HDL were prepared by sequential ultracentrifugation in the density range 1.019 -1.063 and 1.125-1.21 g/ml, respectively, and HDL was depleted of apoE and apoB by heparin-agarose chromatography (20). Trypsinized HDL was prepared as previously described (21) by treating HDL with trypsin for 30 min at 37°C at an HDL/trypsin protein ratio of 40:1. This procedure digests ϳ20% of the total HDL protein content of HDL without disturbing its lipid composition. ApoA-I was purified from HDL, delipidated, and labeled with 125 I as described previously (20). LDL was acetylated by the me...

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