Biosynthetic radiolabeling of bacterial lipopolysaccharide to high specific activity

Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo,both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS in 10 min, this was not accompanied by extensive release of the LPS from the cells. The cross-linking data suggest that much of the LPS that remained bound to the cells was associated with LBP. The ability of LBP to inhibit cell responses could not be explained by its effect on LPS internalization, because LBP did not significantly increase the internalization of the cell-bound LPS. In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the Toll-like receptor 4-MD-2 signaling receptor.Lipopolysaccharide (LPS 1 ; endotoxin), an abundant component of the outer membrane of Gram-negative bacteria, is one of the most potent of the known bacterial agonists in animal host cells. Whereas LPS recognition benefits the host by sensing the presence of bacteria and mobilizing defense mechanisms, an exaggerated response to LPS may contribute to the harmful sequelae of severe sepsis, which include coagulation disorders, organ failure, shock, and death. The host, therefore, has numerous mechanisms that down-regulate responses to LPS and remove it from the circulation and tissues (1-7).The potency of LPS is due to the sensitivity of the host recognition system, which requires the Toll-like receptor 4 (Tlr4) (8) signaling receptor. At least three LPS-binding proteins (LBP (9), CD14 (10), and MD-2 (11)) are required for sensitive Tlr4-mediated LPS recognition. Plasma LBP potentiates LPS recognition by transferring it from bacterial membranes (6) to CD14 (12, 13), which is expressed as a glycosylphosphatidylinositol-anchored protein on the surfaces of myeloid lineage cells (mCD14) and as an anchorless plasma protein, soluble CD14 (sCD14) (14, 15). CD14 presents LPS to Tlr4, presumably by transferring the LPS to MD-2, an accessory protein that is constitutively associated with the extracellular domain of Tlr4 (16). MD-2 binds LPS with high affinity (17), and it is required for Tlr4 signaling (11). Although normal expression of the Tlr4 and MD-2 are too low for direct measurements...

Section: Methodsmentioning

confidence: 99%

Lipopolysaccharide (LPS)-binding Protein Inhibits Responses to Cell-bound LPS

Thompson

Tobias

Viriyakosol

et al. 2003

“…3 H]LPS (1.5 ϫ 10 6 dpm/g) was biosynthetically labeled and isolated as described (34). BODIPY-LPS was prepared from E. coli LCD25 LPS as described (28).…”

Section: Methodsmentioning

confidence: 99%

Plasma Lipoproteins Promote the Release of Bacterial Lipopolysaccharide from the Monocyte Cell Surface

Kitchens¹,

Wolfbauer

Albers

et al. 1999

Self Cite

116

127

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...

“…LPS-Tritiated Escherichia coli LCD25 LPS (1500 3 H disintegrations/ng) was produced as described (28) and stored at Ϫ70°C. Aliquots were suspended in HNEB (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 300 g/ml BSA) (29) and briefly sonicated prior to use.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositides Bind to Plasma Membrane CD14 and Can Prevent Monocyte Activation by Bacterial Lipopolysaccharide

Wang¹,

Kitchens²,

Munford³

1998

Self Cite

Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS-and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo.