Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

Abstract

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...

Native matrix-based human lung alveolar tissue model in vitro: studies of the reparatory actions of mesenchymal stem cells

“…In corroboration with other studies, the up-regulated secretion of soluble CD14 by CSE was detected in our model (Tsutsumi-Ishii and Nagaoka 2003). CD14 is a known activator of inflammation in epithelial cells (Frey et al 1992;Hailman et al 1994;Ulevitch and Tobias 1995;Cario et al 2000). In addition, up-regulation of cystatin C in CSE-exposed cells was also detected.…”

Bacterial DNA activates human neutrophils by a CpG‐independent pathway

“…1A). These results are in agreement with previous reports demonstrating that under serum-free conditions (those employed throughout our studies), LPS fails to activate neutrophils [18,19]. Furthermore, E. coli DNA digested with DNase I for 2 h at 37°C did not stimulate neutrophil activation (not shown).…”

Kupffer cell activation by lipopolysaccharide in rats: Role for lipopolysaccharide binding protein and toll-like receptor 4

“…This property of LBP is consistent with previous reports describing LBP as a lipid-transferring protein greatly accelerating the transfer of monomeric LPS to mCD14 and sCD14 as well as high density lipoprotein. [23][24][25] The LPS receptor on Kupffer cells that mediates the effects of LPS and LBP remains uncertain. Although membrane CD14 is important in mediating the effects of LPS/LBP complexes on peripheral blood monocytes, it is unlikely to be the receptor that can transmit LPS-induced signals across a cell membrane.…”