Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14 (original) (raw)
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Lipopolysaccharide (LPS)-Binding Protein Accelerates the Binding of LPS to CD14
Journal of …, 1994
Sumlnary CD14 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.
Journal of Clinical Investigation, 1994
Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 011 1:B4 LPS on movement of '4C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations 2 0.5 ng/ml, and LPS (10 ng/ml) exposures of 2 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 Ag/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of. 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.
Cellular Binding of Soluble CD14 Requires Lipopolysaccharide (LPS) and LPS-binding Protein
Journal of Biological Chemistry, 1997
The stimulation of nonmyeloid cells by lipopolysaccharide (LPS) is mediated by the serum protein, soluble CD14 (sCD14). We have examined the interaction of sCD14 with whole cells using a biologically active radiolabeled sCD14 molecule as a ligand. Specific binding of sCD14 to nonmyeloid cells is detected only when it is first incubated with both LPS and the serum LPS-binding protein (LBP). Through the use of an anti-CD14 monoclonal antibody, we demonstrate that sCD14 must interact with LPS in order for cellular binding to occur. Although LBP is traditionally known to function as a catalyst in the transfer of LPS to sCD14, our results reveal that LBP is actually a physical part of sCD14containing,cell-associatingcomplexes.TheLPS-andLBPdependent cell surface binding of sCD14 appears to be distinct from events leading to cell stimulation, since certain anti-CD14 and anti-LBP monoclonal antibodies have different effects on cellular binding versus cellular activation. Bound sCD14 is internalized, indicating that the LBP-and LPS-dependent binding of sCD14 may represent a novel general mechanism by which nonmyeloid cells clear LPS.
Journal of immunology (Baltimore, Md. : 1950), 1999
Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLB...
Structure-Function Analysis of CD14 as a Soluble Receptor for Lipopolysaccharide
Journal of Biological Chemistry, 2000
CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.
Journal of Biological Chemistry, 1994
Lipopolysaccharide (LPS) binding protein (LBP), a 58-60 kDa glycoprotein, binds to the lipid A region of LPS. The resulting LPSeLBP complex is recognized by both the membrane-bound (mCD14) and soluble forms of CD14 (sCD14), thereby enhancing the ability of LPS to activate myeloid, endothelial, and epithelial cells. To begin to characterize the structure-function relationships within LBP, we have created and expressed a truncated form of human LBP (herein called NH-LBP) comprising amino acid residues 1-197 of the parent molecule. Ex
Scandinavian Journal of Immunology, 1997
Haziot A, Katz I, Rong G-W, Lin X-Y, Silver J, Goyert SM. Evidence that the Receptor for Soluble CD14:LPS Complexes may not be the Putative Signal-Transducing Molecule Associated with Membrane-Bound CD14. Scand J Immunol 1997;46:242-245 Membrane-bound CD14 acts as a receptor for lipopolysaccharide (LPS) on monocytes/macrophages and neutrophils. Studies have suggested that the activation of monocytes/macrophages by the binding of LPS to membrane-bound CD14 may require the association of a signal-transducing molecule with membrane-bound CD14. The observation that non-CD14 expressing cells, such as endothelial cells, can nevertheless be activated by a complex of LPS and a soluble form of CD14 (sCD14) suggests that the receptor for this complex may be identical to the signal transducing molecule associated with membrane-bound CD14. The studies described show that two CD14-specific MoAb are able to block the LPS-induced activation of endothelial cells but do not affect the response of monocytes to LPS. This suggests that the interaction of the sCD14:LPS complex with endothelial cells is distinct from the interaction of membrane-bound CD14 with its putative signal-transducing molecule.
European Journal of Immunology, 1994
The two soluble forms of the lipopolysaccharide receptor, CD14: characterization and release by normal human monocytes* CD14, a glycolipid-anchored membrane glycoprotein, acts as a high affinity lipopolysaccharide receptor on leukocytes. We previously reported that the Mono-Mac-6 cell line releases two different soluble forms of CD14 (sCD14) (Labeta et al., Eur. J. Zmmunol. 1993. 23: 2144. Here we show that the two sCD14,which we now refer to as sCD14a (low M,) and sCD14P (high Mr), are also synthesized and released by normal human monocytes and present in normal plasma. Their mechanism of release was examined by using the Mono-Mac-6 cell line, Chinese hamster ovary cell (CHO)/CD14+ transfectants and plasma from paroxysmal nocturnal hemoglobinuria (PNH) patients. It was found that: (1) sCD14f3 is released faster than sCD14a and that the release of the latter is a lengthy process.
Endothelial CellInjury andActivation: RoleofSoluble CD14
Vascular endothelial cell (EC) injury by lipopolysaccharides (LPS) plays a major role in the pathogenesis of gram-negative bacterial sepsis and endotoxic shock. The studies described here were performed to define further the molecular mechanisms involved in the EC responses to LPS. We showed that serum was required for LPS-mediated cytotoxicity for bovine brain microvessel, pulmonary, and aortic ECs and that anti-human CD14 antibodies completely blocked LPS-mediated cytotoxicity for ECs in the presence of human serum. The addition of a recombinant soluble form of human CD14 to serum-free medium restored the LPS-mediated cytotoxicity, whereas the addition of LPS binding protein (LBP), a serum protein that potentiates LPS-induced responses to monocytes, had no effect. A similar dependency on serum or recombinant soluble CD14 (under serum-f-ee conditions) was observed for LPS-induced secretion of interleukin-6 by human umbilical vein ECs. These findings indicate that soluble CD14 is required for LPS-mediated EC responses independently of LPB, suggesting that serum soluble CD14 represents a naturally occurring agonist for EC responses to LPS.