Evidence that the Receptor for Soluble CD14:LPS Complexes may not be the Putative Signal-Transducing Molecule Associated with Membrane-Bound CD14 (original) (raw)
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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.
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.
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 Cellular Biochemistry, 1991
The 52 kD myeloid membrane glycoprotein CDI 4 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rlFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rlL-I, rlL-2, rlL-3, rIL-4, rlL-5, rlL-6, rTNFa, rGM-CSF, rM-CSF, rTGFbl, rlFNa, lipopolysaccharide (LPS), and, as a control, rlFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. lFNg concentrations were determined in PBL culture supernatants by ELISA. rlFNa and rlL-2 reduced CDI 4 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rlFNa and rlL-2 were ineffective in purified monocytes or macrophages. rlL-4 strongly reduced CDI 4 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CDI 4, but when added in combination with rlFNg the effect on CD14 downregulation was more pronounced. The effect of rlFNg on CD14 in PBL cultures was dose-dependently inhibited by rlL-4 and this inhibition is probably due to an IL-4 mediated blockade of lFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced lFNg secretion. LPS strongly enhanced CDI 4 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rlFNg and rlL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.
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.
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.
Interactions of lipopolysaccharide with neutrophils in blood via CD14
Journal of leukocyte biology, 1993
The functional characteristics of neutrophils are exceedingly sensitive to physiological conditions as well as the details of isolation. Exposure to lipopolysaccharide (LPS) or even contamination of the isolating media with traces of LPS is known to play an important role in regulating cell function and expression of receptors. Because of the suspected role of CD14 as a receptor for LPS, we used anti-CD14 monoclonal antibodies both to identify CD14 in the cell surface of polymorphonuclear leukocytes and to inhibit functional changes elicited by LPS. Cytometric techniques were used to investigate the regulation of CD14 and CR3 on the neutrophil cell surface in whole blood to minimize any effects of isolation. In whole blood neutrophil express low levels of formyl peptide receptor, CD14, and CR3, which increase substantially in response to formyl peptide and LPS. The increases in CR3 and CD14 occurred in parallel and were independent of protein synthesis and tumor necrosis factor (TNF...
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.
Soluble CD14 activates monocytic cells independently of lipopolysaccharide
Infection and immunity, 1998
The glycoprotein CD14 acts as a receptor for lipopolysaccharide (LPS), either when anchored in the myeloid cell membrane (mCD14) or as a soluble molecule (sCD14) in serum. sCD14-LPS complexes activate cells devoid of mCD14. However, the role of sCD14 independent of LPS is unknown. Therefore, the effect of sCD14 on monocyte functions was investigated in the monocytic cell lines THP1 and Mono Mac 6 and in fresh human monocytes. Under serum-free conditions, endotoxin-free human recombinant sCD14(1-348), (rsCD14(1-348)) induced tumor necrosis factor alpha (TNF-alpha). The TNF-alpha effect was stronger in THP1 cells than in Mono Mac 6 cells or monocytes. It was dose dependent, with a maximum at 1 microg/ml, and time dependent, with a maximum after 2 h. sCD14 purified from urine had the same cytokine-activating capacity. In contrast, C-terminally truncated rsCD14(1-152) was inactive. The rsCD14 effect was not due to LPS contamination, since it was resistant to polymyxin and lipid IVa but ...