Showing papers by "Richard J. Ulevitch published in 1993"

Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14.

Abstract: Myeloid cell activation by lipopolysaccharides (LPS) involves two proteins, plasma LPS-binding protein (LBP) and cell-membrane CD14. Cell membrane CD14, anchored by a glycerophosphatidylinositol tail, is the cellular receptor for LPS-LBP complexes. Another form of CD14, without the lipid tail, circulates as a soluble plasma protein. In this work we show that soluble CD14 (sCD14) is required for activation of endothelial and epithelial cells by LPS. We propose that LPS-LBP complexes transfer LPS to sCD14, and the LPS-sCD14 complexes then bind to a cellular receptor. Support for this pathway comes from experiments in which LBP and CD14 in normal human serum are blocked by specific antibodies, experiments in which serum is replaced by purified LBP and sCD14, and experiments in which specific binding of [3H]LPS to epithelial cells is quantitated.

Glycosyl-phosphatidylinositol-anchored or integral membrane forms of cd14 mediate identical cellular-responses to endotoxin

Abstract: Endotoxin stimulates leukocytes to release cytokines that initiate septic shock in humans and animals. CD14, a glycosyl-phosphatidylinositol-anchored membrane glycoprotein, is an endotoxin receptor on leukocytes, and endotoxin binding to CD14 induces cytokine production. Here we show that glycosyl-phosphatidylinositol-anchored or integral membrane CD14 mediates identical cellular responses to endotoxin, including NF-kappa B activation and protein tyrosine phosphorylation. We also show that an anti-CD14 monoclonal antibody that does not block endotoxin binding to CD14 nonetheless inhibits cell activation by endotoxin. These findings suggest that binding of endotoxin to cell-surface CD14 is followed by subsequent interactions of the endotoxin-CD14 complex with additional membrane component(s) that enable transmembrane signaling. This function of CD14 may be prototypic for other members of the glycosyl-phosphatidylinositol-anchored family of proteins that do not play a primary role in signal transduction but rather are the principal ligand-binding units of membrane-bound receptor complexes.

Recognition of bacterial endotoxins by receptor-dependent mechanisms.

Abstract: Research performed during the past 5 years has provided a considerable amount of evidence to support the contention that the initial interaction of LPS (lipid A) with cells is mediated by distinct plasma membrane proteins. Some of these interactions may be solely involved in removal and eventual degradation of LPS whereas others may play a critical role in transmembrane signaling. Interactions that appear to be limited to a removal function have been assigned to the lipoprotein scavenger receptor or CD18 where R-form LPS, lipid A, or partial lipid A structures such as lipid IVa appear to be the preferred ligands; S-form LPS appears not to interact with these membrane proteins. Whether these interactions reflect events that occur in vivo remains to be definitively established. Moreover, the scavenger receptor and CD18 do not have a role in mediating LPS-induced transmembrane signaling. Photochemical crosslinking studies performed by Morrison and colleagues and by Dziarski (1991a,b) have revealed an LPS-binding membrane protein with an apparent molecular weight 70,000-80,000. This protein binds the lipid A of LPS as well as the carbohydrate backbone of peptidoglycan. Studies with monoclonal antibodies to this protein show that the presence of antibody blocks LPS binding, suggesting that engagement of this protein leads to transmembrane signaling. However, a definitive evaluation of the role of this protein in mediating LPS effects will require complete purification and/or gene cloning. Perhaps the most important advance in our understanding of how LPS acts is derived from the studies of Ulevitch, Tobias, and colleagues wherein the LBP/CD14-dependent pathway of cell stimulation has been identified. This pathway has particular importance for LPS recognition and signaling by cells such as monocytes/macrophages or polymorphonuclear leukocytes that constitutively express CD14. The importance of the LBP/CD14-dependent pathway has been definitively demonstrated by experiments using immunologic, biochemical, and molecular biologic approaches. Available data are consistent with a model for a heterodimeric LPS receptor that consists of CD14 and an as yet unidentified additional protein(s). Clearly a major goal for future research will be to elucidate fully the additional proteins involved in recognition of LPS.

A critical role for monocytes and CD14 in endotoxin-induced endothelial cell activation.

Abstract: Vascular endothelium activated by endotoxin (lipopolysaccharide [LPS]) and cytokines plays an important role in organ inflammation and blood leukocyte recruitment observed during sepsis. Endothelial cells can be activated by LPS directly, after its interaction with LPS-binding protein and soluble CD14 in plasma. LPS-LPS-binding protein complexes in blood also interact with monocytes and neutrophils bearing glycosyl-phosphatidylinositol (GPI) anchored membrane CD14 (mCD14), promoting the release of cytokines such as tumor necrosis factor and interleukin 1 (IL-1). These molecules, in turn, have the capacity to activate endothelial cells providing an indirect pathway for LPS-dependent endothelial cell activation. In this work, we address the relative importance of the direct and the indirect pathway of in vitro LPS-induced human umbilical vein endothelial cell (HUVEC) activation. Substituting whole blood for plasma resulted in a 1,000-fold enhancement of HUVEC sensitivity to LPS. Both blood- and plasma-dependent enhanced activation of HUVEC were blocked with an anti-CD14 monoclonal antibody. Blood from patients with paroxysmal nocturnal hemoglobinuria, whose cells lack mCD14 and other GPI anchored proteins, was unable to enhance LPS activation of HUVEC above the level observed with plasma alone. IL-10, an inhibitor of monocyte release of cytokines, decreased the blood-dependent enhancement of HUVEC activation by LPS. Blood adapted to small doses of LPS was also less efficient than nonadapted blood in producing this enhancement. Addition of purified mononuclear cells to HUVEC or the transfer of plasma from whole blood incubated with LPS to HUVEC, duplicated the enhancement effect observed when whole blood was incubated with HUVEC. Taken together, these data suggest that the indirect pathway of LPS activation of endothelial cell is mediated by monocytes and mCD14 through the secretion of a soluble mediator(s). The indirect pathway is far more efficient than the direct, plasma-dependent pathway.

Cross-linking of lipopolysaccharide (LPS) to CD14 on THP-1 cells mediated by LPS-binding protein.

Abstract: Recent work has established that bacterial endotoxin (LPS) binds to the plasma protein LPS-binding protein (LBP) forming high affinity complexes (LPS-LBP), that LBP is an opsonin for LPS-bearing particles, and that LPS-LBP complexes are potent agonists for monocytic cells (MO). mAb to the MO plasma membrane protein, CD14, inhibit LBP-dependent binding of LPS to MO, and LPS-LBP-dependent stimulation of cytokine release from MO. These data suggest that CD14 functions as a membrane receptor for LPS but do not demonstrate a direct association of LPS with CD14. Calcitriol was used to induce a high level of CD14 expression in the human monocyte-like cell line THP-1, resulting in enhanced responses of these cells to LPS-LBP complexes manifested by enhanced binding of LPS and a decrease in the amount of LPS needed to induce IL-8 release. An Re595 LPS derivative containing a radioiodinated, photoreactive, phenyl azide (125I-ASD-LPS) was used in cross-linking experiments to identify membrane proteins in calcitriol-treated THP-1 cells that interact with LPS. 125I-ASD-LPS was added to calcitriol-induced THP-1 cells in the presence or absence of LBP, the mixture photolyzed, and the resultant radioiodinated proteins analyzed by SDS-PAGE and autoradiography. We observed strong cross-linking of 125I-ASD-LPS to a 55-kDa membrane protein when LBP was present, but failed to observe radiolabeling of any other proteins with apparent molecular masses distinct from CD14. The cross-linked product was identified as CD14 by immunoprecipitation with anti-human CD14 mAb. Studies with human CD14 expressing transfectants of the murine B cell line 70Z/3 also revealed LBP-dependent cross-linking of 125I-ASD-LPS to CD14. These data document binding of LPS to a specific membrane protein that serves as an LPS receptor.

Lipopolysaccharide (LPS) recognition in macrophages. Participation of LPS-binding protein and CD14 in LPS-induced adaptation in rabbit peritoneal exudate macrophages.

Abstract: Exposure of rabbit peritoneal exudate macrophages (PEM) or whole blood to picomolar concentrations of LPS induces adaptation or hyporesponsiveness to LPS. Because of the importance of plasma LPS-binding protein (LBP) and the macrophage cell membrane protein CD14 in recognition of LPS, we examined the effect of LBP on LPS-induced adaptation in PEM. PEM exposed to LPS in the presence of LBP for 8 h were markedly less responsive to subsequent stimulation by LPS than monocytes/macrophages (M phi) adapted in the absence of LBP. LPS-induced expression of TNF was sharply reduced in LBP-LPS-adapted PEM, but in contrast these cells remained fully responsive to Staphylococcus aureus peptidoglycan. We considered that specific hyporesponsiveness in LPS-adapted M phi or in blood monocytes could be due to decreased expression of CD14 or diminished binding of LBP-LPS complexes to CD14. However, flow cytometry analysis revealed only minimal reduction of CD14 expression or CD14-dependent binding of a fluorescent LPS derivative when normo- and hyporesponsive cells were compared. These results show that complexes of LPS and LBP are more effective than LPS alone in inducing adaptation to LPS, and LPS-induced hyporesponsiveness probably results from changes in cellular elements distinct from CD14 that are involved in either LPS recognition or LPS-specific signal transduction.

Interactions of lipopolysaccharide with neutrophils in blood via CD14.

Abstract: The functional characteristics of neutrophils are exceedingly sensitive to physiological conditions as well as the details of isolation. Exposure to lipopolysac- charide (LPS) or even contamination of the isolating me- dia 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 sur- face in whole blood to minimize any effects of isolation. In whole blood neutrophils express low levels of formyl peptide receptor, CD14, and CR3, which increase sub- stantially in response to formyl peptide and LPS. The in- creases in CR3 and CD14 occurred in parallel and were independent of protein synthesis and tumor necrosis fac- tor (TNF) production. The increase in CR3 was inhibited by antibodies MY4, 3C10, and 28C5 against CD14. These findings are consistent with the notion that in blood the observed receptor up-regulation is in direct response to the action of LPS on neutrophils through CD14 and does not require products from macrophages such as TNF or the production of C5a from the plasma. J. Leukoc. Biol. 53: 518-524; 1993.

Purification and characterization of murine lipopolysaccharide-binding protein.

Abstract: The serum protein lipopolysaccharide (LPS)-binding protein (LBP) seems to play an important role in regulating host responses to LPS. Complexes of LPS and LBP form in serum and stimulate monocytes, macrophages, or polymorphonuclear leukocytes after binding to CD14. Previous reports have described the structure and properties of LBP from human and rabbit sera. Since mice are used in some experimental models of endotoxemia or gram-negative bacterial infections, information is needed about the properties of murine LBP. Murine LBP was purified by ion-exchange chromatography and high-pressure liquid chromatography; its NH2-terminal sequence (TNPGLVTRIT) was very similar to those of human and rabbit LBPs (80 to 90% amino acid identity). Murine LBP resembled LBPs from other species in that it promoted the binding of LPS to monocytes and enhanced the sensitivity of monocytes to LPS at least 100-fold. Mouse LBP, like rabbit and human LBPs, was found to be an acute-phase protein. Further in vivo studies with mice and anti-CD14 or anti-LBP reagents should help determine the role of LBP in response to LPS challenges.

Molecular Mechanisms of Lipopolysaccharide-Induced Cytokine Production by Monocytes

Abstract: Gram-negative sepsis is a greatly feared complication that occurs predominantly in patients that are traumatized, immune-suppressed, or under chemotherapy. Little effective therapy is available as now, and the overall mortality of septic shock is approximately 50%. The course of events starting with the undetectable entry of gram-negative bacteria into the organism and leading to the symptoms of full sepsis is so rapid that antibiotic treatment is usually of little use. Scientific interest thus has been focused lately on the host reaction that is triggered during the event of septic shock so that therapeutic intervention might become possible. It is known that endotoxin and its biologically active compound lipid A that enters the bloodstream in the event of sepsis is the cause of the complications of hypotension, disseminated intravascular coagulation (DIC), multiorgan failure, and eventually death [1, 2]. An important mediator in the cascade of events has recently been identified with tumor necrosis factor (cachectin/TNF), secreted by cells of monocytic origin (Mo) [3]. In vitro experiments confirm that lipopolysaccharide (LPS) can stimulate Mo to produce TNF and other monokines, so the Mo is considered to be a key cell in septic shock and related diseases.