Biomedical subjects

R J Ulevitch

Publications and source records attributed to R J Ulevitch.

At least 91 records · Page 5Linked to original sources

PubMed1993

Analysis of lipopolysaccharide binding by CD14.

The cell surface protein CD14 binds bacterial lipopolysaccharide (LPS) in the presence of the serum protein, LPS-binding protein (LBP). This interaction is important for LPS-induced activation of mammalian myeloid cells. We performed quantitative studies of 3H-labeled LPS binding to human CD14 expressed on Chinese hamster ovary cells and on a human macrophage cell line (THP-1). At the concentrations studied (20-100 nM) LPS binding required the expression of CD14 and could be inhibited by a subset of anti-CD14 monoclonal antibodies. LBP was required for LPS binding to CD14. The binding occurred within 10 min and was relatively unaffected by temperature over the range of 4-37 degrees C. Quantitative binding assays were performed at 10 degrees C, or at 37 degrees C, using Chinese hamster ovary cells depleted of ATP. In both cases, 75-90% of the LPS could be released by treatment with phosphatidylinositol-specific phospholipase C, suggesting that it remains associated with the glycosyl phosphatidylinositol-anchored CD14. The apparent dissociation constant of recombinant human CD14 expressed on Chinese hamster ovary cells for LPS at 10 degrees C was 2.74 (+/- 0.99) x 10(-8) M; the apparent dissociation constant of CD14 expressed on THP-1 cells at 10 degrees C was 4.89 (+/- 1.42) x 10(-8) M. In both cell lines, at saturating LPS concentrations, the molar ratio of LPS bound per surface CD14 was approximately 20:1. At 37 degrees C the apparent dissociation constant of recombinant human CD14 for LPS at 37 degrees C was 2.7 (+/- 1.2) x 10(-8) M, and the molar ratio of LPS bound per surface CD14 was approximately 8:1. Although the difference in molar ratio of LPS bound per surface CD14 at the two temperatures is difficult to interpret, it is clear that at both temperatures the molar ratio is not 1:1. The basis of this phenomenon is unclear, but may involve the repeated leucine-rich motifs, which are found within CD14.

Animals↗

PubMed1993

Endotoxin induces rapid protein tyrosine phosphorylation in 70Z/3 cells expressing CD14.

CD14, a glycosylphosphatidylinositol-anchored glycoprotein of leukocytes, binds endotoxin (lipopolysaccharide (LPS)) with high affinity. After the murine pre-B cell line 70Z/3 is transfected with DNA encoding human CD14 (hCD14), the resultant stably transfected cell line, 70Z/3-hCD14, responds to 1000-fold lower LPS concentrations than the parental CD14-negative line. We have used 70Z/3-hCD14 cells, RAW264.7 cells, and elicited murine peritoneal exudate macrophages (PEM) to study LPS-induced protein tyrosine phosphorylation. LPS induces the rapid tyrosine phosphorylation of a 38-kDa protein (p38) in 70Z/3-hCD14 cells, PEM, and RAW264.7 cells and of two isoforms of mitogen-activated protein kinases (MAPK) in only RAW264.7 cells and PEM. p38 can be distinguished from the MAPK isoforms based on differences in mobilities on SDS-polyacrylamide gel electrophoresis and the lack of reactivity of p38 with anti-MAPK antibody even after dephosphorylation with potato acid phosphatase. Synthetic lipid A induces p38 phosphorylation in 70Z/3-hCD14 cells, whereas phorbol 12-myristate 13-acetate and interferon-gamma fail to induce tyrosine phosphorylation of p38. Pretreatment of 70Z/3-hCD14 cells with anti-hCD14 monoclonal antibody or the tyrosine kinase inhibitor herbimycin A inhibits LPS-induced tyrosine phosphorylation of p38. These results suggest that increased protein tyrosine phosphorylation occurs rapidly after LPS binds to CD14 and is likely to be an important event in mediating LPS-induced cell activation.

Animals↗

PubMed1993

Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin.

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.

Amino Acid Sequence↗

PubMed1993

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

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.

Acute-Phase Proteins↗

PubMed1993

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

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.

Acute-Phase Proteins↗

PubMed1993

Interactions of lipopolysaccharide with neutrophils in blood via CD14.

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) 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.

Antibodies, Monoclonal↗

PubMed1993

CD14: cell surface receptor and differentiation marker.

In the past, CD14 has been viewed simply as a useful marker molecule for monocytes and macrophages. Now, new findings on its role in binding of LPS-LBP complexes and in signal transduction have engendered renewed interest in the properties of CD14. Here, CD14 function, its expression in different cell types and the regulation of expression, including the generation of soluble CD14, are described, and the diagnostic value of CD14 in various diseases is discussed.

Animals↗

PubMed1993

Recognition of bacterial endotoxins by receptor-dependent mechanisms.

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.

Acute-Phase Proteins↗

PubMed1993

Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation.

The activation of monocytes, macrophages, and neutrophils by lipopolysaccharides (LPS) involves a serum protein, LPS binding protein (LBP), and a membrane protein, CD14. Evidence to date suggests a pathway in which serum LPS first binds to LBP. The LPS-LBP complexes then interact with CD14, leading to cellular activation. This scheme is supported by experiments in which either LBP or CD14 are blocked by antibodies as well as experiments in which LBP is added to serum free media and CD14 is expressed on cells which normally lack CD14. Discovery of this pathway suggests novel approaches to anti-LPS therapy.

Acute-Phase Proteins↗

PubMed1993

Purification and characterization of murine lipopolysaccharide-binding protein.

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.

Acute-Phase Proteins↗

PubMed1992

Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14-mediated pathway.

Lipopolysaccharides (LPS) that lack acyloxyacyl groups can antagonize responses to LPS in human cells. Although the site and mechanism of inhibition are not known, it has been proposed that these inhibitory molecules compete with LPS for a common cellular target such as a cell-surface binding receptor. In the present study, we used an in vitro model system to test this hypothesis and to evaluate the role of CD14 in cellular responses to LPS. Cells of the THP-1 human monocyte-macrophage cell line were exposed to 1,25 dihydroxyvitamin D3 to induce adherence to plastic and expression of CD14, a binding receptor for LPS complexed with LPS-binding protein (LBP). The uptake of picograms of [3H]LPS (agonist) and enzymatically deacylated LPS [3H]dLPS (antagonist) was measured by exposing the cells to the radiolabeled ligands for short incubation periods. The amounts of cell-associated LPS and dLPS were then correlated with cellular responses by measuring the induction of nuclear NF-kappa B binding activity and the production of cell-associated interleukin (IL)-1 beta. We found that similar amounts of [3H]LPS or [3H]dLPS were taken up by the cells. The rate of cellular accumulation of the ligands was greatly enhanced by LBP and blocked by a monoclonal antibody to CD14 (mAb 60b), yet no cellular responses were induced by dLPS or dLPS-LBP complexes. In contrast, LPS stimulated marked increases of NF-kappa B binding activity and IL-1 beta. These responses were enhanced by LBP and inhibited by mAb 60b. dLPS and its synthetic lipid A counterpart, LA-14-PP (also known as lipid Ia, lipid IVa, or compound 406) strongly inhibited LPS-induced NF-kappa B and IL-1 beta, yet neither antagonist inhibited the uptake of LPS via CD14. dLPS did not inhibit NF-kappa B responses to tumor necrosis factor (TNF) alpha or phorbol ester. Our results indicate that (a) both stimulatory and nonstimulatory ligands can bind to CD14 in the presence of LBP; (b) the mechanism of inhibition by dLPS is LPS-specific, yet does not involve blockade of LPS binding to CD14; and (c) in keeping with previous results of others, large concentrations of LPS can stimulate the cells in the absence of detectable binding to CD14. The findings indicate that the site of dLPS inhibition is distal to CD14 binding in the LPS signal pathway in THP-1 cells, and suggest that molecules other than CD14 are important in LPS signaling.

Acute-Phase Proteins↗

PubMed1992

Plasma lipopolysaccharide (LPS)-binding protein. A key component in macrophage recognition of gram-negative LPS.

LPS-binding protein (LBP) binds with high affinity (Kd approximately equal to 10(-9) M) to lipid A of LPS isolated from rough (R)- or smooth (S)-form Gram-negative bacteria as well as to lipid A partial structures such as precursor IVA. To define the role of LBP in regulating responses to LPS we have examined TNF release in rabbit peritoneal exudate macrophages (M phi) stimulated with LPS or with complete or partial lipid A preparations in the presence or absence of LBP. In the presence of LBP, M phi showed increased sensitivity to S- and R-form LPS as well as synthetic lipid A. Compared with LPS or lipid A, up to 1000-fold greater concentrations of partial lipid A structures were required to induce TNF production. However, consistent with our previous observations that these structures bind to LBP, TNF production was increased in the presence of LBP. In contrast, LBP did not enhance or inhibit TNF production produced by heat-killed Staphylococcus aureus, peptidoglycan isolated from S. aureus cell walls, or PMA. Potentiated M phi responsiveness to LPS was observed with as little as 1 ng LBP/ml. Heat-denatured LBP (which no longer binds LPS), BPI (an homologous LPS-binding protein isolated from neutrophils), or other serum proteins were without effect. LBP-treated M phi also showed a more rapid induction of cytokine mRNA (TNF and IL-1 beta), higher steady-state mRNA levels and increased TNF mRNA stability. These data provide additional evidence that LBP is part of a highly specific recognition system controlling M phi responses to LPS. The effects of LBP are lipid A dependent and importantly, extend to LPS preparations isolated from bacteria of R- and S-form phenotype.

Acute-Phase Proteins↗

PubMed1992

Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

Acute-Phase Proteins↗

PubMed1992

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.

Acute-Phase Proteins↗

PubMed1992

HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma.

The role that inflammatory cytokines may play in the life cycle of the hepatitis B virus and in the pathogenesis of its associated liver disease has not been carefully delineated. In this report, we demonstrate that bacterial lipopolysaccharide, a potent inducer of inflammatory cytokines in vivo, causes a severe acute liver disease in transgenic mice whose hepatocytes produce the hepatitis B virus large envelope polypeptide and retain HBsAg within the endoplasmic reticulum. In contrast, 100-fold higher doses of bacterial lipopolysaccharide do not induce liver cell injury in nontransgenic littermate controls or in transgenic mice whose hepatocytes secrete HBsAg rather than retain it. Coincident with the hepatocellular injury and the influx of inflammatory cells into the liver, a marked reduction occurs in the intrahepatic content of hepatitis B virus steady-state messenger RNA, thereby confirming the selectivity of this process for the HBsAg-positive hepatocyte. Bacterial lipopolysaccharide-induced hepatocellular injury appears to be principally mediated by interferon-gamma because it can be markedly reduced by the prior administration of neutralizing interferon-gamma-specific monoclonal antibodies and because recombinant interferon-gamma is also selectively cytotoxic for the HBsAg-positive transgenic hepatocyte in vivo. Tumor necrosis factor-alpha is also involved in this process because bacterial lipopolysaccharide-induced liver cell injury is significantly reduced by tumor necrosis factor-alpha specific monoclonal antibodies. The role of tumor necrosis factor-alpha in bacterial lipopolysaccharide-induced liver cell injury is less clear than interferon-gamma, however, because unlike interferon-gamma it is also toxic for nontransgenic hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine Transaminase↗

PubMed1992

Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein.

We have studied the effects of highly purified rabbit lipopolysaccharide (LPS)-binding protein (LBP) on the ability of murine bone marrow-derived macrophages to respond to bacterial LPS. Macrophage responses studied include the secretion of tumor necrosis factor alpha, production of arginine-derived nitrite (NO2-), and killing of an intracellular pathogen, Leishmania enriettii. Macrophages from either CBA or LPS-hyporesponsive C3H/HeJ mice exhibited significantly greater sensitivity to LPS in the presence of LBP. Furthermore, both CBA and C3H/HeJ macrophages demonstrated an LBP-dependent enhancement of LPS binding. These results suggest that C3H/HeJ macrophages are capable of binding LPS-LBP complexes and support the hypothesis that hyporesponsiveness in this strain involves a step subsequent to LPS binding. Furthermore, these findings provide additional evidence of the important role played by the acute-phase plasma protein LBP in modifying host response to LPS.

Acute-Phase Proteins↗

PubMed1992

Tumour necrosis factor alpha antibody protects against lethal meningococcaemia.

Tumour necrosis factor alpha (TNF-alpha) has been shown to be the principal mediator of Gram-negative bacterial endotoxin-induced shock. Nevertheless, evidence suggests that TNF-alpha plays a beneficial role in controlling bacterial infections when multiplication of the microorganism is required to kill the host. Using an infant rat model of Neisseria meningitidis infection, we found that blood TNF-alpha concentration reaches a peak three hours after intraperitoneal injection of 3 x 10(6) bacteria. Thereafter, the level of TNF-alpha decreased and was undetectable six to eight hours after infection. A correlation was observed between the magnitude of initial TNF-alpha response and a fatal outcome. Pretreatment of the animals with polyclonal anti-TNF antiserum significantly reduced mortality relative to animals pretreated with control serum. However, pretreatment of animals with anti-TNF antibody did not alter the bacterial invasion of the cerebrospinal fluid. Injection of heat-killed bacteria did not cause death and induced lower TNF-alpha levels than the same number of live bacteria. This excludes the possibility that the role of TNF-alpha is to mediate a shock induced by the endotoxin component of the bacterial inoculum. These results indicate that TNF-alpha has a deleterious effect in this model of bacteraemia. Identification of the critical factors that determine the action of TNF-alpha during lethal bacteraemia will lead to a better understanding of these diseases and the development of appropriate therapeutic intervention.

Animals↗

PubMed1992

Participation of lipopolysaccharide-binding protein in lipopolysaccharide-dependent macrophage activation.

Only recently has the mechanism for lipopolysaccharide (LPS) recognition by macrophages been elucidated. In contrast to many ligand receptor interactions, the interaction of LPS with its receptor, CD14, on myeloid cells is greatly enhanced by prior complexation of LPS with LPS-binding protein (LBP), a recently discovered plasma glycoprotein. LBP is found in normal serum or plasma in the 5 to 10 micrograms/ml range. In plasma, it reacts rapidly but transiently with LPS. LPS-LBP complexes then react with CD14 bearing cells. Blocking CD14 with monoclonal antibodies or removal of LBP from plasma blocks the ability of the cells to react with LPS-LBP complexes and also blocks release of cytokines and other mediators from the cells. In the normal lung, bronchoalveolar lavage fluid contains low levels of LBP. However, during acute lung injury, LBP levels may rise by transudation and enhance activation of alveolar macrophages to release injurious mediators. Description of this pathway for LPS recognition by macrophages and other leukocytes offers the possibility of developing new reagents to block LPS recognition and prevent the development of endotoxemia.

Acute-Phase Proteins↗