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R J Ulevitch

Publications and source records attributed to R J Ulevitch.

At least 109 records · Page 6Linked to original sources
PubMed1992

Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.

A plasma lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of rabbit peritoneal macrophages and human blood monocytes to endotoxin (LPS). We investigated whether LBP is present in lung fluids and the effects of LBP on the response of lung macrophages to LPS. Immunoreactive LBP was detectable in the lavage fluids of patients with the adult respiratory distress syndrome by immunoprecipitation followed by Western blotting, and also by specific immunoassay. In rabbits, the LBP appeared to originate outside of the lungs, inasmuch as mRNA transcripts for LBP were identified in total cellular RNA from liver, but not from lung homogenates or alveolar macrophages. Purified LBP enhanced the response of human and rabbit alveolar macrophages to both smooth form LPS (Escherichia coli O111B:4) and rough form LPS (Salmonella minnesota Re595). In the presence of LBP and LPS, the onset of tumor necrosis factor-alpha (TNF alpha) production occurred earlier and at an LPS threshold dose that was as much as 1,000-fold lower for both types of LPS. In rabbit alveolar macrophages treated with LBP and LPS, TNF alpha mRNA appeared earlier, reached higher levels, and had a prolonged half-life as compared with LPS treatment alone. Neither LPS nor LPS and LBP affected pHi or [Cai++] in alveolar macrophages. Specific monoclonal antibodies to CD14, a receptor that binds LPS/LBP complexes, inhibited TNF alpha production by human alveolar macrophages stimulated with LPS alone or with LPS/LBP complexes, indicating the importance of CD14 in mediating the effects of LPS on alveolar macrophages. Thus, immunoreactive LBP accumulates in lung lavage fluids in patients with lung injury and enhances LPS-stimulated TNF alpha gene expression in alveolar macrophages by a pathway that depends on the CD14 receptor. LBP may play an important role in augmenting TNF alpha expression by alveolar macrophages within the lungs.

Acute-Phase Proteins↗
PubMed1991

Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction.

The lipid A domain of lipopolysaccharide (LPS) is a unique, glucosamine-based phospholipid that makes up the outer monolayer of the outer membrane of most gram-negative bacteria. Because of its profound pharmacological effects on animal cells, especially those of the immune system, lipid A is also known as endotoxin. Despite decades of earlier work, the precise chemistry of endotoxins and the biochemical pathways for their enzymatic synthesis have been elucidated only within the past 5 years. In this review, we summarize the essentials of endotoxin biochemistry and also present recent experiments aimed at identifying surface receptors, signal-transducing elements, transcriptional factors, and key intracellular targets involved in the response of animal cells to endotoxins.

Endotoxins↗
PubMed1991

Modulation of endotoxic activity of lipopolysaccharide by high-density lipoprotein.

Unlike agonists such as cytokines or hormones, the biological activity of bacterial lipopolysaccharide (LPS) is substantially modified by serum proteins. One such interaction in serum is with high-density lipoprotein (HDL) forming LPS-HDL complexes. LPS-HDL complexes have been previously shown to have reduced endotoxic activity, for example pyrogenicity, when compared to other forms of LPS in animal models. In this study, we report results of studies comparing the potency of LPS-HDL complexes with uncomplexed LPS as agonists for interleukin-1 (IL-1) production by two different sources of monocytes. LPS-HDL complexes were purified by ultracentrifugation in sodium bromide gradients. The human monocytic cell line THP-1 and the freshly isolated human monocytes, purified by adherence or elutriation from venous blood from healthy donors, were exposed to medium alone containing 1 mg/ml bovine serum albumin, HDL, LPS (parent LPS) and LPS-HDL complexes. mRNA level was analyzed on Northern blot, and cell-associated protein and supernatants were tested for IL-1 production using immunologic and biologic assays. LPS stimulates substantially more IL-1 mRNA and cell-associated IL-1 protein when the monocytes are stimulated with LPS alone versus LPS-HDL. These data suggest that LPS-HDL complexation may contribute to a reduction in endotoxic activities in vivo by preventing LPS (lipid A) from generating important transmembrane signals after binding to cells.

Blotting, Northern↗
PubMed1991

Hyperexpression of interferon-gamma-induced MHC class II genes associated with reorganization of the cytoskeleton.

Class I and class II major histocompatibility complex (MHC) gene products are key recognition units in the induction and regulation of the immune response. Expression of class I and class II may be constitutive or inducible by cytokines such as interferon-gamma (IFN-gamma). A key step in the induction of MHC genes is recognition of IFN-gamma by its membrane receptor. The work described here examines the regulation of the occupied IFN-gamma receptor by the cytoskeleton. To do this the authors have used the fungal metabolites dihydrocytochalasin B (DHCB) and cytochalasin D (CD), substances that bind to actin filaments and thereby disrupt the cytoskeleton. The authors have studied the effect of DHCB and CD on IFN-gamma-induced MHC gene expression in 143 B cells, a human osteosarcoma-derived cell line. Herein the authors demonstrate that alterations in the cytoskeleton induced by DHCB and CD can lead to increases in IFN-gamma-induced MHC gene expression. Dihydrocytochalasin B added up to 3 hours after IFN-gamma results in a threefold to sixfold increase in levels of class II mRNA while producing minimal enhancement of class I gene expression. In contrast, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was unaltered by IFN-gamma or by the cytochalasins. The increased amount of class II mRNA can be accounted for by a concomitant increase in transcription rate of this gene. Studies using 125I-IFN-gamma demonstrate that the occupied IFN-gamma receptor associates with a Triton X-100 insoluble fraction of 143 B cells and that DHCB and CD markedly inhibit this association. The results described here provide evidence that is consistent with the hypothesis that the activity of the occupied IFN-gamma receptor may be modulated by interactions with the cytoskeleton of the cell. This receptor may be one of a group of plasma membrane receptors that are sensitive to the action of cytochalasins after ligand binding.

B-Lymphocytes↗
PubMed1990

CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

Acute-Phase Proteins↗
PubMed1990

Structure and function of lipopolysaccharide binding protein.

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

Acute-Phase Proteins↗
PubMed1990

Identification of lipopolysaccharide-binding proteins in 70Z/3 cells by photoaffinity cross-linking.

A radioiodinated, photoactivatable derivative of Salmonella minnesota Re595 lipopolysaccharide (LPS) was used to label LPS-binding proteins in 70Z/3 cells. The labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. 125I-Labeled-2-(p-azidosalycylamido)1,3'-dithiopropionamide S. minnesota Re595 LPS (125I-ASD-Re595) labeled a limited number of proteins. The most prominent of these had a apparent molecular mass of 18 kDa. Less prominent labeling of 25- and 28-kDa proteins was also seen. Labeling was saturated by 5 micrograms/ml 125I-ASD-Re595 and was inhibited by a 10-100-fold excess of unlabeled LPS or lipid A. Labeling was maximal within 30 min at 37 degrees C; much less labeling occurred at lower temperatures. The proteins labeled with 125I-ASD-Re595 appear to be on the surface of the cell, since they can be digested by trypsin and were found in the membrane fraction of the cell but not in the cytosol. Studies with competitive inhibitors suggested that the proteins bind to the lipid A region of the LPS molecule. Biologically inactive lipid A analogs were poor inhibitors of labeling, suggesting that the LPS-binding proteins could discriminate between active lipid A and inactive analogs. These studies suggest that the 18- and 25-kDa proteins bind specifically to the lipid A region of the LPS molecule and should be considered as candidates for a functional LPS receptor.

Affinity Labels↗
PubMed1990

Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein.

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.

Acute-Phase Proteins↗
PubMed1990

Biosynthesis of lipopolysaccharide-binding protein in rabbit hepatocytes.

Studies reported here show that the recently discovered acute-phase protein, lipopolysaccharide-binding protein (LBP), is synthesized by hepatocytes. For these studies, explanted rabbit hepatocytes were grown in the presence of 35S-methionine. Biosynthetically labelled LBP in the cells and supernatant was identified using immunoprecipitation with rat anti-rabbit LBP antibody. This antibody immunoprecipitates both the LBP polypeptide and the glycosylated protein. With a cell-free translation system a comparison of RNA from normal rabbit liver with that isolated from acute-phase rabbit liver indicated that a translatable LBP message is only found in the RNA from acute-phase liver. Studies with explanted rabbit hepatocytes showed that several forms of LBP distinguishable by migration in SDS-PAGE are secreted into the extracellular medium. This heterogeneity probably is a result of differences in glycosylation of LBP since pretreatment of the hepatocytes with tunicamycin results in accumulation of a single polypeptide with an apparent mass of 50 kD in SDS-PAGE. Explanted rabbit hepatocytes spontaneously synthesize and secrete LBP and express LBP mRNA as detected by cell-free translation; thus, it was not possible to upregulate the expression of LBP. Nevertheless, these studies form the basis for future investigations on the regulation of LBP biosynthesis.

Acute-Phase Proteins↗
PubMed1990

Adaptation to bacterial lipopolysaccharide controls lipopolysaccharide-induced tumor necrosis factor production in rabbit macrophages.

These experiments provide an explanation for the observation that two intravenous injections of lipopolysaccharide (LPS) spaced 5 h apart in rabbits cause tumor necrosis factor/cachectin (TNF) levels to rise in the blood only after the first LPS injection. Herein we show that treatment of elicited peritoneal exudate rabbit macrophages (PEM) with two doses of LPS given 9 h apart results in a marked reduction in TNF production by the second LPS exposure. This state of hyporesponsiveness is a result of adaptation to LPS, is induced by LPS concentrations that are 1,000-fold less than required to induce TNF production (picograms vs. nanograms), is characterized by a decrease in LPS-induced TNF mRNA without any change in TNF mRNA half-life, is not changed by including indomethacin in cultures, and is specific for LPS since LPS-adapted cells display a TNF response to heat-killed Staphylococcus aureus that is at least as good as that observed in control PEM.

Adaptation, Physiological↗
PubMed1990

A new model of macrophage stimulation by bacterial lipopolysaccharide.

Infection occurring in patients suffering from severe trauma or burns often leads to hypotension, disseminated intravascular coagulation, multiorgan failure, and death. These latter pathophysiologic changes often are associated with Gram-negative sepsis and endotoxemia. Substantial progress has been made in understanding the effector mechanisms for endotoxin (LPS) action with the recognition of the importance of LPS-inducible products of cells of monocytic lineage in mediating LPS-induced injury. Here we will review recent evidence that supports a model for monocyte/macrophage activation by LPS that involves a plasma protein known as lipopolysaccharide binding protein (LBP) and the monocyte differentiation antigen, CD14.

Acute-Phase Proteins↗
PubMed1989

Lipopolysaccharide induces hyporesponsiveness to its own action in RAW 264.7 cells.

Bacterial endotoxin (lipopolysaccharide, LPS) has the property of inducing hyporesponsiveness or tolerance to its own effects. This phenomenon has been demonstrated in man and experimental animals. The cellular changes that contribute to LPS tolerance are not understood. One mechanism of tolerance could involve a diminished response to LPS by key effector cells such as macrophages. Here we describe experiments designed to determine the mechanism whereby LPS produces a hyporesponsive state to its own effects. Because of the importance of the monokine known as tumor necrosis factor-alpha (TNF-alpha) in mediating many of the diverse effects of LPS, we have studied induction of TNF-alpha at the mRNA and activity level in the murine macrophage-like cell line RAW 264.7. Hyporesponsiveness can be induced by exposure of RAW 264.7 cells to low doses of LPS for more than 6 h prior to challenge with a second, normally stimulatory dose of LPS. This hyporesponsiveness is characterized by a diminished ability of LPS to increase steady state levels of TNF-alpha mRNA, is not due to an increased rate of TNF-alpha mRNA degradation, and is specific for LPS since LPS-pretreated and control cells produce similar amounts of TNF-alpha in response to challenge with heat-killed Staphylococcal aureus. The presence of indomethacin during the primary and/or challenge LPS treatment has no effect on the induction of acquired hyporesponsiveness. Thus, cyclooxygenase products are probably not involved in the development of LPS-induced hyporesponsiveness. These studies provide the basis for a better understanding of the cellular mechanisms that contribute to LPS tolerance.

Animals↗
PubMed1989

Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages.

Lipopolysaccharide binding protein (LBP) is an acute-phase reactant that binds bacterial LPS. We show that LBP binds to the surface of live Salmonella and to LPS coated erythrocytes (ELPS), and strongly enhances the attachment of these particles to macrophages. LBP bridges LPS-coated particles to macrophages (MO) by first binding to the LPS, then binding to MO. Pretreatment of ELPS with LBP enabled binding to MO, but pretreatment of MO had no effect. Moreover, MO did not recognize erythrocytes coated with LBP unless LPS was also added, thus suggesting that interaction of LBP with LPS results in a conformational change in LBP that allows recognition by MO. Binding of LBP-coated particles appears to be mediated by a receptor found on blood monocytes and MO but not on other leukocytes or umbilical vein endothelium. The receptor is mobile in the plane of the membrane since binding activity on MO was downmodulated upon spreading of cells on surfaces coated with LBP-LPS complexes. The receptor appears to be distinct from other opsonic receptors since downmodulation of CR1, CR3, Fc gamma RI, Fc gamma RII, and Fc gamma RIII with mAbs did not affect binding of LBP-coated particles, and leukocytes from CD18-deficient patients bound LBP-coated particles normally. Coating of erythrocytes with LBP-LPS complexes strongly enhanced phagocytosis observed in the presence of suboptimal amounts of anti-erythrocyte IgG. However, binding mediated by LBP-LPS complexes alone caused neither phagocytosis of the LBP-coated erythrocytes nor initiation of an oxidative burst. The results of our studies define LBP as an opsonin. During the acute phase, LBP can be expected to bind gram-negative bacteria and bacterial fragments and promote the interaction of coated bacteria with phagocytes.

Acute-Phase Proteins↗
PubMed1989

Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein.

Lipopolysaccharide (LPS) binding protein (LBP), a recently discovered 60-kDa acute phase protein, is present in the acute phase serum of many species including human, rabbits, mice, and rats. Using either highly purified LBP from acute phase rabbit serum or unfractionated acute phase rabbit serum as a source of LBP, we examined the binding of LBP to LPS immobilized on plastic microtiter plates and to LPS electrotransferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of LBP bound to LPS was detected with goat anti-rabbit LBP and peroxidase-conjugated rabbit anti-goat IgG. LBP was found to bind to a variety of LPS types from both rough and smooth strains of Gram-negative bacteria, to lipid A, and to the tetraacyl glucosamine disaccharide diphosphate precursor IVA, but bound very poorly to the diacyl glucosamine phosphate, lipid X. No binding to 3-deoxyoctulosonic acid was observed. Binding affinities for LPS are near 10(9) M-1. The data presented here support the concept that LBP contains a binding site for lipid A.

Acute-Phase Proteins↗
PubMed1989

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide.

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.

Acetophenones↗