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R J Ulevitch

Publications and source records attributed to R J Ulevitch.

At least 19 recordsLinked to original sources
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

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↗
PubMed1979

New function for high density lipoproteins. Their participation in intravascular reactions of bacterial lipopolysaccharides.

The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 or Salmonella minnesota R595 to plasma (or serum) resulted in a marked reduction of the hydrated buoyant density of the parent LPS (0111:B4 [d = 1.44 g/cm3] and R595 [d = 1.38 g/cm3]), to d less than 1.2 g/cm3. This reduction in buoyant density to less than 1.2 g/cm3 of the LPS required plasma (or serum) lipid. Delipidation of plasma (or serum) by extraction with n-butanol/diisopropyl ether (40/60, vol:vol) prevented the conversion of the parent LPS to a form with d less than 1.2 g/cm3. Reversal of the effect of delipidation was accomplished by the addition of physiologic concentrations of high density lipoprotein (HDL). In contrast, as much as two times normal serum concentration of low density or very low density lipoprotein were ineffective. The ability of normal plasma (or serum) to inhibit the pyrogenic activity of LPS, lost after delipidation, was also restored after the addition of HDL. Preliminary results suggested that prior modifications of the LPS, probably disaggregation, may be required before interaction with HDL.

Animals↗
PubMed1978

Two distinct mechanisms for the initiation of mast cell degranulation. II. A specific inhibition of amine release by serum proteins.

Our experiments have provided additional data in support of the concept that different mast cell activators follow distinct biochemical path-ways in the initiation of secretion and degranulation. To do this we have taken advantage of the observation that some serum proteins, and albumins in particular, have the capacity to inhibit selectively the release of amines from rat peritoneal mast cells initiated by some, but not all, stimuli. We show that the relative inhibition of release obtained is independent of the concentration of activator but dependent upon the concentration of albumin, indicating that the inhibitory process does not involve a direct activator--inhibitor interaction. Finally, our data demonstrate that the inhibition does not interfere with the ability of the acitivator to interact with the mast cell. Thus, cells incubated with activator in the presence of inhibitor become increasingly unresponsive, or desensitized, to subsequent challenge with activator in the absence of inhibitor. These combined data therefore provide evidence, that, in addition to a selectivity in the activation/desensitization process initiated by different mast cell stimuli, at least one of the biochemical steps subsequent to the activation step is also not shared by all mast cell stimuli.

Animals↗
PubMed1978

Role of complement in lethal bacterial lipopolysaccharide-induced hypotensive and coagulative changes.

The effect of C3 depletion on the multiple pathophysiological changes produced by a lethal dose of Serratia marcescens lipopolysaccharide (LPS) was evaluated. The injection of this LPS into rabbits resulted in biphasic hypotensive changes and thrombocytopenia. These changes were characterized by an acute, transient decrease occurring within minutes after injection followed by a second more gradual decrease beginning 30 to 60 min post-LPS. Prior depletion of C3, with the anticomplementary protein from cobra venom (CoF), did not alter the extent of either the gradual hypotensive and platelet changes or the coagulative and metabolic changes when normal and C3-depleted rabbits were compared. Importantly, the lethal effects of S. marcescens LPS were not reduced by prior depletion of C3. Only the immediate, reversible thrombocytopenia and hypotension were abrogated by C3 depletion.

Animals↗
PubMed1978

Distribution of endotoxin (lipopolysaccharide) in the tissues of lipopolysaccharide-responsive and -unresponsive mice.

We examined the distribution of bacterial lipopolysaccharide (LPS) in LPS-responsive (C3H/St) and LPS-unresponsive (C3H/HeJ) mice. The results reported here demonstrate that the rates of removal of an immunological or a toxic dose of LPS from the circulation are the same in both strains of mice. C3H/St spleens accumulated significantly more LPS than C3H/HeJ spleens after the intravenous injection of either an immunogenic or a toxic dose of LPS. There was also a greater amount of LPS associated with cells teased from C3H/St spleens compared to those from C3H/HeJ spleens. After a toxic dose of LPS, there was more LPS in C3H/St lymph nodes, adrenals, lungs, kidneys, and heart than in the corresponding C3H/HeJ tissues. The accumulation of more LPS in tissues from C3H/St mice compared to C3H/HeJ mice suggests that these tissues are involved in the pathophysiological and, ultimately, the toxic effects of LPS. The differential accumulation of LPS in the tissues of these two strains may be the reason for the decreased responses of C3H/HeJ mice to LPS.

Animals↗
PubMed1978

The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum.

Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.

Animals↗
PubMed1978

Phospholipase A2 contamination of cobra venom factor preparations. Biologic role in complement-dependent in vivo reactions and inactivation with p-bromophenacyl bromide.

Cobra venom factor (CoF), the anticomplementary protein in Naja naja cobra venom, is usually purified by sequential ion exchange and gel filtration chromatography. CoF prepared in this manner contains small but significant quantities of phospholipase A2 activity. This acyl hydrolase activity can be simply and efficiently removed on a large scale by treatment of CoF with p-bromophenacyl bromide (BPB), an irreversible modifier of the histidine residue in the active site of phospholipase A2. BPB treatment does not alter the anticomplementary activity of CoF. In vivo experiments utilizing intratracheal injections of control and BPB-treated CoF, as well as pure phospholipase A2, revealed that contaminating phospholipase A2, and not the anticomplementary protein, was responsible for the observed acute neutrophil-associated lung injury. However, phospholipase A2 had no effect on the hypotensive and thrombocytopenic effects of CoF infected intravenously into rabbits. Depletion of circulating C3-C9 by intraperitoneal injections of CoF was not altered by removal of phospholipase A2 activity with BPB.

Acetophenones↗
PubMed1978

The effect of complement depletion on bacterial lipopolysaccharide (LPS)-induced hemodynamic and hematologic changes in the Rhesus monkey.

Lipopolysaccharide (LPS) isolated from Salmonella minnesota R595 or from Escherichia coli 0111:B4 induces hypotension in rhesus monkeys with normal complement levels. This hypotension is accompanied by decreased total peripheral resistance. The depletion of C3 and terminal complement components by prior intraperitoneal administration of the anticomplementary protein cobra factor did not alter the hemodynamic changes which occur following the rapid injection of 5 mg/kg of R595 LPS, the infusion of 500 microgram/kg of R595 LPS, or the injection of 500 microgram/kg of 0111:B4 LPS. We conclude that the LPS-induced hemodynamic changes in the subhuman primate are medicated by pathways which do not require the participation of C3. The kinetics and extent of the neutropenia and thrombocytopenia resulting from the injection of 0111:B4 or R595 LPS were not latered by prior depletion of greater than 95% of the plasma C3.

Animals↗
PubMed1977

Complement-dependent hemodynamic and hematologic changes in the rabbit.

The intravenous injection of the anticomplementary protein from cobra venom, cobra factor (CoF),2 induces decreases in mean arterial blood pressure and circulating platelets in rabbits. The changes are rapidly reversed. Both changes require the presence of C3 and occur in rabbits genetically deficient in the sixth component of complement. The hypotensive effects of CoF were blocked by the histamine H2-receptor antagonist burimamide. An acute C3-dependent change in blood pressure and circulating platelets also was demonstrated following the intravenous injection of S. marcescens endotoxin. However, abrogation of these acute changes by C3 depletion did not alter the extent of a second, prolonged fall in blood pressure and platelets induced by S. marcescens endotoxin occurring after 60--90 min. C3 depletion also did not alter the lethal effects of the S. marcescens endotoxin.

Animals↗