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R J Ulevitch

Publications and source records attributed to R J Ulevitch.

At least 73 records · Page 4Linked to original sources
PubMed1995

Lipopolysaccharide (LPS) signal transduction and clearance. Dual roles for LPS binding protein and membrane CD14.

Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.

Acute-Phase Proteins↗
PubMed1995

Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide.

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.

Animals↗
PubMed1995

Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.

Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

Amino Acid Sequence↗
PubMed1995

CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia.

This study was carried out to determine the role of CD14 receptors in the uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Treatment of microglial cell cultures with antibodies to CD14 or with soluble CD14 significantly blocked infection by M. tuberculosis H37Rv, suggesting that CD14 receptors could facilitate entry of nonopsonized tubercle bacilli into macrophages within the brain.

Antigens, CD↗
PubMed1995

Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin.

In humans and experimental animals the presence of bacterial lipopolysaccharide (endotoxin, LPS) signals the presence of gram-negative bacteria. Recognition of LPS triggers gene induction by myeloid and nonmyeloid lineage cells. These inducible genes encode proteins that include cytokines, adhesive proteins, and enzymes that produce low molecular weight proinflammatory mediators. Together the products of these inducible genes upregulate host defense systems that participate in eliminating the bacterial infection. Unfortunately, these same mediators contribute to a serious human disease known as septic shock. Considerable progress has been made during the past decade in determining the sources, identities, and sequence of release of these mediators. In contrast, until recently, marked gaps in our knowledge existed regarding the identity of the LPS receptor and intracellular signaling pathways responsible for LPS-induced cell activation. The discovery in 1986 of a plasma protein termed LPS binding protein (LBP) led to the discovery of unanticipated mechanisms of LPS-induced cell activation. CD14 was found as a soluble serum protein or as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells; it now occupies a key role in LPS-induced cell activation as we understand it today. Here we discuss how LBP enables LPS binding to CD14 and how complexes of LPS and soluble or GPI-anchored CD14 participate in cell activation. We also review the evidence supporting a model for a functional LPS receptor of myeloid cells, which is multimeric, comprised of GPI-anchored CD14 and a presently unidentified transmembrane protein that together bind LPS and initiate cell activation via kinase cascades.

Acute-Phase Proteins↗
PubMed1995

Tumor necrosis factor-alpha and interleukin-1 beta mediate human endothelial cell activation in blood at low endotoxin concentrations.

Activation of endothelial cells by endotoxin (lipopolysaccharide, LPS) may occur through two different pathways. LPS can directly activate endothelial cells through its interaction with soluble CD14 or indirectly via cytokines produced in blood in response to LPS. Substitution of whole blood for plasma apparently increases the endothelial cells responses to LPS by a factor of 1,000, rendering them sensitive to subpicomolar quantities of LPS. This shift in sensitivity is dependent on the presence of monocytes or conditioned plasma from whole blood incubated with small concentrations of LPS. Herein, using agents that block the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we demonstrate that TNF-alpha and IL-1 beta are the two LPS-induced cytokines responsible for the activation of endothelial cells, produced in blood in response to picomolar quantities of LPS. Anti-TNF-alpha monoclonal antibodies (mAbs) and IL-1 receptor antagonist separately had partial inhibitory effects. Complete and sustained inhibition of endothelial cell activation was obtained only when the two inhibitors were added together. We conclude that TNF-alpha and IL-1 beta induced in whole blood by picomolar concentrations of LPS mediate endothelial cell activation to these small quantities of LPS and that blocking of both cytokines is necessary to inhibit LPS-induced blood-dependent endothelial cell activation.

Antibodies, Monoclonal↗
PubMed1995

Activation of endothelial cells by endotoxin: direct versus indirect pathways and the role of CD14.

Optimal activation of endothelial cells by nanomolar quantities of endotoxin (lipopolysaccharide, LPS) requires the presence of plasma or serum. We and others have demonstrated that soluble CD14 (sCD14) and LPS binding protein (LBP) were the key plasma proteins mediating endothelial cell responses to LPS. The role of LBP is to transfer LPS to sCD14 and newly formed LPS-sCD14 will in turn activate endothelial cells via an as yet unknown surface receptor. This plasma-dependent pathway of endothelial cells activation is referred as to the direct pathway. However, endothelial cells are in constant contact with whole blood and not only with plasma. In experiments where whole blood was substituted for plasma, we showed that endothelial cells became sensitive to picomolar, rather than nanomolar quantities of LPS. The fact that endothelial cell responses were amplified by the presence of whole blood prompted us to search for the responsible blood cell(s) and mediator(s) for this effect. Blood cell fractionation experiments, experiments with blood from PNH patients and the use of anti-CD14 antibodies pointed to the monocyte as the blood cell responsible for the amplification effect. Moreover, the blood effect could be entirely reproduced by cells from a CD14-expressing cell line, such as calcitriol-differentiated HL-60 cells. Inhibitors to TNF and to IL-1 blocked LPS-induced activation of endothelial cells partially when added separately to whole blood, but abrogated endothelial cell responses when added together. Thus, the whole blood effect begins with LPS activation of monocytes via cell membrane CD14 and results in endothelial cell activation by the effects of TNF and IL-1. The monokine-mediated endothelial cell activation is referred as to the indirect pathway.

Cells, Cultured↗
PubMed1994

A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.

Mammalian cells respond to endotoxic lipopolysaccharide (LPS) by activation of protein kinase cascades that lead to new gene expression. A protein kinase, p38, that was tyrosine phosphorylated in response to LPS, was cloned. The p38 enzyme and the product of the Saccharomyces cerevisiae HOG1 gene, which are both members of the mitogen-activated protein (MAP) kinase family, have sequences at and adjacent to critical phosphorylation sites that distinguish these proteins from most other MAP kinase family members. Both HOG1 and p38 are tyrosine phosphorylated after extracellular changes in osmolarity. These findings link a signaling pathway in mammalian cells with a pathway in yeast that is responsive to physiological stress.

Amino Acid Sequence↗
PubMed1994

Lipopolysaccharide (LPS) binding protein, truncated at Ile-197, binds LPS but does not transfer LPS to CD14.

Lipopolysaccharide (LPS) binding protein (LBP), a 58-60 kDa glycoprotein, binds to the lipid A region of LPS. The resulting LPS-LBP complex is recognized by both the membrane-bound (mCD14) and soluble forms of CD14 (sCD14), thereby enhancing the ability of LPS to activate myeloid, endothelial, and epithelial cells. To begin to characterize the structure-function relationships within LBP, we have created and expressed a truncated form of human LBP (herein called NH-LBP) comprising amino acid residues 1-197 of the parent molecule. Experiments were done to characterize the ability of NH-LBP to bind LPS and to promote LPS binding to CD14. We found that NH-LBP efficiently binds LPS but does not transfer the LPS to either mCD14 or sCD14. Additionally, NH-LBP inhibited LPS binding to LBP, inhibited the LBP-promoted binding of LPS to CD14, and inhibited the LBP-dependent activation of rabbit peritoneal exudate macrophages. The apparent dissociation constant for LPS-NH-LBP complexes is less than 1 x 10(-8) M which compares well with the dissociation constant for LPS-LBP complexes of approximately 1 x 10(-9) M. We conclude from these studies that the LPS binding site of LBP resides in the amino-terminal half of LBP and that the CD14 interaction site resides in the carboxyl-terminal half of LBP. These data suggest that appropriately modified fragments of LBP might provide novel reagents with high LPS binding affinity that could be useful in inhibiting LPS-dependent cellular activation in vivo.

Acute-Phase Proteins↗
PubMed1994

Lipopolysaccharide binding protein expression in primary human hepatocytes and HepG2 hepatoma cells.

Lipopolysaccharide (LPS)-binding protein (LBP) is a normal plasma protein and an acute phase reactant important for host responses to Gram-negative bacteria and LPS. LBP forms high affinity complexes with LPS which bind to CD14, a monocyte surface protein, to initiate the release of inflammatory mediators. We found that human primary hepatocytes synthesize LBP and that the synthesis is up-regulated by interleukin (IL)-6. To examine this phenomenon in more detail, we evaluated the capacity of IL-6, IL-1, and tumor necrosis factor to induce LBP synthesis in HepG2 cells in the presence or absence of dexamethasone. IL-6 induced LBP synthesis. Dexamethasone, IL-1, and tumor necrosis factor had a synergistic effect when combined with IL-6, but demonstrated minimal effect independently. LBP biosynthesis was evaluated by immunoprecipitation of 35S-labeled LBP from HepG2 supernatants, measurement of steady-state LBP mRNA levels, and analysis of LBP-dependent LPS binding to CD14 positive cells. An 35S-labeled, 60-kDa protein was immunoprecipitated with anti-LBP antibody from IL-6-stimulated HepG2 cell supernatants. Northern blot analysis of cellular RNA revealed an increase in LBP mRNA in IL-6-stimulated cells. CD14 expressing cells bound fluoresceinated LPS in the presence of supernatants from HepG2 cells treated with IL-6. These data provide the first information about specific cytokine and dexamethasone regulation of LBP expression in HepG2 cells. LBP behaves like a Type 1 acute phase protein.

Acute-Phase Proteins↗
PubMed1994

The CD14 differentiation antigen mediates the development of endotoxin responsiveness during differentiation of mononuclear phagocytes.

The CD14 antigen was originally described as a differentiation antigen on mononuclear cells. The purpose of this study was to investigate the relationship between the appearance of surface CD14 and the acquisition of lipopolysaccharide (LPS) responsiveness during maturation of mononuclear phagocytes. Immature THP-1 cells responded poorly to LPS in the absence or presence of serum. Treatment with the maturational agent calcitriol caused a dose- and time-dependent increase in CD14 mRNA and surface CD14 and enhanced the responsiveness of THP-1 cells to smooth and rough form LPS, complexes of LPS and lipopolysaccharide-binding protein (LBP), and LPS in low concentrations of serum. Monoclonal antibodies to CD14 blocked the responses of THP-1 to LPS, LPS-LBP complexes and LPS in serum. Immunodepletion of LBP from serum also inhibited the effect of LPS in serum. The data show that maturation of the response of THP-1 cells to LPS and LPS-LBP complexes depends on the appearance of CD14 on the cell surface. Maturation of the response to LPS in serum depends in large part on the appearance of CD14 on the cell surface and the presence of LBP in serum.

Actins↗
PubMed1994

Recognition of endotoxin by cells leading to transmembrane signaling.

Bacterial endotoxins act at picomolar to nanomolar concentrations to stimulate a wide variety of cell types including phagocytic and endothelial cells. The major elements identified to date that are crucial for recognition of endotoxin are lipopolysaccharide (LPS)-binding protein, membrane-bound CD14 and, most recently, soluble CD14. Recent results also indicate that membrane-bound CD14 is probably one part of a multi-component LPS receptor. An immediate consequence of engagement of this functional LPS receptor is protein tyrosine phosphorylation and initiation of the multiple intracellular events associated with LPS-induced cell activation.

Animals↗
PubMed1994

CD14 is a pattern recognition receptor.

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Animals↗
PubMed1994

Role of the lipopolysaccharide (LPS)-binding protein/CD14 pathway in LPS induction of tissue factor expression in monocytic cells.

Endotoxic shock is associated with a coagulopathy, organ failure, and death. Tissue factor (TF) expression by monocytes exposed to bacterial endotoxin (lipopolysaccharide [LPS]) may mediate the coagulopathy and contribute to the high mortality of this disease. We examined the role of the LPS-binding protein (LBP)/CD14 receptor pathway in the LPS induction of TF expression in human monocytic THP-1 cells and peripheral blood monocytes. In THP-1 cells, the threshold concentration of LPS required to induce TF activity in serum-free medium was reduced 20-fold by purified LBP, which also enhanced TF mRNA synthesis. Similarly, monocytes cultured in the presence of serum were induced to express TF antigen at LPS concentrations 100 times lower than monocytes cultured in serum-free medium. An anti-LBP monoclonal antibody indicated that this effect was dependent on the presence of LBP in serum. LPS/LBP induction of TF activity and TF antigen expression in these monocytic cells were also inhibited by an anti-CD14 monoclonal antibody, indicating a requirement for the CD14 receptor. Thus, we suggest that low levels of LPS (5 to 100 pg/mL) present during sepsis induce TF expression in monocytes via the LBP/CD14-dependent pathway.

Acute-Phase Proteins↗
PubMed1993

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

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.

Antigens, CD↗