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R R Schumann

Publications and source records attributed to R R Schumann.

49 records · Page 3Linked to original sources
PubMed1996

Lipopolysaccharide induces the rapid tyrosine phosphorylation of the mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14.

Human vascular endothelial cells (HUVECs), which do not display the lipopolysaccharide (LPS) receptor CD14, were examined for protein tyrosine phosphorylation after LPS stimulation in the presence and absence of soluble CD14 (sCD14). By phosphotyrosine Western blotting and immunocomplex kinase assays we show that LPS was capable of inducing in these cells rapid protein tyrosine phosphorylation and kinase activation of two members of the mitogen-activated protein kinase (MAPK) family erk-1 and the newly discovered p38, requiring the presence of sCD14. LPS-induced tyrosine phosphorylation of MAPK was associated with increased transcript- and surface protein expression of intracellular adhesion molecule-1 by HUVECs. MAPK phosphorylation and activation was induced by LPS in concentrations as little as 30 ng/mL and as early as 15 minutes after stimulation. Furthermore, tyrosine kinase inhibitors such as Genistein partially inhibited this effect. These results show that LPS triggers similar signaling events in both CD14+ myelo-monocytic cells and cells lacking the putative LPS-receptor CD14, suggesting the presence of a common, yet unidentified element in LPS-signaling in both cell types.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Transcript synthesis and surface expression of the interleukin-2 receptor (alpha-, beta-, and gamma-chain) by normal and malignant myeloid cells.

Expression of the interleukin-2 receptor alpha-(IL-2Ralpha-), IL-2Rbeta-, and the recently identified IL-2Rgamma-chain was examined on a wide range of cells of myeloid origin including neutrophils, monocytes, normal bone marrow-derived myeloid progenitors enriched for CD34+ cells, bone marrow blasts obtained from acute myelogenous leukemia (AML) patients, and permanent myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction and surface membrane analysis using receptor chain-specific monoclonal antibodies and flow cytometry. Expression of the p75 IL-2Rbeta- and the p64 IL-2Rgamma-chain was a common finding in most of the myeloid cell samples investigated, whereas IL-2Ralpha-chain was less frequently expressed. Although the high-affinity IL-2R form (ie, the alpha+, beta+, gamma+ IL-2R form) was detectable in a small minority of primary AML samples as well as the KG-1 cell line and IL-2 binding to these cells was sufficient to initiate signal transduction as evidenced by an increase in overall protein tyrosine phosphorylation and more specifically in tyrosine phosphorylation of the Janus kinase (JAK) 3, in none of these cell types did exposure to IL-2 affect cell growth kinetics. These results suggest that, in myeloid cells, the IL-2R may not stimulate mitogenic responses or that its components may be expressed in a combinational association with receptors for other cytokines and that IL-2Rgamma may play a regulatory role in normal and malignant myelopoiesis possibly independent from IL-2. Because recent studies by others have indicated that the IL-2Rgamma- chain may be shared by the IL-4R, the IL-7R, and most likely the IL-9R, expression of mRNA of these receptor types was also investigated in these cell samples. Surprisingly, in a substantial part of the myeloid lineage cells examined, an IL-2Rgamma+, IL-4R-, IL7R- configuration was noted that was, however, frequently associated with expression of IL-9R. Sharing of IL-9R/IL-2R components was furthermore suggested by inhibition of 125I-IL-2 binding to primary AML cells with excess of unlabeled IL-9.

Acute Disease↗
PubMed1996

The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins.

Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1), IL-6, and glucocorticoids, involving transcriptional gene activation. Lipopolysaccharide-binding protein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms by which the LBP gene is activated, we have investigated the regulation of expression of its mRNA in vitro and in vivo as well as the organization of 5' upstream regulatory DNA sequences. We show that induction of LBP expression is transcriptionally regulated and is dependent on stimulation with IL-1beta, IL-6, and dexamethasone. By definition, LBP thus has to be viewed as a class 1 acute-phase protein and represents the first APR identified which is capable of detecting pathogenic bacteria. Furthermore, cloning of the LBP promoter revealed the presence of regulatory elements, including the common APR promoter motif APRE/STAT-3 (acute-phase response element/signal transducer and activator of transcription 3). Luciferase reporter gene assays utilizing LBP promoter truncation and point mutation variants indicated that transcriptional activation of the LBP gene required a functional APRE/STAT-3 binding site downstream of the transcription start site, as well as an AP-1 and a C/EBP (CCAAT enhancer-binding protein) binding site. Gel retardation and supershift assays confirmed that upon cytokine stimulation APRF/STAT-3 binds to its recognition site, leading to strong activation of the LBP gene. Unraveling of the mechanism of transcriptional activation of the LBP gene, involving three known transcription factors, may contribute to our understanding of the acute-phase response and the pathophysiology of sepsis and septic shock.

Acute-Phase Proteins↗
PubMed1995

Mechanisms of transcriptional activation of lipopolysaccharide binding protein (LBP).

The Lipopolysaccharide Binding Protein (LBP) is of high importance for endotoxin recognition, presentation and subsequent cytokine induction in immune cells. LBP, which is a member of a growing family of structurally and functionally related proteins, is synthesized in hepatocytes and secreted into the blood stream constitutively. During the acute phase response, however, LBP levels rise substantially and here the mechanisms of induction of LBP protein synthesis were reviewed. The induction of LBP in hepatocytes is due to transcriptional and posttranscriptional mechanisms as we have shown by nuclear run-on and RNA half-life experiments. Cloning of the 5'-flanking region of the LBP gene, furthermore revealed a typical acute phase protein promoter. Reporter gene assays employing the luciferase gene and mutation variants of the LBP promoter revealed that integrity of a common acute phase promoter motif, named APRE/STAT-3 is essential for activation of the LBP promoter. Elucidation of the transcriptional activation mechanism could point the way to a therapeutically lowering of LBP levels in high risk patients for reducing their susceptibility to Gram-negative septic shock.

Acute-Phase Proteins↗
PubMed1994

Regulation of MCP-1/JE gene expression during monocytic differentiation.

We examined the expression of the MCP-1/JE gene in freshly prepared human monocytes and again after either in vitro or in vivo maturation of these cells. We show that previously unstimulated blood monocytes of healthy individuals prepared by adherence procedures display high levels of MCP-1/JE mRNA and protein. Monocytes that were not previously exposed to activational plastic surfaces but were separated from other blood cells by counter-flow centrifugal elutriation expressed severalfold lower MCP-1/JE transcript and protein levels. Treatment of these cells with endotoxin was associated with the down-regulation of MCP-1/JE mRNA and protein levels. In contrast, exposure to IFN-gamma resulted in increased MCP-1/JE gene expression. During the process of in vitro maturation of monocytes into macrophages, a gradual decrease of MCP-1/JE mRNA and protein expression was noted. Both mature (day 8) blood monocyte-derived culture macrophages and peritoneal macrophages completely failed to express the MCP-1/JE gene, which could be restored after exposure to IFN-gamma and cross-linking of macrophage Fc gamma R with murine solid phase IgG2a mAbs; however, endotoxin failed to induce MCP-1/JE expression in these cells.

Blotting, Northern↗
PubMed1994

Discordant adaptation of human peritoneal macrophages to stimulation by lipopolysaccharide and the synthetic lipid A analogue SDZ MRL 953. Down-regulation of TNF-alpha and IL-6 is paralleled by an up-regulation of IL-1 beta and granulocyte colony-stimulating factor expression.

Human peritoneal macrophages were exposed to increasing doses of LPS or a synthetic lipid A analogue (SDZ MRL 953) and production of the cytokines IL-1 beta, IL-6, TNF-alpha, and G-CSF was assessed at the protein and mRNA level. Cells were also prestimulated with low doses of LPS and SDZ MRL 953 to study their adaptation to a secondary challenge with high doses of LPS. The ability of macrophages to produce high levels of TNF-alpha and IL-6 after stimulation with LPS could be relieved almost completely by preincubating cells with low doses of LPS. Decreases of TNF-alpha and IL-6 production resulted from inhibition of gene transcription and/or changes in mRNA stability, as transcript levels of these cytokines were down-modulated by the process of LPS adaptation. Surprisingly, however, adapted cells were able to synthesize even larger quantities of G-CSF and IL-1 beta when exposed to a secondary LPS challenge. mRNA levels of the adapted cells remained unaltered for IL-1 beta, but were slightly increased for G-CSF as assessed by Northern blot analysis. High doses of the synthetic lipid A analogue SDZ MRL 953 were also able to adapt macrophages to a secondary LPS challenge by down-regulating TNF-alpha and IL-6 production, whereas priming secretion of G-CSF and IL-1 beta as well. We describe here the discordant adaptation of human peritoneal macrophages to a secondary LPS stimulus in vitro. These findings appear to have ramifications for the in vivo endotoxin response during inflammation and also Gram-negative septicemia.

Dinoprostone↗
PubMed1992

Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, the receptor for LPS/LBP complexes: a short review.

With the recent discovery and cloning of the lipopolysaccharide-binding protein (LBP), the "adapter-molecule" for LPS-binding to the cell surface receptor CD14 was found. The ligand-receptor pair LPS/LBP-CD14 seems to be one important element in LPS-mediated activation of monocytic cells and possibly granulocytes and B cells. Here, some of the known functions of the proteins involved, LBP and CD14, are reviewed in the context of other endotoxin recognition studies, and the outlook for ongoing and future investigations is described.

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

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

Monocyte long-term cultivation on microvascular endothelial cell monolayers: morphologic and phenotypic characterization and comparison with monocytes cultured on tissue culture plastic.

Human monocytes were cultured on monolayers of a newly established microvascular endothelial cell strain. As compared to monocytes cultured on plastic, these endothelium-"derived" monocytes (EDM) showed distinct morphology, higher motility, and different antigen-expression pattern for several surface markers, as detected by cytofluorimetry. The MO-1- and the Leu-M1-marker were maintained on EDMs while they were lost on plastic-cultured cells. The MAX 1-26-termed markers failed to increase on EDMs, in contrast to plastic-cultured monocytes. For seven additional markers, expression after two weeks in vitro was higher on EDMs than on plastic-cultured monocytes. Functionally EDMs showed typical monocyte/macrophage behavior and were easily removable from the culture system for further experimentation. Our data suggest that monocytes cultured on microvascular endothelial cells are maintained for several weeks in a more physiologic state than monocytes cultured on plastic.

Animals↗