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H Loppnow

Publications and source records attributed to H Loppnow.

At least 37 records · Page 2Linked to original sources
PubMed1997

Tumor necrosis factor alpha (TNF alpha) is cardiodepressant in pathophysiologically relevant concentrations without inducing inducible nitric oxide-(NO)-synthase (iNOS) or triggering serious cytotoxicity.

Cardiac hypertrophy and heart failure are frequently accompanied by elevated plasma levels of tumor necrosis factor alpha (TNF alpha), the pathogenetic relevance of this finding being a matter of debate. In human acute septic cardiomyopathy, on the other hand, the negative inotropic impact of TNF alpha on the heart is well documented and frequently ascribed to the induction of inducible nitric oxide (NO) synthase (iNOS) and an enhanced production of NO in the heart. Yet the present study presents evidence that in cardiomyocytes TNF alpha in non-toxic concentrations specifically depresses contractile performance independent of NO. In spontaneously beating neonatal rat cardiomyocytes, TNF alpha in a low, pathophysiologically relevant concentration (10 U/ml, 1-3 days) does not alter basal pulsation amplitude, but blocks alpha- and beta-adrenoceptor-stimulated increase in contractility and beating irregularity and impairs the impact of high extracellular calcium on contractile performance. However, this low TNF alpha-concentration does not suffice to induce iNOS - documented by reverse transcriptase polymerase chain reaction - or enhance nitrite concentrations in the cell culture supernatants as a measure of cellular NO production, neither in the presence nor absence of dexamethasone (0.1 micro M). Only in high concentration - the specific proinflammatory action being documented by an enhanced release of interleukin-6 from cardiomyocytes - TNF alpha (1000 U/mol; 6, 24 h) weakly induces the mRNA for iNOS, with a consecutive moderate rise in cellular nitrite production. TNF alpha-incubation (10-1000 U/ml) does not alter the morphological appearance of the cells displayed by phase contrast microscopy or evoke gross cytotoxicity.

Animals↗
PubMed1997

Beta-adrenoceptor-mediated effects in rat cultured thymic epithelial cells.

1. Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid-induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. 2. In rat cultured thymic epithelial cells mRNA for beta 1- and beta 2-adrenoceptors was detected by reverse transcription-polymerase chain reaction by use of sequence-specific primers. Specific, saturable binding to the cultivated cells was observed with the beta-adrenoceptor agonist CGP 12177. 3. Adrenaline, noradrenaline or the beta-adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose-dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the beta 1- and the beta 2-subtypes. The selective beta 3-adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of beta 1-/beta 2-adrenoceptors to the response. 4. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum-stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T-lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Animals↗
PubMed1996

Human follicular dendritic cells and vascular cells produce interleukin-7: a potential role for interleukin-7 in the germinal center reaction.

Interleukin-7 (IL-7) has long been known as a potent growth factor in lymphocyte development. However, recent data obtained in vitro revealed additional functions for this cytokine, e.g. IL-7 influences the generation of cytotoxic T cells and NK cells, and in higher concentrations, activates monocytes in a manner similar to bacterial lipopolysaccharide. Furthermore, human tonsillar B cells are able to proliferate upon stimulation by IL-7 and cross-linking of the B cell receptor. Considering the latter role of IL-7 for B cell proliferation, we investigated which cells of the human immune system express IL-7 in vivo. mRNA expression of tonsillar cells was analyzed using the reverse transcription polymerase chain reaction, and protein expression was assessed by immunohistochemical staining of frozen sections. Among a variety of different immune cell types isolated from human tonsils, only follicular dendritic cells (FDC) expressed specific IL-7 products, whereas B and T cells were consistently negative. Immunohistochemical staining of tonsillar sections revealed the expression of immunoreactive IL-7 protein on FDC, and a pronounced expression could also be detected in oral mucosa and vascular endothelial cells. For the latter, we could also demonstrate mRNA expression in primary cultured cells. In light of the previous finding that IL-7 can act as a co-stimulus for the induction of proliferation in tonsillar B cells, these data suggest a role of IL-7 in the germinal center reaction. It is tempting to speculate that FDC may function as regulatory cells in B cell development in the tonsil, as epithelial nurse cells are thought to govern T cell development in the thymus.

B-Lymphocytes↗
PubMed1995

IL-8 specifically binds to endothelial but not to smooth muscle cells.

The proinflammatory cytokine and potent chemoattractant IL-8 is involved in regulation of infectious or inflammatory processes. Human vascular endothelial cells (EC) and smooth muscle cells (SMC) probably contribute to these responses by recognition and/or production of rIL-8. We demonstrate here in competitive binding studies with radiolabeled rIL-8 that EC and fibroblasts, but not SMC, specifically bind IL-8 with low affinity. The binding was not saturated by ligand concentrations up to 80 nM 125I-rIL-8. Unlabeled neutrophil-activating peptide-2 competed the binding of 125I-rIL-8, although less potently than unlabeled rIL-8, as reported previously for polymorphonuclear neutrophils. In contrast, connective tissue-activating peptide III, platelet factor 4, or lysozyme did not reduce binding of 125I-rIL-8 to EC or fibroblasts. In accordance with these binding studies, EC and fibroblasts, but not SMC, expressed human IL-8 receptor type I mRNA. Neither cell type expressed mRNA for IL-8 receptor type II. Stimulation with IL-1 alpha or LPS did not alter the results obtained in PCR or binding studies. Although SMC did not express specific binding sites for IL-8, Western blot experiments showed that IL-1 alpha-, TNF-, or LPS-stimulated SMC released two major immunoreactive isoforms of IL-8 in a time- and dose-dependent manner. The m.w. were similar to IL-8 isoforms released by EC or mononuclear cells. The differential capacity of EC and SMC to produce IL-8 and express IL-8 binding sites indicates that vascular cell-derived IL-8 may contribute to differential regulation of infectious and inflammatory responses in the vessel wall.

Base Sequence↗
PubMed1995

Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14.

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.

Antigens, CD↗
PubMed1995

Binding of lipopolysaccharide (LPS) to an 80-kilodalton membrane protein of human cells is mediated by soluble CD14 and LPS-binding protein.

Activation of cells by bacterial lipopolysaccharide (LPS) plays a key role in the pathogenesis of gram-negative septic shock. The 55-kDa glycoprotein CD14 is known to bind LPS and initiate cell activation. However, there must be additional LPS receptors because CD14 is linked by a glycosylphosphatidyl inositol anchor to the cell membrane and therefore unable to perform transmembrane signalling. Searching for potential LPS receptors, we investigated the binding of LPS to membrane proteins of the human monocytic cell line Mono-Mac-6. Membrane proteins were electrophoretically separated under reducing conditions, transferred to nitrocellulose, and exposed to LPS, which was visualized with anti-LPS antibody. Smooth- and rough-type LPS, as well as free lipid A, bound to a variety of proteins in the absence of serum. However, in the presence of serum, additional or preferential binding to a protein of approximately 80-kDa was observed. Experiments with differently acylated lipid A structures showed that the synthetic tetraacyl compound 406 was still able to bind, whereas no binding was detected with the bisacyl compound 606. The 80-kDa membrane protein was also detected on human peripheral blood monocytes and endothelial cells. The serum factors mediating the binding of lipid A to the 80-kDa membrane protein were identified as soluble CD14 and LPS-binding protein. From these results, we conclude that this 80-kDa protein is a candidate for the hypothetical molecule for LPS and/or LPS-CD14 recognition and signal transduction.

Acute-Phase Proteins↗
PubMed1995

Cytokine production by mononuclear cells following stimulation with a peptide-containing, endotoxin-free Escherichia coli extract.

The beneficial effects of the E. coli extract Colibiogen inj. N (Cb) observed in therapy of inflammatory bowel diseases, allergies, or gastrointestinal tumors are possibly mediated by the induction of cytokines in human leukocytes or vascular cells. Thus, the induction of the cytokines interleukin 1 (IL1), IL6 and tumor necrosis factor (TNF) in human mononuclear cells (MNC) and vascular cells was investigated in vitro. Various administration forms of the extract (including Cb-inj. N, Cb-oral, and Cb-infantibus N) induced the release of IL1 and IL6 from MNC. The compounds stimulated TNF production less potently, possibly due to a lower sensitivity of the TNF assay system, as compared to the IL1 and IL6 detection system. The MNC produced the cytokines with a kinetics similar to that observed with other stimuli. Monospecific antibodies abolished the respective cytokine activity in the biological assays. Addition of submaximal amounts of endotoxin potently enhanced the IL1- and IL6-inducing activity of the bacterial extract, indicating synergism of the extract and endotoxin. These results provide evidence that cytokines produced by MNC following administration of the tested bacterial extract may contribute to the regulation of the immune response during therapy of gastrointestinal tumors. At present the in vivo production of cytokines following treatment with the bacterial extract tested is under investigation in a phase III study.

Cytokines↗
PubMed1994

Specific binding of bacillus Calmette-Guérin to urothelial tumor cells in vitro.

Intravesical immunotherapy with bacillus Calmette-Guérin (BCG) against recurrences of superficial bladder cancer and carcinoma in situ is a highly effective regimen in urology. Despite intensive efforts to clarify the immunological mechanisms of the most successful immunotherapy known today, the cellular mechanism of its antitumor activity remains unknown. In our approach to elucidate the way of action of intravesical BCG, we applied an in vitro adhesion assay to investigate the interaction of radiolabeled BCG with urothelial bladder-tumor cells. We demonstrated a BCG dose-dependent binding to bladder-tumor cell lines derived from tumors of different gradings. The binding of BCG is apparently specific, since competition experiments showed an inhibition by nonradioactive BCG but not by Escherichia coli. We also found that there was no difference between the binding of living or heat-killed mycobacteria. Control experiments showed only a low affinity of BCG for fibroblasts, smooth-muscle cells, and endothelial cells in comparison with the tumor cells. Furthermore, we investigated the role of fibronectin as an adhesion molecule that is also present in the bladder wall. We demonstrated that BCG was capable of binding to fibronectin-coated surfaces in a dose-dependent manner. However, competitive binding assays failed to reveal an inhibition of the binding of BCG to bladder-tumor cells by anti-fibronectin. Furthermore, binding was not influenced by soluble fibronectin. These data suggest that the in vitro attachment of BCG to bladder-tumor cells appears not to be mediated by fibronectin. In electron microscope studies an adhesion of BCG to bladder-tumor cells was observed after an incubation period of ony 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

BCG Vaccine↗
PubMed1994

Bacterial endotoxin: molecular relationships of structure to activity and function.

Endotoxins of Gram-negative microbes fulfill as components of the outer membrane a vital function for bacterial viability and, if set free, induce in mammalians potent pathophysiological effects. Chemically, they are lipopolysaccharides (LPS) consisting of an O-specific chain, a core oligosaccharide, and a lipid component, termed lipid A. The latter determines the endotoxic activities and, together with the core constituent Kdo, essential functions for bacteria. The primary structure of lipid A of various bacterial origin has been elucidated and lipid A of Escherichia coli has been chemically synthesized. The biological analysis of synthetic lipid A partial structures proved that the expression of endotoxic activity depends on a unique primary structure and a peculiar endotoxic conformation. The biological lipid A effects are mediated by macrophage-derived bioactive peptides such as tumor necrosis factor alpha (TNF). Macrophages possess LPS receptors, and the lipid A regions involved in specific binding and cell activation have been characterized. Synthetic lipid A partial structures compete the specific binding of LPS or lipid A and antagonistically inhibit the production of LPS-induced TNF. LPS toxicity, in general, and the ability of LPS to induce TNF are also suppressed by a recently developed monoclonal antibody (IgG2a), which is directed against an epitope located in the core oligosaccharide. At present we determine molecular and submolecular details of the specificity of the interaction of lipid A with responsive host cells with the ultimate aim to provide pharmacological or immunological therapeutics that reduce or abolish the fatal inflammatory consequences of endotoxicosis.

Animals↗
PubMed1994

LPS, recIL1 and smooth muscle cell-IL1 activate vascular cells by specific mechanisms.

Vascular endothelial (EC) or smooth muscle cells (SMC) cultured in vitro respond to cytokines, such as interleukin 1 (IL1), or endotoxin (LPS) by production of IL6 or expression of adhesion molecules. Activation of the cells is mediated by specific recognition mechanisms as shown by inhibition experiments with IL1 receptor antagonists (IL1-Ra) or endotoxin antagonists in this report. IL1Ra reduced the activation of EC and SMC by recombinant IL1, as well as the activation of SMC with vascular cell-derived membrane IL1 in coculture experiments. Radiolabelled IL1 bound specifically to EC and SMC, indicating that both cell types expressed IL1 binding sites. LPS antagonists, such as lipid A precursor Ia or nontoxic LPS of Rhodobacter capsulatus reduced the LPS-stimulated IL6 production of EC and SMC and the adhesion of leukocytes to EC, but not the cytokine-induced activation. The antagonists blocked the LPS-mediated activation of vascular cells both in the presence of fetal calf or human serum. The activation of cells by LPS was dependent on the serum concentration. In contrast, activation of cells by IL1 was not serum dependent. Incubation of EC with CD14 antibodies prior to LPS stimulation in medium containing human serum reduced the activation of the cells. CD18 antibody (IB4) did not reduce activation. S-form LPS and IL1 stimulated IL6 production and adhesion differentially. Compared to IL1 stimulation (100%), S-form LPS induced adhesion to the same degree (96.4 +/- 15.5%; n = 6), however, IL6 production to a lower degree (33.7 +/- 15%; n = 6). These results show that both EC and SMC cultured in vitro respond specifically to both IL1 and LPS and that IL6 production and adhesion are regulated differentially.

Antibodies↗
PubMed1993

Agonists and antagonists for lipopolysaccharide-induced cytokines.

Agonistic and antagonistic properties of LPS and partial structures in the induction of cytokines are reviewed. Studies on structure-activity relationships of LPS and lipid A with human mononuclear cells reveal that S- and notably R-form LPS are very potent cytokine inducers. Synthetic E. coli lipid A is somewhat less active, whereas synthetic S. minnesota-type lipid A is significantly less active. Pentaacylated forms of lipid A are less potent than hexaacylated forms, and tetraacylated synthetic precursor Ia and bisacylated disaccharides and monosaccharides are completely inactive, indicating that a structure-dependent hierarchy of LPS and lipid A partial structures determines the monokine-inducing capacity in human mononuclear cells. Precursor Ia is a potent LPS antagonist. The mechanism of its inhibitory activity is shown to be due to competitive binding to cellular binding sites (receptors). Proinflammatory and antiinflammatory cytokines, receptor antagonists, and soluble cytokine receptors influence the cytokine-inducing activity of LPS, suggesting a complex regulatory network.

Acute-Phase Reaction↗
PubMed1993

[Bacterial endotoxins: relationship between chemical structure and biological effect].

Gram-negative bacteria carry on their surface endotoxins, which are essential for bacterial growth and survival. If released from the bacterial cell, endotoxins induce in higher organisms a great variety of pathophysiological effects. Chemically, endotoxins constitute lipopolysaccharides (LPS), the lipid component (termed lipid A) of which is responsible for the induction of endotoxin effects. The structural and conformational parameters, endowing lipid A with its potent bioactivity, have been well characterized. The toxic effects of endotoxins are initiated by the specific interaction of lipid A with macrophages/monocytes resulting in the production of peptide or lipid mediators. This interaction is governed by a unique (toxic) conformation of lipid A on the one hand, and by specific cellular receptors on the other. The interaction and subsequent mediator production can be specifically and antagonistically inhibited by lipid A partial structures. A recently developed monoclonal anti-LPS-antibody cross-reacts with endotoxins of various bacterial origin, and it cross-protects against harmful endotoxin effects such as pyrogenicity and lethality.

Animals↗
PubMed1992

Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways.

Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself ("activated SMC") elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1 alpha inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilitate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli.

Animals↗
PubMed1992

Atherogenic diets enhance endotoxin-stimulated interleukin-1 and tumor necrosis factor gene expression in rabbit aortae.

Cytokine-induced vascular changes may contribute to the atherogenic process. We examined the effect of atherogenic diets on the aortic expression of genes for the cytokines interleukin-1 alpha and beta and tumor necrosis factor alpha. Male New Zealand White rabbits were fed one of the following diets for 10 wk: nonpurified diet, a semipurified diet with 10% corn oil, a semipurified diet with 1% corn oil and 9% partially hydrogenated coconut oil, or the 9% coconut oil diet supplemented with either 0.1, 0.3 or 0.9% added cholesterol (n = 6/group). At 10 wk, 3 rabbits per group received lipopolysaccharide (200 micrograms/kg) intravenously. After 1.5 h the rabbits were killed and their aortae removed and analyzed. Histologic examination showed that the 0.3% and 0.9% cholesterol-fed rabbits developed appreciable aortic lesions. Diet had no major effect on the basal levels of aortic cytokine mRNA as determined by polymerase chain reaction analysis of DNAs. However, aortic tissue from rabbits fed 0.3% and 0.9% cholesterol diets showed significantly enhanced lipopolysaccharide-evoked levels of mRNA encoding interleukin-1 alpha (392 +/- 91 for saturated fat vs. 759 +/- 191 and 800 +/- 120 fmoles/reaction for 0.3% and 0.9% cholesterol), interleukin-1 beta (99 +/- 10 vs. 353 +/- 80 and 355 +/- 86) and tumor necrosis factor alpha (195 +/- 15 vs. 594 +/- 78 and 667 +/- 97). Extracts of aortae from rabbits injected with lipopolysaccharide contained interleukin-1 and tumor necrosis factor alpha activity but differences in biological activity due to diet were not detectable at the 1.5 h time point (chosen for maximal mRNA expression). Increased local cytokine gene expression in response to acute stimulus might influence the evolution of the vascular response to diets rich in cholesterol and saturated fats.

Animals↗