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Biomedical subjects

A Wendel

Publications and source records attributed to A Wendel.

At least 19 recordsLinked to original sources
PubMed1996

Endotoxin-inducible granulocyte-mediated hepatocytotoxicity requires adhesion and serine protease release.

In primary cultures of Kupffer cells and hepatocytes, human granulocytes potentiated toxicity of endotoxin about 1000-fold. Granulocyte elastase activity was found to correlate with toxicity. The serine protease inhibitors alpha1-antitrypsin, eglin C, and aprotinin protected against toxicity. Tumor necrosis factor-alpha (TNF-alpha) induced cytotoxicity and elastase release, whereas neutralization of TNF-alpha blocked both events. We conclude that TNF-alpha formed by Kupffer cells activates granulocytes. Experiments in cultures where cells were separated by membranes permeable to mediators indicated that cell contact is needed for toxicity. Scanning electron microscopy showed granulocytes adhering to and interdigitating with hepatocytes. Using liver cells from ICAM-1-deficient mice had no effect on toxicity. However, neutralizing CD31 inhibited toxicity and elastase release but not granulocyte adhesion. Our findings demonstrate that adhesion of granulocytes is a necessary but not sufficient condition for the synergistic interaction of endotoxin-stimulated liver macrophages and granulocytes in the proteolytic killing of hepatocytes.

Androstadienes↗
PubMed1996

Effect of urodilatin on platelet-activating factor-induced bronchoconstriction, vasoconstriction and edema formation in isolated rat lung.

In the isolated perfused rat lung, perfusion with platelet-activating factor causes bronchoconstriction, vasoconstriction and edema formation. The bronchoconstriction and vasoconstriction are largely mediated by thromboxane, whereas the edema formation is due to enhanced vascular permeability unrelated to eicosanoids. Since natriuretic peptides are known to relax smooth muscle and were suggested to attenuate enhanced vascular permeability, we investigated the effect of urodilatin on the PAF-induced alterations in lung function. Pretreatment with urodilatin (0.25 microM or 0.75 microM) reduced the PAF-induced increase in airway and vascular resistance by approximately 50%. Urodilatin pretreatment, however, was completely ineffective against the PAF-induced increase in weight gain and in vascular permeability, as assessed by the vascular filtration coefficient. Furthermore, urodilatin failed to affect the release of thromboxane into the perfusate in PAF-exposed lungs. Thus, urodilatin relaxes airway and vascular smooth muscle, but fails to reduce edema formation in PAF-perfused rat lungs.

Analysis of Variance↗
PubMed1996

Isolation of rat primary lung cells: characterization of an improved method.

Lungs from female Wistar rats were enzymatically digested by stepwise recirculating perfusion through the pulmonary artery with various enzymes. Lung tissue was micro-dissected, resuspended and the cells obtained were washed by centrifugation. The results showed that our primary rat lung cell culture exceeded the quality of other isolation methods with regard to cell yield and viability and that these lung cultures might be representative of the cell mixture found in the organ.

Animals↗
PubMed1996

Control of fecal peritoneal infection in mice by colony-stimulating factors.

Granulocyte colony-stimulating factor (G-CSF) recruits and primes neutrophilic granulocytes. The role of endogenous and exogenous G-CSF was examined in a murine fecal peritoneal infection model characterized by rapid production of high levels of circulating G-CSF. Pretreatment with anti-murine G-CSF for 5 days reduced neutrophil counts by 50% and sensitized mice to sublethal peritonitis. There were more aerobic bacteria in livers of antiserum-pretreated animals but fewer neutrophils in peritoneal cavities. Pretreatment with 100 micrograms/kg recombinant murine G-CSF intravenously for 2 days raised neutrophil counts 5-fold and significantly protected animals against lethal peritonitis. A similar prophylactic administration of murine granulocyte-macrophage (GM)-CSF neither augmented leukocyte numbers nor protected infected mice. These results show a dissociation between the pharmacologic properties of GM-CSF and G-CSF and demonstrate the crucial role of endogenous G-CSF in controlling neutrophil-dependent defense against bacterial invasion in infection.

Animals↗
PubMed1996

Enhanced release of interleukin-10 and soluble tumor necrosis factor receptors as novel principles of methylxanthine action in murine models of endotoxic shock.

The immunomodulating capacity of the methylxanthine A802715 (5-hydroxy-5-methyl)hexyl-3-methyl-7-propylxanthin) was investigated in various murine models of endotoxemia and compared with that of the chemically related reference compound pentoxifylline. At a dose of 180 mg/kg both compounds protected mice against a lethal shock dose of lipopolysaccharide (LPS) (5 mg/kg) in nonsensitized mice and against LPS (5 micrograms/kg)-initiated liver failure in D-galactosamine (700 mg/kg)-sensitized animals. The methylxanthines attenuated systemic release of endogenous tumor necrosis factor (TNF) and interferon-gamma during endotoxic shock, and potently up-regulated early production of circulating interleukin-10 and interleukin-6. Treatment of mice with A802715 alone induced levels of circulating soluble TNF receptors (sTNF-R p55 and p75) 3- to 4-fold higher than those of controls. This increase was additive to the one elicited by LPS. Moreover, pentoxifylline and A802715 prevented liver injury due to intravenous injection of recombinant TNF in D-galactosamine-sensitized mice. In primary cultures of murine hepatocytes, A802715 (500 microM) as well as other cAMP-raising compounds conferred protection from TNF cytotoxicity. We concluded that, in addition to a direct target cell protection via an increase in intracellular cAMP, methylxanthines prevented the systemic toxicity of LPS in mice by a further principle, i.e., by a shift of the humoral response to LPS in favor of an enhanced release of immunosuppressive cytokines.

Animals↗
PubMed1996

Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin.

BACKGROUND: Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles. The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied. MATERIALS AND METHODS: Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS. RESULTS: LPS induced the release of TX and caused increased airway resistance after about 30 min. Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide. LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177. The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles. COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D. In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238. CONCLUSIONS: LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance. In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

Testing of immunomodulatory properties in vitro.

The immune response of different species to a given stimulus varies considerably. The in vitro evaluation of immunomodulatory properties of test compounds therefore prompts the use of human cells. We have conducted experiments on human whole blood incubations which offer the advantages of few preparation artefacts, natural cell environment and easy performance. Ten different immune stimuli were used to initiate leukocyte mediator release. Out of > 20 factors as readout, each and every stimulus released a unique set of factors with different kinetics and concentration dependences. We also used liver macrophages as an alternative cellular model. In this model, over-activation of the macrophages by endotoxin released a toxic combination of factors which killed co-cultured hepatocytes. Co-culture experiments were carried out with primary rat as well as with human liver cells to check for common mechanisms. Furthermore, we added human neutrophil granulocytes to these co-cultures which synergized with the macrophages in killing hepatocytes. Since a similar cellular interaction exists in vivo, this extended cell system bears additional characteristics of the in vivo situation. Therefore, these in vitro models of basic mechanisms of inflammation might be suitable for the evaluation of pro- and anti-inflammatory properties of test compounds.

Adjuvants, Immunologic↗
PubMed1996

Tumor necrosis factor production in the perfused mouse liver and its pharmacological modulation by methylxanthines.

The liver contains the largest pool of cytokine-producing macrophages in the body and may therefore play an important role in the development and outcome of systemic inflammatory response syndromes. Therefore, we investigated the tumor necrosis factor-alpha (TNF) releasing capacity of the in situ perfused mouse liver and its modulation by methylxanthines, i.e., by a class of well-established inflammatory cytokine-suppressing drugs. We have shown that pretreatment of mice with either lipopolysaccharide or TNF elicited a dose-dependent TNF release into the perfusate which was inhibited by in vivo pretreatment of mice with pentoxifylline or A-802715 [1-(5-hydroxy-5-methyl)hexyl-3-methyl-7-propylxanthin]. Infusion of these methylxanthines into livers from mice pretreated with lipopolysaccharide or TNF also inhibited TNF release in an immediate and reversible way even after TNF production had been initiated. The inhibitory effect of methylxanthines was prevented by pretreatment of mice with the adenylate cyclase inhibitor dideoxyadenosine, suggesting upregulation of the cyclic adenosine monophosphate system as a possible mechanism of action of these drugs. Our findings demonstrate that the liver is a potent cytokine producer and identify it as one of the target organs of methylxanthines or other phosphodiesterase inhibitors in murine models of shock and inflammatory liver failure.

Animals↗
PubMed1996

DNA fragmentation in mouse organs during endotoxic shock.

The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice.

Animals↗
PubMed1996

The 55-kD tumor necrosis factor receptor and CD95 independently signal murine hepatocyte apoptosis and subsequent liver failure.

BACKGROUND: Activation of either the 55-kD tumor necrosis factor receptor (TNF-R1) or CD95 (Fas/Apo-1) causes apoptosis of cells and liver failure in mice, and has been associated with human liver disorders. The aim of this study was first to clarify the association between CD95 activation, hepatocyte apoptosis, and fulminant liver failure. Next, we investigated whether TNF-R1 and CD95 operate independently of each other in the induction of hepatocyte apoptosis. MATERIALS AND METHODS: Using both mice and primary liver cell cultures deficient in either TNF-R1 or functional CD95, the induction of apoptosis and hepatocyte death following activation of TNF-R1 or CD95 were studied in vitro and in various in vivo models of acute liver failure. RESULTS: In vivo or in vitro stimulation of CD95 caused apoptosis of wild-type (wt) murine hepatocytes which had not been sensitized by blocking transcription. Time course studies showed that DNA fragmentation and chromatin condensation preceded, respectively, membrane lysis in vitro and necrosis in vivo. Similar results were obtained after CD95 activation in hepatocytes or livers lacking TNF-R1. Conversely, hepatocytotoxicity due to endogenous or exogenous TNF was not affected in animals or liver cell cultures lacking the expression of functional CD95. CONCLUSIONS: TNF-R1 and CD95 are independent and differentially regulated triggers of murine apoptotic liver failure.

Alanine Transaminase↗
PubMed1995

Quinine inhibits release of tumor necrosis factor, apoptosis, necrosis and mortality in a murine model of septic liver failure.

We investigated the effect of quinine on liver injury induced by lipopolysaccharide in mice sensitized with D-galactosamine. This model is characterized by high systemic release of tumor necrosis factor, which mediates hepatic apoptosis and necrosis. Pretreatment with quinine, a K+ channel blocker, prevented formation of tumor necrosis factor (TNF) as well as the subsequent hepatic DNA fragmentation and liver enzyme leakage. Thus, inhibition of K+ channels may be a novel therapeutic approach in cytokine-related organ damage.

Animals↗
PubMed1995

Effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers.

We explored the ex vivo alteration in the cytokine release of stimulated blood taken from healthy volunteers treated subcutaneously with 480 micrograms granulocyte colony-stimulating factor (G-CSF). In a double-blind, controlled, randomized study with 21 volunteers who received G-CSF once or twice 24 hours apart, we measured lipopolysaccharide (LPS)-inducible release of various cytokines and soluble receptors at different times after treatment. At day 1 after a single dose of G-CSF, mediator release was also initiated with muramyl dipeptide, Staphylococcus aureus enterotoxin A, lipoteichoic acid, streptolysin O, complement factor C5a, phytohemagglutinin, or phorbol myristate acetate. In blood from G-CSF-treated subjects, our major findings were (1) a maximal 12-fold increase in interleukin-1 receptor antagonist (IL-1ra) release and an increase of both the p55 and p75 soluble tumor necrosis factor (TNF) receptors; (2) a reduction in TNF release when using all the various stimuli described except LPS; (3) an increase in G-CSF and, to lesser extent, in IL-6, IL-8, and IL-10 release; and (4) an attenuation of interferon-gamma (IFN-gamma) and granulocyte-macrophage (GM)-CSF release. Our findings demonstrate that the major effect of G-CSF treatment is a change in the responsiveness of blood towards a variety of stimuli, which we interpret as a shift toward an antiinflammatory cytokine response.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1995

Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release.

The systemic inflammatory response is characterized by release of circulating TNF which may cause multiorgan failure including septic liver failure. We studied TNF signaling in an appropriate in vitro system with primary murine hepatocyte cultures from normal and genetically altered animals. Either one of the three different TNF species, huTNF-alpha, huTNF-beta, or muTNF-alpha (at concentrations > 1 ng/ml) induced direct hepatocytotoxicity preceded by DNA fragmentation in cells prepared from wild-type C57BL mice. TNF-induced cytotoxicity was preceded by oligonucleosomal DNA fragmentation. Further cellular responses to TNF exposure were induction of nitric oxide synthase and secretion of serum amyloid A. None of the above events occurred in hepatocytes lacking the gene for the 55-kDa TNF receptor (TNF-R1), even after stimulation with > 1 micrograms/ml TNF. However, selective stimulation of the TNF-R1 in wild-type hepatocytes with huTNF-alpha elicited a pattern of responses essentially similar to that seen with muTNF-alpha. We obtained analogous results when we examined the hepatotoxicity of TNF in D-galactosamine-sensitized mice, i.e., DNA fragmentation and liver failure was noted in wild-type mice, whereas TNF-R1-deficient mice were completely resistant. We conclude that the TNF-R1 is not only necessary, but also sufficient for TNF signaling in murine hepatocytes.

Animals↗
PubMed1995

Tunicamycin potently inhibits tumor necrosis factor-induced hepatocyte apoptosis.

The protein glycosylation inhibitor tunicamycin protected male BALB/c mice from tumor necrosis factor alpha-induced liver failure. Tunicamycin also inhibited tumor necrosis factor-induced cell death in primary hepatocyte cultures with a median inhibitory concentration of 8 nM, but not in the tumor cell line WEHI 164 clone 13. Hepatocyte death in our culture system was characterized by DNA fragmentation and apoptotic changes. These two characteristic signs of programmed cell death were also inhibited by tunicamycin treatment. These data suggest that protein glycosylation is an early and causal event of tumor necrosis factor (TNF)-induced parenchymal cell death in the liver.

Animals↗
PubMed1995

Lipopolysaccharide-induced interleukin-10 in mice: role of endogenous tumor necrosis factor-alpha.

Interleukin (IL)-10 is known to protect mice against the lethal effects of lipopolysaccharides (LPS) and is considered to be an anti-inflammatory cytokine which suppresses the production of pro-inflammatory cytokines. We have examined the interactions of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) with IL-10. Neutralization of TNF-alpha in murine bone marrow-derived macrophages resulted in a significant reduction of LPS-inducible IL-10 production. In mice, injection of 5 mg/kg LPS induced circulating IL-10 with a biphasic time course exhibiting an early peak 1.5 h after challenge (synchronous with TNF-alpha) and, after a nadir at 6 h, a second increase between 8 and 12 h. Treatment of mice with neutralizing anti-mouse TNF-alpha antiserum significantly increased LPS-induced IL-10 plasma levels between 1.5 and 6 h but diminished those at 12 h, while circulating IL-6, interferon-gamma (IFN-gamma) and granulocyte colony-stimulating factor (G-CSF) concentrations were attenuated overall, without a biphasic response. Analysis of LPS-induced IL-10 mRNA expression in different tissues 1 h and 8 h after LPS or LPS plus anti-TNF-alpha revealed that the amount of transcripts in the liver correlated with circulating early and late IL-10 levels. Our findings suggest that endogenous TNF-alpha down-regulates the early and up-regulates the late LPS-induced IL-10 synthesis in vivo and that the liver is the major source of circulating IL-10 after stimulation with LPS.

Animals↗
PubMed1995

Isolation and characterization of rat primary lung cells.

Lung cell culture may be useful as an in vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2 x 10(8) cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of > 70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC50 = 5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC50 = 0.6 mM). All cell types were equally sensitive to the more potent toxicant tert-butylhydroperoxide (EC50 = 0.1 mM). Paraquat was more toxic to lung cells (EC50 = 0.03 mM) than to rat (EC50 = 2.8 mM) and mouse (EC50 = 0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC50 = 2.6 mM) compared to rat (EC50 = 0.2 mM) and mouse (EC50 = 0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC50 = 0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC50 = 0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants.

Animals↗