Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.
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CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently. (+info)
Effects of Ro 31-8220 on lipopolysaccharides-induced hepatotoxicity and release of tumor necrosis factor from rat Kupffer cells.
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AIM: To investigate protein kinase C (PKC) functions on lipopolysaccharide (LPS)-induced hepatotoxicity, a new potent PKC inhibitor Ro 31-8220 (Ro) was used to detect its effect on LPS-induced hepatotoxicity in rat hepatocytes and tumor necrosis factor (TNF) release from rat Kupffer cells (KC). METHODS: Hepatocytes (containing KC) were incubated with LPS (10 mg.L-1) and Ro (0.1-10 mumol.L-1) for 24 h, alanine aminotransferase (AlaA) leakage in the culture as indication of hepatotoxicity. The TNF activity in the supernatant of rat KC culture with LPS in the presence of Ro (0.1-10 mumol.L-1) was monitored by the L929 target cell lytic assay. RESULTS: Ro (0.1-10 mumol.L-1) reduced AlaA leakage in the hepatocyte culture. Ro inhibited dose-dependently the LPS-induced TNF production from rat KC. CONCLUSION: PKC inhibitor Ro protects the hepatocytes from LPS-induced cytotoxicity and inhibits the LPS-induced TNF production from rat KC. (+info)
Influences of Kupffer cell stimulation and suppression on immunological liver injury in mice.
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AIM: To study the possible involvement of Kupffer cells (KC) in immunological liver injury in mice. METHODS: Liver injury was induced by i.v. injection of Bacillus Calmette-Guerin (BCG) 5 x 10(7) viable bacilli followed by i.v. injection of lipopolysaccharides (LPS) 7.5 micrograms to each mouse. Indian ink and silica were i.v. injected to suppress KC and retinol was given po to stimulate KC in these mice. Plasma alanine aminotransferase (AlaAT), aspatate aminotransferase (AspAT), nitric oxide (NO), and liver tissue were examined. RESULTS: Injection of LPS following BCG injection resulted in a remarkable elevation of plasma NO, AlaAT, and AspAT levels, and severe liver damage. The damages were enhanced by the activation of KC with retinol and reduced by suppression of KC with silica and Indian ink. CONCLUSION: The degree of liver injury induced by BCG + LPS is closely correlated with the status of KC, and NO from KC plays an important role in the pathogenesis of the liver damage in mice. (+info)
Febrile-range temperature modifies early systemic tumor necrosis factor alpha expression in mice challenged with bacterial endotoxin.
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Fever improves survival in acute infections, but the effects of increased core temperature on host defenses are poorly understood. Tumor necrosis factor alpha (TNF-alpha) is an early activator of host defenses and a major endogenous pyrogen. TNF-alpha expression is essential for survival in bacterial infections but, if disregulated, can cause tissue injury. In this study, we show that passively increasing core temperature in mice from the basal (36.5 to 37.5 degrees C) to the febrile (39.5 to 40 degrees C) range modifies systemic TNF-alpha expression in response to bacterial endotoxin (lipopolysaccharide). The early TNF-alpha secretion rate is enhanced, but the duration of maximal TNF-alpha production is shortened. We identified Kupffer cells as the predominant source of the excess TNF-alpha production in the warmer animals. The enhanced early TNF-alpha production observed at the higher temperature in vivo could not be demonstrated in isolated Kupffer cells or in precision-cut liver slices in vitro, indicating the participation of indirect pathways. Therefore, expression of the endogenous pyrogen TNF-alpha is regulated by increments in core temperature during fever, generating an enhanced early, self-limited TNF-alpha pulse. (+info)
Intravenous glycine improves survival in rat liver transplantation.
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In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation. (+info)
A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease.
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The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant beta-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37 degrees C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, beta-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days). (+info)
Infection of primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but cytokine production is evident.
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We investigated the ability of dengue virus to invade human primary Kupffer cells and to complete its life cycle. The virus effectively penetrated Kupffer cells, but the infection did not result in any viral progeny. Dengue virus-replicating Kupffer cells underwent apoptosis and were cleared by phagocytosis. Infected Kupffer cells produced soluble mediators that could intervene in dengue virus pathogenesis. (+info)
Prevention of Kupffer cell-induced oxidant injury in rat liver by atrial natriuretic peptide.
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The generation of reactive oxygen species (ROS) by activated Kupffer cells contributes to liver injury following liver preservation, shock, or endotoxemia. Pharmacological interventions to protect liver cells against this inflammatory response of Kupffer cells have not yet been established. Atrial natriuretic peptide (ANP) protects the liver against ischemia-reperfusion injury, suggesting a possible modulation of Kupffer cell-mediated cytotoxicity. Therefore, we investigated the mechanism of cytoprotection by ANP during Kupffer cell activation in perfused rat livers of male Sprague-Dawley rats. Activation of Kupffer cells by zymosan (150 microgram/ml) resulted in considerable cell damage, as assessed by the sinusoidal release of lactate dehydrogenase and purine nucleoside phosphorylase. Cell damage was almost completely prevented by superoxide dismutase (50 U/ml) and catalase (150 U/ml), indicating ROS-related liver injury. ANP (200 nM) reduced Kupffer cell-induced injury via the guanylyl cyclase-coupled A receptor (GCA receptor) and cGMP: mRNA expression of the GCA receptor was found in hepatocytes, endothelial cells, and Kupffer cells, and the cGMP analog 8-bromo-cGMP (8-BrcGMP; 50 microM) was as potent as ANP in protecting from zymosan-induced cell damage. ANP and 8-BrcGMP significantly attenuated the prolonged increase of hepatic vascular resistance when Kupffer cell activation occurred. Furthermore, both compounds reduced oxidative cell damage following infusion of H2O2 (500 microM). In contrast, superoxide anion formation of isolated Kupffer cells was not affected by ANP and only moderately reduced by 8-BrcGMP. In conclusion, ANP protects the liver against Kupffer cell-related oxidant stress. This hormonal protection is mediated via the GCA receptor and cGMP, suggesting that the cGMP receptor plays a critical role in controlling oxidative cell damage. Thus ANP signaling should be considered as a new pharmacological target for protecting liver cells against the inflammatory response of activated Kupffer cells without eliminating the vital host defense function of these cells. (+info)