Lead stimulates intercellular signalling between hepatocytes and Kupffer cells

The role of intercellular signalling between liver cells in lead (Pb)(1)-induced liver toxicity was investigated in cocultures of freshly isolated and cultured rat hepatocytes and Kupffer cells. The Kupffer cells (seeded onto culture dish inserts), the hepatocytes or the two in cocultures were exposed to Pb acetate (2-50 microM) in combination with lipopolysaccharide (0.1-1000 ng/ml). In hepatocyte cultures, the combined Pb/lipopolysaccharide treatment induced no significant increase in the release of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) whereas in Kupffer cell cultures and in cocultures, at low lipopolysaccharide levels (0.1 and 1 ng/ml), TNF-alpha release was synergistically increased (up to 30-fold) when compared to lipopolysaccharide exposure alone. This stimulation of Kupffer cell-derived TNF-alpha release was specific for Pb or not detectable with mercury and cadmium. As a response to the Pb/lipopolysaccharide induced release of TNF-alpha, the cocultured hepatocytes increased their nitric oxide (NO) content sixfold when compared with lipopolysaccharide alone and downregulated the negatively regulated acute phase protein albumin. This downregulation was also detectable without lipopolysaccharide and without TNF-alpha release, indicating that Pb induces additional thus far unidentified Kupffer cell-derived factors, which interact with the cocultured hepatocytes. At the time of TNF-alpha release, the viability of the hepatocytes and the Kupffer cells was not affected. However, after a 48-h treatment period, Pb induced a Kupffer cell specific toxicity without affecting the hepatocytes. Loss of hepatocyte viability after lipopolysaccharide/Pb stimulation was only detectable in the presence of cocultured Kupffer cells together with human-derived granulocytes. It is concluded that Pb stimulates intercellular signalling between Kupffer cells and hepatocytes which is synergistically enhanced in the presence of low lipopolysaccharide levels. The released Kupffer cell-derived signals (e.g. cytokines) promotes most likely proteolytic hepatocyte killing in combination with a direct cellular interaction between the granulocytes and the hepatocytes.     

Kupffer cell-mediated IL-2 suppression of CYP3A activity in human hepatocytes.

Interleukin (IL)-2 administration has been shown to decrease CYP3A enzyme activity in vivo. To determine whether IL-2 suppression of human hepatocyte CYP3A activity is direct or whether it is facilitated by the presence of Kupffer cells, primary human hepatocytes were cultured alone or cocultured with primary human Kupffer cells at physiologic hepatocyte/Kupffer cell ratios of 10:1 or 10:4. Using proinflammatory cytokines as positive controls, IL-1 (0.2-20 ng/ml) and IL-6 (2-200 ng/ml) exposure resulted in a 70 to 90% decrease in CYP3A activity after 72 h in hepatocyte cultures. In the hepatocyte/Kupffer cell cocultures, an 80% decrease in CYP3A activity was observed with IL-1 (2 ng/ml) or IL-6 (20 ng/ml), suggesting that direct suppressive effects of proinflammatory cytokines on hepatocyte CYP3A activity are not substantially altered by Kupffer cells. In contrast to the effects of these proinflammatory cytokines, no sustained suppression of CYP3A activity was observed with IL-2 (2-200 ng/ml) in hepatocyte cultures. However, in hepatocyte/Kupffer cell cocultures, a concentration-dependent 50 to 70% suppression of CYP3A activity was observed with IL-2 at 72 h. In summary, these data suggest that Kupffer cells are required to reconstitute the suppressive effects of IL-2 on CYP3A activity that are observed in vivo and that hepatocyte/Kupffer cell cocultures may provide a useful model for investigating mechanisms of CYP3A4 regulation by cytokines. Of particular relevance to certain hepatic diseases, these findings suggest potential mechanisms whereby cytokines released from infiltrating blood mononuclear cells might modulate intercellular signaling and controls on hepatocyte function by various cell types that reside in liver.     

Role of nitric oxide in down-regulation of CYP2B1 protein, but not RNA, in primary cultures of rat hepatocytes.

There are conflicting reports about the role of nitric oxide in the down-regulation of cytochrome P450 that occurs when animals or cultured hepatocytes are exposed to inflammatory stimuli. Here, we investigated the participation of NO in the down-regulation of CYP2B1 by bacterial endotoxin (LPS) in rat hepatocytes cultured on Matrigel. LPS caused the down-regulation of CYP2B1 mRNA to 20% of control values within 12 h of treatment, and this was not reversed by concentrations of NO synthase inhibitors that completely blocked NO production. LPS was several orders of magnitude more potent in the down-regulation of CYP2B1 mRNA than in induction of NO production. In contrast, concentrations of LPS in the 1 to 100 ng/ml range induced NO production and produced a rapid down-regulation of CYP2B1 protein to 30% and <5% of control at 6 and 24 h, respectively, that could be completely prevented both by inhibitors of NO synthase and by LY83583, which prevents NO synthase-2 induction. The blockade of CYP2B1 down-regulation by NO synthase inhibitors was reversed by arginine, and the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine mimicked CYP2B1 protein suppression. Taken together, these experiments demonstrate two independent mechanisms of CYP2B1 down-regulation by LPS: a rapid, NO-dependent suppression of the protein occurring at high concentrations of LPS and a slower, NO-independent pretranslational suppression occurring at low concentrations of LPS.     

Modeling inflammation–drug interactions in vitro: A rat Kupffer cell-hepatocyte coculture system

Xenobiotic–inflammation interactions lead to hepatotoxicity in vivo. Selected xenobiotic agents (acetaminophen, APAP; chlorpromazine, CPZ; allyl alcohol, AlOH; monocrotaline, MCT) for which this occurs were evaluated for ability to elicit the release of Kupffer cell (KC)-derived inflammatory mediators and to modulate lipopolysaccharide (LPS)-stimulated release of these mediators. Using KCs and hepatocytes (HPCs) isolated from rat, KC/HPC cocultures were treated with either LPS, xenobiotic, vehicle or a combination. Six hours later, the release of inflammatory mediators was assessed. LPS alone caused a concentration-dependent increase in TNF- release but had no significant effect on the release of PGE₂. APAP by itself did not alter release of TNF-, PGE₂, IL-10, Gro/KC or IFN-γ; however, in the presence of LPS, APAP enhanced LPS-induced TNF- and Gro/KC release. APAP also attenuated LPS-induced increases in IL-10 and MCP-1. CPZ alone caused a concentration-dependent increase in TNF- release, which was approximately additive in the presence of LPS. AlOH alone did not affect TNF- release, but decreased TNF- production in the presence of LPS. AlOH increased PGE₂ production, and this effect was potentiated in the presence of LPS. MCT by itself did not affect release of TNF- but increased the response to LPS. Neither MCT, LPS, nor the combination affected production of PGE₂. These results demonstrate that KC/HPC cocultures can be used to evaluate interactions of xenobiotics with LPS. Furthermore, data from these studies qualitatively mirror reported data from whole animal studies, suggesting that this model could be useful for predicting aspects of xenobiotic–inflammation interactions in vivo.     

Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.

Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from na?ve rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.     

Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase

Multiple hepatic cytochrome P450 enzymes are down-regulated at the mRNA and protein levels during inflammation and infection. A body of evidence suggests that nitric oxide (NO) produced from inducible NO synthase (NOS2) is responsible for some of these effects. The current study was designed to examine the NO dependencies of the down-regulation of phenobarbital-induced CYP2B mRNAs and proteins by bacterial endotoxin (lipopolysaccharide, LPS) treatment in vivo, using an NOS2-null mouse model. Treatment of C57/BL6 mice with 0.3 mg/kg of LPS maximally suppressed phenobarbital-induced CYP2B9 and 2B10 mRNAs measured 12 hr after injection, whereas 1-10 mg/kg of LPS was required to elevate NO production. Down-regulation of CYP2B mRNAs by 1 mg/kg of LPS was equivalent in wild-type and NOS2-null mice. No effect of LPS in the dose range of 0.3 to 10 mg/kg was observed on microsomal CYP2B protein levels measured 12 hr after treatment, whereas 1 mg/kg of LPS suppressed CYP2B proteins 24 hr after treatment in both wild-type and NOS2-null mice. We conclude that the main mechanism for the down-regulation of CYP2B proteins in mouse liver following moderate- or high-dose LPS treatment is via NO-independent suppression of CYP2B9 and 2B10 mRNAs. Unlike rat hepatocytes, the contribution of a rapid, NO-dependent mechanism of CYP2B protein suppression in mouse liver appears to be minor or non-existent.     

Differential Cell Sensitivity between OTA and LPS upon Releasing TNF-α

The release of tumor necrosis factor α (TNF-α) by ochratoxin A (OTA) was studied in various macrophage and non-macrophage cell lines and compared with E. coli lipopolysaccharide (LPS) as a standard TNF-α release agent. Cells were exposed either to 0, 2.5 or 12.5 μmol/L OTA, or to 0.1 μg/mL LPS, for up to 24 h. OTA at 2.5 μmol/L and LPS at 0.1 μg/mL were not toxic to the tested cells as indicated by viability markers. TNF-α was detected in the incubated cell medium of rat Kupffer cells, peritoneal rat macrophages, and the mouse monocyte macrophage cell line J774A.1: TNF-α concentrations were 1,000 pg/mL, 1,560 pg/mL, and 650 pg/mL, respectively, for 2.5 μmol/L OTA exposure and 3,000 pg/mL, 2,600 pg/mL, and 2,115 pg/mL, respectively, for LPS exposure. Rat liver sinusoidal endothelial cells, rat hepatocytes, human HepG2 cells, and mouse L929 cells lacked any cytokine response to OTA, but showed a significant release of TNF-α after LPS exposure, with the exception of HepG2 cells. In non-responsive cell lines, OTA lacked both any activation of NF-κB or the translocation of activated NF-κB to the cell nucleus, i.e., in mouse L929 cells. In J774A.1 cells, OTA mediated TNF-α release via the pRaf/MEK 1/2-NF-κB and p38-NF-κB pathways, whereas LPS used pRaf/MEK 1/2-NF-κB, but not p38-NF-κB pathways. In contrast, in L929 cells, LPS used other pathways to activate NF-κB. Our data indicate that only macrophages and macrophage derived cells respond to OTA and are considered as sources for TNF-α release upon OTA exposure.     

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2′-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10–1000 μM MTF and 100–500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5–50 ng/ml epidermal growth factor or 5–100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.     

Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (VIMANG). Role of mangiferin isolated from the extract.

The present study illustrates the effects of a standard aqueous extract, used in Cuba under the brand name of VIMANG, from the stem bark of Mangifera indica L. on the production of tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO) in in vivo and in vitro experiments. In vivo was determined by the action of the extract and its purified glucosylxanthone (mangiferin) on TNFalpha in a murine model of endotoxic shock using Balb/c mice pre-treated with lipopolysaccharide (LPS) 0.125 mg kg(-1), i.p. In vitro, M. indica extract and mangiferin were tested on TNFalpha and NO production in activated macrophages (RAW264.7 cell line) and microglia (N9 cell line) stimulated with LPS (10ng ml(-1)) and interferon gamma (IFNgamma, 2U ml(-1)). M. indica extract reduced dose-dependently TNFalpha production in the serum (ED50 = 64.5 mg kg(-1)) and the TNFalpha mRNA expression in the lungs and livers of mice. Mangiferin also inhibited systemic TNFalpha at 20 mg kg(-1). In RAW264.7, the extract inhibited TNFalpha (IC50 = 94.1 microg ml(-1)) and NO (IC50 = 64.4 microg ml(-1)). In microglia the inhibitions of the extract were IC50 = 76.0 microg ml(-1) (TNFalpha) and 84.0 microg ml(-1) (NO). These findings suggest that the anti-inflammatory response observed during treatment with M. indica extract must be related with inhibition of TNFalpha and NO production. Mangiferin, a main component in the extract, is involved in these effects. The TNFalpha and NO inhibitions by M. indica extract and mangiferin on endotoxic shock and microglia are reported here for the first time.     

In vitro and in vivo studies of the toxic effects of perfluorononanoic acid on rat hepatocytes and Kupffer cells

This study investigated the toxic effects of perfluorononanoic acid (PFNA), a persistent organic pollutant, on rat hepatocytes and Kupffer cells in vitro and in vivo. The results showed that administration of 5μM PFNA increased the viabilities of the hepatocytes and the Kupffer cells. An exposure of 50μM PFNA did not alter the viabilities of both cells, as well as the release of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the primary cultured hepatocytes or the hepatocytes co-cultured with Kupffer cells. An exposure of 100μM PFNA only decreased the viability of the hepatocytes. The administration of PFNA changed the hepatocyte expression of several genes related to lipid metabolism in vitro and in vivo. Oil Red O Staining revealed that 5mg PFNA/kg/D treatment lead to dramatic accumulation of lipids in rat liver. At the same dose PFNA damaged hepatocytes histopathologically. Up-regulated expressions of the inflammatory cytokines occurred in the Kupffer cells treated with 50μM PFNA and in the livers of the rat receiving a 5mg PFNA/kg/D treatment. In addition, these cytokines also increased in serum of the rat receiving higher dose of PFNA. In summary, on the one hand, PFNA exposure affected the viability of the hepatocytes, hepatic lipid metabolism and lead to lipid accumulation in liver. On the other hand, for the first time, PFNA exposure was demonstrated to affect the viability of the Kupffer cells as well as their expression of cytokines, which involved in regulation of various liver functions. Therefore, we conclude that both the hepatocyte and the Kupffer cell contribute to the observed hepatotoxicity of PFNA.     

Effect of Pyrethrins on cytochrome P450 forms in cultured rat and human hepatocytes

High doses of Pyrethrins produce liver and thyroid gland tumours in rats by modes of action involving the induction of hepatic xenobiotic metabolising enzymes. The aim of this study was to compare the effects of Pyrethrins with those of the rat liver and thyroid tumour promoter sodium Phenobarbital on some cytochrome P450 (CYP) forms in cultured rat and human hepatocytes. The treatment of female Sprague-Dawley rat and human (both male and female) hepatocytes for 72 h with 0-1000 microM Pyrethrins and 0-1000 microM Phenobarbital did not result in any marked cytotoxicity. In rat hepatocytes both Pyrethrins and Phenobarbital produced an induction of 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase activity (a CYP1A/2B form marker) and CYP2B1 and CYP2B1/2 mRNA levels. Pyrethrins and Phenobarbital also induced CYP3A-dependent testosterone 6beta-hydroxylase activity in rat hepatocytes. In human hepatocytes Pyrethrins and Phenobarbital induced both testosterone 6beta-hydroxylase activity and CYP3A4 mRNA levels and also increased CYP2B6 mRNA levels. The effects of Pyrethrins and Phenobarbital were concentration-dependent and exhibited a threshold. These results demonstrate that the effects of Pyrethrins on CYP forms in cultured rat and human hepatocytes are qualitatively similar to those of Phenobarbital. Pyrethrins induce CYP2B and CYP3A forms in cultured rat hepatocytes and can induce CYP3A and CYP2B forms in human hepatocytes. While CYP form induction by Pyrethrins, Phenobarbital and related compounds can be associated with liver and thyroid gland tumour formation in rodents, epidemiological data for Phenobarbital suggests that such effects do not occur in humans.     

Lipopolysaccharide-induced paw edema model for detection of cytokine modulating anti-inflammatory agents

Cytokines are critical to pathogenesis of inflammatory disorders. So inhibition of their action provides therapeutic benefits in various diseases. Although inhibition of inflammation caused by intraperitoneally administered LPS can identify cytokine modulators, this inflammatory test-agent does not allow one to determine overall anti-inflammatory potential. Functional characteristics of Carrageenan (Cara)-induced edema were valuable for identification of nonsteroidal anti-inflammatory drugs (NSAIDS). Hence, the potential of LPS-induced paw inflammation was investigated and compared to that by Cara. Stimulation of isolated rat peritoneal exudates cells (PEC) with 10 ng/ml LPS, but not Cara, induced IL-6 (3.04+/-0.2 ng/ml) and TNFalpha (1.030.09 ng/ml). At least 100 mg/ml Cara was necessary for detection of IL-6 (2.03+/-0.1 ng/ml) and TNFalpha (0.6+0.09 ng/ml) in PEC. Similar to Cara, subplantar administration of LPS-induced inflammatory paw edema in rats. LPS, but not Cara, induced TNFalpha (2.14+/-0.6 ng/ml) and IL-6 (2.9+/-0.5 ng/ml) in serum at 1 and 3 h, respectively, which returned to basal levels by 5 h. LPS-induced serum TNFalpha (sTNFalpha) levels closely paralleled paw swelling and its neutralization by anti-TNFalpha antibody or inhibition by pentoxifylline and nimesulide correlated with inhibition of inflammation. Similar to earlier reports, rofecoxib induced sTNFalpha at 30 mg/kg and exhibited pro-inflammatory effect by enhancing paw swelling. LPS-induced edema provides a useful functional model for identification of cytokine modulating anti-inflammatory agents.     

Nitric oxide inhibits T cell adhesion and migration by down-regulation of beta1-integrin expression in immunologically liver-injured mice

Our previous study has reported that nitric oxide (NO) exerts a protective role in immunologically liver-injured mice induced by delayed-type hypersensitivity to picryl chloride. To explore the mechanism of the protection, we have now examined the effect of NO on T cell adhesion and migration. First, we isolated hepatocytes and nonparenchymal cells from the liver-injured mice and separated the nonparenchymal cells into Kupffer cell-enriched and lymphocyte-enriched populations. When these hepatocytes or the fractions of nonparenchymal cells were co-cultured with spleen T cells of the liver-injured mice in a Transwell system, the adhesive potential of the T cells was significantly inhibited in the presence of hepatocytes or the Kupffer cell-enriched population but not the lymphocyte-enriched population of nonparenchymal cells. This effect was dependent on NO production. The NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) could reverse this inhibition of cell adhesion and also decrease NO production. To confirm this effect of NO on T cells, we further examined the role of exogenous or endogenous NO on the adhesive activity of the Jurkat T cell line. As a result, the NO donor, S-nitroso-N-acetyl penicillamine (SNAP) caused a dose-dependent inhibition of the adhesion of Jurkat T cells. Furthermore, the binding ability of Jurkat T cells to collagen decreased gradually after co-incubation with macrophages stimulated by LPS+IFN-gamma, an effect which correlated well with the increasing NO level in the medium. Such opposite changes in cell adhesion and in NO production were also markedly reversed by L-NMMA. Moreover, treatment with SNAP reduced adhesion, transmigration, matrix metalloproteinase-9 production and beta1-integrin expression of spleen T cells of the liver-injured mice. Taken together, these findings suggest that NO can function as a down-regulator of T cell mobility, which might be one of the mechanisms by which NO exerts its protective effect in T cell-mediated liver injury.     

当归多糖对大鼠Kupffer细胞免疫功能的激活作用当归多糖对大鼠Kupffer细胞免疫功能的激活作用

目的观察当归多糖(ASP)对大鼠Kupffer细胞免疫功能的影响。方法ASP体外作用于正常Kupffer细胞,以吞噬中性红A₅₄₀、分泌NO和TNF-α量评价其免疫功能;以LDH漏出量,评价ASP对细胞的直接损害情况。大鼠ig ASP后,检测Kupffer细胞上述指标及胞内酸性磷酸酶(ACP)活性;以sALT和sGST为肝毒性指标。结果ASP增强Kupffer细胞对中性红吞噬作用、增加ACP活性及NO和TNF-α的产生。ASP在体外不增加肝实质细胞LDH漏出,而体内使sGST升高。结论适当剂量ASP可激活Kupffer细胞免疫功能。大剂量ASP出现的轻度肝功能改变可能与NO和TNF-α等免疫因子过度分泌间接有关。     

Drug-metabolizing enzymes in rat, mouse, pig and human macrophages and the effect of phagocytic activation

Aryl hydrocarbon hydroxylase (AHH) activity mediated by cytochrome P-450 is present in pig hepatic microsomes [10 nmol.3 mg protein-1.hr-1]. AHH activity was detectable in both hepatocytes and Kupffer cells isolated from pig liver biopsy material. These cells were isolated from needle or wedge biopsy material by collagenase perfusion and incubation with collagenase at 37 degrees. The two cell types were separated from the resulting cell suspension as previously described for whole liver. Kupffer cells were enriched by adherence and were cultured for 24 hr prior to harvesting. Cells were harvested, and cell viability was determined. AHH activity was assayed in Kupffer cell and hepatocyte homogenates. Kupffer cell AHH activity was approximately one-eighth the level detected in hepatocytes. To determine whether this enzyme was present in other macrophages, monocytes were isolated from 10 ml of heparinized peripheral blood using Ficoll-Hypaque and were enriched by adherence. After 24 hr in culture, cell viability was assessed and monocytes were identified by by cytochemical staining. AHH activity was detectable in pig monocyte homogenates, and the AHH level was similar to that in pig Kupffer cells. AHH was also easily detectable in human monocytes. This macrophage AHH activity was compared with AHH activity in rat monocytes, mouse Kupffer cells and mouse peritoneal macrophages. Monocyte AHH was relatively stable in cell culture but decreased rapidly upon storage at -70 degrees. Macrophage AHH activity was depressed following phagocytic activation in vitro by latex beads with a concomitant increase in heme oxygenase activity.     

Inhibition of endotoxemia-induced nitric oxide synthase expression by cyclosporin A enhances hepatocyte injury in rats: amelioration by NO donors.

The goals of the present study were to provide information into the controversy about nitric oxide (NO) status of the liver during endotoxemia and to assess the role of the phosphatase inhibitor cyclosporin A (CsA) during the insult. Rats were injected with saline, lipopolysaccharide (LPS, 10 mg/kg i.p.) or cyclosporin A (CsA, 5 mg/kg. i.p.) + LPS, S-nitroso-N-acetyl penicillamine (SNAP, 0.1 mMikg) + CsA + LPS or molsidomine (molsid, 0.2 mg/kg) + CsA + LPS. Rat hepatocytes were isolated and tested for metabolic competence by the rate of urea synthesis and for lipid peroxidation. Hepatocytes were cultured under various treatments as LPS or cytokine mixture (CM, TNF-alpha 500 U/ml, INF-gamma 100 U/ml, IL-1beta 200 U/ ml) with or without CsA and iNOS expression was evaluated by NO productivity and by RT-PCR. Twenty-four hours after LPS dosing in vivo, the mortality rate was 15%, while CsA pretreatment increased mortality rate to 30% and reduced hepatocyte viability, increased ALT leakage and reduced urea synthesis. SNAP and Molsid resulted in complete survival of rats, increased urea synthesis, increased cell viability and reduced alanine aminotransferase leakage. LPS or CM increased iNOS expression while CsA pretreatment reduced iNOS expression. There was no correlation between lipid peroxide levels in hepatocytes and functional status of hepatocytes under various treatments. This study demonstrates that NO produced during endotoxemia and under the present conditions is protective to the liver and may function as an adaptive mechanism and that the inhibition of iNOS by compounds like CsA produce unfavorable effects.