The impact of Kupffer cell-derived cytokines outside the liver during the unspecific immune response has not yet been elucidated. To abolish cytokine expression specifically in these liver cells, antisense oligo-deoxyribonucleotides (oligos) directed towards TNF-alpha mRNA were tested for their ability to inhibit TNF-alpha release. Employment of carriers with high affinity to Kupffer cell-specific receptors should address the oligos to these cells.Using FITC-labeled phosphorothioate 21-mer oligos tailored against the start region of pro-TNF-alpha synthesis, uptake, stability and biological efficiency could be demonstrated in the macrophage cell line P388D1. Anionic or cationic carriers including various liposomes did not improve significantly these performances. The inclusion of oxidized low-density lipoprotein to direct the liposomes preferentially to the Kupffer cells failed because of the toxicity of oxLDL for the macrophages.In contrast to the P388D1 cells, rat Kupffer cells degraded the oligos rapidly; an intracellular concentration sufficient for suppression of TNF-alpha synthesis could not be achieved.

The activation of NF-kappa B and AP-1 in sinusoidal endothelial cells (SEC) in response to lipopolysaccharide (LPS), proinflammatory cytokines (tumor necrosis factor, TNF-alpha, interleukin-1 beta, IL-1 beta), transforming-growth-factor-beta (TGF-beta), endothelin-1 (ET-1), A-23187, a Ca2+-ionophore and phorbol myristate acetate (PMA) was investigated. There was a differential response of sinusoidal endothelial cells to the different stimuli used.While LPS, ET-1, TGF-beta and PMA activate both transcription factors, TNF-alpha; IL-1 beta and A-23187 induce the activation of NF-kappa B only. Inhibition of NF-kappa B activation by pyrrolidinium dithiocarbamate (PDTC) suppressed the expression of TNF-alpha, induced by LPS, indicating that the transcription factor NF-kappa B controlled the expression of TNF-alpha. This finding was very similar to that observed with Kupffer cells. To exclude contamination of SEC by Kupffer cells (KC) the specificity of different activators for SEC and KC was tested. While LPS, TNF-alpha and A-23187 activated NF-kappa B in both KC and SEC, PMA, IL-1 beta, TGF-beta and ET-1 activated NF-kappa B in SEC but not in KC.The results indicate that NF-kappa B in SEC responds to proinflammatory substances (LPS, TNF-alpha and IL-1 beta), and controls the expression of TNF-alpha. This suggests an involvement of this transcription factor in the inflammatory response of SEC. Substances which are known to regulate cell differentiation or proliferation, like PMA, ET-1 and TGF-beta elicit the dual activation of AP-I and NF-kappa B, implicating that both transcription factors are required in these processes.

Liver macrophages (Kupffer cells) respond to many stimulations with the production of bioactive substances including cytokines, eicosanoids, and inorganic radicals. In this study the activation of transcription factors by substances inducing cytokine gene expression or superoxide formation in rat Kupffer cells was examined. Using primary cultures of rat Kupffer cells the role of NF-kappa B and activator protein 1 (AP-1) in the expression of the tumor necrosis factor-alpha (TNF-alpha) gene by lipopolysaccharide (LPS) was investigated. Both transcription factors were strongly activated but with different kinetics. Maximal DNA-binding activity was induced with 50 ng of LPS/mL of medium and persisted for at least 24 hours. At that time, NF-kappa B- as well as AP-1-DNA complexes decreased their mobilities in native gels. Among the cytokines tested only TNF-alpha and macrophage colony-stimulating factor (M-CSF) were able to activate NF-kappa B in Kupffer cells. Phorbol ester and zymosan activated AP-1 but not NF-kappa B; the treatment of zymosan yielding a modified form of AP-1. Of all substances found to interfere with TNF-alpha production by Kupffer cells (pyrrolidine dithiocarbamate, dexamethasone, prostaglandin E2, interleukin [IL]-4, IL-10, and transforming growth factor-beta [TGF-beta]) only pyrrolidine dithiocarbamate was able to completely inhibit the activation of NF-kappa B by LPS. Although not abrogating the LPS activation of NF-kappa B, dexamethasone inhibited that of AP-1. The results indicate a direct participation of NF-kappa B in the regulation of TNF-alpha synthesis and a differential effect of LPS on NF-kappa B and AP-1, respectively.

SummaryThe genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6‐hydroxy‐L‐niotine oxidase (6‐HLNO) gene. NDH is composed of three subunits (A, B and C) of Mr 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe‐S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum‐containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron‐sulphur cluster, that the middle‐sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin‐binding domain of XDH. Expression of both the ndh and the 6‐hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane‐associated form. In the presence of tungsten the fraction of membrane‐associated NDH increased.

Nitric oxide (NO) production in the regenerating liver was estimated from the intensity of the electron paramagnetic resonance (e.p.r.) signal of the mononitrosyl complexes of iron and diethylthiocarbamate (DETC). Preformed complexes of intracellular non-heme Fe2+ and added DETC served as a trap for endogenously produced NO. The time-dependent changes of NO production were connected with the periodicity of liver regeneration. The first increase in NO production occurred ca. 1 h after partial hepatectomy (PHE). The second and more pronounced peak of NO production was observed about 6 h after PHE, when the hepatocytes entered the first cell cycle; it originated mainly from these cells. The following minimum of NO synthesis coincided with the maximal rate of DNA synthesis. The third gradual rise of NO production was seen at the end of the investigated period that covered the G2+M phases, the transit from the first to the second cell cycle of the hepatocytes and the entrance of the nonparenchymal cells into proliferation.

Platelet-activating factor (PAF) has been shown to play an important role in the generation of tumor necrosis factor-alpha (TNF-alpha) and superoxide in guinea pig peritoneal macrophages. In this study, the effects of the PAF receptor antagonists, WEB 2170 and RP 59277, and of a PAF analogue, HAGPT, on TNF-alpha and superoxide production by rat Kupffer cells was investigated. The liver macrophages produced very little TNF-alpha and superoxide when exposed to PAF, but released substantial amounts of superoxide following treatment with zymosan or phorbol 12-myristate 13-acetate (PMA). WEB 2170 not only inhibited the generation of superoxide by PMA but also suppressed the LPS-induced TNF-alpha synthesis by Kupffer cells in a concentration-dependent manner. Northern blot analysis revealed that the expression of TNF-alpha mRNA induced by lipopolysaccharide (LPS) in Kupffer cells was partially abrogated by WEB 2170 or RP 59227. Furthermore, WEB 2170 reduced the PMA-induced leakage of lactate dehydrogenase (LDH) from Kupffer cells in a dose-dependent manner. These data suggest that TNF-alpha and superoxide syntheses in Kupffer cells are rather insensitive to exogenous PAF. On the other hand, the PAF antagonists used in this study interfere with the transduction of the signals induced by LPS, PMA or zymosan. It is questionable whether the PAF receptor of the plasma membrane is involved in the inflammatory response of rat Kupffer cells.

Tumor necrosis factor-alpha is an important mediator of various inflammatory and immune responses. Its biological action is crucially dependent on interaction with specific cell surface receptors. Two different receptors for TNF-alpha with molecular masses of 55 and 75 kDa have been described. Here, the presence of a 55 kDa TNF receptor mRNA and the expression of its protein is demonstrated in rat liver Kupffer cells. TNF-alpha receptor was purified from detergent-solubilized rat Kupffer cells by adsorption to recombinant human TNF-alpha-Sepharose. One band of approx. 55 kDa was seen in SDS PAGE. An antibody raised against the 55 kDa TNF receptor bound specifically to the purified receptor as revealed by immunoblot analysis. Using Northern blotting, neither LPS nor TNF-alpha altered the expression of 55 kDa TNF-R mRNA, although the exposure of Kupffer cells to LPS decreased the binding of 125I-labelled TNF-alpha. Interferon-gamma clearly enhanced the level of 55 kDa TNF-R mRNA; this effect was abolished by transcriptional but not by translational inhibitors. The increase in 55 kDa TNF-R mRNA was maximal at 2-4 h of exposure of IFN-gamma. This cytokine also increased the binding of 125I-TNF-alpha to Kupffer cells. On the other hand, the amount of 55 kDa TNF-R transcripts was reduced after treatment with dexamethasone. These data suggest that in Kupffer cells the expression of the 55 kDa TNF-R is regulated at the transcriptional level.

We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric‐oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone‐rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate‐cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin‐dependent formation of inositol phosphates was apparent; values of Insp, InsP2, and InsP3 were 881±99%, 1965±368%, and 791±120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+]i in stellate cells from a basal value of 121±9 nM to maximal 1015±86nM. These endothelin‐1 effects were much stronger than those of the thromboxane‐A2 analogue U46619 and of prostaglandin F2a. In contrast, IIoprost, prostaglandin E2, and sodium nitroprusside promoted stellate‐cell relaxation; for example, 82, 83 and 71% of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500μM sodium nitroprusside to contracted cells. Prostaglandin E2 and Iloprost led to elevation of cAMP levels in stellate cells from a basal value of 9.2±0.8 pmol/well to 55.1±8.0 and 122.2±12.2 pmol/well 10 min after addition of prostaglandin E2 (5μM), and Iloprost (5μM), respectively, in the presence of 3‐isobutyl‐1‐methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9±0.007 pmol/well 13.4±6.7 pmol/well 10min after addition of 500 μM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F‐actin stress fibers became less numerous and de‐aggregated; more than 90% of stellate cells were void of stress fibers after 10 μM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells. The effect of endothelin 1 and prostaglandin F2a seems to be mediated by inositol‐phosphate formation and increase of the intracellular Ca2+ level, that of Iloprost and prostaglandin E2 by cAMP. cGMP may be a second messenger for the action of sodium nitroprusside.