Journal of Hepatology
Volume 37, Issue 5 , Pages 678-680, November 2002

Are nitric oxide synthases new players in the pathophysiology of fulminant hepatic failure?

Laboratoire d'Hémodynamique Splanchnique et de Biologie Vasculaire, INSERM U-481, and Service d'Hépatologie, Hôpital Beaujon, F-92118 Clichy, France

See Article, pages 613–619

Article Outline

 

Nitric-oxide synthases (NOSs) are hemoproteins that catalyze oxidation of l-arginine to nitric oxide (NO) and l-citrulline [1]. The three isoforms of NOS are regulated by distinct genes (see reviews in refs. [2], [3]). Neuronal NOS (nNOS), also known as NOS-1, is found in neuronal and some non-neuronal tissues. Inducible NOS (iNOS or NOS-2) was first found in macrophages but has been identified in other cell types (e.g. hepatocytes). Endothelial NOS (eNOS or NOS-3) was first identified as the enzyme producing endothelium-derived relaxing factor. Both nNOS and eNOS are constitutively expressed. iNOS is not a constitutive enzyme and its expression may be induced by stimuli such as lipopolysaccharide (LPS) or proinflammatory cytokines (i.e. tumor necrosis factor alpha (TNF-α), interleukin-1, and interferon-γ) [3]. These stimuli induce iNOS by stimulating the transcription factor, nuclear factor-κB (NF-κB) [4]. iNOS produces much higher levels of NO than nNOS and eNOS, which is thought to be the reason for the bacterial cytotoxic characteristics of cells expressing iNOS [3].

In target cells, NO may signal by stimulating soluble guanylyl cyclase to produce 3′,5′-cyclic guanosine monophosphate (cGMP) [2] and also independent of cGMP, i.e. via S-nitrosylation of cysteine residues [5]. In addition, under certain conditions, NO may react with the superoxide anion to produce the powerful oxidant peroxynitrite [3].

NOS-derived NO plays important physiological functions such as smooth muscle relaxation, inhibition of platelet activation, neurotransmission, and immune response [2], [3]. In addition, NO has also been shown to play a role in the pathophysiology of inflammatory diseases (arthritis, myocarditis, colitis, and nephritis), amyotrophic lateral sclerosis, neurodegenerative diseases, cancer, and diabetes [2], [3].

In this issue of the Journal, Leifeld and colleagues from the University of Bonn (Germany) provide results suggesting that iNOS may also play a role in the pathophysiology of fulminant hepatic failure [6]. Leifeld et al. used intrahepatic in situ staining and semi-quantification of iNOS by immunochemistry in 25 patients with fuminant hepatic failure (due to hepatitis B virus in 50% of cases and various causes in the remaining patients) and ten normal subjects. Normal subjects had slight iNOS expression in Kuppfer cells and hepatocytes only. In contrast, patients with fulminant hepatic failure had marked iNOS expression in macrophage/Kuppfer cells and hepatocytes and also expressed iNOS in bile ducts, vascular endothelial cells and lymphocytes [6].

Before commenting on iNOS it should be emphasized that, in patients with fulminant hepatic failure, massive destruction of the liver is frequently triggered by immune-mediated mechanisms involving TNF-α or Fas ligand, which both may cause hepatic apoptosis [7]. Patients with fulminant hepatic failure have increased serum levels of TNF-α and TNF receptors [7]. In the livers of patients with fulminant hepatic failure, infiltrating mononuclear cells express high amounts of TNF-α and hepatocytes overexpress the death receptor, TNF receptor 1 [7]. Apoptotic hepatocytes are significantly increased in fulminant hepatic failure, and there is a strong correlation with TNF-α expression, which is even more marked in areas of mononuclear infiltrates [7]. TNF receptor 1 engagement leads to activation of the initiator caspase-8 via the intracellular adapter molecule Fas-associated death domain (FADD) [8], [9]. Activated caspase-8 then activates effector caspases (such as caspase-3) which are proteolytic engines of cell death [8], [9]. In addition, caspase-8 may activate Bid, a BH3-only proapoptotic protein of the ‘Bcl2 clan’ [9]. Bid then translocates to the mitochondria to induce cytochrome c release which contributes to the activation of the initiator caspase-9 leading to caspase-3 activation [9]. In a murine model of fulminant hepatic failure, selective blockade of FADD has been shown to inhibit mitochondrial cytochrome c release, caspase-3 activation and subsequent hepatocyte apoptosis [7].

Engaged TNF receptor 1 has also been shown to activate NF-κB which induces iNOS [8], [10]. TNF-α-induced, NF-κB-mediated, iNOS induction may explain the increased iNOS expression found by Leifeld et al. in livers from patients with fulminant hepatic failure [6].

The effects of iNOS in hepatocytes (and other cell systems) are controversial [11]. On one hand, evidence has been provided that iNOS may have an antiapoptotic action in vivo. Hepatic apoptosis was increased following partial hepatectomy in iNOS knockout mice [12]. Pharmacological inhibition of iNOS increased hepatic apoptosis in endotoxemia in the rat [13]. In d-galactosamine-sensitized mice, LPS challenge [14] or TNF-α administration [11] induced hepatic apoptosis. Animals carrying a iNOS transgene were protected against d-galactosamine/LPS-induced liver damage [15]. Selective pharmacological delivery of NO to the liver decreased apoptosis induced by TNF-α in d-galactosamine-sensitized rats [16]. In addition, in primary mouse hepatocytes, engagement of TNF receptor 1 (by TNF-α) or Fas (by a Fas agonist antibody) induced caspase-8 activation, Bid activation, cytochrome c release, caspase-3 activation and apoptosis only in cells in which NF-κB activation was inhibited by different tools [10]. In these experiments, lack of iNOS induction (which is a consequence of NF-κB inhibition) partly explained the occurrence of TNF-α- or Fas-induced hepatocyte death [10]. Moreover, in primary hepatocytes with inhibited NF-κB, exposure to an NO donor blocks TNF-α- and Fas-induced proapoptotic signaling [10]. In different cell systems, including hepatocytes, NO has been shown to inhibit caspases (including caspase-8 and -3) through S-nitrosylation [17], [18], [19], [20], [21], [22]. As yet unidentified cGMP-dependent mechanisms may also contribute to NO-induced antiapoptotic effects [18], [23]. Finally, NO may induce antiapoptotic heat shock proteins, including heme oxygenase-1 [24], [25].

On the other hand, in vivo studies have provided evidence that iNOS may elicit apoptosis. Hemorrhagic shock induced liver and lung injury associated with induction of iNOS in these organs [26]. In hemorrhagic shock, iNOS inhibition or iNOS deficiency was associated with a marked decreases in liver and lung injury [26]. Administration of the T-cell mitogenic plant lectin concanavalin caused severe liver apoptosis in mice [27]. iNOS was induced in Kuppfer cells and hepatocytes in the livers of concanavalin-treated mices [27]. iNOS knockout mice were protected from concanavalin-induced liver damage [27]. Concanavalin-elicited intrahepatic DNA fragmentation was significantly lower in iNOS knockout mice than in wild-type mice. iNOS knockout mice were also protected from liver damage induced by LPS administration following sensitization with d-galactosamine [27]. In addition, pretreatment of wild-type mice with an iNOS inhibitor significantly reduced transaminase release after concanavalin or d-galactosamine/LPS [27]. Interestingly, in iNOS knockout mice sensitized with d-galactosamine, administration of recombinant TNF-α induced liver injury. In addition, pretreatment of wild-type mice with an iNOS inhibitor did not prevent hepatic injury after d-galactosamine/recombinant TNF-α treatment. Moreover, in concavalanin-treated mice, TNF-α levels in the liver and the plasma were found to be significantly lower in iNOS knockout mice than in wild-type mice [27]. Although a direct hepatotoxicity of NO cannot be excluded (e.g. via formation of peroxynitrite [3] or by activating cytochrome c [28]), the above-mentioned findings suggest that iNOS-derived NO may regulate proinflammatory genes in vivo, and thus contribute to inflammatory liver injury in mice by stimulation of TNF-α production.

There is currently no clear theory to explain the contradicting results of studies showing the antiapoptotic action of NO in hepatocytes and those showing proapoptotic action of NO in these cells [11]. It is thought that low NO concentrations protect the cell while high NO concentrations induce cell death [3]. It should be emphasized that concavalalin induced iNOS in both Kuppfer cells and hepatocytes while d-galactosamine/LPS induced iNOS in Kuppfer cells only [27]. Thus, Kuppfer cell-derived NO may play an important role in death of hepatocytes adjacent to Kuppfer cells. Kuppfer cell/macrophage-derived NO may also decrease survival of hepatocytes in the livers of patients with fulminant hepatic failure. On the other hand, in concanavalin-treated mice, it has been shown that there was no iNOS immunoreactivity in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive liver cells, indicating that iNOS expressing hepatocytes were not damaged [27]. Therefore, NO produced by iNOS within hepatocytes may protect them against death. Together these findings suggest that, in hepatocytes in patients with fulminant hepatic failure, the spatial relationships between iNOS expressing cells and hepatocyte apoptosis should be investigated.

Leifeld et al. also showed that in fulminant hepatic failure eNOS was overexpressed in sinusoidal endothelial cells and Kuppfer cells/macrophages [6]. The mechanisms of this overexpression are unclear. Several stimuli have been shown to regulate eNOS expression [29]. Moreover, an overexpressed enzyme may be not hyperactive, due to posttranslational inhibition [30], [31]. Even if eNOS is hyperactive in fulminant hepatic failure, the functional consequences of this must be elucidated. As suggested by Leifeld et al., overproduction of eNOS-derived NO may have beneficial effects on hepatic microcirculation [6].

The finding by Leifeld et al. that iNOS and eNOS are overexpressed in livers of patients with fulminant hepatic failure suggest that NOSs may be new players in the pathophysiology of fulminant liver failure. Clearly, further studies are needed in this field.

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PII: S0168-8278(02)00317-3

Journal of Hepatology
Volume 37, Issue 5 , Pages 678-680, November 2002

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