Journal of Hepatology 37 (2002) 613–619 www.elsevier.com/locate/jhep
Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure Ludger Leifeld 1,*, Magdalene Fielenbach 1, Franz-Ludwig Dumoulin 1, Nicola Speidel 2, Tilman Sauerbruch 1, Ulrich Spengler 1 1
Department of Internal Medicine I, University of Bonn, Sigmund Freud Strabe 25, D-53105 Bonn, Germany 2 Department of Surgery, University of Bonn, Sigmund Freud Strabe 25, D-53105 Bonn, Germany
See Editorial, pages 678–680
Background/Aims: Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. Methods: Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). Results: Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P ¼ 0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P ¼ 0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P , 0.001, P ¼ 0.017). Conclusions: Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Fulminant hepatic failure; Liver disease; Kupffer cells
1. Introduction A balanced release of nitric oxide (NO) is essentially involved in several important physiological functions including blood vessel relaxation, inhibition of platelet aggregation, and neuronal communication . On the other hand, high NO concentrations are cytotoxic, because NO is an unstable molecule that triggers formation of oxidaReceived 19 March 2002; received in revised form 26 June 2002; accepted 23 July 2002 * Corresponding author. Tel.: 149-228-287-5259; fax: 149-228-2874323. E-mail address: [email protected]
(L. Leifeld). Abbreviations: FHF, fulminant hepatic failure; CLD, chronic liver disease; NC, normal controls; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase.
tive free radicals such as peroxynitrite (ONOO 2). NO is generated from l-arginine by a reaction which is catalysed by three different NO synthases. The constitutive NO synthases are the endothelial NO synthase (eNOS) and the neuronal NO synthase. Both synthesise low amounts of NO and are supposed to regulate physiological NO homeostasis and cellular signalling. In contrast, the inducible form of NO synthase (iNOS) produces high amounts of NO induced by cytokines such as interferon gamma, tumor necrosis factor alpha (TNF-a) or interleukin 1a [2–4]. NO is also involved in the control of programmed cell death. Its effects on apoptosis depend on its concentration in one hand and the cell types in the other [5,6]. Low concentrations block apoptosis via inhibition of the main mediators of cell death-caspases (caspase-3 and -8), while higher concentrations are toxic via the formation of reactive
0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S 0168-827 8(02)00271-4
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products like peroxynitrite and dinitrogen trioxide . Nonetheless, the effects of NO with respect to liver damage are discussed controversially, since also protective functions of NO have been described [7–11]. For example, at the single cell level, NO protects against inflammatory and pro-apoptotic stimuli by upregulation of heat shock protein 70 , or by inhibiting caspase-3 and -8 activity [13–15] as well as Bcl-2 cleavage and cytochrome c release . In animal models NO synthases are involved in the pathogenesis of hyperdynamic circulation [17–19]. A functional role of iNOS in causing liver damage up to fulminant hepatic failure (FHF) has been demonstrated in the models of concanavalin-A and of galactosamine-lipopolysaccharide induced fulminant hepatitis  as well as acetaminophen induced liver injury and lethality . Importantly, iNOS knock-out mice were protected in these murine models of fulminant hepatic failure [20,21]. Until now the role of NO synthases in human FHF has not been analysed. Therefore, we examined the intrahepatic expression of iNOS and eNOS in human fulminant hepatic failure in liver specimens from patients with fulminant hepatic failure compared to the specimens from patients with liver cirrhosis and from normal controls. 2. Materials and methods 2.1. Patients Explant tissue from 25 patients obtained at transplantation because of fulminant hepatic failure were included into the study (FHF due to hepatitis B virus (HBV) n ¼ 13, cryptogenic n ¼ 7, toxic n ¼ 3, autoimmune hepatitis n ¼ 2). All patients fulfilled the King’s College criteria  for urgent transplantation. Furthermore, explant specimens from 40 patients with endstage chronic liver diseases (CLD) were included (chronic hepatitis B: n ¼ 19, chronic hepatitis C: n ¼ 8, primary biliary cirrhosis: n ¼ 10, primary sclerosing cholangitis n ¼ 3). Non-tumor-bearing liver tissue obtained during the resection of hepatic malignancies in four patients as well as wedge biopsies from six donor livers at the time of hepatectomy were included as normal controls (NC).
2.2. Immunostaining procedures Liver tissue was immediately embedded in ‘Tissue Tek OCT‘ compound (Miles Laboratories, Inc. Naperwille, IL, USA) and snap frozen in liquid nitrogen. Frozen tissue was kept at 2808C until examined. Sections of 6mm were stained by an indirect immunoperoxidase technique. Endogenous peroxidase activity was blocked by 0.03% H2O2/NaNO3 (Peroxidase Blocking Reagent, Dako Carpinteria, USA). The sections were incubated with primary antibodies in antibody diluent with background reducing components (Dako) at room temperature for 90 min. After washing in phosphate buffered saline, peroxidase-coupled secondary antibody (Dianova, Hamburg, Germany) was applied for 30 min. Bound antibody was detected with 3-amino-9-ethylcarbazole (Sigma Chemicals, Munich, Germany). All sections were then counterstained with Meyer’s hematoxylin.
2.3. Double staining eNOS and iNOS producing cells were identified by double labelling experiments with iNOS/eNOS and cell type-specific antibodies. After performing the eNOS or iNOS specific immunoperoxidase reaction we incubated the sections for 30 min with fluorescein conjugated cell type-
specific antibodies for sinusoidal endothelial cells, macrophages and stellate cells. Analysis of double labelling experiments was done by bright field and fluorescent photomicrographs on a Leica DMLB fluorescence microscope with a MPS60 photo camera.
2.4. Antibodies Staining for iNOS and eNOS was performed with the rabbit polyclonal antibody NOS 2 (N-20) (Santa Cruiz) and with the polyclonal rabbit antibody NOS 3 (C-20) (Santa Cruiz). To ensure specificity of the observed staining patterns we also used the monoclonal mouse anti-human iNOS, clone 6 (R&D Systems, Minneapolis, USA) and the monoclonal mouse anti-human eNOS Clone 3 (R&D Systems, Minneapolis, USA), which confirmed the staining pattern of iNOS and eNOS expression. In the double labelling experiments we used the FITC labelled monoclonal antibody JC/ 70A (DAKO, Carpinteria, USA) to identify CD31 positive sinusoidal endothelial cells and FITC labelled KP1 (DAKO, Carpinteria, USA) to identify CD68 positive macrophages/Kupffer cells, respectively. Stellate cells were identified by Cy3-conjugated smooth muscle actin antibodies (anti-SMA, Sigma Chemicals). Irrelevant mouse monoclonal antibodies (IgG1 and IgG2; Becton Dickinson, Heidelberg, Germany) were used as isotype controls.
2.5. Statistical analysis eNOS and iNOS positive cells were counted in ten randomly selected high power fields at 400-fold magnification by two observers who were blinded with respect to the aetiology of the underlying liver disease. The results of each patient group are given as mean ^ standard deviation of all evaluated visual fields. Using the SPSS PC 1 software package, we analysed the differences between the groups for statistical significance by the non-parametric Mann–Whitney U-test. Furthermore, Pearson’s correlation-coefficients were calculated.
3. Results 3.1. Intrahepatic iNOS expression In NC, expression of iNOS was predominantly noted on a small number of CD68 positive Kupffer cells/macrophages. Additional faint staining was found on a small proportion of hepatocytes. In a single normal control specimen approximately 10% of the bile ducts also expressed iNOS. As shown in Table 1 5-fold higher total numbers of liver cells expressed iNOS in CLD (P , 0:001) and the highest numbers of iNOS expressing cells were found in FHF (FHF versus NC: P , 0:001; FHF versus CLD: P ¼ 0:001; Fig. 1, Table 1). The main cell types which expressed iNOS in CLD and FHF were Kupffer cells/macrophages (Fig. 2) and hepatocytes (Fig. 2). In FHF and in CLD additional iNOS expression was found on bile ducts, vascular endothelial cells and lymphocytes, while sinusoidal endothelial cells and stellate cells (Fig. 2) did not express iNOS. No significant differences were found regarding iNOS expression in the different aetiologies of FHF and CLD, respectively. 3.2. Intrahepatic eNOS expression The intrahepatic expression pattern of eNOS was different from iNOS expression. While hepatocytes were consis-
L. Leifeld et al. / Journal of Hepatology 37 (2002) 613–619 Table 1 Total numbers of iNOS and eNOS positive cells per high power field ( £ 400) Disease
No iNOS eNOS (cells/high power field) (cells/high power field)
Fulminant hepatic failure HBV 13 40 ^ 18 Cryptogenic 7 27 ^ 14 Toxic (MDMA) 3 34 ^ 1 Autoimmune 2 27 ^ 10 hepatitis Total 25 35 ^ 16 a Chronic liver disease HBV 19 HCV 8 Primary biliary 10 cirrhosis Primary sclerosing 3 cholangitis Total 40 Normal controls
35 ^ 12 27 ^ 13 33 ^ 6 26 ^ 9 32 ^ 11 b
19 ^ 16 16 ^ 12 25 ^ 14
22 ^ 5 21 ^ 7 25 ^ 5
25 ^ 20
21 ^ 7
20 ^ 15a
22 ^ 6b
5 ^ 4a
22 ^ 8b
a iNOS: FHF versus CLD: P ¼ 0.001; FHF versus NC: P , 0.001; CLD versus NC: P , 0.001. b eNOS: FHF versus CLD: P ¼ 0.001; FHF versus NC: P ¼ 0.017; CLD versus NC: P ¼ 0.64.
tently negative for eNOS in all groups of patients, sinusoidal endothelial cells expressed eNOS (Fig. 2) together with Kupffer cells/macrophages (Fig. 2), vascular endothelial cells and lymphocytes. Bile ducts stained negative for eNOS with the exception of one single patient with primary biliary cirrhosis. In NC the numbers of eNOS expressing cells exceeded the numbers of iNOS positive cells approximately 4-fold. The cell types, which expressed eNOS, were identical in CLD and FHF. It is noteworthy that hepatocytes remained eNOS negative. Moreover, no significant difference was found between the numbers of eNOS expressing cells in NC and CLD (P ¼ 0:64). In contrast, in FHF, the number of eNOS expressing cells was significantly higher than in CLD or in NC (FHF versus CLD: P ¼ 0:001; FHF versus NC: P ¼ 0:017). Enhanced eNOS expression in FHF was especially caused by higher numbers of eNOS expressing Kupffer cells/macrophages and sinusoidal endothelial cells in the liver. No significant differences in eNOS expression were found between the different aetiologies of FHF and CLD, respectively.
4. Discussion In this study we found different expression patterns of iNOS versus eNOS. In normal controls eNOS was apparently expressed constitutively on sinusoidal and vascular endothelial cells as well as on Kupffer cells/macrophages but not on hepatocytes. No difference in eNOS expression
was found between normal controls and cirrhotic human livers. Only in FHF higher levels of eNOS was found caused by increased numbers of eNOS expressing infiltrating macrophages in FHF. In contrast to eNOS, iNOS was limited to a few Kupffer cells/macrophages and hepatocytes in physiological conditions. In cirrhosis and in FHF iNOS expression was induced significantly on hepatocytes and Kupffer cells/macrophages. Moreover, we also found iNOS expression on bile ducts, vascular endothelial cells and lymphocytes. Sinusoidal endothelial cells and stellate cells, however, remained iNOS negative. The total numbers of iNOS positive cells were incrementally increased in CLD (4-fold) and FHF (7-fold), (see Table 1). For the first time our data describe systematically the intrahepatic in situ expression of iNOS and eNOS in human livers of FHF and liver cirrhosis. Expression of eNOS by sinusoidal endothelial cells confirmed the findings reported in rat [23,24], and iNOS expression by hepatocytes and by Kupffer cells is in line with previous reports on murine and human livers [4,20,25–30]. However, our data also indicate species specific differences in iNOS and eNOS expression between humans and animals: in particular, we could not find any iNOS expression by sinusoidal endothelial cells, which had been demonstrated in rat . Also rat stellate cells were found to produce NO after endotoxin stimulation  but stained consistently negative for iNOS and eNOS in our human liver specimens. The different patterns of iNOS and eNOS expression most likely reflect the special functions of both enzymes, while eNOS seems to be of importance in the physiological vasoregulation, iNOS operates in inflammatory liver damage. We demonstrated distinct basal expression of eNOS in sinusoidal endothelial cells and Kupffer cells/macrophages in normal liver tissue, which was not significantly altered in CLD. Our finding of constitutive eNOS expression probably reflects the important role of eNOS in the regulation of physiological vascular tone. Involvement of eNOS from sinusoidal lining cells in the regulation of intrahepatic resistance and perfusion has been analysed in rats: Shah  demonstrated in rat that intrahepatic blood flow and the resulting sinusoidal shear stress is a pivotal mechanism of NO regulation by eNOS from sinusoidal lining cells. Increased shear stress by enhanced sinusoidal perfusion leads to rising NO production, which in turn promotes dilatation of the sinusoids, resulting in an increase in vessel caliber and, in consequence, to a normalisation of shear stress to which sinusoidal endothelial cell are exposed. Previous studies could not detect altered eNOS protein expression in cirrhotic rats with portal hypertension . Our study confirms this finding also for human liver specimens from patients with chronic liver diseases and cirrhosis, indicating that altered eNOS expression levels are unlikely to contribute to portal hypertension in this condition. This finding is in line with concepts that eNOS activity is regulated by post-translational modifications, such as its binding
L. Leifeld et al. / Journal of Hepatology 37 (2002) 613–619
Fig. 1. Representative intrahepatic stainings of iNOS (1A, 1C, 1E) and eNOS (1B, 1D, 1F) in patients with FHF, CLD and NC as well as numbers of positive cells per visual field ( £ 400) (1G, 1H). Strong differences were found between the number of iNOS and eNOS positive cells in the livers of patients with FHF, CLD and NC. In FHF significant higher numbers of iNOS (1A, 1G) as well as eNOS expressing cells (1B) were found than in CLD (1C, 1D) and NC (1E, 1F). The example 1E demonstrates the most prominent iNOS expression seen in a NC. While iNOS expression was higher in CLD than in NC (P ¼ 0.001), eNOS expression did not differ between these groups (P ¼ 0.64).
L. Leifeld et al. / Journal of Hepatology 37 (2002) 613–619
to caveolin-1 [17,18], which down-regulates eNOS activity independent from the level of eNOS protein expression in cirrhotic rats. In contrast to eNOS, we found only minimal physiological hepatic expression of the enzyme iNOS. However, iNOS was significantly upregulated in CLD and even more conspicuously in FHF. The functional consequence of this finding on inflammatory processes in FHF seems to be complex, since both pro- and antiapoptotic effects of NO
are described, dependent on the involved cell types and on the concentration of NO . While high levels of NO promote cell damage, antiapoptotic effects on different cell types, including hepatocytes, are described for low levels of NO . Herein, functional studies on the influence of NO on cell damage in FHF are of special importance. Commonly used animal models demonstrate a predominant injurious function of NO in FHF: iNOS knock-out mice or mice in which iNOS activity was inhib-
Fig. 2. In situ localisation of iNOS and eNOS to different intrahepatic cell types. (A) Double staining of iNOS (left, immunoperoxidase) and CD68 positive macrophages (right, immunofluorescence). Macrophages/Kupffer cells are a main source of iNOS in the liver. (B) Double staining of iNOS (immunoperoxidase) and SMA-Cy3 positive stellate cells (immunofluorescence). While hepatocytes (‘H’) express iNOS, stellate cells (‘S’) are iNOS negative. (C) Double staining of eNOS (left, immunoperoxidase) and CD68 positive macrophages (right, immunofluorescence). (D) Double staining of eNOS (left, immunoperoxidase) and CD31 positive sinusoidal endothelial cells (right, immunofluorescence).
L. Leifeld et al. / Journal of Hepatology 37 (2002) 613–619
ited by l-N 6-(1-iminoethyl)-lysine were protected against fulminant liver damage induced by concanavalin-A or galactosamine-LPS application . Herein, nitric oxide seems to act as a second messenger for TNF release in FHF, since liver damage in iNOS knock-out animals could not be prevented, when TNF was used in galactosamine primed mice instead of LPS . Similar to concanavalin A and galactosamine-LPS induced liver damage, iNOS knock-out mice were also protected against multiple organ failure induced by zymosan application  and against liver injury induced by application of acetaminophen . In light of the animal models our findings of upregulated intrahepatic iNOS expression in human liver specimens of patients with FHF support the idea that a similar iNOS mediated mechanism of organ damage may also apply to human FHF. In summary, we could demonstrate major differences in the in situ expression of eNOS versus iNOS apparently corresponding to their putatively different roles in liver homeostasis. eNOS seems to be particularly involved in the physiological regulation of hepatic perfusion. In contrast, iNOS is closely related to liver pathology. iNOS probably leads to the generation of NO, contributing to severe inflammation and to liver damage in human FHF, as been suggested in animal models of FHF.
References  Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med 1994;120:227–237.  Curran RD, Billiar TR, Stuehr DJ, Ochoa JB, Harbrecht BG, Flint SG, et al. Multiple cytokines are required to induce hepatocyte nitric oxide production and inhibit total protein synthesis. Ann Surg 1990;212:462–469 discussion 470-471.  Billiar TR, Curran RD, Harbrecht BG, Stadler J, Williams DL, Ochoa JB, et al. Association between synthesis and release of cGMP and nitric oxide biosynthesis by hepatocytes. Am J Physiol 1992;262:C1077–C1082.  Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA 1993;90:522–526.  Li J, Billiar TR. Nitric oxide. IV. Determinants of nitric oxide protection and toxicity in liver. Am J Physiol 1999;276:G1069–G1073.  Kim PK, Zamora R, Petrosko P, Billiar TR. The regulatory role of nitric oxide in apoptosis. Int Immunopharmacol 2001;1:1421–1441.  Billiar TR, Curran RD, Harbrecht BG, Stuehr DJ, Demetris AJ, Simmons RL. Modulation of nitrogen oxide synthesis in vivo: NGmonomethyl-l-arginine inhibits endotoxin-induced nitrate/nitrate biosynthesis while promoting hepatic damage. J Leukoc Biol 1990;48:565–569.  Bohlinger I, Leist M, Barsig J, Uhlig S, Tiegs G, Wendel A. Tolerance against tumor necrosis factor alpha (TNF)-induced hepatotoxicity in mice: the role of nitric oxide. Toxicol Lett 1995;82–83:227–231.  Bohlinger I, Leist M, Barsig J, Uhlig S, Tiegs G, Wendel A. Interleukin-1 and nitric oxide protect against tumor necrosis factor alphainduced liver injury through distinct pathways. Hepatology 1995;22:1829–1837.  Ou J, Carlos TM, Watkins SC, Saavedra JE, Keefer LK, Kim YM, et al. Differential effects of non-selective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis,
ICAM-1 expression, and neutrophil accumulation during endotoxemia. Nitric Oxide 1997;1:404–416. Saavedra JE, Billiar TR, Williams DL, Kim YM, Watkins SC, Keefer LK. Targeting nitric oxide (NO) delivery in vivo. Design of a liverselective NO donor prodrug that blocks tumor necrosis factor-alphainduced apoptosis and toxicity in the liver. J Med Chem 1997;40:1947–1954. Kim YM, de Vera ME, Watkins SC, Billiar TR. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70 expression. J Biol Chem 1997;272:1402–1411. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem 1997;272:31138–31148. Kim YM, Kim TH, Chung HT, Talanian RV, Yin XM, Billiar TR. Nitric oxide prevents tumor necrosis factor alpha-induced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8. Hepatology 2000;32:770– 778. Li J, Billiar TR, Talanian RV, Kim YM. Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem Biophys Res Commun 1997;240:419–424. Kim YM, Kim TH, Seol DW, Talanian RV, Billiar TR. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem 1998;273:31437–31441. Shah V, Toruner M, Haddad F, Cadelina G, Papapetropoulos A, Choo K, et al. Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat. Gastroenterology 1999;117:1222–1228. Petermann H, Vogl S, Schulze E, Dargel R. Chronic liver injury alters basal and stimulated nitric oxide production and 3H-thymidine incorporation in cultured sinusoidal endothelial cells from rats. J Hepatol 1999;31:284–292. Rockey DC, Chung JJ. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: endothelial dysfunction in portal hypertension. Gastroenterology 1998;114:344–351. Sass G, Koerber K, Bang R, Guehring H, Tiegs G. Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice. J Clin Invest 2001;107:439–447. Bourdi M, Masubuchi Y, Reilly TP, Amouzadeh HR, Martin JL, George JW, et al. Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase. Hepatology 2002;35:289–298. Grady KM, Alexander R, Hayllar KM, Williams R. Early indicators of prognosis in fulminant hepatic failure. Gastroenterology 1989;97:439–445. Shah V, Haddad FG, Garcia-Cardena G, Frangos JA, Mennone A, Groszmann RJ, et al. Liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance in the hepatic sinusoids. J Clin Invest 1997;100:2923–2930. Yokomori H, Oda M, Ogi M, Kamegaya Y, Tsukada N, Ishii H. Endothelial nitric oxide synthase and caveolin-1 are co-localised in sinusoidal endothelial fenestrae. Liver 2001;21:198–206. Adamson GM, Billings RE. Cytokine toxicity and induction of NO synthase activity in cultured mouse hepatocytes. Toxicol Appl Pharmacol 1993;119:100–107. Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang SC, et al. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc Natl Acad Sci USA 1993;90:3491–3495. Osei SY, Ahima RS, Fabry ME, Nagel RL, Bank N. Immunohistochemical localisation of hepatic nitric oxide synthase in normal and transgenic sickle cell mice: the effect of hypoxia. Blood 1996;88:3583–3588. Buttery LD, Evans TJ, Springall DR, Carpenter A, Cohen J, Polak JM.
L. Leifeld et al. / Journal of Hepatology 37 (2002) 613–619 Immunochemical localisation of inducible nitric oxide synthase in endotoxin-treated rats. Lab Invest 1994;71:755–764.  Romero M, Garcia-Monzon C, Clemente G, Salcedo M, Alvarez E, Majano PL, et al. Intrahepatic expression of inducible nitric oxide synthase in acute liver allograft rejection: evidence of modulation by corticosteroids. Liver Transpl 2001;7:16–21.  Majano PL, Garcia-Monzon C, Lopez-Cabrera M, Lara-Pezzi E, Fernandez-Ruiz E, Garcia-Iglesias C, et al. Inducible nitric oxide synthase expression in chronic viral hepatitis. Evidence for a virusinduced gene upregulation. J Clin Invest 1998;101:1343–1352.
 Helyar L, Bundschuh DS, Laskin JD, Laskin DL. Induction of hepatic Ito cell nitric oxide production after acute endotoxemia. Hepatology 1994;20:1509–1515.  Kim YM, Bombeck CA, Billiar TR. Nitric oxide as a bifunctional regulator of apoptosis. Circ Res 1999;84:253–256.  Cuzzocrea S, Mazzon E, Dugo L, Barbera A, Centorrino T, Ciccolo A, et al. Inducible nitric oxide synthase knockout mice exhibit resistance to the multiple organ failure induced by zymosan. Shock 2001;16:51–58.