Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes

ABB Archives of Biochemistry and Biophysics 449 (2006) 104–114 www.elsevier.com/locate/yabbi

Inflammatory cytokines induce protein tyrosine nitration in rat astrocytes Boris Go¨rg a, Hans J. Bidmon b, Verena Keitel a, Natalie Foster a, Roland Goerlich c, Freimut Schliess a,*, Dieter Ha¨ussinger a a

Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, Du¨sseldorf, Germany b C&O Vogt Institute for Brain Research, Heinrich-Heine-University, Du¨sseldorf, Germany c Institute of Biology VII, Molecular Biotechnology, RWTH University, Aachen, Germany Received 12 October 2005, and in revised form 1 February 2006 Available online 9 March 2006

Abstract Protein tyrosine nitration may be relevant for the pathogenesis of hepatic encephalopathy (HE). Infections, sepsis, and trauma precipitate HE episodes. Recently, serum levels of tumor necrosis factor (TNF)-a were shown to correlate with severity of HE in chronic liver failure. Here the effects of inflammatory cytokines on protein tyrosine nitration in cultured rat astrocytes and rat brain in vivo were studied. In cultured rat astrocytes TNF-a (50 pg/ml–10 ng/ml) within 6 h increased protein tyrosine nitration. TNF-a-induced tyrosine nitration was related to an increased formation of reactive oxygen and nitrogen intermediates, which was downstream from a NMDAreceptor-dependent increase of intracellular [Ca2+]i and nNOS-catalyzed NO production. Astroglial tyrosine nitration was also elevated in brains of rats receiving a non-lethal injection of lipopolysaccharide, as indicated by colocalization of nitrotyrosine immunoreactivity with glial fibrillary acidic protein and glutamine synthetase, and by identification of the glutamine synthetase among the tyrosine-nitrated proteins. It is concluded that reactive oxygen and nitrogen intermediates as well as protein tyrosine nitration by inflammatory cytokines may alter astrocyte function in an NMDA-receptor-, Ca2+-, and NOS-dependent fashion. This may be relevant for the pathogenesis of HE and other conditions involving cytokine exposure the brain.  2006 Elsevier Inc. All rights reserved. Keywords: Astrocytes; Calcium; Hepatic encephalopathy; Inflammation; Interferon; Lipopolysaccharide; Liver; nNOS; NMDA receptor; Sepsis; Tumor necrosis factor

Hepatic encephalopathy (HE)1 is a frequent complication of chronic liver disease. It is thought to reflect the clinical manifestation of a low grade chronic cerebral edema [1,2] and to be a primary disorder of astrocytes with consequences for neuronal function [3]. The molecular mechanisms underlying the pathogenesis of HE are poorly understood. There is consensus that ammonia plays a key role [3]. However, HE-precipitating factors are heteroge*

Corresponding author. Fax: +49 211 811 18752. E-mail address: [email protected] (F. Schliess). 1 Abbreviations used: HE, hepatic encephalopathy; GFAP, anti-glial fibrillary acidic protein; FITC, fluorescein isothiocyanate; L-NMMA, NG-mono-methyl-L-arginine; TRIM, L-(2-trifluoromethylphenyl) imidazole; PBS, phosphate-buffered saline; LPS, lipopolysaccharide. 0003-9861/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.02.012

neous and include also hyponatremia, sedatives, trauma, and inflammation [2]. Recent data point to an important role of oxidative and nitrosative stress for the pathogenesis of HE. Ammonia [4,5], hypoosmotic swelling [6] or benzodiazepins [7], in cultured astrocytes cause oxidative stress and protein tyrosine nitration. Among the tyrosine-nitrated proteins glutamine synthetase, peripheral-type benzodiazepine receptor, and the glyceraldehyde 3-phosphate dehydrogenase have been identified, i.e., proteins potentially involved in the pathogenesis of HE [4,5,7]. Most importantly, astroglial protein tyrosine nitration was also observed in the brains from rats which underwent portocaval anastomosis or received an acute ammonia [5] or benzodiazepine [7] load.

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In acute and chronic liver failure impaired Kupffer cell function and/or the presence of portosystemic shunts frequently lead to elevated plasma endotoxin levels, which were suggested to importantly contribute to the pathogenesis of HE [8–10]. Elevation of tumor necrosis factor (TNF)-a accounts for multiple endotoxin effects and was recently shown to significantly correlate with the severity of HE due to chronic liver disease [9]. Interestingly, the association of high TNF-a serum levels with HE was not specific for the presence of infection or other HE-precipitating factors [9]. In rats with fulminant hepatic failure the severity of behavioral abnormalities was related to TNF-a plasma levels [11]. The assumption that astroglial protein tyrosine nitration plays a role in the pathogenesis of HE would imply that inflammatory cytokines might also increase protein tyrosine nitration in astrocytes. Because HE is considered as a primary gliopathy cultured astrocytes are a frequently used and well recognized model to study HE-relevant molecular effects of precipitating factors [3]. In the present study the effect of TNF-a, IFN-c, and IFN-a, which is frequently used in antiviral therapy, on protein tyrosine nitration was studied in cultured rat astrocytes. In addition, cerebral protein tyrosine nitration was examined in rats acutely challenged with lipopolysaccharide (LPS). Materials and methods Materials TNF-a was from Roche Diagnostics (Mannheim, Germany) and IFN-a from Serotec (Du¨sseldorf, Germany). 5- (and 6-) Carboxy-2 0 ,7 0 -dichlorodihydrofluorescein (DCFDA) and 1,2-bis(o-aminophenoxy)ethaneN,N,N 0 ,N 0 -tetraacetic acid (BAPTA-AM) were from Molecular Probes Inc (Eugene, OR, USA). Cell culture media were from Gibco Life Technologies (Gaithersburg, MD), fetal calf serum was from PAA Laboratories GmbH (Linz, Austria). LPS, IFN-c, catalase, superoxide dismutase (SOD), uric acid, MK-801, and polyclonal anti-glial fibrillary acidic protein (GFAP) antibody were from Sigma (Deisenhofen, Germany). Monoclonal antibodies raised against glutamine synthetase and eNOS, and polyclonal antibodies against iNOS were from BD Biosciences (San Diego, CA), respectively. NG-mono-methyl-L-arginine (L-NMMA), and the monoclonal anti-3 0 -nitrotyrosine antibody were from Upstate (Charlottesville, Virginia, USA). N5-(L-imino-3-butenyl)-L-ornithine (L-VNIO) was from Axxora (Gru¨nberg, Germany). L-(2-Trifluoromethylphenyl) imidazole (TRIM) was from Merck (Bad Soden, Germany). The polyclonal antibody raised against nNOS was a gift from Dr. Bernd Mayer (Institute for Pharmacology and Toxicology, Graz, Austria) [12]. Polyclonal antibodies against eNOS were purchased from Affinity BioReagents (Golden, USA). Peroxynitrite and monoclonal anti-3 0 -nitrotyrosine antibodies were purchased from

Calbiochem–Novabiochem Germany).

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GmbH

(Bad

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Preparation and cultivation of rat brain astrocytes Primary astrocytes were prepared from cerebral hemispheres of newborn Wistar rats and cultured for 2–3 weeks as described [13,14]. Purity of the cell culture as determined by glial fibrillary acidic protein (GFAP), and S-100 immunohistochemical staining was >95%. Immunoreactivity of the neuron-specific nuclear, NeuN, was not detectable in the astrocyte culture. After starvation for 24 h in serumfree medium experimental treatment of the astrocytes was started without re-addition of serum. If indicated cells were pre-incubated with inhibitors which remained present during the course of the experimental treatment of the cells. Western blot analysis At the end of the experimental astrocyte treatment, cells were lysed at 4 C using 10 mmol/L Tris–HCl buffer (pH 7.4), containing 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 0.2 mmol/L PMSF, and 0.5% NP-40. The homogenized lysates were centrifuged at 20,000g at 4 C. Protein concentration was estimated by using the BioRad protein assay (BioRad, Munic). For dot blot analysis equal amounts of proteins (1.5–3.0 lg) in a volume of 2 ll were applied to a nitrocellulose membrane. For SDS–gel electrophoresis and Western blot analysis the supernatant was added to an identical volume of 2 · gel loading buffer (pH 6.8), containing 200 mmol/L dithiothreitol. After heating to 95 C for 5 min, the samples were subjected to gel electrophoresis (20–40 lg protein/lane, 7.5 or 10% gels). Following electrophoresis, gels were equilibrated with transfer buffer (39 mmol/L glycine, 48 mmol/L Tris–HCl, 0.03% SDS, and 20% methanol). Proteins were transferred to nitrocellulose membranes using a semi-dry transfer apparatus (Pharmacia, Freiburg, Germany). Dot- and Western blots were blocked overnight in 1% fetal calf serum solubilized in 20 mmol/L Tris–HCl pH 7.5, containing 150 mmol/L NaCl and 0.1% Tween 20, and then incubated for 2 h with antiserum against 3 0 -nitrotyrosine (mAb, Calbiochem, 1:5000). Following washing and incubation with horseradish peroxidase-coupled anti-mouse-IgG antibody diluted 1:10,000 at room temperature for 2 h, blots were washed again and developed using enhanced chemiluminescent detection (Amersham, Braunschweig, Germany). Reduction of nitrotyrosine to aminotyrosine was achieved by incubation of the nitrocellulose membrane in freshly prepared 100 mmol/L sodium dithionite in 100 mmol/L disodiumtetraborate, pH 9.0, for 1 h at room temperature under gentle shaking. At the end of the incubation period the membrane was washed excessively and blocked with 1% fetal calf serum for 30 min before probing with the anti 3 0 -nitrotyrosine antibody. Densitometric analysis was performed with the Kodak Image Station 440CF, using the

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Kodak 1D software or Scion Image (Scion Corporation, Maryland, USA). Analysis of eNOS and nNOS mRNA expression by quantitative reverse transcription polymerase chain reaction Total RNA was isolated using the RNA extraction kit (Qiagen, Hilden, Germany), and cDNA was obtained with the QuantiTect reverse transcription kit (Qiagen). The level of gene expression in astrocytes was measured by real-time SYBR Green PCR with the 7500 Real-time PCR System (Applied Biosystems, Foster City, CA) as described previously [15]. Data were produced in duplicates for each gene. Mean values of cycle numbers of the target gene were subtracted from the mean of cycle numbers of the house-keeping gene hypoxanthine-phosphoribosyl transferase (hprt1) for the respective sample. This value taken to the power of 2 is the mRNA expression of the target gene (respective nos) in relation to hprt1 expression, termed ‘‘relative mRNA unit’’ (RRU). Real-time primers were as follows: rat nos1-for, 5 0 -cctgcaaagccctaagtccag-3 0 ; rat nos1-rev, 5 0 gtgtggagacgcacgaagatg-3 0 ; rat nos3-for, 5 0 - gtgctggcataca gaaccca-3 0 ; rat nos3-rev, 5 0 -gaccccatagtgcagagggc-3 0 ; rat hprt1-for, 5 0 -tgctcgagatgtcatgaagga-3 0 ; rat hprt1-rev, 5 0 cagagggccacaatgtgatg-3 0 . Lipopolysaccharide treatment of rats Lipopolysaccharide (LPS, dissolved in 0.9% saline) was injected to male Wister rats intraperitoneally at a nonlethal dose of 4 mg/kg body wt and animals were housed individually. Control rats were injected with saline only. After 2, 6, or 24 h, respectively, rats were anesthesized with pentobarbital (50 mg/kg) and transcardially perfused with 20 ml physiological saline containing heparin (10,000 iU/ L, Liquemin, Hoffmann La Roche, Basel, Switzerland) to remove blood cells, followed by 100 ml physiological saline containing 20% sucrose as a cryoprotectant. Brains were quickly dissected and 4 mm thick sections from the middle of each hemisphere were prepared. One of these thick sections was frozen for protein extraction. The other was immersion fixed in modified Zambonis fixative [16]. These experiments were approved by the national animal welfare legislation. Immunofluorescence staining of 3 0 -nitrotyrosine residues, GFAP, glutamine synthetase, eNOS, and nNOS Confocal laser scanning microscopy was performed using the LSM510 (Carl Zeiss AG, Oberkochen, Germany) or the Leicar TSC NT (Leica, Bensheim, Germany). For immunofluorescence, astrocytes were cultured on glass coverslips with a diameter of 10 mm. Cells were fixed with modified Zamboni fixative for 10 min at 4 C and washed three times with ice-cold phosphate-buffered saline (PBS). Subsequently, cells were incubated with Triton X-100 (0.1% in PBS) for 10 min. Cells were washed again and

incubated with 25% goat-Ig in the case of staining with anti-3 0 -nitrotyrosine (mAb, 1:85), and anti-GFAP (pAb, 1:200) antibodies for at least 2 h at room temperature. The cells were extensively washed and incubated for 2 h with fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG or Cy3-conjugated anti-mouse IgG at room temperature. Images were acquired from one or two channels at 488 and 568 nm wavelengths. For immunofluorescence of eNOS and nNOS cells cells were blocked with 5% bovine serum albumin in PBS for 30 min before incubating with polyclonal anti-eNOS or nNOS antibody (1:100 and 1:500, respectively), or monoclonal anti-GFAP antibody (1:200) in PBS containing 5% bovine serum albumin for 2 h at room temperature. Cells were extensively washed and incubated for 2 h with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG or Cy3-conjugated anti-rabbit IgG (1:200, Jackson Corp., West Grove, USA). Coverslips were mounted in Vectashield mounting medium for fluorescence containing DAPI (Vector Laboratories, Burlingame, USA). Images were acquired from three channels at 405, 488, and 568 nm wavelengths. Zamboni-fixed (4 C, 24 h) brain hemispheres were submerged in 20% sucrose in phosphate-buffered saline (PBS, 24 h, 4 C) and further processed as described [16]. As primary antibodies the anti-3 0 -nitrotyrosine antibody (polyclonal, final dilution 1:100), anti-GS antibody (monoclonal, final dilution 1:100), and anti-GFAP antibody (polyclonal, final dilution 1:100) were used. As second antibodies were used fluorochrome-coupled antichicken-FITC, anti-rabbit-Cy5, and anti-mouse Cy3 (1:200, Jackson Corp., West Grove, USA). Detection of reactive oxygen and nitrogen intermediates Cultured astrocytes of the first passage were seeded on a 24 well culture plate until confluency. Cells were starved overnight in culture medium without FCS and incubated for 30 min with 100 lmol/L H2DCFDA. After repeated washing with culture medium astrocytes were stimulated for 6 h with the respective cytokine without further pretreatment or after a 30 min pretreatment with an inhibitor. After the experimental treatment cells were washed with PBS and fluorescence was measured by using a VictorFluorimeter (EG&G Wallac, Wellesley, USA) or an Ascent Fluorescent FL-Fluorimeter (Thermo Electron Corporation, Dreieich, Germany) with an excitation wavelength of 485 nm and emission at 535 nm. Detection of intracellular [Ca2+] changes Measurements were performed with astrocytes grown on coverslips as described above. Cells were incubated in DMEM in absence of phenol red but containing 5 lmol/ L of the fluorescent Ca2+-chelator Fluo-4 for 1 h at room temperature. After repeated washings coverslips were mounted on an inverted fluorescence microscope (Zeiss Axiovert, Germany). Cells were excited at 488 nm by a

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Fig. 1. Inflammatory cytokines induce protein tyrosine nitration in cultured rat astrocytes. Astrocytes remained untreated or were exposed (6 h) to TNF-a (10 ng/ml or lower concentrations where indicated), IFN-c (100 U/ml) and IFN-a (100 U/ml), respectively. Protein tyrosine nitration was analyzed by Western blot using an anti-3 0 -nitrotyrosine-specific antibody. Overall protein tyrosine nitration was analyzed by dot blot of protein lysates. For loading control blots and dots were stained with an antibody recognizing GAPDH or glutamine synthetase, respectively. (A) Western blot analysis of protein tyrosine nitration by TNF-a, IFN-c, and IFN-a. A representative of three independent experiments is given. (B) Dot blot analysis of protein tyrosine nitration by TNF-a, IFN-c, and IFN-a (lanes 1–4). For specificity control of nitrotyrosine detection, purified glutamine synthetase and glutamine synthetase treated with peroxynitrite in vitro were also dotted (lanes 5 and 6). In addition, dots were probed with the second antibody only, or with the antibodies recognizing 3 0 -nitrotyrosine or GS after reductive treatment with sodium dithionite. (C) Dose dependence of TNF-a-induced protein tyrosine nitration. A representative dot blot is shown. The diagram is based on Pthree independent experiments.

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monochromator, and emission was measured at 515– 565 nm using a CCD camera provided by the QuantiCell 2000-calcium imaging setup (VisiTech, Sunderland, UK). Emission intensity of unstimulated cells was set to 1, and increased emission intensity after the addition of TNF-a is expressed as relative increase compared with unstimulated emission intensity. All measurements were performed in a bathing solution at room temperature. Analysis of results Results from n independent experiments are expressed as means ± SD. Data were compared by using one-way analysis of variance (ANOVA) followed by Dunett’s multiple comparison post hoc test. p < 0.05 was considered statistically significant. Results Effect of inflammatory cytokines on protein tyrosine nitration in cultured rat astrocytes As shown by Western blot analysis (Fig. 1A), a 6 h exposure of rat astrocytes to TNF-a (10 ng/ml), IFN-c (100 U/ml), and IFN-a (100 U/ml), respectively, increased tyrosine nitration of distinct proteins in the range of 20–148 kDa. Similar patterns of tyrosine-nitrated proteins were observed after treatment with each of these cytokines. Dot blot analysis unravelled a 1.98 ± 0.68-fold increase of protein tyrosine nitration in presence of 10 ng/ml TNF-a

after 6 h (n = 19, p < 0.01). Similarly, IFN-a (100 U/ml) and IFN-c (100 U/ml) after 6 h induced a 2.74 ± 1.16-fold (n = 6, p < 0.01) and 2.30 ± 0.83-fold (n = 6, p < 0.01) increase of protein tyrosine nitration, respectively, compared to control cells (Fig. 1B, lanes 1–4). Protein tyrosine nitration by TNF-a, IFN-a, and IFN-c was transient. After 9 h protein tyrosine nitration returned to 1.44 ± 1.03, 1.49 ± 0.72, and 1.55 ± 0.92-fold of protein tyrosine nitration found in the respective controls (n = 3–5, p > 0.05). To control the specificity of 3 0 -nitrotyrosine detection by the antibody recognizing 3 0 -nitrotyrosine, purified sheep glutamine synthetase exposed to peroxynitrite in vitro was dotted. As shown in Fig. 1B (lanes 5 and 6) peroxynitrite treatment of glutamine synthetase increased 3 0 -nitrotyrosine immunoreactivity which was absent in the untreated sample. No signals were obtained when dots were probed with the second antibody only or with the anti-nitrotyrosine antibody after reductive treatment with dithionite, which was without effect on GS immunoreactivity (Fig. 1B, lanes 1–6). This further supports the specificity of nitrotyrosine detection by the anti3 0 -nitrotyrosine antibody. As shown in Fig. 1C, TNF-a concentrations of 50 pg/ml–10 ng/ml, which are in the range of TNF-a serum concentrations found in patients with chronic liver disease [9] and acute liver failure [17,18], induced a pronounced protein tyrosine nitration in cultured astrocytes. Fig. 2 shows the immunocytochemical analysis of 3 0 -nitrotyrosine immunoreactivity in cultured rat astrocytes. Whereas very little protein tyrosine nitration (red staining) was detectable under control

Fig. 2. TNF-a-induced 3 0 -nitrotyrosine immunoreactivity in cultured rat astrocytes. Immunoreactivities of 3 0 -nitrotyrosine and GFAP were visualized by confocal laser scanning microscopy. Cultured rat astrocytes were maintained in the absence of TNF-a (A, C, and E) or were exposed to 10 ng/ml TNF-a for 6 h (B, D, and F). Green: GFAP immunoreactivity, red: 3 0 -nitrotyrosine immunoreactivity, yellow: colocalization of 3 0 -nitrotyrosine and GFAP. A representative of three independent experiments is shown.

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conditions, tyrosine nitration increased in response to TNF-a. Astrocytes were counterstained with anti-GFAP antibody (green colour). Pharmacological characterization of TNF-a-induced protein tyrosine nitration in cultured astrocytes Nitrated tyrosine residues are indicative for the presence of reactive nitrogen species, which may derive from nitric oxide (NO) as metabolic precursor [19]. To evaluate a role of iNOS in protein tyrosine nitration induced by TNF-a, IFN-c, and IFN-a, iNOS expression in cultured astrocytes was monitored. As shown in Fig. 3A none of these cytokines induced iNOS expression when added individually to cultured astrocytes for 3, 6, or 9 h, respectively. However, combined treatment of the astrocytes with TNF-a (10 ng/ml), IL-1b (100 U/ml), IFN-c (100 U/ml), and

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LPS (1 lg/ml) increased iNOS expression (positive control, Fig. 3A). These findings suggest that iNOS expression is not required for induction of protein tyrosine nitration by TNF-a, IFN-c, or IFN-a in cultured rat astrocytes. Expression of the Ca2+-dependent isoforms nNOS and eNOS by the cultured astrocytes was analyzed by real-time PCR, Western blot, and immunocytochemistry. Both, nNOS and eNOS mRNA expression relative to HPRT mRNA expression was 0.0013 ± 0.0004 RRU and 0.0006 ± 0.0004, respectively (n = 4). However, at the protein level only nNOS expression was detectable in cultured astrocytes (Fig. 3B), whereas both, nNOS and eNOS were detected in rat hippocampus (positive control, Fig. 3B). nNOS expression was confirmed by immunocytochemistry (Fig. 4). The findings suggest a major role of mRNA translation and/or protein degradation as regulatory sites of nNOS/eNOS expression in cultured astrocytes. To further examine a contribution of NOS activity to protein tyrosine nitration induced by TNF-a, L-NMMA, a NOS inhibitor of broad specificity [20] was used. L-NMMA at a concentration of 100 lmol/L blocked the TNF-a-induced protein tyrosine nitration in cultured astrocytes (Table 1). Buffering intracellular Ca2+ with BAPTA-AM diminished protein tyrosine nitration in response to TNF-a (Table 1), indicating a Ca2+-dependence of protein tyrosine nitration by TNF-a. In addition, the peroxynitrite scavenger uric acid [21] at 200 lmol/L blocked TNF-a-induced protein tyrosine nitration in cultured astrocytes (Table 1), suggesting a major contribution of peroxynitrite. MK-801 affects glutamate signaling by N-methyl-D aspartate (NMDA) receptor inhibition [22] and its specific binding to NMDA receptor sites on cultured astrocytes was recently demonstrated [7]. At a concentration of 100 lmol/L, MK-801 largely abolished the TNF-a-induced protein tyrosine nitration (Table 1), suggesting the involvement of NMDA receptors. As shown in Fig. 5 and consistent with earlier findings [23] TNF-a in cultured astrocytes stimulates a slow increase of intracellular Ca2+ ([Ca2+]i). MK-801 (100 lM) impaired the TNF-a-induced [Ca2+]i increase, suggesting that [Ca2+]i-elevation by TNF-a plays a role for the contribution of NMDA receptor activation to protein tyrosine nitration. TNF-a, IFN-c, and IFN-a-induced generation of oxidative stress in cultured rat astrocytes

Fig. 3. Western blot analysis of NOS expression in cultured rat astrocytes (A) iNOS expression. Protein lysates of cultured astrocytes were analysed by Western blot for the presence of iNOS. Astrocytes were treated with TNF-a (10 ng/ml), IFN-c (100 U/ml), and IFN-a (10 ng/ml) for 3, 6, or 9 h, respectively. To produce a positive control for iNOS expression astrocytes were exposed for 24 h to TNF-a (10 ng/ml) plus IL-1b (100 U/ ml) plus IFN-c (100 U/ml) plus LPS (1 lg/ml). (B) nNOS and eNOS expression by cultured astrocytes. Serum-starved astrocytes were analyzed for eNOS and nNOS expression by Western blot. For positive control lysates of rat hippocampus were analyzed. eNOS expression in cultured astrocytes was analyzed by using two different antibodies showing almost identical results. Representatives of at least n = 3 astrocyte preparations are shown.

To address the generation of oxidative stress more directly, experiments with DCFDA-loaded astrocytes were performed. As shown in Table 2, TNF-a increased DCF fluorescence within 6 h. Also IFN-a (100 U/ml) and IFN-c (100 U/ml) after 6 h significantly increased DCF fluorescence to 1.32 ± 0.10 (n = 3) and 1.10 ± 0.04 (n = 11)-fold of control, respectively, suggesting an increased production of reactive oxygen/nitrogen species by the cytokines. The TNF-a-induced oxidative stress was analyzed by pharmacological means (Table 2). Presence of the NMDA

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Fig. 4. Immunocytochemical analysis of eNOS and nNOS in cultured rat astrocytes. Immunoreactivities of eNOS or nNOS and GFAP and 4 0 , 6-diamidino-2-phenylindole (DAPI)-counterstained nuclei were visualized by confocal laser scanning microscopy. Green: GFAP immunoreactivity, red: nNOS/eNOS immunoreactivity as indicated, yellow: colocalization of nNOS and GFAP, blue; nuclear staining. While all astrocytes showed a strong cytosolic nNOS immunoreactivity, in some cells in addition a nearby nuclear localization was noted. A representative staining from three independent astrocyte preparations is shown.

Table 1 Pharmacological characterization of TNF-a-induced protein tyrosine nitration in cultured rat astrocytes Fold PTN of control Control TNF-a +MK-801 +Uric acid +L-NMMA +BAPTA-AM

1.00 ± 0.00 1.98 ± 0.68a 1.11 ± 0.34b 1.16 ± 0.59b 1.15 ± 0.56b 1.01 ± 0.40b

Astrocytes were exposed to TNF-a (10 ng/ml) for 6 h without further pretreatment or after preincubation with MK-801 (100 lmol/L), uric acid (200 lmol/L), L-NMMA (100 lmol/L) or BAPTA-AM (10 lmol/L) 30 min prior to TNF-a addition. Dot blots probed with an 3 0 -nitrotyrosine-recognizing antibody were quantified by densitometry. Tyrosine nitration induced by TNF-a was normalized to that found under control conditions which was set to one. Data are given as means ± SD (n P 3 different experiments). a Level of protein tyrosine nitration significantly (p < 0.05) different from the respective control. b Significantly (p < 0.05) different to the level of protein tyrosine nitration in presence of 10 ng/ml TNF-a alone.

receptor antagonist MK-801 (100 lmol/L) abolished the generation of oxidative stress by TNF-a. MK-801 (1 mmol/L) did not affect oxidative stress produced by treatment of the astrocytes with 500 lmol/L peroxynitrite [increase of DCF fluorescence measured after bolus

Fig. 5. TNF-a-induced increase of [Ca2+]i in cultured astrocytes: sensitivity to MK-801 [Ca2+]i was followed by monitoring Fluo4 fluorescence as described in the Materials and methods section. Astrocytes were exposed to 10 ng/ml TNF-a for about 15 min without further treatment or after addition of MK-801 (100 lmol/L, 30 min pretreatment). Representative traces of three independent experiments are shown.

addition of peroxynitrite: 6.0 ± 4.2 in the absence of MK-801 vs. 9.8 ± 4.3 in the presence of MK-801, (n = 4)]. Further, MK-801 (100 lmol/L, 1 mmol/L) was without effect on the fluorescence increase of H2DCFDA (100 lmol/L in KH2PO4 pH 7.0) stimulated in vitro by peroxynitrite (50 or 100 lmol/L, data not shown), suggesting that a scavenging of reactive oxygen and nitrogen intermediates by MK-801 does not account for its inhibitory effect on the TNF-a-induced generation of oxidative stress in

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Table 2 Pharmacological characterization of TNF-a-induced generation of reactive oxygen/nitrogen intermediates in cultured rat astrocytes DCF-fluorescence Control TNF-a +MK-801 +Uric acid +BAPTA-AM +L-VNIO +TRIM

1.00 ± 0.00 1.41 ± 0.42a 0.95 ± 0.33 1.04 ± 0.21 1.09 ± 0.23 0.98 ± 0.07 0.98 ± 0.04

Astrocytes were loaded with H2DCFDA and fluorescence emission at 535 nm was taken as a measure for the generation of oxidative stress. Astrocytes were exposed to TNF-a (10 ng/ml) for 6 h without further pretreatment or after preincubation with MK-801 (100 lmol/L) uric acid (200 lmol/L), or BAPTA-AM (10 lmol/L), L-VNIO (20 lmol/L), TRIM (100 lmol/L) for 30 min. Data are given as means ± SD (n P 3). a DCF fluorescence significantly (p < 0.05) different from that observed under the respective control condition.

cultured astrocytes. Also BAPTA-AM (10 lmol/L) and uric acid (200 lmol/L) abolished the generation of oxidative stress by TNF-a in cultured astrocytes. The contribution of nNOS to the TNF-a-induced increase in oxidative stress was examined by using the nNOS inhibitors L-VNIO and TRIM [24,25]. Both, L-VNIO (20 lmol/L) and TRIM (100 lmol/L) prevented the increase of oxidative stress induced by TNF-a (Table 2). The data indicate a TNF-ainduced increase of reactive oxygen and nitrogen intermediates, which depends on NMDA receptor activation, changes of intracellular Ca2+ and nNOS-catalyzed NO formation and probably involves the formation of peroxynitrite. In vivo relevance of cytokine-induced protein tyrosine nitration Cerebral protein tyrosine nitration was studied in the in vivo model of inflammation induced by lipopolysaccharide (LPS) injection. Rats were injected with LPS (4 mg/kg) and sacrified 2, 6, or 24 h later. As revealed by dot blot analysis, LPS increased protein tyrosine nitration in brain by 2.45 ± 1.46-fold after 6 h (n = 9, p < 0.05) whereas no increase of protein tyrosine nitration was observed after 2 h (0.83 ± 0.15-fold, n = 3). After 24 h LPS-induced protein tyrosine nitration was reduced to 1.49 ± 0.15-fold (n = 3), respectively. This indicates that cerebral protein tyrosine nitration induced by a single LPS administration in vivo is of transient nature. Western blot analysis unravelled increased tyrosine nitration of distinct proteins in rat brain 6 h after LPS treatment (Fig. 6). Among the tyrosinenitrated proteins, glutamine synthetase was identified by immunoprecipitation experiments (Fig. 6). Because glutamine synthetase in brain is localized predominantly in astrocytes [26] this indicates astroglial protein tyrosine nitration in response to LPS in vivo. Fig. 7A shows increased protein tyrosine nitration in brain slices obtained from LPS-treated rats. As shown in

Fig. 6. LPS-induced protein tyrosine nitration in rat brain in vivo. Rats were injected with lipopolysaccharide (LPS, 4 mg/kg) or saline and sacrified 6 h later. Protein extracts were analyzed for protein tyrosine nitration by Western blot using antibodies recognizing 3 0 -nitrotyrosine. In addition, anti-3 0 -nitrotyrosine immunoprecipitates were analyzed in Western blot for the presence of glutamine synthetase (GS), and, vice versa, anti-GS-immunoprecipitates were analyzed for the presence of 3 0 nitrotyrosine. Representatives of three experiments are given.

Fig. 7B(B1–B6) by confocal laser-scanning microscopy, cortical tyrosine nitration was enriched along the blood vessels and colocalizes in part with glutamine synthetase. Fig. 7B(B7–B12) focuses on perivascular astrocytes and shows a triple staining of 3 0 -nitrotyrosine residues, GFAP, and glutamine synthetase. There was a high degree of colocalization of 3 0 -nitrotyrosine with glutamine synthetase and GFAP, respectively (Fig. 7B, B10–B12), supporting protein tyrosine nitration in astrocytes of the blood–brain barrier. Discussion Endotoxemia and elevated levels of inflammatory cytokines in cirrhotic patients were suggested to essentially contribute to the pathogenesis of HE [8–10]. Recently, astroglial protein tyrosine nitration was found to be increased under HE-relevant conditions including

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B A

Fig. 7. Immunohistochemical analysis of protein tyrosine nitration in brain of LPS-treated rats. Rats were injected with a non lethal dose (4 mg/kg) lipopolysaccharide (LPS) or saline and sacrified 6 h later. (A) Diaminobenzidine staining showed a LPS-induced increase in cerebral 3 0 -nitrotyrosine (NO2Tyr) immunoreactivity (B) Confocal laserscanning microscopy (B1–B12). (B1–B6), cellular localization of 3 0 -nitrotyrosine (red) and glutamine synthetase (GS, green) immunoreactivities. Subsequent to LPS treatment many cells in the parenchyma show increased 3 0 -nitrotyrosine immunoreactivity which in part (yellow staining, B6) colocalizes with GS-positive astrocytes. Some enrichment of nitrotyrosine immunoreactivity was found in astrocytes localized along the blood vessels (B1–B6). (B7–B12), Triple staining of 3 0 -nitrotyrosine (blue), glial fibrillary acidic protein (GFAP, green), and GS (red). Astroglial 3 0 -nitrotyrosine immunoreactivity colocalizes to a high degree with GS and GFAP. 3 0 -Nitrotyrosine immunoreactivity underneath the astrocytes in B12 may indicate tyrosine nitration in vascular endothelial cells.

hyperammonemia, diazepam treatment and hypoosmolarity [5–7]. As shown here, TNF-a, IFN-c, and IFN-a induce oxidative stress and protein tyrosine nitration in cultured rat astrocytes. Astroglial protein tyrosine nitration was also identified in brain of LPS-challenged rats, supporting the in vivo relevance of astroglial protein tyrosine nitration under inflammatory conditions. TNF-a-induced generation of reactive oxygen/nitrogen intermediates and protein tyrosine nitration in cultured astrocytes was dependent on a NMDA-receptor-dependent elevation of [Ca2+]i (this paper). TNF-a in astrocytes induces glutamate release and simultaneously blocks glutamate uptake [27], which may contribute to NMDA receptor stimulation and the [Ca2+]i increase observed in

TNF-a-treated astrocytes (Fig. 5). [Ca2+]i elevation is sufficient to increase astroglial formation of NO [28]. Because only nNOS was detectable in the cultured astrocytes (Figs. 3 and 4) and specific nNOS inhibitors block the entire oxidative stress generation in response to TNF-a (Table 2) it is well conceivable that a Ca2+-dependent and nNOS-catalyzed NO production is crucial for the overall increase of reactive oxygen and nitrogen species in response to TNFa. This suggestion is supported by the recent finding that already low NO concentrations in rat astrocytes stimulate mitochondrial O2  production, which even outlasts the presence of NO [29]. Recombination of O2  with NO to the nitrating species peroxynitrite [19] may account for the accumulation of tyrosine-nitrated proteins.

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TNF-a induces astrocyte swelling [30]. Interestingly, swelling of cultured astrocytes by hypoosmolarity was sufficient to produce an NMDA-receptor- and Ca2+-dependent generation of protein tyrosine nitration [6], raising the possibility that in HE astrocyte swelling induced by TNF-a together with other HE precipitating factors plays a role for astroglial protein tyrosine nitration. Protein tyrosine nitration was also found in brains of LPS-treated rats (this paper, Figs. 6 and 7). Astroglial tyrosine nitration in this model is demonstrated by the strong tyrosine nitration of glutamine synthetase, which is specific for astrocytes. Nitration of the glutamine synthetase is associated with a loss of activity [5,31], which, on the one hand, may alleviate astrocyte swelling by ammonia, but on the other, promote neurotoxicity of ammonia due to its reduced detoxication. Astroglial protein tyrosine nitration in LPS-treated rats was most pronounced in the perivascular area (Fig. 7B). Astrocytes are known to be important constituents of the blood–brain barrier and tyrosine nitration may therefore impair blood–brain barrier integrity [32]. TNF-a alters the permeability of the blood–brain barrier [33] and it is tempting to speculate that cytokine-induced tyrosine nitration in perivascular astrocytes may contribute to the specific blood brain barrier permeability changes observed in HE [34]. Recently plasma TNF-a levels were shown to correlate with the severity of HE in cirrhotics [9]. Depending on HE severity, serum levels of TNF-a in cirrhotics were reported to fluctuate between 5 and 60 pg/ml [9]. Patients with fulminant hepatic failure even exhibit up to 350 pg/ ml serum TNF-a [17,18]. As shown here, TNF-a concentrations in this range increase protein tyrosine nitration in cultured astrocytes, supporting the potential HE-relevance of the cell culture findings. In the in vivo rat model used in this study serum TNF-a levels were 337, 52, and 18 pg/ml at 3, 6, and 12 h following LPS administration [35], which fits well into the HE-relevant plasma concentration range. Similar concentrations were achieved in the brain of rats after peripheral LPS administration [36,37]. In summary, TNF-a (this paper) like ammonia [5], diazepam [7] and hypoosmolarity [6] induces astroglial generation of oxidative stress and protein tyrosine nitration in a NMDA-receptor- and Ca2+-dependent manner. In the clinical situation TNF-a in synergism with other HE-precipitating factors may contribute to the full pattern of protein tyrosine nitration. The finding that NMDA receptor antagonists abolish protein tyrosine nitration by TNFa (this paper), diazepam [7], and ammonia [5] in cultured astrocytes and in brains of acutely ammonia-loaded rats [38] on the one hand, and were reported on the other to improve the neurological outcome in ammonia-intoxicated animals [39,40] argue for a possible role of astroglial production of oxidative stress and protein tyrosine nitration in the pathogenesis of HE. The astroglial mechanisms of reactive oxygen and nitrogen intermediate production described here may be of relevance also for other conditions involving cytokine exposure of the brain.

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