Increased Nitric Oxide Synthesis and Inducible Nitric ... - Clinical Science

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Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, Avenida C a m p Charro sin, 37007. Salamanca, Spain, *Departamento de Medicina, ...
Clinical Science (1998) 9 4 , 6 3 7 4 3 (Printed in Great Britain)

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Increased nitric oxide synthesis and inducible nitric oxide synthase expression in patients with alcoholic and non=alcoholicliver cirrhosis A. SANCHEZ-RODR~GUEZ*,M. CRIADO, A. M. RODR~GUEZ-LOPEZ, A. ESTELLER, A. MARTiN DE ARRlBAt and J. M. LOPEZ-NOVOA lnstituto Reina Sofia de lnvestigaciones Nefrolbgicas, Departamento de Fisiologia y Farmacologia, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, Avenida C a m p Charro sin, 37007 Salamanca, Spain, *Departamento de Medicina, Universidad de Salamanca, Avenida Campa Charra sln,

37007 Salamanca. Spain. and tDepartamento de Medicina Nuclear. Universidad de Salamanca, Avenida Campa Charro sln, 37007 Salamanca, Spain

1. The synthesis and release of nitric oxide may play a role in the pathogenesisof peripheral vasodilatation and hyperdynamic circulation observed in liver cirrhosis. In this work, we analysed the synthesis of nitric oxide by the lympho-mononuclear cells of peripheral blood from patients with chronic alcoholic and non-alcoholic liver disease and we identified the isoform of nitric oxide synthase involved in the increased nitric oxide synthesis. 2. Patients were classified following clinical and histological criteria in non-alcoholic cirrhotic, alcoholic cirrhotic and non-cirrhotic chronic liver disease. We studied clinical and analytical characteristics, haemodynamic parameters and endotoxin levels in these patients. 3. Cirrhotic patients showed an increase of cardiac output and a decrease of peripheral vascular resistance. These patients had higher levels of plasma endotoxin than those observed in the control group. W-Nitro-Larginine methyl ester (L-NAMEbinhibitable nitrite production from mononuclear lymphocyte cells was higher in patients than in the control group, the highest levels being in non-alcoholic cirrhotic patients, and the lowest levels in patients with non-cirrhotic alcoholic liver

production in hepatic cirrhosis. Thus, this increased nitric oxide synthesis could be implicated in the pathogenesis of the haemodynamic disturbances frequently found in cirrhotic patients. This increase seems to be induced, at least in part, by activation of an inducible isoform of nitric oxide synthase.

INTRODUCTION

In man and experimental animals, portal hypertension is associated with a hyperdynamic circulation elicited by peripheral vasodilatation, mainly in the splanchnic bed [11. This peripheral vasodilatation is clinically relevant since it is thought to be responsible for the pathogenesis of many of the alterations associated with hepatic cirrhosis and its consequences [l-31. Due to its vasodilator effect, nitric oxide (NO) has been suggested to be involved in the haemodynamic alterations observed in alcoholic patients with hepatic damage [4,5]. . Furthermore, several authors have shown that the inhibition of NO synthesis, by the administration of disease. competitive antagonists of nitric oxide synthase 4. Immunocytuchemistry studies revealed a positive (NOS), increases arterial pressure, renal vasoimmunoreactivity for the inducible isoform of nitric constriction and natriuresis in control conditions [6,7 oxide synthase in lympho-mononuclear cells that was and in experimental models of cirrhosis [8-111. In more evident in non-alcoholic than in alcoholic cirrhotic addition, it has been reported that leucocytes from patients. By Northern blot, inducible nitric oxide syncirrhotic patients show an increased NO synthesis thase mRNA expression was observed only in lympho[10-12]. mononuclear cells from non-alcoholic cirrhotic patients. The aim of the present experimentswas to assess the 5. Our patients show a correlation between nitric oxide synthesis of NO by the lympho-mononuclear (LMN) synthesis, endotoxin levels and haemodynamic paracells from patients with chronic alcoholic and nonmeters. alcoholic liver disease and to identify the isoform of 6. These findings indicate that lympho-mononuclear NOS involved in the increased NO synthesis. cell stimulation may play a role in elevated nitric oxide Key words: cardiac output, lympho-mononuclear cells, mRNA. nitric oxide, nitric oxide synthase. NO-nitro-L-arginine methyl ester, peripheral vascular resistances. Abbreviations: IL. interleukin; LMN, lympho-mononuclear; L-NAME. NO-nitro-L-arginine methyl ester; NO, nitric oxide; iNOS. inducible nitric oxide synthase; TNF-a, tumour necrois factor-a. Correspondence: Professor JosC M. L6pez-Novoa.

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A. Sanchez-Rodriguez and others

PATIENTS A N D METHODS Patients

A total of 63 subjects, 51 men and 12 women (mean age 54.5i-2 years for men and 60.1 k 3 years for women), from the Department of Medicine in the University Hospital were studied. This study abides by the rules of the Ethics Committee of the Hospital and the Regulations for Clinical Studies in Spain. Patients were classified into different groups following clinical (stigmata of liver disease) and histological (biopsy proven) criteria. A total of 51 patients with chronic liver disease were selected for this study. Ten of these patients presented with non-alcoholic cirrhosis (five men and five women, age 6 2 k 4 years; seven had chronic hepatitis C infection and three had chronic hepatitis B infection), 23 patients presented with alcoholic cirrhosis (23 men, age 5 7 k 2 years) and 18 patients presented with chronic alcoholic liver disease (17 men and 1 woman, age 47.8 f3 years). Clinical evaluation

We selected from clinical history chronic alcoholic patients who consumed at least 80 g of ethanol daily for more than 10 years. In all of the patients diagnosis was established on the basis of liver biopsy. . We classified the patients with hepatic cirrhosis, viral or alcoholic, using the Child-Pugh score [ 131, including degree of encephalopathy, presence or absence of ascites, total bilirubin concentration, serum albumin and prothrombin time. We took into account all symptoms and signs whether non-specific or specifically derived from portal hypertension and/or from hepatic failure, a global study of hepatic function tests, viral markers and an image diagnosis (echography, fibrogastroscopy and computer-assisted tomography). All patients underwent liver biopsy. Chronic hepatitis B infection. The group with chronic hepatitis B infection comprised three subjects seropositive for hepatitis B surface antigen for at least 6 months. All patients, displayed markers for viral replication. At the time of the investigation, none of these patients received immunomodulatory or antiviral therapy. All patients had undergone liver biopsy within the previous 6 months with evidence of cirrhosis. Chronic hepatitis C infection. The group with chronic hepatitis C infection comprised seven subjects. Patients were entered into the study if they had biopsy findings consistent with chronic hepatitis C and were shown to be positive for antibodies to hepatitis C virus. None of these patients received immunomodulatory or antiviral therapy. None of the patients showed combined liver disease. None of the patients had a case history of diabetes, renal disease, arterial hypertension, ischaemic cardiopathy, congestive heart failure or familial heterozygous hyperlipoproteinaemia. Serum creatinine levels

were below 2 mg/ 100 ml. Administration of diuretics, vasoactive drugs and non-steroidal anti-inflammatory drugs was interrupted 2 weeks before the study. Stimulants or caffeine-containing drinks were not allowed 12 h before extraction of blood. Each patient had to undergo an 8 h fast before extraction at approximately 22 "C. The same conditions applied for controls. Endotoxin assay

Endotoxin concentration in plasma samples from cirrhotic patients and normal individuals was determined using the kinetic quantitative chromogenic Limulus Amoebocyte Lysate (LAL) system, KINETIC-QCL- 1000@(Boehringer-Ingelheim). This method is based on the observations of Levin and Bang [ 141. Escherichia coli 055 :B5 endotoxin was used as standard. Endotoxin concentration for samples was determined from a standard curve (0-250 enzyme units/ml) through a photometer (KINETIC-QCL, Boehringer-Ingelheim)at 405 nm. The calculated coefficient of variation was 4 O h . Haemodynamic study

Absolute ventricular volumes and cardiac output were determined by isotopic ventriculography using a variant of the Maurer technique to calculate the specific attenuation factor in each subject. Stroke volume was obtained from absolute systolic and diastolic volumes. The cardiac output of each subject, expressed in litres/min, was obtained from stroke volume and heart rate. Peripheral vascular resistances, expressed as mmHg min-' .l-l, were determined from the mean arterial pressure and cardiac output. L M N cell extraction and culture

The blood samples from patients were processed after clinical and biochemical studies and hepatic biopsy had been concluded. Venous blood was collected into sterile endotoxinfree vacuum blood collection tubes containing lithium heparin. LMN cell separation was made by differential centrifugation in a Histopaque@-lO77(Sigma) gradient following the method of Boyum [15]. Cells were counted in a Neubauer chamber and cellular viability was studied by Trypan Blue exclusion (more than 95 % remained viable). The final preparation consisted of 15-20 YOmonocytes and 80-85 YOlymphocytes and no other type of cell. Cells were resuspended in a culture medium consisting of RPMI- 1640, Phenol-Red-free (Gibco), supplemented with 10 % fetal calf serum (Gibco), insulin (Sigma, 5 pg/ml), transferrin (Sigma, 5 pglml), glutamine (Panreac, 2 mM), selenium (Sigma, 5 ng/ml), penicillin (10.66 pg/ml), streptomycin (50 pg/ml) and polymyxin B (2.5 pg/ml) (all three from BoehringerIngelheim). The L-arginine content of the culture

Nitric oxide synthase expression in liver cirrhosis

medium was 1.14 mM and the medium was plated out in 6 x 4 well plates (1 x 106cellsper well). These and all subsequent procedures required specific preventive precautions in order to minimize contamination. The study was made in a sterile laminar vertical flow cabin (Cabin TC-48, Gelaire, Cultek, Flow Laboratories, U.K.) in sterile conditions using pyrogen-free materials. In every case incubation with ZP-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) was performed to inhibit NO-dependent nitrite production. All samples were incubated for 6h at 37 "C with 5 % CO,. Nitrite determination

NO synthesis was assessed in the cell supernatant at the end of the incubation. When the incubation medium is under constant oxidoreduction conditions, NO is oxidized to nitrites and then to nitrates in a constant ratio between their concentrations [161. For nitrite measurement, cells were plated in 6 x 4 well culture plates (Nunc, Denmark) and incubated with the medium above described. After 6 h of incubation in tested conditions, nitrite concentration was determined in the supernatant by a colorimetric quantitative method based on the Griess reaction [171. Briefly, 500 p1 of sample were mixed with 250 pl of Griess reagent [ 1 YO sulphanilamide, 0.1 % naphthyl ethylenediamine dihydrochloride in 2.5 % orthophosphoric acid (Sigma)] and incubated for 15 min at room temperature. The absorbance was read at 540 nm. Standard NO,- calibration was carried out using sodium nitrite. All samples were assayed against a blank comprising RPMI- 1640 medium incubated under the same conditions but in the absence of cells. lmmunocytochemistry

LMN cells were washed in phosphate buffer and incubated for 48 h at 4 "C with mouse monoclonal antibody specific for macrophage inducible NOS (mac-iNOS, isotype Ig2a) (Mnity, Nottingham, U.K.), followed by an indirect biotin-streptavidin amplified detection system (BioGenex, San Ramon, CA, U.S.A.). The chromogen used was an alkaline phosphatase kit (BioGenex), and cells were then counterstained with haematoxylin. The specificity of the immunostaining was controlled by omitting the specific antibody in the first incubation step [18]. Northern blot analysis

Total cellular RNA was isolated from LMN cells by the guanidinium-thiocyanate-phenol-chloroform method [ 191, size-fractionated by electrophoresis (10 pg/lane) through denaturing 1% agarose4.66 M formaldehyde gels, transferred to Hybond hybridization transfer membranes (Amersham, U.K.), and UV cross-linked before hybridization as previously

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described [20]. A 700-bp PCR fragment of the murine macrophage iNOS cDNA (MAC-NOS, generous gift of Drs Lowenstein and Snyder) was obtained using the following primers: sense, 5'-GAG AGA TCC GAT TTA GAG TCT-3'; antisense, 5'-GCA GAT TCT GCT GGG ATT TCA-3', and used as a probe for Northern blot analysis. Hybridization was performed at 42 "C for 12-16 h; membranes were then washed in final stringency conditions of 55 "C, 0.2 x SSC (0.03 M NaC1/0.003 M sodium citrate), and exposed on SAR Kodak film using an intensifying screen in order to correct for uneven loading and to make sure that differences in mRNA were obtained before transfer. Densitometric analysis was performed on a Phosphorimager apparatus (Molecular Dynamics, Sunnydale, CA, U.S.A.). Results are expressed in arbitrary units as the ratio of iNOS/28 S expression. Statistical analysis

The statistical study of biochemical parameters is based on a multiple analysis of variance, by the computer programme STATVIEW 11. P values less than 0.05 were considered statistically significant in the tests used, Fisher's and Scheffe's test. Statistical significance was 95%. The other parameters are expressed as means +_ S.E.M. For comparison of means, one-way analysis of variance was performed, followed by the comparison of multiple means (Scheffe's test). The coefficient of correlation was evaluated by linear regression. RESULTS

The distribution of the Child-Pugh score shows statistically significant differences between both groups of cirrhotic patients for the signs that determine their classification in groups A, B and C : 1. Non-alcoholic cirrhotic patients. Type A = 50 % ; B = 27%; C = 23%. 2. Alcoholic cirrhotic patients. Type A = 12.5% ;B = 43.7%; c = 43.7%. Table 1 summarizes analytical data for the different groups of patients. Table 2 shows plasma endotoxin levels of the two groups of cirrhotic patients and the control group. The detection limit of the assay was 21 f 4 pg/ml. Plasma endotoxin levels were almost undetectable in the control group. Both groups of cirrhotic patients showed higher values of plasma endotoxin than those observed in control subjects. Non-alcoholic cirrhotic patients had higher levels of endotoxin than alcoholic cirrhotic patients. Haemodynamic parameters are shown in Figure 1. In all the groups studied, ejection fraction was inside the normal range, although cirrhotic patients presented higher values. These patients also showed an increase in cardiac output and a decrease in peripheral vascular resistance. None of the patients studied showed significant changes in mean arterial pressure nor in heart rate with respect to controls.

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Table I Analytical characteristics of t h e different groups of patients Values are given as means and range. AST/ALT: aminotransfense ratio. Statistical significance: *P < 0.05 compared with control; tP < 0.05 compared with alcoholic cirrhosis; SP < 0.05 compared with non-alcoholic cirrhosis.

Haematocrit (%) Mean corpuscular volume (fl) Albumin (g/l) Prothrombin activity (%) Total bilirubin @mol/l) AST/ALT @kat/l)

Alcoholic cirrhosis (n= 17)

39.0 (32-52) 87.0 (63-96)

35.0 (2243)

36.0 (2447)

lo1.m(8cI I I)

86.V(69-94)

42.V (29-50) 98.m(89-106)

49.7 (38.9-62.5) 98.0 (*loo) 6.88 (6.02-7.05) i.4 (I.2-1.6)

32. I S (21.345) 61.m (23400) 77.U(5.5-206.4) 2.3 (I.2-3.7)

32.7S (19.242) 73.m (15-100) 29.24 (2.92-79.12) I.4* (0.54-3.75)

41.3V (25-53) 87.V (43-1 00) 27.52* (4.81-101.48) I.5 (0.83-2.5)

Table 2 Plasma endotoxin levels

Values are meansfS.E.M. Data are the average (S.E.M.) of five different measurements, each in triplicate. Statistical significance: *P 0.05 compared with control; tP < 0.05 compared with alcoholic cirrhosis and non-alcoholic cirrhosis; W < 0.05 compared with alcoholic cirrhosis.

-=

Endotoxin (pg/ml) Control Alcoholic cirrhosis Non-alcoholiccirrhosis

25*3 52&4* 78&6*t

Nitrite production (nmoi/io6 cells)

81

*

20

0

* t

T

t

60 40

*

0.34+0.01* 0.64+ 0. IVt 0. l4+0.01 *tS

Peripheral Vascular Resistance

Cardiac Output

80

0.0*0.09

Control Alcoholic cirrhosis Non-alcoholiccirrhosis Alcoholic liver disease

L-NAME-inhibitable nitrite production from LMN cells of peripheral blood is shown in Table 3. The values were higher in cirrhotic patients than in the Ejection Fraction

Non-alcoholiccirrhosis (n = 10)

Table 3 ~-NAME-lnhlbltablen i t r i t e productionby LMN cells from different groups Incubated for 6 h w i t h RPMI164

Values are meansfS.E.M. Statistical significance: *P < 0.05 compared with control; tP < 0.05 compared with alcoholic cirrhotic patients.

%

[

nil

C

AC

Alcoholic liver disease (n = 16)

Control (n = 12)

NAC

NAC ALD

Mean Arterial Pressure

Heart Rate

100

bPm

-g

I5

75

50

50

25

25

n "

-

n C

AC

NAC ALD

C

AC

NAC ALD

Figure I Haemodynamlc parameters Valuer are meansf5E.M. C, control group; AC. alcoholic cirrhotic patients: NAC. non-alcoholic cirrhotic patients; ALD. patients with chronic alcoholic liver disease. Statistical significance: *P < 0.05 compared with C: tP < 0.05 compared with AC and NAC.

Nitric oxide synthase expression in liver cirrhosis

64 I

Table 4 Correlation c d c l e n t between endotoxin levels. nitrite production and hwmodynamic parameters The numbers presented are the correlationcoefficients ( r values). *P c 0.05.

Cardiac output Peripheral vascular resistance Mean arterial pressure Heart rate NO

NO

Endotoxin

0.82* 0.W 0.97* 0.28

0.W 0.88*

-

0.98* 0.26 0.W

control group, and the difference between the nonalcoholic and the alcoholic cirrhotic group after 6 h incubation was statistically significant. There was a statistically significant decrease of NO production in patients with chronic alcoholic liver disease compared with cirrhotic groups. Table 4 summarizes the correlation coefficients between endotoxin levels, nitrite production and haemodynamic parameters when analysis was performed on cirrhotic patients and controls combined. The analysis indicated a strong correlation of endotoxin levels, nitrite production and cardiac output, peripheral vascular resistance and mean arterial pressure. There was no correlation between either endotoxin or NO production and heart rate. In immunocytochemistry studies (Figure 2), cells with positive immunoreactivity for the iNOS isoform can be observed in both alcoholic and non-alcoholic cirrhotic patients. The protein is visualized as reddishbrown spots, more evident in non-alcoholic cirrhotic patients. Control cells did not show positive immunoreactivity. Northern blot analysis (Figure 3) shows iNOS mRNA expression in LMN cells from non-alcoholic cirrhotic patients. This expression was not observed in either alcoholic cirrhotic patients or in controls.

A

B

DISCUSSION

The present results demonstrate an increased NO production by peripheral blood LMN cells from patients with chronic alcoholic liver disease compared with healthy subjects. These data are in agreement with results from Hunt and Goldin [21] demonstrating an increased NO synthesis by monocytes from patients with alcoholic hepatitis. Our study also identifies a greater increased NO synthesis in cirrhotic patients than in patients with chronic alcoholic liver disease. This increased NO synthesis was also higher in patients with non-alcoholic compared with alcoholic cirrhosis. This increased NO synthesis in cirrhotic patients is probably based on activation of an inducible-type NOS, as demonstrated by immunocytochemistryand Northern blot experiments. Plasma levels of endotoxin have been found to be increased in patients with liver cirrhosis. Furthermore, plasma nitrite levels in these patients have been shown to correlate with endotoxin levels [22-241. The same

C Figure 2 Light micrographs showing lmmunoqtochemical expression of i N O S (red marks) In the L M N calls from alcoholic Cirrhotic (A), non-alcoholic Cirrhotic ( 8 )and control patients (C)

A greater degree of iNOS labelling can be observed in monocytes from non-alcoholic cirrhotic than alcoholic cirrhotic patienu. Original magnification x 521.

endotoxin is an activator of monocytes [25]. In addition, functional properties of those cells :3re enlarged from cytokines secreted by T-cells. Both cxll types have been implicated in immune or inflammatc'ry

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Kb

NOS

+ 4.4

11.3

Figure 3 Northern blot analysis for I N O S m R N A expression (4.4 kb) in LMN cells Lane 5 shows a positive control (pulmonary macrophages). An increase of iNOS mRNA was shown in L M N cells from nonalcoholiccirrhotic patients (lane 4). N o iNOS mRNA was detected in alcoholic cirrhotic (lane 3) or control patients (lanes I and 2). Glyceraldehyde )-phosphate dehydrogenase (GAPDH) mRNA was used as a control.

response modulation [26]. Increased NO production by LMN cells from patients with chronic liver disease is probably based on its activation by bacterial endotoxin of intestinal origin, and cytokines from the liver and other cells. This increase is explained by the splanchnic haemodynamic alterations and the increased intestinal permeability to endotoxin observed in those patients [22]. Although in this study we have not performed measurements of cytokine plasma levels, these have been reported to be higher in cirrhotic than in non-cirrhotic patients [27]. Peripheral blood monocytes from patients with alcoholic cirrhosis or hepatitis produce higher amounts of tumour necrosis factor-a (TNF-a), interleukin (1L)-la and IL-6 after lipopolysaccharide or infection stimulation compared with healthy patients [27,28]. Moreover, TNF-a, IL-1B and interferon-y are cytokines that activate inflammatory processes not only in monocyte-macrophages, but also in other cells such as hepatocytes, Kupffer cells, etc. Interferon-y has been shown to be the principal priming signal for in vivo and in vitro activation of monocytes by lipopolysaccharide [29]. TNF-a has been shown to increase the production of oxygen free-radicals such as superoxide anion and hydroxyl radical [30]. Through this effect, TNF-a can deplete the cells of reduced glutathione (GSH), the main defence mechanism against oxidative stress [31,32]. Oxygen radicals and oxidative stress in turn can stimulate the cells to produce TNF-a [33]. Oxygenfree radicals and GSH depletion can activate cell proteases involved in activation of nuclear factor-lcB, a transcription factor that plays a major role in the induction of TNF-a and iNOS gene expression [34]. This may explain the results obtained in patients with chronic alcoholic liver disease. From a haemodynamic point of view, hepatic cirrhosis is associated with a decreased arterial press-

ure, an increased cardiac output and a decreased peripheral vascular resistance. Previous studies indicate the involvement of NO in the splanchnic and systemic vasodilatation observed in hepatic cirrhosis [1,4]. Moreover, our patients show a good correlation between NO synthesis, endotoxin levels and haemodynamic parameters and these data are in agreement with previous results from this and other laboratories [ 12,351. Therefore, these results suggest that the increased NO synthesis is related to the development of hyperdynamic circulation in the cirrhotic patients and the elevated plasma levels of some iNOS-stimulating substances like endotoxin. The increased NO synthesis was also higher in non-alcoholic cirrhotic than in alcoholic cirrhotic patients which can be explained by the fact that the latter have been shown to have higher levels of IL-6 [28]. IL-6 and elevated alcohol levels may act as a negative regulator of the cytokine cascade and can suppress endotoxin and TNF-a-induced IL- 1 production in macrophages [36,37] and IL- I#?-induced iNOS expression in hepatocytes [38]. These effects may also explain why NO production by monocytes from non-alcoholic cirrhotic patients is higher than in alcoholic cirrhotic patients. Another explanation of the higher NO production in non-alcoholic cirrhotic patients is that a higher percentage of these patients have a chronic hepatitis C infection, and it has been shown that monocytes from patients with hepatitis C produce increased amounts of TNF-a and IL-lB [27]. Thus, our data add to the mounting evidence that endotoxin or cytokine alterations may play a role in the LMN cell stimulation with elevated NO production in hepatic cirrhosis, which is higher in nonalcoholic cirrhotic patients. These results support a role for an enhanced synthesis of NO after the induction of iNOS implicated in the inflammatory mechanisms and the hyperdynamic circulation of cirrhotic patients.

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