Increased apoptosis dependent on caspase-3 activity inpolymorphonuclear leukocytes from patients with cirrhosis

and ascites

Marıa Jose Ramırez1, Esther Titos2, Joan Claria2, Miguel Navasa1,*, Javier Fernandez1,Juan Rodes1

1Liver Unit, Hospital Clınic Universitari, Institut d’Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Barcelona, Spain2DNA Unit, Hospital Clınic Universitari, Institut d’Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Barcelona, Spain

Background/Aims: Patients with decompensated cirrhosis are prone to develop neutropenia. Although hypersplenism

and increased clearance of polymorphonuclear leukocytes (PMN) in the spleen are thought to contribute to neutropeniain these patients, other factors cannot be excluded. The aim of the current study was to investigate whether the presence

of increased PMN apoptosis could also contribute to the appearance of neutropenia in these patients.

Methods: PMN were isolated by Ficoll-Hypaque gradient centrifugation from 17 patients with decompensated

cirrhosis (CH group) and 13 patients with compensated chronic liver disease (CT group). PMN were incubated in

RPMI 1640 medium at 37 8C in a 5% CO2 atmosphere and viability and frequency of apoptosis were evaluated after 0,

10, 20 and 40 h of culture. Viability was determined by the MTT assay and apoptosis by microscopic examination of cell

morphology (Diff-Quik staining), DNA fragmentation by agarose gel electrophoresis (DNA laddering) and caspase-3

activity by DVDE-p-nitroanilide cleavage.Results: Compared to CT patients, PMN isolated from CH patients exhibited a decreased PMN viability and a

marked accelerated apoptosis as revealed by an increased number of condensed nuclei, increased DNA laddering and

significantly higher caspase-3 activity.

Conclusions: These findings indicate that shortening of PMN survival via apoptosis may explain in part the

neutropenia present in decompensated cirrhotic patients with ascites, thus favoring the development of bacterial

infections in these patients.

q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: Neutropenia; Chronic liver disease; Apoptosis; Bacterial infections; Cirrhosis; Ascites

1. Introduction

Patients with chronic liver disease have an increased

susceptibility to develop severe and recurrent infections,

especially from gram-negative bacteria [1,2]. Most of these

infections are associated with a poor prognosis and clearly

jeopardize the evolutive course of the disease. Among

patients with liver disease, those with decompensated

cirrhosis are particularly predisposed to suffer from

bacterial infections [1,2]. In addition, some of these patients

develop the life-threatening spontaneous bacterial perito-

nitis, a spontaneous infection of the ascitic fluid. Further-

more, bacterial infections may trigger hepatic

decompensation in cirrhotic patients [3].

The exact mechanisms by which patients with decom-

pensated cirrhosis frequently exhibit bacterial infections are at

present unknown, but they are probably related to an

impairment in the host–defense system [4,5]. In this regard,

patients with decompensated cirrhosis are prone to develop

neutropenia and depleted phagocyte function [1,2,4,5].

Although neutropenia in cirrhotic patients is associated with

the presence of splenomegaly and increased clearance of

granulocytes in the spleen [6], the complete sequence of events

leading to neutropenia in cirrhosis is at present unknown.

Journal of Hepatology 41 (2004) 44–48

www.elsevier.com/locate/jhep

0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.jhep.2004.03.011

Received 29 September 2003; received in revised form 26 February 2004;

accepted 12 March 2004; available online 12 April 2004* Corresponding author. Address: Liver Unit, Institut de Malaties

Digestives, Hospital Clınic, Villarroel 170, 08036 Barcelona, Spain. Tel.:

93-227-5400x2344; fax: 93-451-5272.

E-mail address: navasa@medicina.ub.es (M. Navasa).

Polymorphonuclear leukocytes (PMN) are the primary

effector cells in the host response to injury and infection.

PMN have a short life-span and spontaneously undergo

apoptosis in the living body [7]. Although neutrophils are

programmed to undergo apoptosis at the time of differen-

tiation, the rate of apoptosis is under the regulation of

external factors. Therefore, changes in the rate of PMN

apoptosis are likely to occur in the setting of cirrhosis.

In the current investigation, we provide evidence that

PMN from decompensated cirrhotic patients have an

enhanced frequency of apoptosis, which is likely to

contribute to explain the observed neutropenia in these

patients.

2. Materials and methods

2.1. Cell isolation and culture

Fresh peripheral blood was obtained by venipuncture using acid citratedextrose as anticoagulant from 17 patients with cirrhosis and ascites (CHgroup). Thirteen patients with compensated chronic liver disease were usedas controls (CT group). Cirrhotic patients PMN were obtained from wholeblood by Ficoll-Hypaque gradient centrifugation. Cells were isolated by theBoyum method [8], including dextran sedimentation and red cell removalby hypotonic lysis [9]. The final pellet was resuspended in RPMI 1640medium supplemented with 10% FBS, 100 U/ml streptomycin–penicillinand 2 mmol glutamine. Cell suspensions routinely contained 96% of PMN,as determined by flow cytometry and microscopic examination ofpreparations excluding trypan blue. Purified PMN were cultured for 0,10, 20, and 40 h at 37 8C in a humidified 5% CO2 incubator. At the end ofthe incubation periods, viability of cells was determined. For apoptosisanalysis, PMN were washed twice in phosphate-buffered saline (PBS)before the assays.

2.2. PMN viability

PMN viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [10]. In this assay, theyellow MTT is reduced to a blue formazan product by the mitochondria ofviable cells. Briefly, 2 £ 105 PMN/well were placed within replicates in 96-well plastic culture plates (Becton Dickinson, New Jersey, NJ) and adjustedto a final volume of 100 ml of RPMI 1640 medium. At the end of eachincubation period (0, 10, 20 or 40 h), 10 ml of MTT in PBS (5 mg/ml) wasadded to the wells and cells further incubated for 3 h at 37 8C in ahumidified 5% CO2 incubator. Thereafter, 100 ml of the solubilization-stopsolution (10% SDS in 0.01 M HCl) was added to each well, incubated over-night and the absorbance read at 570 nm in an automatic 96-well platereader (Molecular Devices, Menlo Park, CA). Results obtained by the MTTassay were confirmed by the trypan blue dye exclusion test. To this end,5 £ 106 PMN were placed in P-60 tissue culture dishes (TPPw, Zurich,Switzerland) and adjusted to a final volume of 2 ml of RPMI 1640 medium.At the end of each incubation period (0, 10, 20 or 40 h), cells were exposedto trypan blue and enumerated in a Neubauer counting chamber.

2.3. Assessment of PMN morphology by light microscopy

For morphological studies, cytospin preparations of PMN wereincubated with Diff-Quikw stain at room temperature. After being washedwith PBS, cells were examined in a light microscope under highmagnification (2000 £ ). Apoptotic cells were identified according to thecriteria of Savill et al. [11]: condensed or fragmented nuclei, cytoplasmicvacuolation and decrease in cell size.

2.4. DNA fragmentation assay

Quantification of DNA fragmentation was performed using a TACSe

Apoptotic DNA Laddering kit (R&D Systems, Minneapolis, MN),according to the manufacturer’s instructions. Briefly, genomic DNA fromPMN (8 £ 106 cells) was obtained with extraction buffer, acetate sodium, 2-propanol and 70% etanol, resuspended in DNase-free water and theconcentration and purity determined by measuring the UV absorbance at260 and 280 nm in an UV spectrophotometer (Uvikon 922, KontronInstruments, Italy). Equal amounts of DNA from each sample (6 mg) wereelectrophoresed on 1.5% TreviGel 500 and fragments visualized byethidium bromide staining (1 mg/ml) under UV transillumination.

2.5. Caspase-3 assay

Caspase-3 activity was measured using the ApoTarget CPP32Colorimetric Protease Assay (BioSource Internacional, Camarillo, CA).This assay is based on the spectrophotometric detection of the chromophorep-nitroanilide after cleavage from the labeled substrate of the enzyme,DVDE-p-nitroanilide (DVDE-pNA). Briefly, PMN (5 £ 106 cells) wereincubated in P-100 tissue culture plates for 0, 10, 20 and 40 h and the cellscollected, washed with PBS and resuspended in 50 ml of chilled lysis buffer.Whole cell lysates were centrifuged at 10,000 £ g for 10 min, and adjustedto a protein concentration of 3 mg/ml. Afterwards, 50 ml of 2X reactionbuffer (containing 10 mM DTT) was added and samples were incubatedwith 5 ml of DEVD-pNA (4 mM) at 378 for 2 h. Released p-NA wasdetermined in a spectrophotometer at 400 nm.

2.6. Statistical analysis

All results were expressed as mean ^ standard error of the mean(SEM). Statistical significance was determined by Mann–Whitney test andmultiple comparisons test ANOVA. Statistical significance was defined asP , 0:05:

3. Results

The clinical and biochemical characteristics of patients

included in the study are summarized in Table 1. The CH

group had severe liver disease revealed by lower prothrom-

bin time and increased levels of serum bilirubin, creatinine

and BUN with respect to CT patients. Table 2 shows the

hematological characteristics of patients included in the

study. CH patients had leukopenia, thrombocytopenia and

decreased red cell count and hematocrit. Active infection

and particularly spontaneous bacterial peritonitis was ruled

out in all patients, ascitic cell count was normal, and ascitic

culture was negative.

MTT analysis revealed that compared to CT patients,

viability of PMN from CH patients was significantly

decreased at 0 and 10 h of culture (Fig. 1). After 20 and

40 h in culture, viability was significantly reduced in both

CH and CT patients and no differences in cell viability were

observed between these two groups (Fig. 1). These results

were confirmed by trypan blue exclusion (% viability CH

group: 60 ^ 2, 60 ^ 9, 49 ^ 1 and 17 ^ 6% and CT group:

90 ^ 6, 77 ^ 11, 52 ^ 2 and 30 ^ 13% at 0, 10, 20 and

40 h, respectively). Aging of PMN for 10, 20 and 40 h were

associated with gradually morphological changes charac-

teristic of apoptosis (Fig. 2A), including nuclei conden-

sation, cytoplasmic vacuolation and decreased cell size.

Interestingly, as compared to the CT group, the number of

apoptotic cells was significantly higher in the CH group

after 20 h of PMN aging (Fig. 2B).

To further define differences between PMN undergoing

M.J. Ramırez et al. / Journal of Hepatology 41 (2004) 44–48 45

apoptosis and those suffering primary necrosis, DNA

fragmentation analysis was performed in PMN from CH

and CT patients. As shown in Fig. 3, a chromatin ladder

pattern of internucleosomal cleavage indicative of endonu-

clease activation was observed after 10, 20 and 40 h of PMN

aging. Again, the degree of apoptosis was greater in PMN

isolated from the CH group than in those from the CT group.

Since caspase-3 is an important transduction factor of

apoptosis, we explored its potential implication in PMN

apoptosis. To this end, PMN were aged for 10, 20 and 40 h

and caspase-3 activity was determined by the DVDE-pNA

colorimetric assay. As shown in Fig. 4, caspase-3 activity

increased over time in both groups of PMN, being this

parameter significantly higher at 10, 20 and 40 h in PMN

from CH patients.

4. Discussion

Neutrophils provide the first line of protection against

bacterial and fungi invasion. Over-recruitment, inappropri-

ate activation or deregulated clearance of these cells results

in the establishment of a wide variety of clinical disorders

[12,13]. In the specific case of cirrhosis, neutropenia (i.e. a

decreased number of circulatory PMN) may play a role in

the pathogenesis of the increased rate of bacterial infections

seen in these patients [14,15].

Several hypotheses regarding the mechanisms under-

lying neutropenia in cirrhosis have been postulated.

Splenomegaly, hypersplenism, increased clearance of

PMN in the spleen and the presence of serum hematopoietic

progenitor cell inhibitors have been suggested as mechan-

isms for the presence of anemia, leukopenia and thrombo-

cytopenia in cirrhotic patients [16–18]. However, to date an

accurate study clearly establishing the mechanism by which

cirrhotic patients usually develop neutropenia is still

lacking.

Neutrophils are constantly produced in the bone marrow,

and therefore a similar number of PMN are required to die

within a defined time period in order to keep cellular

homeostasis under physiologic conditions [19]. Apoptosis

or programmed cell death is a critical process regulating the

life-span of inflammatory cells. Thus, the rate of apoptosis

rapidly changes cell number in such systems. For instance,

in many bacterial and autoimmune inflammatory diseases,

delayed apoptosis is an important mechanism leading to

PMN accumulation [20,21]. In contrast, in the current study

we demonstrate an increased rate of apoptosis in PMN

from decompensated cirrhotic patients. Because in vivo,

Table 1

Clinical and biochemical characteristics of patients included in the study

Normal values CH ðn ¼ 17Þ CT ðn ¼ 13Þ P

Mean age (years) – 61.8 ^ 2.5 52.1 ^ 5.1 NS

Sex (M/F) – 10/7 8/5 NS

Etiology (HCV, HBV, OH, other) – 10/1/5/1 11/0/0/2 NS

ASAT (U/l) (5–40) 64.4 ^ 9.2 67.5 ^ 22.6 NS

ALAT (U/l) (5–40) 44.3 ^ 6.4 107.8 ^ 25.7 , 0.05

GGT (U/l) (5–40) 66.4 ^ 13.8 100.0 ^ 43.9 NS

Prothrombin activity (%) (80–100) 63.2 ^ 6.9 96.8 ^ 1.8 , 0.001

Serum bilirubin (mg/dl) (0.2–1.2) 2.7 ^ 0.6 0.8 ^ 0.1 , 0.01

Serum creatinine (mg/dl) (0.3–1.3) 1.3 ^ 0.1 1.0 ^ 0.0 , 0.05

BUN (mg/dl) (10–25) 32.6 ^ 6.1 15.4 ^ 2.9 , 0.01

Total protein (g/l) (60–80) 63.7 ^ 3.5 75.2 ^ 2.0 , 0.005

Medications (Furosemide/ aldactone/ lactitol/ omeprazol) 11/9/8/5 0/0/1/1

Abbreviations: HCV, hepatitis C virus; HBV, hepatitis B virus; OH, alcohol; ASAT, aspartate amino transferase; ALAT, alanine amino transferase; GGT,

gamma glutamyl transferase; BUN, blood urea nitrogen.

Fig. 1. Viability of PMN from patients with decompensated cirrhosis

(CH) (B) and CT patients with compensated chronic liver disease (A).

PMN viability was determined by the MTT assay after 0, 10, 20 and

40 h in culture. Data represent the mean 6 SEM. aP < 0:05 vs. CT.

*P < 0:05 and **P < 0:005 vs. values at 0 h.

Table 2

Hemathological characteristics of patients included in the study

CH CT P

Leukocyte count (103 cell/mm3) 4.9 ^ 0.4 6.4 ^ 0.6 ,0.05

Neutrophil count (103 cell/mm3) 3.2 ^ 0.3 4.2 ^ 0.5 ,0.05

Platelet count (103 cell/mm3) 93.3 ^ 12.2 203.5 ^ 19.4 ,0.001

Red cell count (106 cell/mm3) 3.3 ^ 0.2 4.7 ^ 0.2 ,0.001

Hematocrit 0.3 ^ 0.0 0.41 ^ 0.0 ,0.001

M.J. Ramırez et al. / Journal of Hepatology 41 (2004) 44–4846

apoptotic PMN are recognized and phagocyted by macro-

phages mainly by Kupffer cells [22], accelerated PMN

apoptosis in cirrhosis may contribute to the development of

neutropenia in liver disease.

The mechanisms underlying the presence of accelerated

PMN apoptosis in decompensated cirrhosis are unknown.

During inflammation, the life-span of neutrophils is

extended by cytokines [23], growth factors [24] and the

activated endothelium [25,26]. On the contrary, PMN

apoptosis is accelerated by at least two different mechan-

isms: engagement of well-described death-inducing

receptors TNF-R or Fas, or phagocytosis of complement

and IgG-opsonized [21,27]. Moreover, the generation of

reactive oxygen species (ROS) has been shown to be an

important apoptotic signal [28] and indeed, neutrophils from

patients with decompensated liver cirrhosis spontaneously

produce more ROS [29]. Most apoptotic signaling pathways

originating from death-receptor engagement or stress

stimuli converge on caspases which are cysteine proteinases

activated by diverse apoptotic stimuli and key executers of

apoptosis [30–34]. Apoptosis triggered by ligation of death

receptors such as Fas and TNF-R is referred to as an

extensive pathway of apoptosis and include caspase-8, 10 or

3 [30,35]. In the current study, PMN from CH patients had

higher caspase-3 activity than PMN from the CT group. In

any event, further work is required to investigate the

potential mechanism by which caspase-3 is activated in

PMN from decompensated cirrhotic patients.

In summary, our findings indicate that PMN from

decompensated cirrhotic patients have a shorter life-span

because of an increased rate of caspase-3 dependent

apoptosis. These findings may contribute to explain the

existence of neutropenia in cirrhosis.

Fig. 2. (A) Diff-Quikw-stained PMN from CH and CT patients at 0 and

20 h in cell culture. Magnification 800 3 . (B) Number of condensed

nuclei (apoptotic cells) in PMN from CH (B) and CT (A) patients. The

number of condensed or fragmented nuclei was determined by light

microscopy in Diff-Quikw stained PMN after 0, 10, 20 and 40 h in

culture. Data represent mean 6 SEM. *P < 0:05 vs. CT.

Fig. 3. DNA fragmentation in PMN from CH and CT patients. DNA

fragmentation was analyzed by gel electrophoresis in PMN cultured for

0, 10, 20 and 40 h. 6 mg of DNA were loaded in each lane. M, molecular

marker (F 3 174 Hae III Digest).

Fig. 4. Time course for caspase-3 activity in aging PMN from CH and

CT patients. Caspase-3 activity was determined by the CPP32

colorimetric protease assay at 0, 10, 20 and 40 h of culture. Data

represent the mean 6 SEM.

M.J. Ramırez et al. / Journal of Hepatology 41 (2004) 44–48 47

Acknowledgements

These studies were supported in part by grants from

Fondo de Investigacion Sanitaria (FIS 00/0921), Ministerio

de Ciencia y Tecnologıa (SAF 03/0586) and Instituto de

Salud Carlos III (C03/02).

References

[1] Rimola A, Navasa M. Infections in liver disease. In: Bircher J,

Benhamou JP, McIntyre N, Rizzetto M, Rodes J, editors. Oxford

textbook of clinical hepatology. Oxford Medical Publications; 1999.

p. 1861–1874.

[2] Wyke RJ. Problems of bacterial infection in patients with liver

disease. Gut 1987;28:623–641.

[3] Navasa M, Follo A, Filella X, Jimenez W, Francitorra A, Planas R,

et al. Tumor necrosis factor and interleukin-6 in spontaneous bacterial

peritonitis in cirrhosis: relationship with the development of renal

impairment and mortality. Hepatology 1998;27:1227–1232.

[4] Laffi G, Carloni V, Baldi E, Rossi ME, Azzari C, Gresele P, et al.

Impaired superoxide anion, platelet-activating factor, and leukotriene B4

synthesis by neutrophils in cirrhosis. Gastroenterology 1993;105:

170–177.

[5] Rimola A, Soto R, Bory F, Arroyo V, Piera C, Rodes J. Reticuloen-

dothelial system phagocytic activity in cirrhosis and its relation to

bacterial infections and prognosis. Hepatology 1984;4:53–58.

[6] Uchida T, Kariyone S. Intravascular granulocyte kinetics and spleen

size in patients with neutropenia and chronic splenomegaly. J Lab

Clin Med 1973;82:9–19.

[7] Squier MK, Sehnert AJ, Cohen JJ. Apoptosis in leukocytes. J Leukoc

Biol 1995;57:2–10.

[8] Boyum A. Isolation of mononuclear cells and granulocytes from

human blood. Isolation of monuclear cells by one centrifugation, and

of granulocytes by combining centrifugation and sedimentation at 1g.

Scand J Clin Lab Invest Suppl 1968;97:77–89.

[9] Papayianni A, Serhan CN, Brady HR. Lipoxin A4 and B4 inhibit

leukotriene-stimulated interactions of human neutrophils and endo-

thelial cells. J Immunol 1996;156:2264–2272.

[10] Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine

DL, et al. Feasibility of drug screening with panels of human tumor cell

lines using a microculture tetrazolium assay. Cancer Res 1988;48:

589–601.

[11] Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C.

Macrophage phagocytosis of aging neutrophils in inflammation.

Programmed cell death in the neutrophil leads to its recognition by

macrophages. J Clin Invest 1989;83:865–875.

[12] Del Fabbro M, Francetti L, Pizzoni L, Weinstein RL. Congenital

neutrophil defects and periodontal diseases. Minerva Stomatol 2000;

49:293–311.

[13] Rokusz L, Liptay L. Infections of febrile neutropenic patients in

malignant hematological diseases. Mil Med 2003;168:355–359.

[14] Rajkovic IA, Williams R. Abnormalities of neutrophil phagocytosis,

intracellular killing and metabolic activity in alcoholic cirrhosis and

hepatitis. Hepatology 1986;6:252–262.

[15] Feliu E, Gougerot MA, Hakim J, Cramer E, Auclair C, Rueff B, et al.

Blood polymorphonuclear dysfunction in patients with alcoholic

cirrhosis. Eur J Clin Invest 1977;7:571–577.

[16] Bashour FN, Teran JC, Mullen KD. Prevalence of peripheral blood

cytopenias (hypersplenism) in patients with nonalcoholic chronic

liver disease. Am J Gastroenterol 2000;95:2936–2939.

[17] Ohki I, Dan K, Kuriya S, Nomura T. A study on the mechanism of

anemia and leukopenia in liver cirrhosis. Jpn J Med 1988;27:

155–159.

[18] Shah SH, Hayes PC, Allan PL, Nicoll J, Finlayson ND. Measurement

of spleen size and its relation to hypersplenism and portal

hemodynamics in portal hypertension due to hepatic cirrhosis. Am J

Gastroenterol 1996;91:2580–2583.

[19] Walker RI, Willemze R. Neutrophil kinetics and the regulation of

granulopoiesis. Rev Infect Dis 1980;2:282–292.

[20] Colotta F, Re F, Polentarutti N, Sozzani S, Mantovani A. Modulation

of granulocyte survival and programmed cell death by cytokines and

bacterial products. Blood 1992;80:2012–2020.

[21] Kasahara Y, Iwai K, Yachie A, Ohta K, Konno A, Seki H, et al.

Involvement of reactive oxygen intermediates in spontaneous and

CD95 (Fas/APO-1)-mediated apoptosis of neutrophils. Blood 1997;

89:1748–1753.

[22] Gregory SH, Wing EJ. Neutrophil–Kupffer cell interaction: a critical

component of host defenses to systemic bacterial infections. J Leukoc

Biol 2002;72:239–248.

[23] Dibbert B,Weber M, Nikolaizik WH, Vogt P,Schoni MH,BlaserK,et al.

Cytokine-mediated Bax deficiency and consequent delayed neutrophil

apoptosis: a general mechanism to accumulate effector cells in

inflammation. Proc Natl Acad Sci USA 1999;96:13330–13335.

[24] Saba S, Soong G, Greenberg S, Prince A. Bacterial stimulation of

epithelial G-CSF and GM-CSF expression promotes PMN survival in

CF airways. Am J Respir Cell Mol Biol 2002;27:561–567.

[25] Tennenberg SD, Finkenauer R, Wang T. Endothelium down-regulates

Fas, TNF, and TRAIL-induced neutrophil apoptosis. Surg Infect

(Larchmt) 2002;3:351–357.

[26] Watson RW, Rotstein OD, Nathens AB, Parodo J, Marshall JC.

Neutrophil apoptosis is modulated by endothelial transmigration and

adhesion molecule engagement. J Immunol 1997;158:945–953.

[27] Murray J, Barbara JA, Dunkley SA, Lopez AF, Van OX, Condliffe

AM, et al. Regulation of neutrophil apoptosis by tumor necrosis

factor-alpha: requirement for TNFR55 and TNFR75 for induction of

apoptosis in vitro. Blood 1997;90:2772–2783.

[28] Buttke TM, Sandstrom PA. Oxidative stress as a mediator of

apoptosis. Immunol Today 1994;15:7–10.

[29] Szuster-Ciesielska A, Daniluk J, Kandefer-Szerszen M. Oxidative

stress in the blood of patients with alcohol-related liver cirrhosis. Med

Sci Monit 2002;8:CR419–CR424.

[30] Daigle I, Simon HU. Critical role for caspases 3 and 8 in neutrophil

but not eosinophil apoptosis. Int Arch Allergy Immunol 2001;126:

147–156.

[31] Fadeel B, Ahlin A, Henter JI, Orrenius S, Hampton MB. Involvement

of caspases in neutrophil apoptosis: regulation by reactive oxygen

species. Blood 1998;92:4808–4818.

[32] Pongracz J, Webb P, Wang K, Deacon E, Lunn OJ, Lord JM.

Spontaneous neutrophil apoptosis involves caspase 3-mediated

activation of protein kinase C-delta. J Biol Chem 1999;274:

37329–37334.

[33] Weinmann P, Gaehtgens P, Walzog B. Bcl-Xl- and Bax-alpha-

mediated regulation of apoptosis of human neutrophils via caspase-3.

Blood 1999;93:3106–3115.

[34] Khwaja A, Tatton L. Caspase-mediated proteolysis and activation of

protein kinase Cdelta plays a central role in neutrophil apoptosis.

Blood 1999;94:291–301.

[35] Wang J, Chun HJ, Wong W, Spencer DM, Lenardo MJ. Caspase-10 is

an initiator caspase in death receptor signaling. Proc Natl Acad Sci

USA 2001;98:13884–13888.

M.J. Ramırez et al. / Journal of Hepatology 41 (2004) 44–4848