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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: [email protected] (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