| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Surgery and
* Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin and Mater Misericordiae Hospital, Dublin 7, Ireland
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: acute pancreatitis • caspases • Fas
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Neutrophils play an important role in the development of many inflammatory disorders [4 ]. Their release of reactive oxygen intermediates and proteolytic enzymes is associated with extensive tissue damage, specifically necrosis [5 ]. In animal models of AP, the neutrophil has been implicated in the destruction of the pancreas and the development of acute lung injury associated with this condition [2 , 6 , 7 ]. The resolution of any neutrophil-mediated inflammatory response is, in part, because of the induction of neutrophil apoptosis, resulting in the disposal of these cells in a controlled process [8 ]. Delayed apoptosis is associated with the development and persistence of inflammatory disorders, including inflammatory bowel disease (unpublished results), acute respiratory distress syndrome (ARDS) [9 ], and systemic inflammatory response syndrome (SIRS) [10 ].
The life span and functional activity of the neutrophil can be extended significantly by the inflammatory microenvironment. Proinflammatory cytokines, cell migration, and neutrophil states of activation mediate survival [11 12 13 ]. However, the mechanisms of this delay are still unknown. Caspase proteases are central executioners of the cell death pathway [14 , 15 ]. Freshly isolated neutrophils have a high expression of procaspase 3 [16 ], which is cleaved during spontaneous apoptosis. Spontaneous and induced neutrophil apoptosis results in caspase activation, which is inhibited by preincubation with inflammatory mediators [17 ].
We hypothesized that neutrophils isolated from patients with AP have a delay in neutrophil apoptosis, which is regulated by altered caspase expression. This may contribute to the persistence and severity of the response.
In this study, delayed neutrophil apoptosis was associated with mild and, to a greater degree, severe AP. Isolated neutrophils were resistant to Fas antibody-inducted apoptosis. This resistance may be explained by altered procaspase 3 expression, but no change in the initiator procaspase 8 was shown. Serum factors were shown to regulate apoptosis of which interleukin (IL)-1ß and granuloctye-macrophage colony-stimulating factor (GM-CSF) were elevated. These effects were independent of altered procaspase 3 expression. Altered apoptosis and procaspase 3 expression in patient neutrophils were associated with an increase in glutathione-S-transferase (GST), an important regulator of glutathione that has antiapoptotic effects. There was no increase in Mcl-1, which is a member of the Bcl-2 family of antiapoptotic proteins. Alterations in the caspase cascade may represent an additional site contributing to delayed apoptosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We studied 10 healthy controls with a mean age of 45 (range, 24–66) with a male:female ratio of 5:5, and 40 patients with AP. This group was divided into mild (n=35), with a mean age of 44 (range, 16–93) and a male:female ratio of 1:1, and severe (n=5), with a mean age of 49 (range, 26–70) and a male:female ratio of 4:1.
Only patients with AP who had developed symptoms (predominantly pain) in the preceding 24 h were enrolled in the study. Systemic venous blood (10 ml) was collected following diagnosis, before the institution of any medical treatment. This was immediately transported to the laboratory, where serum and neutrophils were isolated for analysis. No further blood samples were obtained from the patients.
Reagents and antibodies
Dulbecco’s modified Eagle’s medium (DMEM), penicillin and
streptomycin solution, L-glutamine, and fetal calf serum (FCS) were
purchased from GIBCO Life Technologies (Paisley, UK). Dextran T-500 and
Ficoll were purchased from Pharmacia (Buckinghamshire, UK). E-lysis was
purchased from Cardinal Associates (Santa Fe, NM). Procaspase 3 and 8
antibodies (mouse antihuman), as well as anti-GST and Mcl-1 antibodies
(mouse antihuman), were purchased from Transduction Laboratories
(Lexington, KY), and Fas antibody (CH-11) was from Immunotech
(Bedfordshire, UK). Blocking monoclonal antibodies (mAbs) to IL-1ß
and GM-CSF were purchased from R&D Systems (Oxon, UK). All remaining
chemicals were purchased from Sigma-Aldrich Company (Dorset, UK), if
not otherwise stated.
Neutrophil isolation
Neutrophils were isolated by dextran (6%) sedimentation and
centrifugation through a discontinuous Ficoll gradient, as described
previously [18
]. Neutrophil-rich pellets were subjected
to hypotonic lysis of the remaining erythrocytes with E-lysis. Cell
pellets were resuspended in DMEM, supplemented with 10% FCS (heat
inactivated), 1% glutamine, and 1% penicillin/streptomycin solution
at a concentration of 1 x 106 cells/ml. Cells were
incubated in polypropylene tubes (Falcon/Becton Dickinson, Cambridge,
UK) to prevent adherence. Neutrophil purity was 95%, as assessed by
size and granularity on flow cytometry, and viability was >98%, as
assessed by trypan blue and propidium iodide exclusion.
Quantification of apoptosis
Propidium iodide DNA staining
Apoptosis was measured by flow cytometry as the percentage of
cells with hypodiploid DNA [18
]. Cell suspensions were
centrifuged at 200 g for 10 min. The cell pellets were
resuspended in 500 µl of hypotonic fluorochrome solution [50 µg/ml
phosphatidylinositol (PI), 3.4 mM sodium citrate, 1 mM
ethylenediaminetetraacetate (EDTA), 0.1% Triton X-100] and were
stored in the dark at 4°C before they were analyzed using a Coulter
Elite cytofluorometer (Coulter, Bedfordshire, UK).
Annexin-V
The expression of phosphatidylserine on the surface of apoptotic
cell was assessed by fluorescein isothiocyanate (FITC) Annexin-V
antibody, detected using flow cytometry. Cell suspensions were washed
with cold phosphate-buffered saline (PBS), resuspended at 1 x
104/100 µl, and stained according to the manufacturer’s
specifications. After staining, cells were stored in the dark at 4°C
before they were analyzed using a Coulter Elite cytofluorometer
(Coulter).
Morphology
Cells were resuspended to 1 x 106/200 µl,
spun onto polysine slides using a cytospin (Shandon, Chestire, UK) at
500 g for 5 min, and stained with Giemsa stain. Apoptotic cells
were identified by nuclear condensation at 40x magnification.
Western blot analysis
Total protein was isolated from 2 x 106 human
neutrophils using Nonidet P-40 (NP-40) isolation solution [0.5%
NP-40, 10 mM Tris, pH 8.0, 60 mM KCl, 1 mM EDTA, pH 8.0, 1 mM
dithiothreitol (DTT), 10 mM phenylmethylsulfonyl fluoride (PMSF), and 1
µM leupeptin and aprotinin]. Isolated protein concentrations were
measured by the Bradford assay Protein Detection Kit (Bio-Rad,
Hercules, CA) and were loaded at 50 µg per well. Samples were then
run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gradient gel (140 V for 60 min) and were electrophoretically
transferred to Immobilon-P (Millipore, Bedford, MA) (100 V, 45 min).
Membranes were stained after transfer with ponceau S solution (2%) to
confirm equal loading. Blots were incubated with mouse antiprocaspase 3
primary antibody (1:1000) in 1% bovine serum albumin (BSA)
Tris-buffered solution and 0.1% Tween 20 for 1 h at room
temperature and were then incubated with horseradish peroxidase
(HRP)-conjugated antimouse immunoglobulin G (IgG) at 1:5000 dilution
for 1 h. Blots were developed using an enhanced chemiluminescence
(ECL) system.
Serum IL-1ß and GM-CSF concentration
Standard enzyme-linked immunosorbent assay (ELISA) technique
(R&D Quantikine, R&D Systems) was used to measure IL-1ß and GM-CSF in
serum isolated from the systemic circulation of control patients and
those with AP. Blood was collected and transported on ice. Serum was
separated within 1 h of collection and stored at -80°C. All
samples were analyzed within 3 months of collection.
Statistics
Statistical analysis was carried out using analysis of variance
(ANOVA) with Student-Newman correction. All results are expressed as
mean ± standard deviation (SD). Significance was
assumed for values of P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
AP delays neutrophil apoptosis
Neutrophils isolated from patients diagnosed with AP had
significant decreases in the number of apoptotic cells after 24 h
in vitro incubation compared with control neutrophils
incubated for the same time. There were no apoptotic cells found in
freshly isolated neutrophils (data not shown). Apoptosis was assessed
by propidium iodide DNA staining and confirmed by morphology
(Fig. 1
). nThe study group was further divided into mild and severe, with delays
in the severe group significantly greater than the mild (Fig. 1)
. Serum
from patients with AP reduced significantly the percentage of apoptotic
cells when incubated in vitro for 24 h with control,
normal neutrophils. However, there was no difference between the mild
and severe groups (Fig. 2
). Apoptosis was assessed by propidium iodide DNA staining and Annexin-V
using flow cytometry.
|
|
|
|
|
|
Delayed apoptosis is reversed by diethylmaleate (DEM)
DEM, a glutathione-depleting agent, mediates neutrophil apoptosis
through direct activation of the caspase cascade (unpublished results).
DEM induced significant apoptosis in normal and AP neutrophils
(Fig. 7
).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Local injury and mortality of AP are, in part, mediated by the neutrophil through the release of reactive oxygen intermediates and proteolytic enzymes [19 ]. Studies in experimental models of AP indicate that pancreatic oxidative stress occurs during an early stage of induction. Scavenger therapy has demonstrated some success, especially when administered before induction [20 ]. Increased lipid peroxidation is increased in human AP in bile and pancreatic tissue [21 ]. Neutrophil infiltration, the source of reactive oxygen intermediates, has been detected in the pancreas as early as 1 h after the induction of experimental AP [6 ]. The depletion of circulating neutrophils has also been demonstrated to decrease the severity of pancreatitis and completely prevents lung injury [7 ]. The resolution of any inflammatory response is, in part, dependent on the induction of neutrophil apoptosis and thus their removal from the inflammatory site. Delays in this apoptotic response may lead to the accumulation of activated neutrophils and increased tissue damage [8 , 22 , 23 ]. This study demonstrates a significant decrease in the number of apoptotic neutrophils after incubation in vitro, when isolated from mild and severe AP patients compared with controls. This indicates a delay in their apoptotic induction. Prolonged neutrophil survival may contribute to the development of MODS in the severe group, but, as yet, this is a small preliminary group of patients, and a larger study will have to be carried out. The development of other inflammatory disorders, such as ARDS and MODS, previously has been associated with delayed rates of neutrophil apoptosis [9 , 10 ].
Serum factors contribute to this delay in apoptosis, however there was no difference in the effect induced by mild and severe serum. Many inflammatory cytokines, including IL-1ß [13 ] and GM-CSF [12 ], have been shown to delay neutrophil apoptosis. These were increased in the serum of the severe group, but other cytokines may also be involved in this effect. This was underlined by the fact that mAbs to IL-1ß and GM-CSF had no effect on inhibiting the antiapoptotic effects of the serum.
Caspase proteases represent a central mechanism mediating apoptosis [15 ]. Fas ligation is an important trigger of this caspase cascade [14 ]. Fas antibodies induce apoptosis in normal neutrophils, however cells isolated from mild and severe groups were resistant to this induction. Resistance to Fas ligation is associated with delayed neutrophil apoptosis in in vivo and in vitro models of inflammation [18 , 24 ]. Neutrophils isolated from the circulation of septic patients demonstrate similar delays in spontaneous apoptosis and are resistant to Fas ligation [10 ]. Cleavage and activation of the initiator caspase 8 and effector caspase 3 are important steps in the cascade, leading to apoptosis of normal neutrophils incubated with Fas antibodies [17 ]. Altered caspase expression has been hypothesized to contribute to differential activation and resistance to apoptosis [25 ]. This study demonstrated no difference in expression of procaspase 8 among the control, mild, and severe groups but a significant decrease in procaspase 3 expression in both AP groups compared with control. This result was unexpected, because decreased procaspase expression would be associated with conversion to the active caspase and induction of apoptosis. Further studies are required to determine if there is a corresponding decrease in mRNA for these caspases. Mechanisms that regulate the expression of these caspases are unknown. This alteration in procaspase 3 was only demonstrated in neutrophils isolated from the study patients and not in normal neutrophils incubated with patient serum. Altered procaspase 3 expression may well be mediated at the level of maturation, but still demonstrates a mechanism for delayed spontaneous and Fas antibody-induced apoptosis. Current studies in our laboratory have now demonstrated that GM-CSF has no effect on procaspase 3 expression in normal neutrophils but does alter the cleavage of procaspase 3 (32 kDa) to its active caspase (17 kDa), thus inhibiting spontaneous and Fas antibody-induced apoptosis.
Bcl-2 and thiols have been shown to regulate the cleavage and activity of the caspases [26 ]. Only one Bcl-2 family member, Mcl-1, is expressed in the neutrophil [27 ]. Lipopolysaccharide (LPS) and GM-CSF have been shown to increase the expression of Mcl-1 at 3 h after stimulation, resulting in delayed apoptosis [27 ]. Despite this observation, there was no increased expression of Mcl-1 in neutrophils isolated from mild or severe AP patients. Glutathione has important antiapoptotic properties, which are mediated through the inhibition of caspase activity [17 , 18 ]. Ethical factors limited the amount of blood collected from the patients, and thus, total intracellular glutathione measurements could not be undertaken. Intracellular GST, which is involved in the synthesis of glutathione [28 ], was measured and was shown to be significantly increased in the mild and severe groups. Neutrophil intracellular reduced glutathione (GSH) is increased by LPS and GM-CSF, possibly as a mechanism to protect against oxidative stress [18 ]. This process may also result in the inhibition of apoptosis through caspase inhibition.
Where increased GSH is associated with resistance to apoptosis, depleting this antioxidant results in the induction of programmed cell death [29 ]. Diethylmaleate induces apoptosis in normal and inflammatory neutrophils [29 ] and in the neutrophils isolated from the mild and severe groups of AP. Previous studies have demonstrated that diethylmaleate increases conversion of procaspase 3 to its active caspase, resulting in the induction of apoptosis (unpublished results). This induction of apoptosis may represent a mechanism by which delayed apoptosis can be reversed, and the resolution of an inappropriate inflammatory response can be triggered.
Understanding how AP leads to the MODS could lead to better detection of patients who are at risk and could then be treated in a more aggressive manner. Severe AP is associated with a significant decrease in neutrophil apoptosis and development of MODS. This delay may be mediated at the level of decreased procaspase 3 expression, which is serum-independent, and secondly, at the level of caspase cleavage to its active protein, which is cytokine-dependent. However, further studies will need to be carried out to clarify these mechanisms and determine the significance of reduced procaspase 3 apoptotic-resistant neutrophils.
|
ACKNOWLEDGEMENTS |
|---|
|
FOOTNOTES |
|---|
Received September 20, 1999; revised January 29, 2000; accepted January 31, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. J. Molloy, A. J. O'Neill, J. J. Grantham, M. Sheridan-Pereira, J. M. Fitzpatrick, D. W. Webb, and R. W. G. Watson Sex-specific alterations in neutrophil apoptosis: the role of estradiol and progesterone Blood, October 1, 2003; 102(7): 2653 - 2659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Frossard, A. K. Saluja, N. Mach, H. S. Lee, L. Bhagat, A. Hadenque, L. Rubbia-Brandt, G. Dranoff, and M. L. Steer In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L541 - L548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-J. Hsu, S.-S. Lee, and W.-W. Lin Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apoptosis in human neutrophils through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway J. Leukoc. Biol., July 1, 2002; 72(1): 207 - 216. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
P < 0.05 vs. mild percent
apoptosis.
