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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-11-0122.
PHAGOCYTES
From the Respiratory Medicine Division, Department of
Medicine, University of Cambridge School of Clinical Medicine,
Addenbrooke's and Papworth Hospitals, Cambridge, United
Kingdom.
Phosphoinositide 3-kinase (PI3-kinase)-dependent phosphorylation
of the proapoptotic Bcl-2 family member Bad has been proposed as an
important regulator of apoptotic cell death. To understand the
importance of this pathway in nontransformed hematopoietic cells, we
have examined the effect of survival cytokines on PI3-kinase activity
and Bad expression and phosphorylation status in human neutrophils. Granulocyte macrophage-colony-stimulating
factor (GM-CSF) and tumor necrosis factor- Human neutrophils undergo spontaneous apoptosis in
vitro and in vivo, and this process has been proposed as a pivotal
mechanism underlying the resolution of granulocytic inflammation. The
process of apoptosis involves the promotion of controlled cell death
while it maintains cellular integrity; this prevents the release of proinflammatory mediators and promotes the recognition and phagocytic elimination of effete cells from the site of
inflammation.1-3 Wide-ranging proinflammatory cytokines
and mediators that serve as neutrophil-priming or -activating agonists
also inhibit the progression of apoptosis in vitro, and both events are
thought to be important in the establishment of the chronic
inflammatory response.4 Granulocyte
macrophage-colony-stimulating factor (GM-CSF), interleukin-8 (IL-8),
lipopolysaccharide (LPS), C5a, leukotriene B4
(LTB4), and hypoxia have been documented to delay neutrophil apoptosis,5-10 whereas tumor necrosis
factor- The differential expression and phosphorylation status of Bcl-2 and
Bcl-2-Like proteins, which serve as a common checkpoint for several
distinct signaling pathways, appear to be important in controlling the
apoptotic threshold of inflammatory cells. Proapoptotic and
antiapoptotic members of the Bcl-2 family appear to regulate death
signaling through their ability to form complex homodimers and
heterodimers that ultimately influence the insertion of Bax and
Bax-like proteins into the outer mitochondrial membrane. This event
plays a key role in triggering neutrophil apoptosis and results in the
release of cytochrome C and the activation of caspase 9/Apaf-1
apoptosome.19,20 Bcl-2 family members contain 4 Bcl-2
homology (BH) domains,21 designated BH1 to BH4, which are
conserved in antiapoptotic members (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, and A1), whereas proapoptotic members (Bax, Bak, and Bok) show
less conservation for the BH4 region.22 Deletion and
mutagenesis analysis of the BH3 domain has revealed this to be the
minimal death domain required for heterodimerization and the promotion of apoptosis.23,24 These reports are supported by
structural data relating to a subgroup of Bcl-2-Like proteins with
only BH3 domain conservation (Bad, Bid, Bik, Bim, Blk, Bmf, and
Hrk).23,25,26 These BH3-only proteins show no
intrinsic or independent cell destructive properties; instead, they
appear to function as dominant inhibitors of the antiapoptotic
Bcl-2-Like proteins. One such complex interaction that may be of
particular importance in mature neutrophils that lack
Bcl-227,28 involves heterodimerization between
Bcl-XL and Bad. In overexpression models, Bad
phosphorylation at serine residues 112, 136, and
1556,29-32 has been shown to promote the association of
Bad with the cytoplasmic protein 14-3-3, allowing Bcl-XL to
associate with proapoptotic proteins. Substitution of Ser112 or Ser136
eliminates 14-3-3 binding and enhances the death-promoting activity of
Bad. However, this proposed sequence of events is based on in vitro
analysis of purified components in overexpression systems, and its
relevance to primary cells has been questioned.33, 34
In this study we have examined the signal transduction pathways
involved in cytokine-mediated survival in human peripheral blood
neutrophils with reference to the influence of GM-CSF and TNF- Neutrophil preparation and culture conditions
Superoxide anion generation
Measurement of PtdIns(3,4,5)P3 mass The effect of GM-CSF on neutrophil PI3-kinase activity was determined by measuring the accumulation of PtdIns(3,4,5)P3 mass in acidified chloroform-methanol cell extracts. This was performed by quantitative conversion of PtdIns(3,4,5)P3 to Ins(1,3,4,5)P4 and measurement of this water-soluble product using a specific radioreceptor assay. Recombinant Ins1,3,4,5P4 binding protein (GAPIP4BP) was obtained from Dr P. Cullen (University of Bristol, United Kingdom) and was purified as described.36,37 The binding characteristics of each batch of GAPIP4BP was confirmed using 0.1 nM InsP6 (phytate), and a maximum [3H]Ins1,3,4,5P4 binding (Bmax) value of 20% was used in subsequent assays. Isolated neutrophils (8 × 106 assay point) were incubated with GM-CSF (100 ng/mL) or buffer for 30 minutes, and the reactions were stopped by the addition of methanol-chloroform (2:1, vol/vol). Lipid extractions were then performed as previously described.38 After drying, the samples were boiled in 1 M KOH for 30 minutes, which converts PtdIns(3,4,5)P3 to Ins(1,3,4,5)P4 with an efficiency of 62%. Samples were neutralized with 1 M acetic acid and washed with water-saturated butan-1-ol, petroleum ether, and ethyl acetate (20:4:1, vol/vol/vol) to remove the cleaved fatty acids. The lower aqueous phase was dried in a vacuum concentrator and stored at 20°C until assayed. Samples were
resuspended in acetic acid to pH 5.0 before assay. The
Ins(1,3,4,5)P4 radioreceptor assay was performed as
described37 using 0.005 µCi (0.000185 MBq)
[3H]Ins(1,3,4,5)P4 per sample. A standard
displacement curve was constructed for each experiment using 0.001 to
120 pmol unlabeled authentic Ins(1,3,4,5)P4.
Morphologic analysis of neutrophil apoptosis Neutrophils were cultured in 96-well, ultralow attachment plates (Costar, Hycombe, Bucks, United Kingdom) at 7.5 × 105 cells/well. Following resuspension the cells were aspirated, cytocentrifuged, fixed, and stained as described above. Morphologic analysis of neutrophil apoptosis was assessed under oil immersion light microscopy with the observer masked to the experimental conditions. Apoptotic neutrophils were defined as cells with darkly stained, condensed nuclei. For each of the conditions investigated, triplicate slides were prepared, and at least 400 neutrophils were counted per slide.Annexin V analysis of neutrophil apoptosis Neutrophils were cultured under identical conditions in the presence or absence of GM-CSF or TNF- as detailed above. Neutrophils were then aspirated and centrifuged (275g, 5 minutes at
4°C), and the cell pellet was resuspended in 200 µL HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (10 mM
HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing Annexin-V-fluorescein isothiocyanate (FITC) (1 µg/mL) and propidium iodide (10 µg/mL). Samples were incubated for 15 minutes at 4°C in the dark, and the volume was increased to 500 µL with HEPES buffer immediately before analysis by
flow cytometry.
Immunoprecipitation of Bad Neutrophils were incubated for varying times with or without GM-CSF (10 ng/mL) or TNF- (200 U/mL) in the presence or
absence of kinase inhibitors (as detailed in the figure
legends) in 6-well, ultralow attachment plates at
2.5 × 107 cells/well. Cells were then pelleted and
resuspended in lysis buffer (10 mM Tris-HCl, pH 7.8), 1.5 mM EDTA
(ethylenediaminetetraacetic acid), 10 mM KCl, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM levamisole, 0.5 mM benzamidine, 0.05%
NP-40, and a proteinase inhibitor cocktail (Boehringer Mannheim,
Germany) and were incubated on ice for 10 minutes before and
after sonication (Soniprep 150, setting 13 for 30 seconds).
Cellular debris was pelleted by centrifugation for 5 minutes at
1850g (4°C), and membrane and cytosolic fractions were
prepared by further centrifugation at 22 000g (20 minutes, 4°C) or 104 000g (30 minutes, 4°C) as indicated in the
figure legends. Membrane fractions were resuspended in the above buffer containing 0.1% Triton-X100. Whole-cell lysates were prepared in an
identical manner, except that the cells were originally lysed in the
presence of 0.1% Triton-X100. After normalization for protein content,
whole-cell, membrane, and cytosolic samples were incubated with a
rabbit polyclonal antibody directed against Bad (H-168; Santa Cruz
Biotechnology, CA). Samples were rotated for 2 hours at 4°C before
the addition of immunoglobulin G (IgG) beads (Amersham Biosciences,
United Kingdom) and subsequently were incubated overnight.
Immunoprecipitates were washed 3 times, initially with 0.1 M Tris-HCl
(pH 7.4), followed by 0.01 M Tris-HCl (pH 7.4), and finally with
dH2O.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting Immunoprecipitates were solubilized with 160 µL Laemmli buffer and were heated for 10 minutes at 100°C. Proteins were separated on 12% (wt/vol) polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes. These were washed overnight in blocking solution containing 5% (wt/vol) dried-milk powder in PBS with 0.1% Tween-20 (PBS-Tw20) at 4°C. The membranes were incubated for 2 hours in PBS-Tw20 containing 0.25% dried-milk powder with polyclonal antibodies to Ser112 or Ser136 phosphorylated-Bad (New England Biolabs, Beverly, MA) or Bcl-X (Oncogene Science, Cambridge, MA) or a monoclonal antibody to Bad (36420; Transduction Laboratories, Lexington, KY), at dilutions of 1:500, 1:1000, 1:40, and 1:1000, respectively. After 3 washes in PBS-Tw20, the membranes were incubated with peroxidase-conjugated secondary antibodies (final dilution, 1:2000) in PBS-Tw20 supplemented with 0.25% dried-milk powder for 1 hour and subsequently were washed as described above. Detection was performed by chemiluminescence using an ECL kit (Amersham Biosciences) and subsequent exposure to x-ray film (XOMAT-AR; Amersham Biosciences).RT-PCR procedures Total RNA was isolated using TRIzol (Life Technologies). RNA (10 µg) was transcribed to cDNA using oligo(dt) primers (Life Technologies) and 50 U reverse transcriptase (Promega). Polymerase chain reaction (PCR) amplification was performed using specific primer sets for Bad (sense, 5'-TCC-CAG-AGT-TTG-AGC-CGA-GT; antisense, 3'-ATG-TGG-AGC-GAA-GGT-CAC-TG; 471-bp product), Bcl-X (sense, 5'-GAA-TCT-TAT-CTT-GGC-TTT-GGA; antisense, 3'-GTA-GAG-TGG-ATG-GTC-AGT-GT; 799-bp product), and BAX (sense, 5'-TGC-TTC-AGG-GTT-TCC-AGG; antisense, 3'-ACC-ACT-GTG-ACC-TGC-TCC-AGA-A; 476-bp product). For control reactions, a specific primer set for -actin (sense,
5'-GTG-GGG-CGC-CCC-AGG-CAC-CA; antisense,
3'-CTC-CTT-AAT-GTC-ACG-CAG-CAC-GAT-TTC; 548-bp product) was used. PCR
(35 cycles) was performed using 2 U ampliTaq DNA polymerase (Bioline).
PCR products were analyzed by agarose gel electrophoresis and imaged
with ethidium bromide under UV light.
Functional assessment of neutrophil priming-activation status following cell isolation We have previously demonstrated that neutrophil priming, for example, by inadvertent exposure of cells to trace amounts of LPS during the isolation procedure, has a significant impact on the rate of constitutive neutrophil apoptosis in vitro and the modulation of this event by cytokines such as TNF- and GM-CSF.4,8,39 To
ensure that our neutrophil isolation technique did not prime cells, we
examined the effect of fMLP on O (200 U/mL), or
fMLP (100 nM) alone had little or no effect on O-primed
cells (Figure 1A).
These data confirm that the isolated neutrophils were in an unprimed,
nonactivated state. As previously reported, the PI3-kinase inhibitors
wortmannin (100 nM) and LY294002 (50 µM) completely blocked
fMLP-stimulated O GM-CSF-stimulated PtdIns(3,4,5)P3 accumulation in human neutrophils To investigate the potential importance of the PI3-kinase signaling pathway to cytokine-mediated neutrophil survival, we examined the effect of GM-CSF on the accumulation of PtdIns(3,4,5)P3 mass, the intermediate metabolic product of PI3-kinase activity, which reflects the action of the class 1a (p85:p110, ,  ) and the class 1b (p101:p110 ) PI3-kinase isoforms in neutrophils.
Stimulation of neutrophils with GM-CSF (100 ng/mL) for 30 minutes caused a significant (approximately 6-fold) accumulation of PtdIns(3,4,5)P3 compared with control (Figure 1B), and this response was completely blocked by preincubation with LY294002 (10 µM) (data not shown). These data signify the ability of GM-CSF to stimulate PI3-kinase in human neutrophils. Effect of PI3-kinase inhibitors on the rate of neutrophil apoptosis in vitro Having demonstrated that GM-CSF induces a PI3-kinase-dependent increase in PtdIns(3,4,5)P3 accumulation and agonist-stimulated OApoptotic neutrophils have a distinct morphologic appearance with cell
shrinkage, cytoplasmic vacuolation, and condensation of chromatin,
leading to the loss of the normal multilobed nucleus. This allows
accurate quantification of the extent of apoptosis by visual
examination of cytospin preparations. The photomicrographs in Figure
2 illustrate the ability of GM-CSF to
inhibit neutrophil apoptosis over a 20-hour incubation period (Figure
2B) and the inhibition of this survival effect by LY294002 (Figure 2C).
The extreme instability of wortmannin in aqueous media precludes the
use of this inhibitor in experiments requiring such prolonged incubation periods. Morphologic assessment revealed that GM-CSF (10 ng/mL) inhibited apoptosis at 6 hours and 20 hours (percentage apoptosis: control 6 hours, 10.7% ± 0.9%; GM-CSF 6 hours,
4.1% ± 0.4%, P < .005; control 20 hours,
72.3% ± 3.2%; GM-CSF 20 hours, 11.9% ± 2.3%,
P < .005, n = 8) (Figure 2E, F). The cytoprotective effect of GM-CSF at 20 hours was significantly attenuated
(64.1% ± 8.4%, P < .005) by preincubation with
LY294002 (10 µM). Of note, LY294002 alone had no effect on the rate
of neutrophil apoptosis at 6 or 20 hours (Figure 2). In contrast,
TNF- Annexin-V-FITC assessment of neutrophil apoptosis Double labeling of neutrophils with Annexin-V-FITC (AnV), which binds to PS on the surfaces of apoptotic cells, and the nuclear stain propidium iodide (PI), which allows assessment of membrane integrity, enables differentiation between viable nonapoptotic cells (AnV , PI), viable apoptotic cells
(AnV+, PI), and late apoptotic-necrotic
cells (AnV+, PI+). Previous studies have
underscored the importance of using complementary but distinct methods
for quantifying neutrophil apoptosis, not least because of the improved
sensitivity of AnV binding over morphology as a marker of early
apoptosis.41 In addition, certain experimental and
pharmacologic interventions can lead to dissociation between the extent
of morphologic apoptosis and the proportion of AnV+
cells.20,41 To address these issues, we examined the
influence of GM-CSF, TNF-, and LY294002 on neutrophil apoptosis
using dual AnV and PI staining.
AnV analysis of neutrophil apoptosis confirmed the cytoprotective
effect of GM-CSF at 20 hours (percentage apoptosis: control 20 hours,
53.2% ± 1.5%; GM-CSF 20 hours, 31.6% ± 2.2%,
P < .005, n = 8) and the near complete inhibition of
this survival effect with LY294002 (Figure
3A-D). Of note, AnV staining (unlike the morphologic quantification above) failed to demonstrate any significant cytoprotective effect of TNF-
Such a discrepancy, though unexplained, has been widely observed in
other studies of neutrophil apoptosis20,42 and underscores the importance of using complementary techniques to assess cell death.
Further analysis of the subpopulation of AnV+ cells
indicated that GM-CSF did not influence the proportion of apoptotic
(AnV+) cells that were PI+ at 20 hours (Table
1). In contrast, TNF-
This most likely reflects the early induction of neutrophil apoptosis
by TNF-
Effect of the MAPK inhibitor PD98059 on the rate of neutrophil apoptosis Previous studies have suggested that activation of the ERK1/2 pathway may be involved in the survival effect of GM-CSF in human neutrophils.6 To compare the relative importance and contribution of ERK1/2 and the PI3-kinase pathways to neutrophil apoptosis, we performed comparative experiments using the ERK1/2 inhibitor PD98059. In contrast to the marked effect of LY294002 on GM-CSF-mediated neutrophil survival, analysis of neutrophil apoptosis using AnV-FITC binding indicated that PD98059 did not attenuate the survival effect of GM-CSF (P = .055) (Figure 5A).
These data were confirmed by independent assessment of cell morphology;
PD98059 again failed to influence the extent of neutrophil apoptosis
under control or GM-CSF-stimulated conditions at 6 and 20 hours (Table
2). PD98059 also had no effect on the
early proapoptotic or the late survival effect of TNF-
To determine whether the simultaneous inhibition of the ERK1/2 and
PI3-kinase pathways induced additive inhibition of GM-CSF-mediated survival, neutrophils were pretreated with both inhibitors (LY294002 10 µM, PD98059 50 µM) before incubation with GM-CSF or TNF- Effect of the PKA inhibitor H-89 on the rate of neutrophil apoptosis Several papers have recently reported an additional Bad phosphorylation site at Ser155, potentially phosphorylated by PKA rather than PI3-kinase/PKB. In view of these observations and our previous demonstration that PGE2 and cell-permeable cAMP analogues inhibit neutrophil apoptosis through a PKA-dependent pathway,43 we extended the above studies to examine the role of the PKA pathway in GM-CSF-induced neutrophil survival. Neutrophils were pretreated with H-89 (1 µM) before incubation with GM-CSF (10 ng/mL) for 6 and 20 hours. AnV analysis of neutrophil apoptosis indicated that H-89 induced a significant inhibition of the GM-CSF survival effect; however, the magnitude of this effect was smaller than that observed with LY294002 (Figure 5B). Together, these data support a dominant role for the PI3-kinase signaling in mediating GM-CSF- and TNF--mediated neutrophil survival in vitro.
PI3-kinase-dependent effect of GM-CSF on Bad localization and Bad phosphorylation To examine the phosphorylation status and cytosolic localization of Bad following GM-CSF or TNF- stimulation in neutrophils, we used
a hypotonic lysis buffer containing a low percentage of detergent to
differentiate cytosolic from membrane-bound Bad and performed Western
blot analysis using anti-Bad and selective anti-phospho-Bad antibodies. Figure 6A shows
representative immunoblots demonstrating the ability of GM-CSF to cause
a marked increase in the amount of Bad protein present in neutrophil
cytosolic fractions and a corresponding decrease in membrane-associated
Bad at 30 minutes.
Moreover, though LY294002 had no effect on the levels of cytosolic or membrane-bound Bad under control conditions, it completely abolished the GM-CSF-stimulated changes in Bad distribution (Figure 6). These data suggest that under control conditions, Bad is largely sequestered to a membrane compartment, possibly in association with Bcl-XL bound to the outer mitochondria membrane. Although this conclusion is supported by a recent report indicating that GM-CSF does not alter Bcl-XL expression in neutrophils,44 we and others45,46 have been unable to identify Bcl-XL protein in neutrophils (data not shown). Because GM-CSF-induced Bad phosphorylation has been speculated to release Bad from its mitochondria-bound partner, the phosphorylation status of Bad was determined using phosphospecific antibodies directed against phosphorylated Bad at Ser112 and Ser136. Figure 6B and C show representative anti-phospho-Bad immunoblots from cytosolic extracts prepared from cells incubated with GM-CSF for 30 minutes. Following GM-CSF stimulation, a marked increase in the phosphorylation status of Bad at Ser112 and Ser136 was observed. As shown in Figure 6B and C, LY294002 significantly attenuated the GM-CSF-stimulated phosphorylation of Bad at Ser112 and Ser136, supporting the view that PI3-kinase-dependent dual phosphorylation of Bad underlies the release of Bad from its membrane-bound partner. Regulation of Bax, Bad, and Bcl-XL transcription in human neutrophils In addition to examining the effects of GM-CSF on Bad phosphorylation, we determined the effects of prolonged cytokine stimulation on Bax, Bad, and Bcl-XL mRNA levels. We confirmed previous reports identifying Bad and Bcl-XL mRNA in freshly isolated human neutrophils and the ability of GM-CSF (10 ng/mL) to reduce Bax mRNA levels (data not shown). The PCR primers used for Bcl-X bind to sequences shared by Bcl-XL and Bcl-XS, thus allowing simultaneous identification of both Bcl-X mRNA isoforms. Of note, we were unable to identify mRNA for the Bcl-XS isoform in freshly isolated neutrophils (data not shown).We report for the first time that levels of Bad mRNA decrease following
GM-CSF stimulation. Densitometry analysis of 6 independent experiments
indicated that GM-CSF reduced Bad mRNA expression by 43.8% ± 5.3%
at 4 hours, compared with time-matched controls (Figure
7A).
In contrast, TNF- To determine whether the PI3-kinase pathway was involved in the
differential regulation of Bad mRNA expression, neutrophils were
pretreated with LY294002 before incubation with GM-CSF or TNF-
The resolution of granulocyte inflammation is controlled by
a number of factors, including the neutralization or removal of the
inciting stimulus, cessation of neutrophil influx, and active removal
of effete cells by the process of programmed cell death. Many of the
inflammatory cytokines involved in initiating the primary inflammatory
response also have major effects on the rate of which the cells
involved in this response undergo apoptosis. Elucidation of the
cellular mechanisms underlying such effects has important implications
for understanding whether inflammation resolves or persists. This study
has investigated the signal transduction pathways used by GM-CSF and
TNF- Our initial observation that GM-CSF stimulation of unprimed neutrophils caused a significant increase in the cellular accumulation of PtdIns(3,4,5)P3 highlights the fact that GM-CSF has the capability to stimulate PI3-kinase activity. Al-Shami et al47 have shown that GM-CSF activates the JAK/STAT pathway, more specifically JAK2, STAT3, and STAT5B,48,49 and they have demonstrated that GM-CSF stimulates PI3-kinase in a tyrosine kinase- and a JAK2-dependent, but a Lyn-independent, manner.47,50 However, the role of Lyn remains controversial. Lyn activation can induce neutrophil survival through stimulation of the ERK1/2 pathway,51 and antisense depletion of Lyn kinase inhibits GM-CSF-induced neutrophil survival.5 Contrary to previous reports, inclusion of the ERK1/2 inhibitor PD98059
caused only a marginal attenuation of the survival effect of either
GM-CSF or TNF- The PI3-kinase/PKB signaling pathway has been implicated in cell survival in a wide range of cells. However, most of these studies, in particular those proposing Bad as a downstream target, have used manipulated cell lines, overexpression models, or artificial protein deletion strategies. We have used primary nontransformed cells to confirm the ability of survival cytokines to activate PI3-kinase and subsequently to enhance Bad phosphorylation at Ser136. Of interest, we also show PI3-kinase-dependent phosphorylation of Bad at Ser112. Previous studies using PKB overexpression systems report Bad phosphorylation of Ser136 only and report that coexpression and activation of ERK1/2 is required to phosphorylate Ser112. Phosphorylation of Ser112 has been suggested not to correlate with PKB activation; therefore, an additional downstream target of PI3-kinase may contribute to Bad phosphorylation at Ser112. We have also identified the ability of GM-CSF to reduce Bad mRNA levels
through a PI3-kinase-dependent mechanism; this could be explained by
decreased transcription or increased transcript instability. In
contrast, TNF- Bad, though showing no intrinsic or independent cell destructive
properties, is believed to promote apoptosis through heterodimerization with antiapoptotic bcl-2 family proteins. We have identified in human
neutrophils the ability of GM-CSF and TNF- Although the expression of other Bcl-2-like proteins in neutrophils, including Mcl-1, A1, Bcl-W, Bid, Bim, and Bax and their involvement in regulating neutrophil apoptosis, has been investigated by a number of groups, several important issues remain unsolved. In particular, the expression and role of Bcl-2 and Bcl-XL in granulocyte apoptosis remains controversial. Bcl-2 protein expression has been reported by Van Der Vliet et al54 in relatively pure populations of mature neutrophils. Most groups have been unable to identify Bcl-2 in neutrophils at either a protein or an mRNA level.10,44 The extent of Bcl-X expression is also uncertain with several groups, including our own, reporting little or no expression of Bcl-X in mature neutrophils.46 In contrast, Weinmann et al44 detected Bcl-X in neutrophils by reverse transcription-PCR (RT-PCR) and Western blot analysis; Bcl-X expression diminished slowly over 22 hours and was proposed as a potential mechanism driving the natural onset of constitutive apoptosis in vitro. In the present study, the PCR primers for Bcl-X were designed to bind sequences shared by Bcl-XL and Bcl-XS and clearly demonstrated the presence of Bcl-XL in freshly isolated neutrophils. Moreover, cytokine stimulation (4 hours) or PI3-kinase inhibition did not alter the expression of either Bcl-X isoforms, suggesting regulation through a PI3-kinase-independent mechanism. Bax mRNA levels in neutrophils have also been reported to decline when these cells are aged in vitro.44 We have shown that the rate of Bax mRNA degradation appears to be enhanced following GM-CSF stimulation, which may further contribute to neutrophil survival. In contrast, the ability of TNF- | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
in addition to causing Bad phosphorylation at Ser112 and Ser136
