From the Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, United Kingdom and ¶ The Cell Biology Unit, Glaxo-Wellcome, Gunnelswood Road, Stevenage, Herts, SG1 2NY, United Kingdom
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ABSTRACT |
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During beneficial inflammation, potentially
tissue-damaging granulocytes undergo apoptosis before being cleared by
phagocytes in a non-phlogistic manner. Here we show that the rate of
constitutive apoptosis in human neutrophils and eosinophils is greatly
accelerated in both a rapid and concentration-dependent
manner by the fungal metabolite gliotoxin, but not by its inactive
analog methylthiogliotoxin. This induction of apoptosis was abolished
by the caspase inhibitor zVAD-fmk, correlated with the inhibition of
nuclear factor-kappa B (NF- Neutrophilic and eosinophilic granulocytes originate from a common
myeloid precursor; neutrophils are particularly active in the defense
against invading micro-organisms whereas eosinophils serve in
anti-parasitic defenses and play a role in allergic inflammation. The
normally beneficial acute inflammatory response can become dysregulated
and result in chronic inflammatory conditions where tissue damage
arises in part due to the inappropriate liberation of inflammatory
cell-derived histotoxic products. We have previously described a
granulocyte clearance mechanism likely to be important in the normal
control and resolution processes of inflammation whereby granulocytes
must first undergo apoptosis (programmed cell death) before being
phagocytosed and cleared by macrophages in situ (1, 2).
Apoptosis also causes functional down-regulation of granulocytes and
the retention of proteolytic granule contents to further limit the
potential for granulocyte-mediated tissue damage (3, 4). While little
is known about the physiological mechanisms involved in controlling
granulocyte apoptosis, many in vitro studies have now shown
that an array of pro-inflammatory cytokines and inflammatory mediators
known to be present at inflamed sites inhibit the process of apoptosis
in granulocytes (2-9). This has led to the suggestion that such agents
act both to attract and activate inflammatory cells and also delay
their removal. A notable exception to this rule, however, is
TNF- Many inflammatory mediators regulate gene expression in target cells by
influencing the activities of transcription factors such as nuclear
factor- Whereas the inhibition of NF- Gliotoxin, a member of the epipolythiodioxoperazine family of compounds
(24), exhibits immunosuppressive activity both in vivo and
in vitro. For example, gliotoxin has been shown to inhibit mitogen-induced proliferation of both T and B cells, induce macrophage and osteoclast apoptosis in vitro (25, 26), and cause
thymocyte and spleen cell apoptosis in vivo (27). However,
the biochemical and molecular mechanisms underlying these effects
remain uncertain. Gliotoxin has, however, recently been shown to be a
potent and specific inhibitor of NF- Neutrophil and Eosinophil Isolation and Culture
Neutrophils and eosinophils were isolated from the peripheral
blood of normal donors by dextran sedimentation followed by centrifugation through discontinuous plasma-Percoll gradients (2, 29).
Only neutrophil preparations with a neutrophil purity of >98% were
used. Eosinophils were separated from contaminating neutrophils using
an immunomagnetic separation step with sheep anti-mouse IgG-Dynabeads
(Dynabeads M-450, Dynal, Merseyside, United Kingdom) coated with the
murine anti-neutrophil antibody 3G8 (anti-CD16; a gift from Dr. J. Unkeless, Mount Sinai Medical School, New York). Cells were mixed with
washed 3G8-coated Dynabeads at a bead:neutrophil ratio of 3:1 on a
rotary mixer at 4 °C for 20 min, and the beads removed magnetically
by two 3-min stationary magnetic contacts (Dynal Magnetic Particle
Concentrator, MPC-1) to yield an a eosinophil population of >98%
purity. After purification, cells were washed twice in
phosphate-buffered saline without calcium and magnesium and once in
phosphate-buffered saline before resuspending in Iscove's DMEM (Life
Technologies, Paisley, UK) with 10% autologous serum. Both cell types
were cultured in flat-bottomed Falcon flexible wells (Becton Dickinson,
Oxford, UK) at 37 °C in a 5% CO2 atmosphere; neutrophils at a concentration of 5 × 106/ml and
eosinophils at 2 × 106/ml. Cells were cultured in the
absence or presence of test agents as described in the figure legends.
All experiments were performed at least 3 times and each treatment done
in triplicate.
Assessment of Granulocyte Apoptosis
Morphology--
Cells were cyto-centrifuged, fixed in methanol,
stained with Diff-QuikTM, and counted using oil immersion
microscopy (×100 objective) to determine the proportion of cells with
highly distinctive apoptotic morphology (5, 7, 10). At least 500 cells
were counted per slide with the observer blinded to the experiment
conditions. The results were expressed as the mean % apoptosis ± S.E.
Annexin V Binding--
A separate and independent assessment of
apoptosis was performed by flow cytometry using fluorescein
isothiocyanate-labeled recombinant human annexin V that binds to
phosphatidylserine exposed on the surface of apoptotic cells. Stock
annexin V (Bender MedSystems, Vienna, Austria) was diluted 1:200 with
binding buffer and then added (25 µl) to 75 µl of the recovered
cell samples. Following a 10-min incubation at 4 °C, these samples
were fixed by the addition of 100 µl of 3% paraformaldehyde in
phosphate-buffered saline before analysis using an EPICS Profile II
(Coulter Electronics, Luton, UK).
DNA Fragmentation Assay--
DNA was extracted as described
previously (10). Briefly, 2 × 106 neutrophils were
taken after the indicated treatment and lysed in 500 µl of lysis
buffer (6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0, and 0.1% N-lauroyl sarcosine) at 4 °C
and the nucleic acids extracted by the addition of an equal volume of 10 mM Tris-HCl, pH 8.0-saturated phenol:chloroform mixture
(50:50, v/v). The resulting emulsion was centrifuged at 12,000 × gav for 10 min at room temperature and the
aqueous phase removed and precipitated with 0.6 volumes of isopropyl
alcohol at room temperature. The precipitated nucleic acids were then
pelleted by centrifugation at 10,000 × gav
for 5 min and re-dissolved in 50 µl of TE buffer (10 mM
Tris-Cl, 1 mM EDTA, pH 8.0) containing 50 µg/ml RNase A. The fragmented DNA was separated by agarose gel electrophoresis on a
1.4% (w/v) agarose (Flowgen, UK) 0.5 × TBE (10 mM
Trizma (Tris base), 10 mM boric acid, and 1 mM
EDTA, pH 8.3) gel. The gel was run for 2 h at 75 V and stained
using ethidium bromide (0.5 µg/ml). The UV transilluminated image was
printed by digital thermal printing using a GS7600 gel documentation
system (UVP Products, UK).
Assessment of Cell Membrane Integrity
Since apoptotic neutrophils and eosinophils maintain the
integrity of their plasma membrane, assessment of granulocyte necrosis can be determined by the ability of cells to exclude the vital dye
trypan blue and also by flow cytometry using propidium iodide staining.
Samples (150 µl of cells at 2 × 106/ml) were
centrifuged and resuspended in 150 µl of propidium iodide solution
(33 µg/ml propidium iodide in phosphate-buffered saline containing
1.67 mg/ml RNase). The profiles of heat-treated (necrotic cells) from
the same samples were used as controls.
Electrophoretic Mobility Shift Assay (EMSA)
EMSAs were carried out as described by the manufacturer (Promega
Corp, Southampton, UK). Nuclear extracts were prepared from 5 × 106 cells using a modification of the method of Dignam
et al. (30). Briefly, pelleted cells were resuspended in 200 µl of hypotonic buffer (buffer A: 10 mM Tris-HCl, pH 7.8, 1.5 mM EDTA, 10 mM KCl, 0.5 mM
dithiothreitol, 1 µg/ml aprotinin, leupeptin, and pepstatin A, 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM sodium orthovanadate, 0.5 mM benzamidine,
and 2 mM levamisole) and placed on ice for 10 min.
Following the addition of 0.1 volumes of 10% Nonidet P-40 (W/v) the
cells were vortexed briefly and centrifuged at 12,000 × gav for 2 min at 4 °C. The supernatant was
discarded and the pellet washed in 100 µl of buffer A minus Nonidet
P-40 and re-centrifuged. The pelleted nuclei were then resuspended in
50 µl of hypertonic buffer (buffer B: 20 mM Tris-HCl, pH
7.8, 150 mM NaCl, 50 mM KCl, 1.5 mM
EDTA, 5 mM dithiothreitol, 1 µg/ml aprotinin, leupeptin,
and pepstatin A, 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM sodium orthovanadate, 0.5 mM
benzamidine, and 2 mM levamisole) and stored at Nuclear extracts (approximately 2 µg of protein, 7 × 105 cell equivalent, in 7 µl) were incubated in binding
buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, with 50 µg/ml poly(dI-dC)·poly(dI-dC) (Pharmacia Biotech, UK) with 17 fmol
of Materials
Further specific materials were obtained as follows: curcumin,
gliotoxin, LPS (Escherichia coli 0127:B8),
methylthiogliotoxin, and PDTC (Sigma Co., Poole, UK); recombinant human
TNF- Statistical Analysis
The results are expressed as mean ± S.E. of the number
(n) of independent experiments each using cells from
separate donors with each treatment performed in triplicate.
Statistical analysis was performed by ANOVA with comparisons between
groups made using the Newman-Kuels procedure. Differences were
considered significant when p < 0.05.
Effect of Gliotoxin on Neutrophil Apoptosis--
As shown in Fig.
1 gliotoxin caused a rapid and profound
induction of neutrophil apoptosis in vitro which was both
concentration (e.g. at 6 h EC50 = 76.1 ± 22.1 ng/ml, Fig. 1A), and time-dependent (Fig. 1, A and B). Hence using a maximally
effective gliotoxin concentration of 1 µg/ml, apoptosis was readily
apparent within 2 h and reached 100% by 6 h. At 20 h,
when the rate of constitutive neutrophil apoptosis was 58.7 ± 2.9%, gliotoxin caused 100% apoptosis at all concentrations greater
than 0.1 µg/ml. The inactive analogue of gliotoxin,
methylthiogliotoxin, did not affect the constitutive rate of neutrophil
apoptosis at any of the time points studied (Fig. 1B).
Neither gliotoxin nor its inactive analogue, methylthiogliotoxin, caused cell necrosis since less than 1% of the cells were permeable to
the vital dye trypan blue.
Gliotoxin Acts Synergistically with TNF-
The genuine nature of both the intrinsic pro-apoptotic effect of
gliotoxin and the dramatic synergy with TNF- Combined Gliotoxin and TNF- Gliotoxin Inhibits the Survival Effect of LPS--
To investigate
whether gliotoxin could modulate the effects of LPS on the rate of
neutrophil apoptosis we performed a series of experiments where
neutrophils were cultured for 2, 3, 4, and 20 h in the presence of
LPS, gliotoxin, and a combination of LPS plus gliotoxin and apoptosis
assessed morphologically (Fig. 4). As
reported previously (7) LPS caused an inhibition of neutrophil apoptosis at 20 h when compared with control cells. Interestingly, the suppressive effect of LPS was prevented by the strong pro-apoptotic effect of gliotoxin. In addition, unlike co-culture of gliotoxin plus
TNF- Gliotoxin Unmasks the Ability of TNF- Gliotoxin Causes Inhibition of an Inducible Isoform of
NF-
In both TNF-
Further evidence that strongly supports the suggestion that NF- Induction of Apoptosis by Gliotoxin Is Dependent on Activation of
the Caspase-cascade Pathway--
We have recently demonstrated that
the early pro-apoptotic effects of TNF- Gliotoxin May Enhance TNF- We have demonstrated that gliotoxin, but not its inactive
derivative methylthiogliotoxin, (a) induces a direct time-
and concentration-dependent increase in the rate of
constitutive apoptosis in both neutrophils and eosinophils,
(b) enhances the pro-apoptotic effect of TNF- Because NF- Several mechanisms, aside from NF- Our results indicate that the inducible isoform of NF- When neutrophils were co-cultured with LPS and gliotoxin, gliotoxin
failed to render LPS pro-apoptotic despite the fact that LPS induced
survival was inhibited by gliotoxin (Fig. 4). These results suggest
that LPS does not trigger a death pathway in neutrophils but stimulates
a NF- In a number of immune cells NF- Enhanced cytotoxic responses to TNF- The mechanism whereby inactivation of NF- The ability of gliotoxin to enhance the cytotoxic effects of TNF-B), and was mimicked by a cell permeable
inhibitory peptide of NF-
B, SN-50; other NF-
B inhibitors,
curcumin and pyrrolidine dithiocarbamate; and the proteasome inhibitor,
MG-132. Gliotoxin also augmented dramatically the early (2-6 h)
pro-apoptotic effects of tumor necrosis factor-
(TNF-
) in
neutrophils and unmasked the ability of TNF-
to induce eosinophil
apoptosis. In neutrophils, TNF-
caused a gliotoxin-inhibitable
activation of an inducible form of NF-
B, a response that may
underlie the ability of TNF-
to delay apoptosis at later times
(12-24 h) and limit its early killing effect. Furthermore,
cycloheximide displayed a similar capacity to enhance TNF-
induced
neutrophil apoptosis even at time points when cycloheximide alone had
no pro-apoptotic effect, suggesting that NF-
B may regulate the
production of protein(s) which protect neutrophils from the cytotoxic
effects of TNF-
. These data shed light on the biochemical and
molecular mechanisms regulating human granulocyte apoptosis and, in
particular, indicate that the transcription factor NF-
B plays a
crucial role in regulating the physiological cell death pathway in granulocytes.
INTRODUCTION
Top
Abstract
Introduction
References
,1 which, at early
time points in neutrophil culture, causes acceleration of the
constitutive rate of apoptosis (10).
B (NF-
B). NF-
B is composed of homo- or heterodimers of
the Rel family proteins (p50/NF
B1, p52/NF
B2, p65/RelA, and cRel)
which are sequestered in the cytoplasm by physical association with
inhibitor proteins referred to as I
B (11). Upon activation, the
I
B subunit is rapidly phosphorylated leading to its proteolytic
breakdown permitting NF-
B to translocate to the nucleus (12, 13)
where it regulates the activity of many genes involved in the
inflammatory response, including those for pro-inflammatory cytokines.
In a number of cell systems TNF-
has been shown to induce rapid
activation of NF-
B, a response known to mediate a number of TNF-
induced cellular responses (reviewed in Refs. 14-16). However, whether
the activation of NF-
B is involved in either the pro- or
anti-apoptotic effects of TNF-
in granulocytes is currently unknown.
B has been shown to induce apoptosis in
murine B cells (17), a completely opposite effect has been observed in
other cell types where apoptosis is associated with activation of
NF-
B (18). In addition, several groups have reported that
inactivation of NF-
B increases the cytotoxic effects of TNF-
(19-21). In granulocytes, it remains uncertain whether NF-
B can be
activated by inflammatory mediators, or is indeed present, in human
neutrophils. For example, while MacDonald et al., (22)
reported that lipopolysaccharide (LPS), TNF-
, and the chemotactic
peptide N-formyl-methionyl-leucyl-phenylalanine all cause a
marked activation of NF-
B, Browning et al., (23) found no
such activity in these cells despite obvious NF-
B activation in
peripheral blood mononuclear cells.
B (28). We therefore used
gliotoxin as a pharmacological tool to investigate the involvement of
NF-
B in the regulation of granulocyte apoptosis. We demonstrate that gliotoxin causes a rapid and major induction of apoptosis in human peripheral blood granulocytes in vitro and up-regulates
TNF-
-induced apoptosis in both neutrophils and eosinophils. In
addition, we present evidence that these effects occur via a specific,
non-toxic and caspase-controlled mechanism that is mediated by the
ability of gliotoxin to inhibit an inducible form of NF-
B. The
ability of other NF-
B inhibitors to cause a similar induction of
apoptosis provides further evidence supporting the involvement of
NF-
B in granulocyte apoptosis. Interestingly, the pro-apoptotic
effect of TNF-
is enhanced by protein synthesis blockade suggesting that NF-
B activation results in the generation of an unidentified survival protein. These data therefore strongly suggest that NF-
B plays a key role in regulating both constitutive and TNF-
stimulated human granulocyte apoptosis.
EXPERIMENTAL PROCEDURES
80 °C
until use.
-32P-labeled double stranded oligonucleotide
containing the decameric
B-binding site (3000 Ci/mmol; Promega
Corp., Southampton, UK) at 22 °C for 20 min prior to addition of 5 µl of loading buffer (0.01% (w/v) bromphenol blue, 20% (w/v) Ficoll
400TM, and 1 mM EDTA). Samples were loaded onto an 8%
(w/v) native acrylamide gel (Protean IIxi, Bio-Rad, Hemel Hempstead,
UK) in 0.5 × TBE buffer and run at 250 V for 2 h. The gel
was then dried onto 3MM paper (Whatman UK, Maidstone, UK) and BioMax
MS-1 x-ray film (Kodak, Anachem, Luton, UK) was exposed to the gel.
Processed films were analyzed using the GrabIt and GelPlate software
(UVP, Orme Technologies, UK) and the data expressed as a percent of the
control value for each experiment.
(R & D Systems, Abingdon, Oxon, UK); zVAD-fmk (Bachem (UK)
Ltd., Saffron Walden, UK). The proteasome inhibitor MG-132
(N-cbz-Leu-Leu-leucinal), and the NF-
B inhibitory peptides SN50 and
SN50M (Biomol, Affinity Research Products, Mamhead, UK). All other
reagents were obtained from Sigma, UK, and were of the highest purity.
RESULTS
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Fig. 1.
Time course and concentration-response curve
for the effect of gliotoxin on human neutrophil apoptosis. Human
neutrophils (5 × 106/ml) were cultured at 37 °C in
Iscove's DMEM containing 10% autologous serum and treated with the
indicated concentrations of gliotoxin. At the time periods indicated,
the cells were resuspended and cytocentrifuge preparations made. These
were fixed and stained, and apoptosis was assessed morphologically.
A, represents the effect of gliotoxin (0.001-30 µg/ml) on
neutrophil apoptosis after 2, 6, or 20 h of culture. B,
represents the effect of methylthiogliotoxin (30 µg/ml) or gliotoxin
(30 µg/ml) on neutrophil apoptosis after 2, 6, or 20 h culture.
All values represent mean ± S.E. of n = 3 experiments, each performed in triplicate. Where not shown, S.E. values
are less than 2% of the mean.
to Stimulate Neutrophil
Apoptosis--
In contrast to many other hematopoetic cells, human
neutrophils appear highly resistant to the induction of apoptosis
induced by certain agents, for example, incubation with
Ca2+ ionophores (31, 32), cAMP elevating agents (33),
corticosteroids (9), and LPS (7) causes inhibition of apoptosis as
does hypoxia (34). Furthermore, while TNF-
and Fas-L can induce neutrophil apoptosis, this effect is modest and transient, and in the
case of TNF-
abolished if the cells are initially primed with
platelet-activating factor or LPS (10, 35). We therefore sought to
determine the effect of gliotoxin on TNF-
-induced apoptosis in
neutrophils. These experiments were performed deliberately at a very
early time point (2 h) when the independent pro-apoptotic effects of
even a maximally effective concentration of TNF-
(10 ng/ml) (10) and
gliotoxin (1 µg/ml; Fig. 1A) are only just apparent. As
shown in Fig. 2A, a major
synergy was observed between these agents for the induction of
apoptosis which was apparent even at gliotoxin concentrations as low as
3 ng/ml. Hence, a concentration of 0.1 µg/ml gliotoxin in combination
with TNF-
(10 ng/ml) caused almost 100% apoptosis at 2 h. With
gliotoxin alone, only 6% apoptosis was noted at 2 h, with just
over 65% at 6 h (Fig. 1A). Again, methylthiogliotoxin
had no effect on constitutive apoptosis or TNF-
-induced
apoptosis at 2 h (Fig. 2B). The level of necrosis in
cells from each treatment was assessed by trypan blue exclusion; all
values were <1% (data not shown).
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Fig. 2.
The effect of gliotoxin, methylthiogliotoxin,
and TNF- on human neutrophil apoptosis.
Human neutrophils (5 × 106/ml) were cultured at
37 °C in Iscove's DMEM containing 10% autologous serum and treated
with the indicated concentrations of gliotoxin, plus or minus TNF-
.
After 2 h of culture, the cells were resuspended and
cytocentrifuge preparations made. These were fixed and stained, and
apoptosis was assessed morphologically. A, represents the
effect of gliotoxin (0.001-30 µg/ml) on TNF-
(10 ng/ml)-induced
neutrophil apoptosis. B, represents the effect of
methylthiogliotoxin (30 µg/ml) or gliotoxin (30 µg/ml), plus or
minus TNF-
(10 ng/ml). All values represent mean ± S.E. of
n = 3 experiments, each performed in triplicate. Where
not shown, S.E. values are less than 2% of the mean.
was assessed by
comparing the quantitative morphological effects of these agents with
their effects on annexin V binding and DNA fragmentation. The changes
from normal cell morphology to apoptotic morphology are clearly
seen in Fig. 3A; where
non-apoptotic
neutrophils contain a multilobed nucleus and the apoptotic cells have a
shrunken appearance with pyknotic nuclei. These data can be compared
with Fig. 3B, where the annexin V "low peak" represents
non-apoptotic cells and the annexin V "high peak" represents
apoptotic cells since the fluorescein isothiocyanate-labeled annexin V
binds in the presence of Ca2+ to phosphatidylserine exposed
on the outer membrane of apoptotic cells. Although control cells at
2 h exhibit low rates of apoptosis, the small increase in annexin
V positive cells observed with TNF-
and gliotoxin alone is again
dramatically augmented when the cells are cultured in the presence of
both reagents together. Analysis by DNA fragmentation also demonstrates
that cells cultured alone or in combination with the above reagents
exhibit the classical "ladder" of DNA fragmentation associated with
apoptosis (Fig. 3C).
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Fig. 3.
The effect of gliotoxin and
TNF- on neutrophil morphology,
phosphatidylserine expression, and cell membrane integrity.
A, cytocentrifuge preparations of human neutrophils
incubated for 2 h in Iscove's DMEM at 37 °C containing 10%
autologous serum alone (control), plus TNF-
, plus
gliotoxin, or with both TNF-
and gliotoxin at the concentrations
shown. Neutrophils treated with both reagents all clearly show
apoptotic morphology. B, after 2 h in culture at
37 °C, cells treated with Iscove's DMEM, TNF-
(10 ng/ml),
gliotoxin (0.1 µg/ml), or with both TNF-
plus gliotoxin were
resuspended and incubated with fluorescein isothiocyanate-labeled
recombinant human annexin V to determine phosphatidylserine expression.
The cells were then fixed and analyzed using an EPICS Profile II. Mean
fluorescence values are shown for a minimum of 5,000 cells for each
condition. C, DNA fragmentation in human neutrophils treated
with TNF-
(10 ng/ml), gliotoxin (0.1 µg/ml), or both reagents
together for 2 h. DNA was prepared as detailed under
"Experimental Procedures"; lane 1, DNA marker
(1-kilobase ladder); lane 2, freshly isolated neutrophils;
lane 3, control; lane 4, TNF-
; lane
5, gliotoxin, 2 h; lane 6, co-culture with
gliotoxin and TNF-
; lane 7, DNA marker (1-kilobase
ladder). D, to assess cell membrane integrity, cells treated
with both TNF-
(10 ng/ml) and gliotoxin (0.1 µg/ml) for 4 h,
which induced 100% apoptosis even by 2 h, were resuspended and
incubated with propidium iodide, fixed, and analyzed using an EPICS
Profile II (solid line). An aliquot of cells from this
preparation was heated as indicated under "Experimental Procedures"
to produce 100% necrosis (dotted line). Mean fluorescence
values are shown for a minimum of 5,000 cells for each condition.
Treatment Does Not Cause
Necrosis--
Although our initial studies using trypan blue as a
marker of plasma membrane integrity indicated that gliotoxin, both in the presence and absence of TNF-
, induced a purely apoptotic form of
cell death, we felt it was important to validate this further by
assessing necrosis in an independent manner using propidium iodide
staining detected by flow cytometry. Fig. 3D shows the profile of neutrophils 4 h following treatment with gliotoxin (0.1 µg/ml) and TNF-
(10 ng/ml) where, despite apoptotic rates of
100%, almost all cells showed low fluorescence indicating that the
cell membrane had remained intact. As a positive control, cells
cultured initially with TNF-
and gliotoxin were then heat-treated (60 °C, 5 min) to ensure 100% necrosis. This resulted in a uniform and major increase in propidium iodide staining (Fig. 3D).
These data coincide completely with the results obtained with trypan blue staining and confirm that these cells had undergone apoptotic cell
death and were not necrotic.
, no synergistic induction of apoptosis was observed when LPS
was cultured in the presence of gliotoxin (Fig. 4).
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Fig. 4.
The effect of gliotoxin alone and in the
presence of LPS on human neutrophil apoptosis. Human neutrophils
(5 × 106/ml) were cultured in Iscove's DMEM at
37 °C containing 10% autologous serum alone and treated with
gliotoxin (0.1 µg/ml), with or without LPS (1 µg/ml). At the time
periods indicated, the cells were resuspended and cytocentrifuge
preparation made. These were fixed and stained, and apoptosis was
assessed morphologically. All values represent mean ± S.E. of
n = 3-5 experiments, each performed in triplicate.
Where not shown, S.E. values are less than 2% of the mean.
to Induce Eosinophil
Apoptosis--
To explore whether the pro-apoptotic effect of
gliotoxin was restricted to neutrophils, a similar set of experiments
were performed using human peripheral blood eosinophils isolated from mildly atopic individuals. While these cells display a similar capacity
to undergo constitutive apoptosis when aged in vitro, this
process is much slower than that observed for the neutrophil and is
differentially regulated being, for example, stimulated rather than
inhibited by corticosteroids (9). We therefore investigated the effect
of gliotoxin on eosinophil apoptosis at 4 h in the presence
and absence of TNF-
. The results, shown in Fig.
5, A and B,
demonstrated that gliotoxin caused a similar induction of apoptosis in
eosinophils (EC50 = 0.37 ± 0.22 µg/ml) and caused a
synergistic increase in the rate of apoptosis when the cells were
co-cultured with TNF-
. This latter observation was all the more
striking since in eosinophils, TNF-
treatment alone had no effect on
the rate of apoptosis (Fig. 5B and data not shown). Hence,
almost 100% apoptosis was observed using a gliotoxin concentration of
0.1 µg/ml plus TNF-
at a time point of 4 h. In comparison,
eosinophils cultured in the absence of gliotoxin would normally show
only 40% apoptosis at a 40-h time point (2, 36). Necrosis in these
cells was <2% and the inactive analogue of gliotoxin had no effect on
either the constitutive rate of apoptosis alone or in conjunction with
TNF-
(data not shown). As with the neutrophils, eosinophils
demonstrated classic apoptotic morphology when treated with these
agents.
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Fig. 5.
The effect of gliotoxin alone and in the
presence of TNF- on human eosinophil
apoptosis. Human eosinophils (2 × 106/ml) were
cultured in Iscove's DMEM at 37 °C containing 10% autologous serum
alone and treated with the indicated concentrations of gliotoxin with
or without TNF-
. At the time periods indicated, the cells were
resuspended and cytocentrifuge preparation made. These were fixed and
stained, and apoptosis was assessed morphologically. A,
represents the effects of gliotoxin (0-3 µg/ml) on eosinophil
apoptosis after 4 h of culture. B, represents the
effects gliotoxin (0-0.3 µg/ml) with or without TNF-
(10 ng/ml) on eosinophil apoptosis after a 4-h culture. All values
represent mean ± S.E. of n = 3 experiments, each
performed in triplicate. Where not shown, S.E. values are less than 2%
of the mean.
B--
Recent studies have indicated that NF-
B may play an
important role in regulating the rate of apoptosis in certain
transformed cells (17, 18). Hence, because gliotoxin has been reported to act as a specific inhibitor of NF-
B (28) experiments were designed to identify and characterize the expression of this
transcription factor in human neutrophils and determine if gliotoxin
could inhibit such activity. Preliminary time course data established
90 min as the optimal time to examine basal, gliotoxin, and TNF-
regulated NF-
B activity in these cells (data not shown). Of note,
this time point also coincided with the onset of the biologically
observable effect of gliotoxin. As shown in Fig.
6, A-C, NF-
B
EMSAs performed on neutrophil nuclear extracts indicated the presence
of 3 discrete bands in these gels. To ascertain which of these bands
were specifically NF-
B, an excess of unlabeled probe was included in
the labeling reaction to displace specific binding; as shown in Fig.
6C, two NF-
B bands were identified and designated A and
B.
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Fig. 6.
Effect of gliotoxin and
TNF- on NF-
B
mobilization. A, EMSA of nuclear extracts from
neutrophils treated with control buffer: lane 1, TNF-
(10 ng/ml); lane 2, gliotoxin (1 µg/ml); lane 3, gliotoxin (0.1 µg/ml); lane 4, and TNF-
(10 ng/ml) plus
gliotoxin (0.1 µg/ml); lane 5, for 90 min at 37 °C.
B, EMSA showing the up-regulation of the inducible isoform
(band A) by LPS (a known inhibitor of neutrophil apoptosis)
after 20 min culture: lane 1, control; lane 2,
LPS (100 ng/ml); and lane 3, LPS (100 ng/ml) plus gliotoxin
(0.1 µg/ml). C, EMSA showing displacement of specific
NF-
B bands by excess cold oligonucleotide probe; lane 1,
control; lane 2, TNF-
(10 ng/ml); lane 3,
TNF-
(10 ng/ml) plus 50-fold excess cold oligonucleotide; lane
4, TNF-
(10 ng/ml) plus 100-fold excess cold oligonucleotide.
Only the bands marked A and B are specific. D, densiometry
scanning of band A from the EMSA shown in A. This shows the
reduction of an inducible isoform of NF-
B by gliotoxin
(a = 1 µg/ml; b = 0.1 µg/ml), and
further inhibition by co-treatment with TNF-
plus gliotoxin
(b).
(10 ng/ml, 0-90 min) and LPS (1 µg/ml, 0-120 min)
treated cells, no change in the intensity of band B was observed (Fig.
5, A and B, and data not shown). This, together
with its strong expression in freshly prepared untreated neutrophils
suggests that this band represents a form of constitutively active
NF-
B. However, band A was markedly up-regulated by TNF-
(Fig. 6,
A and C), and as shown in Fig. 6A,
gliotoxin caused both a concentration-dependent inhibition
of this NF-
B activity and abolished the TNF-
stimulated increase
in this band. As shown in Fig. 6D, densiometric analysis of
these data confirmed that co-treatment of neutrophils with TNF-
and
gliotoxin at a maximal effective functional concentration of 0.1 µg/ml (Fig. 2A) inhibited this band more than treatment with gliotoxin alone. The results in Fig. 6, A and
D, confirm that the basal level of NF-
B activity in
control samples at 90 min was inhibited by gliotoxin treatment; a
finding that was observed at all other time points tested.
B
inhibition is linked to the induction of neutrophil apoptosis is
provided by the fact that the cell permeable NF-
B inhibitory peptide, SN50 (37), also increased the rate of constitutive neutrophil
apoptosis despite the fact that less than 5% of the peptide reportedly
enters the cell (37). Hence at 6 h, the SN50 peptide increased
neutrophil apoptosis from 4.7 ± 1.2 to 15.0 ± 3.2%
(n = 3; p < 0.05), whereas
the less active peptide SN50M only increased apoptosis from 4.7 ± 1.2 to 5.6 ± 0.8% (n = 3). Similar effects on
neutrophil apoptosis were seen at 20 h and eosinophil apoptosis at
20 and 40 h (Table I and data not shown). Other agents known to inhibit NF-
B similarly induced neutrophil apoptosis. The proteasome inhibitor, MG-132 (38) and the
NF-
B inhibitor curcumin (39) caused a time-dependent induction of neutrophil apoptosis (Fig.
7A). PDTC, that acts as both a
radical scavenger and inhibitor of NF-
B activation (40), also caused
a significant induction of apoptosis when cultured with neutrophils for
20 h (Fig. 7B). Furthermore, treating neutrophils with
LPS (100 ng/ml, 20 min) which we have previously reported to induce a
profound inhibition of neutrophil apoptosis (7) was found to cause the
appearance of this inducible isoform of NF-
B (Fig. 6B);
and this induction could be inhibited by gliotoxin (0.1 µg/ml).
The effect of NF-B inhibitory peptides on human neutrophil apoptosis
View larger version (15K):
[in a new window]
Fig. 7.
Effect of other NF-kB inhibitors on
neutrophil apoptosis. Human neutrophils (5 × 106/ml) were cultured at 37 °C in Iscove's DMEM
containing 10% autologous serum and treated with the indicated
reagent. A, neutrophils were treated with MG 132 (20 µM) and curcumin (20 µM) at the time
periods indicated; B, neutrophils were treated with MG-132
(100 µM), curcumin (20 µM), and PDTC (300 µM) for 20 h. After incubation, the cells were
resuspended and cytocentrifuge preparations made. These were fixed and
stained, and apoptosis was assessed morphologically. All values
represent mean ± S.E. of n = three to six
experiments, each performed in triplicate. Where not shown, S.E. values
are less than 2% of the mean.
in human neutrophils
requires activation of both TNF-55 and TNF-75 receptor subtypes and
thereby differs significantly from the priming effect of TNF-
which
is signaled via the TNF-p55 receptor alone (10). To determine whether
the pro-apoptotic effects of gliotoxin and the marked synergism
displayed by TNF-
and gliotoxin were mediated via activation of the
caspase pathway, we co-incubated neutrophils with TNF-
, gliotoxin,
and zVAD-fmk. At 2 h, zVAD-fmk completely inhibited the increase
in apoptosis induced by gliotoxin, TNF-
and by both factors together
(Fig. 8A). This demonstrates
that apoptosis induced by both factors alone, or together, is dependent
on caspase activation.
View larger version (12K):
[in a new window]
Fig. 8.
Effect of the caspase inhibitor, zVAD-fmk,
and the protein synthesis inhibitor, cycloheximide, on gliotoxin,
TNF- , or gliotoxin plus
TNF-
-induced human neutrophil apoptosis.
Human neutrophils (5 × 106/ml) were cultured in DMEM
containing 10% autologous serum and treated with the indicated
reagents. After 2 h of culture, the cells were resuspended and
cytocentrifuge preparation made. These were fixed and stained, and
apoptosis was assessed morphologically. A, represents the
effects of zVAD-fmk (100 µM) on gliotoxin (0.1 µg/ml)-,
TNF-
(10 ng/ml)-, or gliotoxin plus TNF-
-induced neutrophil
apoptosis. B, represents the effects of cycloheximide (5 µM) on gliotoxin (0.1 µg/ml)-, TNF-
(10 ng/ml)-, or
gliotoxin plus TNF-
-induced neutrophil apoptosis. All values
represent mean ± S.E. of n = three experiments,
each performed in triplicate. Where not shown, S.E. values are less
than 2% of the mean.
-induced Apoptosis by Inhibiting
Production of a Survival Factor--
Taken together, the above results
suggest that activation of an inducible form of NF-
B may inhibit or
restrain the pro-apoptotic effects of TNF-
which are mediated by the
parallel activation of the caspase pathway. The possibility that this
most likely reflects the production of a protein or proteins which act
to suppress the activation of the caspase pathway and thus protect granulocytes from the cytotoxic effects of this cytokine was
investigated by incubating neutrophils with TNF-
and cycloheximide,
a protein synthesis inhibitor. As illustrated in Fig. 8B,
cycloheximide used specifically at a concentration (5 µM)
that alone had almost no effect on neutrophil apoptosis at 2 h,
caused a synergistic increase in the level of apoptosis when
neutrophils were co-cultured with TNF-
. This supports the view that
TNF-
treatment indeed results in the generation of a survival
protein that protects these cells from the TNF
receptor-caspase-dependent induction of apoptosis by TNF-
.
DISCUSSION
in
neutrophils, and (c) reveals the cytotoxic effects of
TNF-
in eosinophils. In these studies extreme care was taken to
ensure that gliotoxin, at all time points and concentrations studied, was non-toxic and caused genuine apoptosis that was indistinguishable from later constitutive apoptosis. The similar effects of gliotoxin in
both neutrophils and eosinophils and the same concentration of
gliotoxin (0.1 µg/ml) required for maximal enhancement of the pro-apoptotic effects of TNF-
, suggests that an identical underlying mechanism is regulating the induction of cell death in both these cell
types. However, although inhibition of basal NF-
B activity may be
involved in neutrophil apoptosis when induced by gliotoxin alone (Fig.
1) since gliotoxin appears to block basal levels of NF-
B activity
(Fig. 6, A and D), only the expression of the
inducible NF-
B isoform is down-regulated before the onset of cell
death driven by the combined effects of TNF-
and gliotoxin (Fig. 6). Even in control neutrophils incubated for 20 h, where the
constitutive rate of apoptosis is approximately 70%, the density of
the constitutive NF-
B band was unaffected (data not shown). These
differences in the inducible and constitutive forms of NF-
B most
likely reflects differential regulation of activation, for example, by
the involvement of different isoforms of the inhibitory I
B subunit,
or that the constitutively active NF-
B is formed from a different
set of dimers from the classical RelA/p50 heterodimer. It has recently been demonstrated that neutrophils contain c-Rel, p50, and p105 (the
p50 precursor protein) as well as Rel-A (22, 41). The inducible band we
observed has also been reported to be up-regulated in neutrophils by
phagocytosis of IgG opsonized yeast particles (42), and has been shown
to consist mainly of Rel-A/p50 heterodimers and possibly a small amount
of c-Rel (42). In that study, phagocytosis of these particles did not
affect the activity of the constitutive complex. In addition, it has
recently been reported that NF-
B becomes activated, via a mechanisms
not involving oxidant generation, when neutrophils phagocytose bacteria
(43).
B is also activated by certain pro-apoptotic stimuli such
as TNF-
, this transcription factor has been considered as a possible
regulator of cell death. Hence, in some T cell clones, activation of
NF-
B appears to correlate with the onset of apoptosis (18). However,
NF-
B activation has clearly been shown to be anti-apoptotic in HT
1080 fibrosarcoma cells (21) and TNF-
induced NF-
B activation
prevents cell death in HeLa and MCF7 cells (44). Here we show for the
first time that in a non-transformed cell namely the neutrophil,
inhibition of an inducible form of NF-
B is related to the induction
of apoptosis.
B inhibition, have been proposed
for the pro-apoptotic actions of gliotoxin in other cells. Sutton
et al. (45) have shown that although this fungal metabolite did not affect intracellular calcium levels, there was a correlation between increases in cAMP levels and apoptosis in gliotoxin-treated splenocytes. However, we and others have previously demonstrated that
agents that elevate intracellular cAMP inhibit apoptosis in both
neutrophils and eosinophils (33, 46). It has also been suggested that
protein kinase A-dependent phosphorylation of histone H3
correlates with gliotoxin-induced apoptosis in thymocytes (47), but
again in neutrophils, activation of protein kinase A inhibits apoptosis
(33). Although gliotoxin has been reported to inhibit protein synthesis
(48) it is highly unlikely that this mechanism is directly responsible
for its pro-apoptotic effects: first, gliotoxin induces apoptosis in
thymocytes whereas inhibition of protein synthesis by cycloheximide
inhibits thymocyte apoptosis. Second, since NF-
B activation is
involved in the control of multiple genes, many of which encode for
inflammatory mediator synthesis, inactivation of NF-
B would
therefore be expected to inhibit protein synthesis. Third, while
protein synthesis inhibitors do up-regulate the rate of constitutive
cell death in granulocytes, the kinetics of this response are very
different to those observed with gliotoxin. For example, Whyte et
al. (49) have reported that cycloheximide (50 µM)
and actinomycin D (1 µM) induce apoptosis in
approximately 30% of neutrophils by 6 h. In our experiments,
gliotoxin (0.1 µg/ml), induces a rate of almost twice this, whereas
1.0 µg/ml gliotoxin induced 100% neutrophil apoptosis by this time
point (see Fig. 1A). Likewise, our own results with
cycloheximide indicate that protein synthesis inhibition alone does not
affect the rate of neutrophil apoptosis at 2 h whereas gliotoxin
alone produced almost 15% apoptosis over this period (Figs.
1A and 8B). While gliotoxin inhibits NF-
B,
cycloheximide and actinomycin D have been shown in several systems to
activate this transcription factor (50, 51). Although both
cycloheximide and gliotoxin give a similar synergistic pro-apoptotic
response with TNF-
, this suggests that different mechanisms must be
involved. However, while gliotoxin may prevent synthesis of a
protective protein inducible by NF-
B activation, cycloheximide would
also preclude synthesis of such a protein so that in both cases the
cells would be sensitive to the pro-apoptotic effects of TNF-
. It is
of interest to note that granulocytes do have the capacity to
synthesize proteins, albeit in a limited capacity (49). We believe that
this synthetic capacity will be directed toward resolution of the
inflammatory response with the generation of protein(s) that affect the
apoptotic program of inflammatory cells.
B disappears
from the gliotoxin-treated granulocyte nucleus just before the onset of
stimulated apoptosis. The possibility that these events are causally
related is supported by the following observations: (i) the synthetic
cell-permeable peptide SN50 (37), a known inhibitor of NF-
B, also
induces apoptosis in neutrophils and eosinophils; (ii) other agents
that inhibit NF-kB activation, namely PDTC and curcumin as well as the
proteasome inhibitor MG-132 also cause an induction of granulocyte
apoptosis; (iii) the kinetics for gliotoxin-mediated inhibition of
NF-
B match those for the onset of induction of apoptosis; (iv)
LPS which stimulates NF-
B activity prolongs neutrophil and
eosinophil survival; and (v) that gliotoxin sensitizes both neutrophils
and eosinophils to the pro-apoptotic effects of TNF-
. Indeed, our
studies provide the first plausible explanation for the modest and
temporally constrained apoptotic response of neutrophils to TNF-
and the observation that pretreatment with LPS, PAF or
granulocyte/macrophage-colony stimulating factor, abolishes the
cytotoxic effect of this cytokine (10). Indeed, this latter point is of
particular relevance when investigating the pro-apoptotic effect of
TNF-
in neutrophils since pre-treatment of these cells causes a
rapid decrease of both TNF-
receptors subtypes from the surface
membrane (10). This phenomenon, together
with the fact that the effects of SN50 are, at best, modest due to
limited access of the peptide to its intracellular target (37) and the
requirement for pretreatment with the peptide, precluded accurate
assessment of SN50 on TNF-
induced apoptosis in neutrophils.
B-mediated survival pathway i.e. when NF-
B activation is blocked, LPS is no longer capable of delaying apoptosis. The precise intracellular mechanisms by which the NF-
B inhibitors used in this study induce apoptosis are unknown and is the subject of
further investigation. For example, it would be of interest to perform
an in-depth analyses of the effect of gliotoxin and the other agents on
the degradation of the inhibitory subunit I
B, especially since Pahl
et al. (28) reported that gliotoxin appeared to prevent
I
B degradation rather than mediate its effect at the level of DNA binding.
B activation by agents such as
TNF-
has been shown to play a central role in regulating the genes
for inflammatory cytokines such as granulocyte/macrophage-colony stimulating factor and TNF-
itself (14). The importance of this
response in vivo is that many of these factors inhibit
granulocyte apoptosis and may therefore delay inflammatory resolution
by enhancing the longevity of these cells. Indeed, a positive-feedback
loop may exist since many of these inflammatory mediators which protect against apoptosis in neutrophils and eosinophils also activate NF-
B
(7). Conversely, we have recently shown that NO, a known inhibitor of
NF-
B (52, 53), is a potent inducer of apoptosis in neutrophils (54).
Our current results suggest that the activation of an inducible form of
NF-
B represents a powerful survival mechanism in granulocytes, and
that when this pathway is inhibited, in both neutrophils and
eosinophils, these cells undergo a greatly augmented rate of apoptotic
cell death. It is possible that NF-
B performs a similar function in
other cell types that undergo apoptosis in response to gliotoxin.
have also been demonstrated in
cells where NF-
B is genetically deficient or inactivated (19-21)
and hepatocytes from Rel-A null mice are known to undergo apoptosis
causing death in utero (55). Embryonic fibroblasts and
macrophages from Rel-A-deficient mice also showed dramatic loss of
viability when treated with TNF-
leading to the suggestion that
Rel-A regulates a protective mechanism against the cytotoxic effects of
TNF-
. It would be of interest to investigate the effects of TNF-
and gliotoxin on granulocytes isolated from mice deficient in Rel A; it
would be reasonable to predict that TNF-
will induce a rapid cell
death and gliotoxin and other NF-
B inhibitors would not have a
dramatic effect on the rate of granulocyte apoptosis. Although our
experiments indicate that gliotoxin does not inhibit the constitutive
form of NF-
B, at least at early time points, it does inhibit the
activation of an inducible isoform of NF-
B, which most likely
consists of heterodimers containing the Rel-A/p65 protein and therefore
could perform a similar anti-apoptotic function in neutrophils and
eosinophils. While in our hands TNF-
does not produce significant
cytotoxic effects in eosinophils, co-treatment with gliotoxin caused
these cells to become highly responsive to this cytokine producing
greatly increased levels of apoptosis. This suggests that both of these
inflammatory cell types could be stimulated to undergo apoptosis and
hence be cleared rapidly by phagocytes at sites of inflammation if
activation of the inducible NF-
B isoform were inhibited.
B induces granulocyte
apoptosis and increases the cytotoxic response to TNF-
is currently
unclear. Since gliotoxin and TNF-
driven apoptosis are both
inhibited by the caspase-inhibitor zVAD-fmk, this, together with the
synergy for apoptosis observed with these agents, implies that NF-
B
or an NF-
B regulated step influences granulocyte apoptosis at an
intermediate step between the TNF-
receptor and caspase activation.
The possibility that NF-
B controls the transcriptional activity of a
gene(s) which induces the synthesis of survival proteins is supported
by the observation that cycloheximide also increases apoptosis in
granulocytes (49). This suggests a strong link between inducible
NF-
B activation and the control of TNF-
-induced apoptosis,
possibly via the production of a protein inhibitor of this pathway.
Indeed, as we have shown, protein synthesis inhibition enhances the
pro-apoptotic effect of TNF-
as early as 2 h of culture.
Indeed, one possible candidate for this protein has already been
suggested: A20, a protein induced by TNF-
activation of NF-
B (56,
57), has been shown to protect against TNF-
induced cell death by
acting at the level of the TNF-
receptor-associated proteins TRAF-1
and TRAF2 (58). Although A20 has yet to be demonstrated in neutrophils
or eosinophils, this would represent an attractive candidate protein to
fulfill such a role.
and itself produce a rapid onset of apoptosis in inflammatory cells
such as neutrophils and eosinophils may suggest NF-
B inhibition as a
logical therapeutic target in the treatment of inflammatory conditions.
In a rat model of lung inflammation, suppression of NF-
B activity
has already been shown to block the development of neutrophil lung
inflammation by inhibiting the synthesis of chemotaxins (59). Our
results suggest that NF-
B inhibition may also be of benefit in
enhancing the resolution of inflammation by allowing a more rapid
clearance of granulocytes. We therefore propose that granulocyte
apoptosis is regulated by an inducible form of the transcription factor
NF-
B and suggest that inhibition of this transcription factor may be
exploited for therapeutic benefit in inflammatory conditions where
granulocytes play a prominent role.
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FOOTNOTES |
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* This work was supported in part by Medical Research Council Program Grant G9016491 and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Glaxo-Wellcome studentship.
§ Wellcome Senior Research Fellow in Clinical Science.
To whom correspondence should be addressed: Respiratory
Medicine Unit, Dept. of Medicine, Rayne Laboratory, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland, United Kingdom. Tel.: 44-131-651-1323; Fax: 44-131-650-4384; E-mail: a.g.rossi{at}ed.ac.uk.
The abbreviations used are:
TNF-, tumor
necrosis factor-
; EMSA, electrophoretic mobility shift assay; LPS, lipopolysaccharide; NF-
B, nuclear factor-
B; PDTC, pyrrolidine
dithiocarbamate; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone: DMEM, Dulbecco's
modified Eagle's medium.
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REFERENCES |
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