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INTRODUCTION |
Interleukin-13 (IL-13)1
is a pleiotropic cytokine secreted by activated Th-2 lymphocytes which
regulates a variety of immune target cells (1, 2). In B lymphocytes,
IL-13 induces proliferation and differentiation, promotes CD23
expression and production of certain immunoglobulins such as IgG4 and
IgE (3-5). In monocytes, IL-13 induces morphological changes (2),
up-regulates expression of members of the integrin superfamily and
major histocompatability complex class II antigen expression, and
down-regulates expression of CD14 and Fc
R receptors (6). In
lipopolysaccharide-stimulated monocytes, IL-13 also acts as a
suppresser of proinflammatory cytokines (e.g. TNF type
,
IL-1, and IL-6), chemokines (e.g. IL-8 and macrophage
inflammatory protein-1
), and hematopoietic growth factors
(e.g. granulocyte/macrophage-colony stimulating factor and
granulocyte-colony stimulating factor) expression by activated
monocytes/macrophages or endothelial cells (1, 6). Another target of
IL-13 is epithelial cells and we have recently demonstrated that IL-13
can modulate chemokine generation from the human colonic epithelial
cell line HT-29 (7, 8) and inhibit iNOS expression and NO generation in
this system (9). Activation of the signaling enzyme
phosphatidylinositol 3-kinase (PI 3-kinase) by IL-13 is important for
mediating these effects (8, 10). PI 3-kinase and its major downstream
effector, the serine/threonine kinase protein kinase B (PKB), have been
shown to be key mediators of growth factor-induced cell survival and protection against c-Myc-induced cell death in fibroblasts (11-13). In
addition, IL-13 has been reported to up-regulate the cell survival factors Bcl-xL and Mcl-1 as well as protect B lymphocytes
from apoptosis (14). Moreover, the related cytokine, IL-4, also
enhances cell survival (15) and this correlates well with observations that both IL-13 and IL-4 stimulate PI 3-kinase (10, 16) and with the
notion that the IL-13 receptor and IL-4 receptor share a common subunit
in signal transduction (17-19).
We have previously shown that a combination of pro-inflammatory
cytokines (IL-1
/IFN-
/TNF-
) up-regulates iNOS expression and
generates NO in a human colonic epithelial cell line HT-29 (20). Also,
it has recently been shown that IFN-
, in combination with TNF-
or
anti-CD95, induces apoptosis in HT-29 cells (21) and an increased
frequency of epithelial apoptosis mediated by the CD95-CD95L system
is seen in ulcerative colitis (22, 23), which is an inflammatory bowel
disease of unknown etiology. However, colonic epithelial cell injury,
resulting in impaired barrier function, could contribute to the
pathogenesis of inflammatory bowel disease (24). It has been postulated
that overproduction of nitric oxide (NO) by inflamed mucosa may play a
role in the pathophysiology of inflammatory bowel disease due to the
increased expression of the inducible form of nitric oxide synthase
(iNOS) found in biopsies taken from patients with active ulcerative
colitis as compared with normal colon (9). The production of NO might play a critical role in the resolution of inflammation (25), possibly
by inducing apoptosis in the leukocytic population recruited to the
area (e.g. neutrophils) (25). While NO has also been reported to inhibit apoptosis in several settings (26-29), it has also
been reported to mediate cell death through mechanisms consistent with
apoptosis in various cells including peritoneal macrophages (30-32),
-cells (33, 34), and thymocytes (35). Also, overproduction of NO may
lead to oxidant-induced injury of the colon epithelial crypt (36),
possibly by the reaction with superoxide to form peroxynitrite which in
turn results in the nitration of proteins on tyrosine residues
(37).
In this study, we sought to ascertain whether there is a relationship
between NO production and apoptosis of HT-29 epithelial cells observed
in response to a combination of cytokines and/or CD95 ligation. In
addition, given that the ability of IL-13 to inhibit iNOS expression
and NO generation in this system is driven by PI
3-kinase-dependent pathway, we investigated whether IL-13 could provide a cell survival signal through PI 3-kinase to protect against cytokine-driven apoptotic signals.
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EXPERIMENTAL PROCEDURES |
Materials--
Human recombinant IL-13 was purified from culture
supernatants of stable transfected CHO cells (1) and generously
provided by Dr. A. Minty (Sanofi Elf Bio Recherche, Labège,
France). Human recombinant IL-1
(specific activity, 5 × 107 units/mg) and TNF-
(specific activity, 6 × 107 units/mg) were generous gifts from Glaxo-Wellcome
(Stevenage, United Kingdom) and Bayer (Slough, UK), respectively. Human
recombinant IFN-
(specific activity, > 2.0 × 107
units/mg) and histone H2B was purchased from Roche Molecular Biochemicals. Anti-human Fas monoclonal antibody (IgM CH11 clone) was
purchased from Upstate Biotechnology. PKB
polyclonal Ab was from
Brian Hemmings (Friedrich Miescher-Institute, Basel, Switzerland). 2,3-Diaminonaphthalene was purchased from Lancaster Synthesis Ltd.
LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) was
purchased from Affiniti (Exeter, UK), Z-VAD-FMK was purchased from
Calbiochem (Nottingham, UK), and [
-32P]ATP (3000 Ci/mmol) was purchased from DuPont NEN, UK. All other reagents were
from Sigma (Poole, UK).
Cell Culture--
The human colonic epithelial carcinoma cell
line HT-29 was obtained from the European Collection of Animal Cell
Cultures. Cells were cultured in humidified incubators at 37 °C, 5%
CO2 in McCoy's 5A medium supplemented with 10% fetal
bovine serum, 10 units/ml penicillin/streptomycin, and 10 µg/ml
fungizone. The cells were passaged weekly and, for experiments, HT-29
cells were aliquoted into 96-well plates (104 cells/well)
and allowed to adhere overnight prior to cytokine stimulation in medium
without serum.
Detection of Apoptosis--
The detection of apoptosis was
performed by determining the histone-associated DNA fragments (mono-
and oligonucleosomes) generated by the apoptotic cells using the
photometric cell death detection ELISAPLUS (Roche Molecular
Biochemicals). Briefly, 104 cells/well were aliquoted into
96-well plates and allowed to adhere overnight. Cells were then treated
and pelleted as indicated, the supernatants were removed and the cell
pellets were lysed and assayed according to the manufacturer's
instructions. Photometric analysis, with
2,2'-azino-di-(3-ethylbenzathiazoline sulfonate) as the substrate, was
performed using a microtiter plate-reader (Dynatech MR5000). The
specific enrichment of mono- and oligonucleosomes released into the
cytoplasm was calculated using the following formula,
|
(Eq. 1)
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Alternatively, apoptosis was determined by direct fluorescence
detection of end-labeled genomic DNA using the "Oncor Apoptag Direct
In Situ Apoptosis Detection Kit Fluorescein" (Appligene, Durham, UK). Treated and untreated cells were spun onto slides, fixed,
and stained according to the manufacturer's instructions. The slides
were observed under a fluorescence microscope. The results were scored
by counting apoptotic cells (green) and viable cells (red) randomly in
various fields. For each condition, 500 to 1000 cells were counted. In
some experiments, apoptosis was detected by measuring the
externalization of phosphatidylserine by fluorescein
isothiocyanate-labeled annexin V binding using the Apoptosis Detection
Kit (R&D Systems, Abingdon, UK). Cells were treated as indicated in the
figure legends and stained according to the manufacturers instructions
before analysis by flow cytometry (Becton Dickinson FACS Vantage).
Percent increases in annexin V fluorescein binding above controls were measured.
Fluorometric Assay for Nitric Oxide--
NO production by HT-29
cells was determined by measuring the stable end-product nitrite in the
cell culture supernatants by fluorometric assay which is based upon the
reaction of nitrite with 2,3-diaminonaphthalene to form the fluorescent
product 1-(H)-naphthotriazole as described previously (10, 38).
Detection of Caspase Activity--
Colorimetric protease assay
kits for both caspase-8 and -3 (Chemicon Intl. Inc., Temecula, CA) are
based on spectrophotometric detection of the chromophore
p-nitroanilide (pNA) after cleavage from the
labeled substrate DEVD-pNA. Briefly, 3 × 106
cells/well were aliquoted into 96-well plates and allowed to adhere
overnight. Cells were then treated as indicated and pelleted after
24 h to include any floating cells. The supernatants were removed
and the cell pellets were lysed and assayed according to the
manufacturers instructions. After a 2-h incubation with DEVD-pNA substrate, the pNA light emission was
quantified using a microtiter plate reader at 405 nm. Comparison of the
absorbance of pNA from the apoptotic sample with uninduced control
determined the fold increase in caspase activity.
Cell Lysis--
107 cells/ml were stimulated and
incubated at 37 °C in McCoys as indicated. Reactions were terminated
by the addition of 1 ml of ice-cold lysis buffer (1% (v/v) Nonidet
P-40, 150 mM NaCl, 50 mM Tris pH 7.5, 5 mM EDTA, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml soybean trypsin inhibitor, 1 µg/ml
pepstatin A, 1 mM sodium orthovanadate, and 1 mM sodium molybdate). Lysates were incubated at 4 °C for 15 min, followed by centrifugation at 14,000 rpm.
Protein Kinase B Assays--
Aliquots of cell lysate supernatant
were boiled in Laemmli buffer and electrophoresed through 12.5% (v/v)
acrylamide gels (with an acrylamide:bis-acrylamide ratio of 37.5:1) by
SDS-PAGE and the proteins were transferred by electroblotting onto
nitrocellulose (Schleicher & Schuell) as described previously (10, 39).
The blots were probed with a phosphospecific PKB antibody (0.5 µg/ml) which only has affinity for the active,
473Ser-phosphorylated forms of PKB (New England BioLabs)
and proteins visualized by ECL with a goat anti-rabbit Ig (0.1 µg/ml)
conjugated with horseradish peroxidase as a secondary antibody. Where
appropriate, blots were completely stripped of antibodies by incubation
at 55 °C for 60 min with stripping solution (62.5 mM
Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-mercaptoethanol).
After extensive washing, blots were reblocked prior to reprobing with
anti-PKB antibody (0.5 µg/ml) (New England Biolabs). Alternatively,
cells were stimulated as described above and PKB
was
immunoprecipitated from cell lysates with 1 µg of anti-PKB (Santa
Cruz) and assayed for in vitro kinase activity using histone
H2B as a substrate as described previously (40).
Immunoprecipitation of Bad--
Bad was immunoprecipitated
from cell lysates with 4 µg of anti-Bad mAb (Transduction Labs).
After addition of 30 µl of protein G-Sepharose beads (50% suspension
in phosphate-buffered saline), immunoprecipitates were rotated for
1 h at 4 °C and then washed three times in lysis buffer,
resuspended in Laemmli buffer, and boiled for 5 min prior to
electrophoresis through 12.5% (v:v) acrylamide gels (with an
acrylamide:bis-acrylamide ratio of 118:1) by SDS-PAGE. The proteins
were transferred by electroblotting onto nitrocellulose (Schleicher & Schuell) as described previously (10, 39). Bad was immunoblotted with
0.5 µg/ml anti-Bad polyclonal antibody (Santa Cruz) and proteins
visualized by a chemiluminescence detection system (ECL, Amersham, UK)
with a goat anti-rabbit Ig (0.1 µg/ml) conjugated with horseradish
peroxidase as a secondary antibody (DAKO).
 |
RESULTS |
Pro-inflammatory Cytokines Induce Apoptosis of Colon Epithelial
Cells Which Is Inhibited by IL-13--
Growth-arrested HT-29
monolayers stimulated with a combination of the pro-inflammatory
cytokines IL-1
(10 ng/ml), IFN-
(300 units/ml)), and TNF-
(100 ng/ml) result in a 25-80% increase in the expression of apoptotic
markers, as assessed by a number of assays which measure early and late
stage markers of apoptotic events (namely externalization of
phosphatidylserine, DNA fragmentation, and DNA-histone association),
over a time course of 4-24 h (Table I
and Fig. 1). In contrast, treatment with
individual cytokines was insufficient to increase cell death above
basal levels (Table I). Pretreatment of the HT-29 cells with the
anti-inflammatory cytokine IL-13 (30 ng/ml) markedly inhibited
apoptotic events stimulated by the pro-inflammatory cytokines (Table I
and Fig. 1). These experiments indicate that IL-13 consistently
inhibited the induction of apoptotic markers in cells treated with the
combined stimuli of IL-1
/IFN-
/TNF-
by 50-65%. Treatment of
HT-29 cells for 24 h with IL-13 had no effect on the level of
expression of TNF receptor-1 as assessed by flow cytometry using a
receptor-specific Ab, thus confirming previous observations (41).
Similarly, flow cytometry using appropriate receptor Abs also revealed
that IL-13 had no effect on the levels of expression of IL-1 receptor
(type-1) or IFN-
receptor (data not shown), indicating that
down-regulation of receptor expression cannot account for the observed
reduction in cell death.
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Table I
IL-13 protects against IL-1 /IFN- /TNF- -induced apoptosis of
HT-29 cells
Growth arrested monolayers of HT-29 cells were either left untreated or
treated with 30 ng/ml IL-13 for 1 h. Where indicated, the cells
were then treated with IL-1 (10 ng/ml), IFN- (300 units/ml), and
TNF- (100 ng/ml) or left untreated. After the times indicated, the
expression of apoptotic markers by HT-29 cells was determined using
either the commercial fluorescein isothiocyanate-labeled annexin V
binding kit or the Apoptag apoptosis detection kits as described under
"Experimental Procedures." The data are the mean ± S.E. of 5 separate experiments.
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Fig. 1.
IL-1 /IFN- /TNF- -induced
apoptosis in HT-29 cells is inhibited by IL-13. HT-29 cells
(104 cells) were either left untreated ( , control) or
treated with 30 ng/ml IL-13 ( , IL-13 + IL-1 /IFN- /TNF-
(3C)) for 1 h. Where indicated, the cells were then
further treated with IL-1 (10 ng/ml) TNF- (100 ng/ml), and
IFN- (300 units/ml) ( , IL-1 /IFN- /TNF- ) or left untreated
( , control). At the times indicated, the supernatants were removed
and the cell pellets were lysed and apoptosis determined using the
ELISAPLUS assay as described under
"Experimental Procedures." The data are the mean ± S.E. of three separate experiments.
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IFN-
Is Required in Combination with the Death-inducing Factors
TNF-
or CD95 Ligation to Induce Apoptosis--
Ligation of CD95
(Fas/APO-1), which is a member of the TNF receptor superfamily, is
associated with the induction of apoptosis in several cell types (42)
and flow cytometry has revealed the presence of CD95 on HT-29 cells
used in this study (data not shown). We therefore performed experiments
to investigate whether or not CD95 ligation stimulates apoptosis of
HT-29 cells. In this respect, ligation of CD95 with the antibody CH11
did not induce apoptosis above control basal levels (Fig.
2). However, treatment of HT-29 cells
with IFN-
in combination with the anti-CD95 mAb CH11, induced an
approximate 9-fold increase in DNA fragmentation over 24 h, which
was comparable to the apoptosis induced by IFN-
and TNF-
(Fig.
2). By contrast, the minimum cytokine combination for the induction of
iNOS and NO generation, namely IL-1
/IFN-
(20), induced only a
5-fold increase in DNA fragmentation above basal levels. Preincubation
with IL-13 again reduced the prevalence of apoptotic markers
induced by IFN
/CH11 and IFN-
/TNF-
by approximately 50%, but
did not inhibit apoptosis driven by IL-1
/IFN-
(Fig. 2).

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Fig. 2.
IFN- is required in
combination with the death-inducing factors TNF-
or CD95 ligation to induce apoptosis. HT-29 cells
(104 cells) were either left untreated or treated with 30 ng/ml IL-13 for 1 h. Where indicated, the cells were then further
treated with combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml),
IFN- (300 units/ml), or CH11 (100 ng/ml) or left untreated. After
24 h, the supernatants were removed and the cell pellets were
lysed and apoptosis determined using the ELISAPLUS assay as
described under "Experimental Procedures." The data are the
mean ± S.E. of three separate experiments.
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Cytokine-driven iNOS Activation and Apoptosis Are Independent
Functional Events--
Several studies have demonstrated that nitric
oxide regulates apoptosis in a number of settings (reviewed in Ref.
43). Given that concentrations of IL-1
/IFN-
/TNF-
that are
known to induce iNOS expression and that NO production can also
stimulate apoptosis, we considered the possibility that this apoptotic
response may be NO-dependent. To investigate this
possibility we pretreated HT-29 cells with the iNOS inhibitor 500 µM aminoguanidine, which markedly inhibited the
concentration of nitrite generated by IL-1
/IFN-
/TNF-
(although
less inhibition was observed with the minimum combination of
IL-1
/IFN-
) over 24 h (Fig.
3A), but had no effect on the apoptotic signals provided by combinations of either
IL-1
/IFN-
/TNF-
or IL-1
/IFN-
(Fig. 3B).

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Fig. 3.
Effect of iNOS inhibitor on cytokine-induced
nitrite production and apoptosis. A, HT-29 cells
(3.5 × 106 cells) were treated for 10 min with
vehicle or with 500 µM aminoguanidine (AG) as
indicated at 37 °C. The cells were then further treated with
combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml), or IFN-
(300 units/ml) as indicated or left untreated. Nitrite production after
the indicated treatments was determined in
supernatants after 24 h at 37 °C as described under
"Experimental Procedures." The data are the mean ± S.E. of
three separate experiments. B, HT-29 cells (104
cells) were treated for 10 min with vehicle or with 500 µM aminoguanidine (AG) as indicated and then
treated with combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml),
and IFN- (300 units/ml) as indicated or left untreated for 24 h. Supernatants were removed and the cell pellets were lysed and
apoptosis determined using the ELISAPLUS assay as described
under "Experimental Procedures." The data are the mean ± S.E.
of three separate experiments.
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Initial apoptotic events are known to result in the activation of the
proteolytic enzyme cascades involving caspases which cleave specific
proteins and irreversibly commit the cell to apoptotic death (44).
Inhibition of this proteolytic cascade can be achieved using a broadly
selective caspase inhibitor Z-VAD-FMK (45). We therefore used this
caspase inhibitor to assess whether cytokine-driven iNOS production was
dependent upon activation of caspases. Hence, pretreatment of cells
with 50 µM Z-VAD-FMK completely abrogates the apoptosis
of HT-29 cells stimulated by IL-1
/IFN-
/TNF-
, IL-1
/IFN-
,
IFN-
/TNF-
, and IFN-
/CH11 (Fig.
4A). However, the caspase
inhibitor did not interfere with the observed ability of HT-29 cells to
generate nitrite in response to IL-1
/IFN-
/TNF-
or
IL-1
/IFN-
(Fig. 4B), as does pretreatment with IL-13.
Furthermore, IFN
/TNF
(Fig. 4B) and IFN
/CH11 (data
not shown) are not able to induce iNOS or stimulate the generation of
nitrite, but are able to stimulate apoptosis (Fig. 4A).
Together, these data indicate that cytokine-driven iNOS activation and
apoptosis are separable, independent events.

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Fig. 4.
Effect of the caspase inhibitor Z-VAD-FMK on
cytokine-induced nitrite production and apoptosis. A,
HT-29 cells (104 cells) were either left untreated or
treated with 50 µM Z-VAD-FMK for 1 h. Where
indicated, the cells were then further treated with combinations of
IL-1 (10 ng/ml), TNF- (100 ng/ml), IFN- (300 units/ml), or
CH11 (100 ng/ml) or left untreated. After 24 h at 37 °C, the
supernatants were removed and the cell pellets were lysed and
apoptosis determined by the ELISAPLUS assay as
described under "Experimental Procedures." The data are the
mean ± S.E. of three separate experiments. B, HT-29
cells (3.5 × 106 cells) were treated for 10 min with
vehicle or with 50 µM Z-VAD-FMK as indicated and then
treated with combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml),
and IFN- (300 units/ml) as indicated or left untreated for 24 h
at 37 °C. Nitrite production after the indicated treatments was
determined in supernatants as described under "Experimental
Procedures." The data are the mean ± S.E. of three separate
experiments.
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Reversal of IL-13-induced Inhibition of Apoptosis by Wortmannin and
LY294002--
We have previously shown that IL-13 strongly activates
the lipid kinase PI 3-kinase (10) which is believed to be a pivotal upstream component of a signaling cascade important in promoting cell
survival events in many cell types (11-13, 46). Hence, to investigate
the role of PI 3-kinase in mediating the anti-apoptotic effects of
IL-13 on IL-1
/IFN-
/TNF-
-stimulated apoptosis of HT-29 cells,
the PI 3-kinase inhibitors wortmannin and LY294002 were used.
Preincubations of wortmannin (10-300 nM) for 10 min before
cytokine treatments were able to dose dependently reverse the ability
of IL-13 to protect HT-29 cells from cytokine-induced apoptosis (Fig.
5A). Equally, the structurally
unrelated PI 3-kinase inhibitor, LY294002 (1-30 µM), was
also able to reverse the IL-13 effect (Fig. 5A). Similarly,
treatment of HT-29 cells with either wortmannin (Fig. 5B) or
LY294002 (data not shown), also prevented the IL-13-mediated inhibition
of apoptosis induced by either IFN-
/TNF-
or IFN-
/CH11

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Fig. 5.
PI 3-kinase inhibitors prevent IL-13
suppression of cytokine-induced apoptosis of HT-29 cells. HT-29
cells (104 cells) were treated for 10 min with vehicle,
wortmannin, or LY294002 as indicated at 37 °C. Cells were then
either left untreated or treated with 30 ng/ml IL-13 for 1 h.
Where indicated, the cells were then further treated with combinations
of IL-1 (10 ng/ml), TNF- (100 ng/ml), IFN- (300 units/ml), or
CH11 (100 ng/ml) or left untreated. After 24 h, the supernatants
were removed and the cell pellets were lysed and apoptosis
determined using the ELISAPLUS assay as described
under "Experimental Procedures." The data are the mean ± S.E.
of three separate experiments.
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IL-13 Activates the PI 3-Kinase Effector Protein Kinase B--
PKB
is a major downstream effector of the PI 3-kinase-dependent
signaling cascade and has been shown to be a key mediator required for
growth factor-induced cell survival and protection against
c-Myc-induced cell death in fibroblasts (11-13). We therefore investigated whether the protective affects of IL-13 on
IL-1
/IFN-
/TNF-
-stimulated apoptosis in HT-29 cells, correlated
with IL-13 activation of PKB. Hence, cell lysates derived from resting
and IL-13-stimulated cells were immunoblotted using a phosphospecific
antibody to the phosphorylated active form of PKB. IL-13 can be shown
to activate PKB within 5 min stimulation, up to a maximum at 10 min,
and is comparable in magnitude with PKB activation observed in response to insulin (5 µg/ml) treatment as a positive control (Fig.
6A). Insulin, however,
provided only a 10-20% protective effect against cytokine-induced
apoptosis (data not shown). The IL-13-stimulated activation of PKB
appears to be sustained for up to 20 min, but has returned to control
levels after 1 h. Both wortmannin and LY294002 were able to
inhibit this signal, adding further evidence that the activation of PI
3-kinase by IL-13 leads to the activation of PKB and that this pathway
is anti-apoptotic in this system. Blots were stripped and reprobed with
an anti-PKB antibody provided in the kit to verify equal loading and
efficacy of protein transfer (Fig. 6A). In addition,
endogenous PKB was immunoprecipitated from IL-13-stimulated cells in
the presence and absence of the PI 3-kinase inhibitor, wortmannin, and
the immunoprecipitates were assayed for in vitro PKB
activity. This approach confirmed that PKB
was activated by IL-13 in
a PI 3-kinase-dependent manner (Fig. 6B).

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Fig. 6.
IL-13 stimulates activation of the PI
3-kinase effector PKB. A, HT-29 cells (107)
were treated for 10 min with vehicle (left panel), 30-300
nM wortmannin (middle panel), or 1-30
µM LY294002 (right panel) as indicated at
37 °C. Cells were then left unstimulated or further treated with 30 ng/ml IL-13 or 5 µg/ml insulin at 37 °C for the times indicated.
The HT-29 cells were lysed and the lysates were immunoblotted with a
phosphospecific PKB antibody with affinity for the
Ser473-phosphorylated active form of PKB as described under
"Experimental Procedures." Data is from a single experiment
representative of at least three others. B, 107
cells were stimulated with 30 ng/ml IL-13 at 37 °C for the times
indicated with or without a 10-min preincubation of 100 nM
wortmannin. After cell lysis and immunoprecipitation with PKB ,
immunocomplexes were subjected to an in vitro kinase assay
as described under "Experimental Procedures." Data are
from a single experiment representative of at least three others.
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The mechanism by which PKB is believed to promote cell survival
involves the serine phosphorylation of the death promoting Bcl-2 family
member Bad causing it to dissociate from Bcl-xL, thus
allowing Bcl-xL to act as a survival factor (47, 48). Immunoblotting of HT-29 whole cell extracts revealed no detectable amounts of Bad (data not shown). However, immunoprecipitation and
immunoblotting of Bad, after electrophoresis through a low-bis 12.5%
acrylamide gel, revealed a barely detectable hyperphosphorylated form
of Bad as characterized by its shift in gel mobility (Fig. 7).

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Fig. 7.
Effect of IL-13 stimulation on Bad in HT-29
cells. HT-29 cells (107) were stimulated with 30 ng/ml
IL-13 at 37 °C for the times indicated. The HT-29 cells were lysed
and the lysates were subjected to immunoprecipitation with anti-Bad
mAb. The immunoprecipitates were washed and proteins separated by
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Bad
antibody as described under "Experimental Procedures." Data are
from a single experiment representative of at least three others.
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Effect of IL-13 on Cytokine-induced Activation of the Caspase
Cascade--
Activation of the caspase cascade is pivotal to the death
execution phase of apoptosis and it appears that caspase-8 is the apical member of the pathway induced by CD95 and TNF receptor-1 with
caspase-3 lying downstream (49-51). Hence, we investigated whether the
induction of apoptotic markers induced by various cytokine
combinations also correlated with activation of caspase-8 and -3. Indeed, treatment of cells with IL-1
/IFN-
/TNF-
and IFN-
/TNF
(Fig. 8A), but
not IL-1
/IFN-
(data not shown) stimulated caspase-8 and caspase-3
activity. In comparison, while treatment with IL-1
did not stimulate
caspase activity, both TNF-
and IFN-
elicited modest activation
of these caspases (Fig. 8A). Interestingly, pretreatment of
HT-29 cells with IL-13 consistently induced a partial inhibition of
IL-1
/IFN-
/TNF-
-stimulated caspase-8 and caspase-3 activity at
early time points (e.g. 12 h) which may constitute a
delay in caspase activation by IL-13 (Fig. 8, B and
C).

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Fig. 8.
Effect of IL-13 on
IL-1 /IFN- /TNF-
induced activation of caspase-8 and caspase-3 in HT-29
cells. A, HT-29 cells (3 × 106 cells)
were either left untreated or treated with combinations of IL-1 (10 ng/ml), TNF- (100 ng/ml), or IFN- (300 units/ml) as indicated.
B and C, HT-29 cells (3 × 106
cells) were either left untreated or treated with 30 ng/ml IL-13 for
1 h. Where indicated, the cells were then further treated with
IL-1 (10 ng/ml), TNF- (100 ng/ml), and IFN- (300 units/ml) or
left untreated. After 24 h (A) or the times indicated
(B and C), cells were pelleted to include any
floating cells, the supernatants were removed and the cell pellets were
lysed and assayed for caspase-8 and caspase-3 activity as described
under "Experimental Procedures." Data are the mean of triplicates
from one experiment ± S.E., representative of three independent
experiments.
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 |
DISCUSSION |
In this report we demonstrate that a combination of
proinflammatory cytokines, namely IL-1
/IFN-
/TNF-
,
stimulates the expression of apoptotic markers in approximately
25-80% of cytokine-treated HT-29 cells (depending on time of
analysis) as evidenced by assays that detect DNA fragmentation and
externalization of phosphatidylserine. Apoptosis can also be stimulated
to varying extents by the combination of IL-1
/IFN-
or
IFN-
/TNF-
. Moreover, we present evidence that the induction of
these apoptotic markers is not dependent on the expression of iNOS and
NO production. Furthermore, pretreatment with the anti-inflammatory
cytokine IL-13 which is known to prevent induction of NO production by
HT-29 cells in response to IL-1
/IFN-
/TNF-
(9, 10), also
protects against IL-1
/IFN-
/TNF-
-, IFN-
/TNF-
-, and
IFN-
/CH11-induced (but not IL-1
/IFN-
-induced) cell death in
this system via a PI 3-kinase-dependent mechanism. This
also correlates with the first demonstration that IL-13 stimulates activation of the major downstream PI 3-kinase effector PKB, which is
thought to mediate the promotion of cell survival by this signaling pathway in a number of cell systems (11-13). However, IL-13-induced phosphorylation of Bad (a known downstream target of PKB) was barely
detectable, suggesting that this is an unlikely target for PKB activity
in this system. In addition, IL-13 pretreatment partially inhibited,
but did not prevent cytokine-stimulated activation of either caspase-8
or caspase-3.
Individually, IL-1
, IFN-
, and TNF-
do not induce apoptosis
of HT-29 cells. However, our observation that combinations of IL-1
,
IFN-
, and TNF-
induce apoptosis of HT-29 cells, correlates well
with recent reports that IFN-
increases the sensitivity of HT-29
cells to pro-apoptotic agents such as TNF-
by directly and
indirectly inducing select apoptosis-related genes (52). In addition,
we have previously reported that this combination of cytokines
stimulates NO production from HT-29 cells and there is considerable
evidence that NO can promote apoptosis in other systems (43). Indeed,
iNOS transcripts can be detected 6 h after cytokine treatment (20)
and this appears to precede cell death which is detectable at 8 h
post-cytokine stimulation. However, there are several lines of evidence
to indicate that cytokine-driven iNOS and apoptosis are independent
functional events. First, the iNOS inhibitor aminoguanidine prevented
IL-1
/IFN-
/TNF-
and IL-1
/IFN-
-induced NO production, but
had no effect on the apoptosis stimulated by these combinations of
cytokines. Second, apoptosis can also be stimulated by IFN-
/TNF-
and IFN-
/CH11 which are unable to stimulate NO production. Third,
inhibition of IL-1
/IFN-
/TNF-
-induced apoptosis by Z-VAD-FMK
had no effect on NO production induced by these cytokines. Although
markers of cytokine-stimulated apoptosis, such as DNA fragmentation and
phosphatidylserine externalization, are detectable from 8 to 24 h,
time course experiments have revealed that other functional responses
continue unabated. For instance, identical cytokine treatment can also
stimulate up-regulation of iNOS and chemokine mRNA up to 24 h
post-stimulation (7, 20) and these responses can be down-regulated by
pretreatment with IL-13 (7, 8, 10). So, cytokine-induced expression of
apoptotic markers and events do not necessarily correlate with abrogated cell function, at least in the time frame studied here.
Activation of the proteolytic cascade by caspases appears to be
essential to cytokine-induced apoptosis of HT-29 cells, given our
observation that pretreatment with the caspase inhibitor Z-VAD-FMK completely prevents apoptosis induced by IL-1
/IFN-
/TNF-
,
IL-1
/IFN-
, IFN-
/TNF-
, and IFN-
/CH11. This is
particularly interesting given that both IL-1
and TNF-
(53) and
CD95 (54) ligation have been reported to activate the ceramide pathway,
which has also been implicated as a signaling pathway involved in
apoptosis (55, 56). However, since the apoptosis of HT-29 cells
stimulated by these cytokine combinations is completely inhibited by
the caspase inhibitor Z-VAD-FMK, this may indicate that ceramide
production is not sufficient for cell death in this system. Indeed, it
has recently been shown that IFN-
was unable to induce changes in sphingolipid levels in HT-29 cells (57), suggesting that
ceramide-mediated signaling pathways may be cell-type specific. It is
also interesting to note that while treatment of HT-29 cells with
TNF-
or IFN-
resulted in modest stimulation of caspases -8 and
-3, this is insufficient to drive cell death, since neither TNF-
nor
IFN-
stimulated apoptosis in this system. This is in marked contrast to the TNF-
-induced apoptosis observed in neutrophils and T
lymphocytes which correlates well with caspase activation (58, 59).
Even though IL-13 exerts a protective effect against cell death induced
by IL-1
/IFN-
/TNF-
, IFN-
/TNF-
, and IFN-
/CH11, IL-13
pretreatment was unable to completely inhibit cytokine-activated caspase-8 and caspase-3. Rather, it appears that IL-13 pretreatment delays activation of these caspases by IL-1
/IFN-
/TNF-
and in this respect it is interesting to note that IL-13 provides only partial
protection against cell death induced by IL-1
/IFN-
/TNF-
and
IFN-
/TNF-
. Caspase activation is required for the execution of
cell death in an apoptotic manner (reviewed in 44), but the order of
caspase activation cascades is not absolute and the commitment to live
or die may originate from the mitochondria (reviewed in Ref. 60).
Hence, while IL-13 partially inhibits and possibly delays activation of
caspase-8 and caspase-3, there may well be additional targets of
IL-13-activated biochemical signals that mediate cell survival at some
point distal to the apical caspase-8 and the downstream caspase-3,
possibly involving mitochondrial activity. It is certainly possible
that other upstream and downstream caspases are activated by the
cytokine combinations used in this study. Indeed, it has recently been
shown that PKB can phosphorylate caspase-9 and inhibit its protease
activity (61). This would fit nicely with our observations that IL-13
can provide only partial protection against cell death induced by
IL-1
/IFN-
/TNF-
and IFN-
/TNF-
, whereas apoptosis
stimulated by IL-1
/IFN-
was unaffected by IL-13. Hence, it
appears that multiple death promoting pathways with different
sensitivity to IL-13-activated cell survival mechanisms are activated
by the cytokine combinations used in this study.
The protective effects of IL-13 against IL-1
/IFN-
/TNF-
-,
IFN-
/TNF-
-, and IFN-
/CH11-stimulated apoptosis are dependent on the PI 3-kinase-dependent signaling pathway, since the
PI 3-kinase inhibitors wortmannin and LY29002 abrogated the protective
effects of IL-13. These observations are consistent with demonstrations that the PI 3-kinase-dependent signaling pathway and in
particular its downstream effector PKB are involved in growth
factor-dependent cell survival (11-13, 46). Indeed, we
have previously shown that IL-13 strongly activates PI 3-kinase as
evidenced by PtdIns (3,4,5)-P3 accumulation (10). Moreover,
data in this study demonstrates that IL-13 also activates PKB and this
activation is abrogated by pretreatment with PI 3-kinase inhibitors.
PKB is now known to promote cell survival by phosphorylating a critical
serine residue (136Ser) on the death-promoting protein Bad,
causing it to dissociate from and thus allow activation of the cell
survival factor, Bcl-xL (47, 48). However, consistent with
observations from other groups (52), Bad is expressed at very low
levels in HT-29 cells, such that the band shift of Bad to the
serine-phosphorylated form was barely detectable. It would seem
unlikely, therefore, that the cell survival effects of IL-13 are solely
mediated by PKB phosphorylation of Bad in the system described here.
However, there are two alternative explanations to account for
IL-13-stimulated PI 3-kinase/PKB-dependent cell survival
mechanisms. First, other death promoting Bcl-2 family proteins may be
regulated by PKB-dependent phosphorylation in a manner
similar to that described for the regulation of Bad (47, 48). Indeed,
expression of the related Bcl-2 family member Bak, which can also
promote cell death, has been reported to be directly induced by IFN-
(52). However, while Bak is expressed, we could not detect any
IL-13-stimulated hyperphosphorylation of Bak by immunoblotting (data
not shown). Nevertheless, it remains possible that other Bcl-2 family
proteins may act as targets for IL-13-activated PKB. Second, an
alternative target for the PI 3-kinase-dependent cell
survival signals provided by IL-13 may be the transcription factors of
the NF-
B family which have been reported to be important in cell
survival by regulating unidentified, antiapoptotic genes (62).
Recent evidence has identified the inhibitor of apoptosis (IAP)
proteins c-IAP1 and c-IAP-2 as gene targets of NF-
B transcriptional
activity (63). The c-IAP1 and c-IAP2 proteins specifically inhibit the
active forms of caspase-3 and caspase-7 (62). In other systems such as
T lymphocytes, activation of NF-
B has been reported to be dependent
on p70 S6 kinase (64), which in turn has been reported to be a target
for phosphorylation by either PKB (65) and/or its upstream kinase(s)
PDK-1 and the putative PDK-2 (66, 67). Hence, one possibility is that
the observed cell survival effects of IL-13 involves PI
3-kinase-dependent activation of NF-
B transcriptional activity, although this hypothesis does not fit easily with the recent
report demonstrating that IL-13 down-regulates TNF-
-mediated activation of NF-
B (41).
In summary, apoptosis of HT-29 epithelial cells observed in response to
a combination of cytokines and/or CD95 ligation is not dependent on NO
production. In addition, IL-13 can provide a PI
3-kinase-dependent cell survival signal to HT-29 cells
which protects against cytokine-driven apoptotic signals. The mechanism underlying this cell survival effect of IL-13 is unclear and apparently distinct from the known cell survival signals provided by
PKB-dependent phosphorylation of Bad. Nevertheless, our
observations indicate a potential role for IL-13 in regulating the
controlled program of cell death and survival, a process which plays an
important role during several stages of normal colonic epithelial cell
development and maturation. Hence, dysregulation of cell survival and
death may be important in the pathogenesis of inflammatory bowel
disease and carcinogenesis in the large bowel.