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and PI 3-kinase
in TNF-
-mediated antiapoptotic signaling in
the human neutrophil
Department of Pediatrics, University of Pennsylvania School of Medicine and the Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The
proinflammatory cytokine tumor necrosis factor (TNF)-
has been
implicated in the attenuation of neutrophil spontaneous apoptosis during sepsis. Antiapoptotic signaling is
principally mediated through the p60TNF receptor (p60TNFR). In
neutrophils, TNF- is an incomplete secretagogue and requires input
from a ligated integrin(s) for neutrophil activation. In adherent
neutrophils, TNF- triggers association of both protein kinase C
(PKC)-
and phosphatidylinositol (PI) 3-kinase with the p60TNFR. In
this study, a role for PKC- and PI 3-kinase in TNF--mediated
antiapoptotic signaling was examined. TNF- inhibited spontaneous
apoptosis in fibronectin-adherent neutrophils, and this
antiapoptotic signaling was blocked by the PKC- inhibitor
rottlerin, but not by an inhibitor of Ca2+-dependent PKC
isotypes, Go-6976. Inhibition of PI 3-kinase by LY-294002 also
inhibited TNF--mediated antiapoptotic signaling. Cycloheximide
blocked TNF--mediated antiapoptotic signaling, suggesting
protein synthesis is required. Inhibition of either PKC- or PI
3-kinase attenuated TNF--mediated activation of the antiapoptotic transcription factor NF
B. Thus both PKC- and PI 3-kinase have essential roles in TNF--mediated antiapoptotic signaling in adherent neutrophils.
sepsis; inflammation; signal transduction; nuclear factor B; protein kinase C-; tumor necrosis factor-
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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HUMAN NEUTROPHILS are important in host defense against bacterial infection but may also contribute to the tissue damage of inflammation. Neutrophils ingest and kill invading microorganisms through the release of toxic oxygen radicals and proteases. Mature human neutrophils have a relatively short life span in the circulation and undergo spontaneous or constitutive apoptosis within 24 h. This apoptotic process is thought to be a protective mechanism that minimizes the risk of host tissue damage caused by the release of neutrophil-derived toxic mediators. Neutrophils undergoing apoptosis have decreased oxygen radical production, degranulation, and phagocytosis in response to stimuli (24, 57).
During inflammatory diseases such as sepsis, systemic inflammatory
response syndrome (SIRS), and adult respiratory distress syndrome
(ARDS), neutrophil apoptosis is attenuated (14, 22, 23,
34, 37). The increased survival of neutrophils may contribute to
the pathophysiology of these inflammatory diseases through excessive release of toxic mediators, resulting in host tissue damage
and organ dysfunction. Tumor necrosis factor (TNF)- and other
proinflammatory cytokines have been implicated as endogenous mediators
responsible for the modulation of neutrophil apoptosis during inflammation (10, 12, 14, 18, 22, 23, 27, 31, 34, 37, 39,
42, 54).
TNF- is a unique proinflammatory cytokine whose signaling pathways
are linked to both antiapoptotic and proapoptotic responses in
neutrophils (3, 10, 12, 23, 39, 42, 54). Neutrophils possess two TNF- receptors, a 55-60 kDa (p60TNFR) and a
75-80 kDa (p80TNFR), and both antiapoptotic and
proapoptotic signaling pathways are regulated principally by the
p60TNFR (4, 33, 49). This seemingly paradoxical situation
suggests that other factors, either intracellular or external,
determine whether TNF- will activate signaling pathways for cell
survival or programmed cell death.
TNF- is an incomplete secretagogue in neutrophils and requires input
from both ligated integrins and TNF- receptors to trigger superoxide
anion generation, degranulation, and activation of enzymes such as
phosphatidylinositol (PI) 3-kinase (26, 28, 41). The
adherence of neutrophils to matrix proteins such as fibronectin
provides signaling from 
-integrins, a requirement for neutrophil
responsiveness to TNF- (40, 41, 53). Neutrophils adherent to matrix proteins, rather than neutrophils in suspension, are
relevant to an inflammatory focus. Because adherent neutrophils respond
differently to TNF- than those in suspension, it is possible that
input from integrin-activated signaling may have a critical role in
regulating TNF--mediated antiapoptotic signaling. Indeed, different extracellular matrix proteins have disparate effects on
neutrophil spontaneous apoptosis and may have an important role
in regulating TNF- antiapoptotic and proapoptotic signaling (47, 56).
Our recent studies demonstrate that both protein kinase C- (PKC-)
and PI 3-kinase associate with the p60TNFR in response to TNF-, an
association that requires engagement of -integrins (26,
28). PI 3-kinase has been implicated in antiapoptotic signaling triggered by proinflammatory cytokines (12, 27). PI 3-kinase activation has been linked to the activation of PKC- in
several cell systems (5, 50, 51). PKC- is a
phosphatidylserine (PS)/diglyceride (DG)-dependent, calcium-independent
PKC isotype. In neutrophils, PKC- has a selective role in the
regulation of TNF--mediated signaling through serine phosphorylation
of the p60TNFR (26). PKC- is an important signaling
component in TNF--mediated proapoptotic signaling (13,
17). Whether PKC- also has an antiapoptotic role in
TNF--mediated signaling remains to be determined. The goal of this
study was to examine the role of PKC- and PI 3-kinase in
TNF--mediated antiapoptotic signaling in neutrophils adherent to
the physiologically relevant matrix protein fibronectin.
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents.
Recombinant human TNF- was obtained from R&D Systems (Minneapolis,
MN). Rottlerin, Go-6976, and KN-62 were obtained from BIOMOL Research
Laboratories (Plymouth Meeting, PA). The PI 3-kinase inhibitor LY-294002 was purchased from Calbiochem (San Diego, CA). Z-VAD-FMK [Z-Val-Ala-Asp(OMe)-FMK] was
obtained from Enzyme Systems Products (Livermore, CA). Neutrophil DNA
fragmentation was quantitated using an In Situ Cell Death Detection Kit
(Boehringer Mannheim, Indianapolis, IN). Caspase-3 activity was
determined using the EnzChek caspase-3 assay kit no. 2 (Molecular
Probes, Eugene, OR). Human plasma fibronectin was purchased from Life Technologies (Gaithersburg, MD), and SuperSignal ULTRA
chemiluminescence substrate was from Pierce (Rockford, IL). Polyclonal
rabbit anti-p65 NFB, NFB gel shift oligonucleotides, TransCruz
Gel supershift p65 NFB reagent, and peroxidase-conjugated goat
anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Polyclonal goat antilactate dehydrogenase was obtained from
Fitzgerald Industries International (Concord, MA), and polyclonal
rabbit antihistone H3 was from Upstate Biotechnology (Lake Placid, NY). BSA, EGTA, leupeptin, protease inhibitor cocktail, and phosphatase inhibitor cocktail were obtained from Sigma (St. Louis, MO).
Preparation of human neutrophils and culture.
Neutrophils were isolated from heparinized venous blood (10 U/ml)
obtained from healthy adult volunteers. Standard isolation techniques
(8) were used employing Ficoll-Hypaque centrifugation, followed by dextran sedimentation and hypotonic lysis to remove residual erythrocytes. Cells were suspended in a HEPES buffer (pH 7.3)
having the following composition (in mM): 150 Na+, 5 K+, 1.29 Ca2+, 1.2 Mg2+, 155 Cl
, and 10 HEPES. For cell culture experiments, cells
were resuspended in RPMI 1640 supplemented with 2 mM
L-glutamine, 1% nonessential amino acids, 1% minimal
essential medium vitamin solution, 0.1% gentamycin, and 10%
heat-inactivated fetal bovine serum. Neutrophils were cultured for
20 h at 37°C in a 5% CO2 atmosphere in
fibronectin-coated 96-well plates at a concentration of 1.5 × 106 cells/200 µl. Fibronectin-coated wells were prepared
according to the method of Nathan (41) using a
concentration of 3.4 µg/well.
TUNEL assay for DNA fragmentation. Neutrophil DNA fragmentation was quantitated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling method (TUNEL). Neutrophils (1.5 × 106) were cultured for 20 h in RPMI 1640 plus 10% heat-inactivated FBS in fibronectin-coated 96-well plates. Cells were harvested, washed once in PBS, and fixed in 2% paraformaldehyde/PBS (pH 7.4). The cells were permeabilized in 0.1% Triton X-100/0.1% sodium citrate, incubated with TUNEL reaction mixture containing Tdt and fluorescein labeled dUTP, washed, and resuspended in PBS. Mean channel fluorescence of labeled cells was determined by flow cytometry using an Epics Elite Flow Cytometer equipped with an argon laser. The data are expressed as a percentage of apoptotic neutrophils, and the data were accumulated and stored as two-parameter histogram files. All photomultiplier tube voltages were adjusted to achieve the same mean channel fluorescence values as determined from previous calculations. For each sample, 10,000-20,000 events were accumulated.
Caspase-3 assay. Neutrophils were cultured for 20 h and harvested as described above. The activities of caspase-3-like proteases were determined using a caspase-3 assay kit. Caspase-3-like protease activity was determined by monitoring the cleavage of rhodamine-110 bis-(N-CBZ-L-aspartyl-L-gluamyl-L-valyl-L-aspartic acid amine) (Z-DEVD-R110). Fluorescence was measured in a Fluorocount Microplate Reader (Packard Instruments) at an excitation wavelength of 485 nm and emission of 530 nm. Background fluorescence was determined measuring substrate cleavage in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Results are expressed as arbitrary fluorescence units (AFU).
Preparation of nuclear fractions.
Neutrophils were incubated at 37°C in fibronectin-coated 12-well
plates at a concentration of 20 × 106 cells/well.
Appropriate inhibitors were added 20 min before the addition of either
TNF- or buffer. Samples were then incubated with either TNF- (25 ng/ml) or buffer for 10 min and placed on ice. Nuclear extracts were
prepared by a modification of the method of Dignam et al.
(11), using KCl instead of NaCl. Briefly, samples were
resuspended in hypotonic buffer containing 10 mM HEPES (pH 7.8), 1.5 mM
MgCl2, 10 mM KCl, 1.5 mM EDTA, 0.5 mM DTT, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail and were placed on ice for 30 min. Cells were disrupted by Dounce homogenization, and the cellular extract was centrifuged. The nuclear pellet was resusupended in high-salt buffer
consisting of 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 5 µg/ml
leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease
inhibitor cocktail and were incubated on ice for 20 min. The extract
was centrifuged, and aliquots of the supernatant fractions (nuclear extract) were frozen at 
70°C. Samples for Western blot analysis were prepared by mixing an aliquot of the nuclear extracts with 2×
sample buffer and heating for 15 min at 65°C. Protein concentration was determined using the Noninterfering Protein Assay Kit
(GenoTechnology, St. Louis, MO). Purity of nuclear fractions was
routinely determined by probing fractions for cytoplasmic (lactate
dehydrogenase) and nuclear (histone) markers.
Western blotting.
Nuclear extracts were run on a 4-12% gradient SDS-PAGE,
transferred to a nitrocellulose membrane, and blocked for 1 h at
room temperature with Tris-buffered saline, pH 7.5, containing 0.1% Tween-20 and 1% BSA/3% casein as described previously
(26). The membranes were incubated with a rabbit
polyclonal anti-p65 NFB antibody, washed, and incubated with
peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive bands were
visualized using Pierce SuperSignal ULTRA chemiluminescence substrate.
Translocation of the p65 NFB subunit to the nucleus was quantitated
by densitometry analysis of Western blots by Scan Pro, and the values
were expressed in arbitrary densitometry units (ADU).
Electrophoretic mobility shift assay.
Reactions for electrophoretic mobility shift assays (EMSAs) were
prepared as follows. Nuclear extracts (5 × 105 cell
equivalents) were resuspended in a buffer having a final concentration
of 10 mM HEPES, pH 7.9, 42 mM NaCl, 120 µM MgCl2, 20 µM
EDTA, 50 µM phenylmethylsulfonyl fluoride, 50 µM DTT, and 12.5%
glycerol. A double-stranded oligonucleotide of sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3' was radioactively labeled with
[
-32P]ATP (3,000 Ci/mmol). Each reaction mixture
contained 10 ng isotope-labeled oligonucleotide probe. For competitions
with unlabeled probe, 50-fold greater unlabeled oligonucleotide was
added to reactions. For supershift assays, nuclear extracts were
pretreated with anti-p65 NFB (C-20). All reagents including 0.5 µg
poly(dI-dC) were combined on ice then incubated at room temperature for
2 min. Labeled probe was added last, and reactions were further
incubated at room temperature for 10 min. DNA-protein complexes were
separated on 7.8% polyacrylamide gels at 4°C with 0.5× TBE as
running buffer. Gels were subsequently dried and subjected to autoradiography.
Statistical analysis. Results are expressed as means ± SE. Data were analyzed by Student's t-test.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of TNF- on spontaneous neutrophil apoptosis: a
role for PKC.
Freshly isolated human neutrophils were cultured for 20 h on
fibronectin-coated wells at 37°C. DNA fragmentation was determined by
flow cytometry using the TUNEL method. Under these experimental conditions, neutrophils undergoing spontaneous apoptosis
comprised 43 ± 12% of the total neutrophil population (Table
1 and Fig. 1A). The addition of
TNF- (25 ng/ml) to neutrophil cultures resulted in a significant
attenuation of spontaneous apoptosis compared with neutrophils
cultured in medium alone. In the presence of TNF-, DNA fragmentation
was significantly reduced by 60% (n = 4, P < 0.01, Table 1 and Fig. 1B).
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, PKC-I, and PKC-II;
PS/DG-dependent but calcium-independent PKC-, and PS-dependent but
DG/calcium-independent PKC-
(29, 35, 36). The effects
of different PKC isotype inhibitors were examined to determine whether
PKC isotypes had a role in TNF--mediated antiapoptotic
signaling. Rottlerin (5 µM), a PKC- inhibitor, abolished the
inhibitory effect of TNF- on spontaneous neutrophil
apoptosis (P < 0.01, Table 1 and Fig. 1C). In contrast, pretreatment with 10 nM Go-6976, an
inhibitor of calcium-dependent PKC isotypes including PKC-, -I,
and -II (16, 25), had no effect on TNF--mediated
attenuation of neutrophil apoptosis (P = NS,
Table 1 and Fig. 1D). Thus PKC-, but not the
calcium-dependent PKC isotypes, is involved in TNF--mediated antiapoptotic signaling.
Effect of TNF- on neutrophil caspase-3 activity: role of PKC.
Activation of caspases is one of the earliest markers of
apoptosis. Caspase-3 is activated during neutrophil spontaneous
apoptosis, and this activation occurs upstream of DNA cleavage
in the apoptotic pathway (3, 15, 30, 48). We next
determined whether PKC- was acting on TNF--mediated signal
transduction at the level of caspase-3 activation. Similar to DNA
fragmentation, caspase-3 activity was increased in human neutrophils
cultured for 20 h compared with freshly isolated neutrophils (data
not shown). As shown in Fig. 2, the
culture of neutrophils for 20 h resulted in caspase-3-like
activity of 9,802 ± 268 AFU/1.5 × 106 (SE)
cells (n = 5). Pretreatment of neutrophils with the
peptide caspase inhibitor Z-VAD-FMK (20 µM) for 30 min
before the addition of TNF- resulted in a >98 ± 1%
inhibition of fluorescence, indicating caspase activity (Fig. 2,
P < 0.01). Similar results were also obtained when the
more specific caspase-3 inhibitor DEVD-CHO was used (data not shown).
The addition of TNF- to neutrophil cultures decreased caspase-3
activity to 50% of neutrophils cultured in buffer alone
(P < 0.01, Fig. 2). Pretreatment with rottlerin before the addition of TNF- abolished the inhibitory effect of TNF- on
caspase-3 activity (P < 0.01, Fig. 2). In contrast,
caspase-3 activity was not significantly different in TNF- + Go-6976-treated samples compared with TNF- treatment alone
(P = NS, Fig. 2). Rottlerin is a relatively specific
inhibitor of PKC-. At a concentration of 5 µM, rottlerin also
inhibits Ca2+/calmodulin kinase II but not
calcium-dependent PKC isotypes (16, 19). To ascertain
whether the effects of rottlerin on caspase-3 activity were
Ca2+/calmodulin kinase II dependent, the effect of KN-62, a
selective inhibitor of Ca2+/calmodulin kinase II, was
examined. Pretreatment with KN-62 (1 µM) had no significant effect on
the antiapoptotic effects of TNF- (93 ± 14% of TNF-
alone, P = NS, n = 3), indicating that the inhibitory effect of rottlerin on TNF--mediated
antiapoptotic signaling was not Ca2+/calmodulin kinase
II mediated. Thus, similar to DNA fragmentation, caspase-3 activity was
increased in fibronectin-adherent neutrophils cultured for 20 h.
The addition of TNF- decreased caspase-3 activity, an effect that
was abolished by rottlerin but not Go-6976 or the Ca2+/calmodulin kinase II inhibitor KN-62. Therefore,
PKC- appears to be selectively involved in TNF--mediated
inhibition of neutrophil spontaneous apoptosis and blocks
caspase-3 activation.
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Effect of cycloheximide on TNF--mediated inhibition of caspase-3
activity.
To further investigate the mechanism of prolonged neutrophil survival
in response to TNF-, the requirement for protein synthesis in
fibronectin-adherent neutrophils was next investigated. Inhibition of
spontaneous apoptosis by TNF- requires long-term exposure, suggesting that protein synthesis is required for the inhibitory responses. Fibronectin-adherent neutrophils were preincubated with 5 µM cycloheximide before addition of either buffer or TNF-. After a
20-h incubation, cells were harvested, and caspase-3 activity was
determined. As shown in Fig. 3,
cycloheximide had no significant effect on spontaneous neutrophil
apoptosis as determined by caspase-3 activity
(P = NS, n = 5). In contrast,
cycloheximide pretreatment before TNF- abolished the
antiapoptotic effects of TNF- on spontaneous neutrophil
apoptosis (P < 0.02, Fig. 3). Thus
TNF--mediated attenuation of neutrophil apoptosis in
fibronectin-adherent neutrophils requires protein synthesis.
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TNF--mediated activation of NFB: a role for PKC-.
TNF- is a potent activator of NFB, a family of transcription
factors thought to have a critical role in cell survival through the
induction of antiapoptotic genes (1, 6, 7, 9, 32, 44,
55). Neutrophil NFB activation requires the translocation of
the p50 and p65 NFB subunits to the nucleus. We next examined whether TNF- activates NFB in fibronectin-adherent neutrophils and whether this activation requires PKC-. As shown in Fig.
4, densitometry analysis of nuclear
fractions showed low levels of the p65 NFB subunit present in the
nucleus in adherent neutrophils treated with buffer alone. There was no
significant increase in translocation of p65 NFB to the nucleus when
neutrophils were pretreated with either Go-6976 or rottlerin alone. The
addition of TNF- to neutrophils resulted in a threefold increase in
p65 NFB translocation (Fig. 4, P < 0.01, n = 5). Pretreatment with the inhibitor Go-6976 had no
significant effect on TNF--mediated translocation of p65 NFB. In
contrast, pretreatment with rottlerin inhibited TNF--mediated
translocation of the p65NFB subunit by 55% (Fig. 4,
P < 0.01).
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B EMSAs were also performed on neutrophil nuclear extracts.
Concordant with our translocation results, incubation of
fibronectin-adherent neutrophils with TNF- significantly enhanced
NFB DNA binding activity compared with neutrophils incubated with
buffer alone (Fig. 5). Pretreatment with
rottlerin before TNF- addition resulted in a significant decrease in
NFB DNA binding activity to 40.0 ± 13.5% of TNF- alone
(n = 3, P < 0.05, Fig. 5). Competition with unlabeled NFB probe reduced the major species, indicating that
DNA binding activity was specific to NFB. Supershift analysis revealed that the p65 subunit of NFB was involved in the DNA/protein binding in neutrophils (Fig. 5). These results indicate that TNF- activates NFB in fibronectin-adherent neutrophils, and this process requires PKC- acting at a site upstream of NFB translocation and
activation.
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TNF--mediated activation of NFB: a role for PI 3-kinase.
In some cell systems, activation of NFB has been linked to PI
3-kinase through a signaling cascade that activates the protein kinase
Akt (43). Our recent studies demonstrated that
TNF--mediated activation of PI 3-kinase requires twin signals from
the p60TNFR and -integrins and that PI 3-kinase is only activated by
TNF- in adherent neutrophils (28). We next determined
whether PI 3-kinase has a role in TNF--mediated activation of NFB
in adherent neutrophils. Pretreatment of neutrophils with the PI
3-kinase inhibitor LY-294002 (10 µM) inhibited TNF--mediated
translocation of the p65 NFB subunit to the nucleus by 60% (Fig.
6, P < 0.02, n = 5). LY-294002 itself did not have any effect on p65
NFB translocation. Thus both PKC- and PI 3-kinase are upstream of
NFB activation in TNF--mediated signaling in fibronectin-adherent
neutrophils.
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Effect of PI 3-kinase inhibition on caspase-3 activity.
We next determined whether PI 3-kinase had a role in TNF--mediated
antiapoptotic signaling in adherent neutrophils. Pretreatment with
the PI 3-kinase inhibitor LY-294002 had no significant effect on
spontaneous neutrophil apoptosis as determined by caspase-3 activity after a 20-h incubation (Fig. 7,
P = NS, n = 6). In contrast, LY-294002
abolished the antiapoptotic effect of TNF- on caspase-3 activity
(P < 0.01, Fig. 7). Similar results were also obtained when wortmannin (100 nM) was used to inhibit PI 3-kinase (data not
shown). These studies indicate that PI 3-kinase is an essential component of TNF--mediated antiapoptotic signaling.
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Effect of inhibition of PKC- and PI 3-kinase on caspase-3
activity.
PI 3-kinase and PKC- are required signaling elements for
TNF--triggered activation of NFB and inhibition of caspase-3. The
effect of combinations of maximal and submaximal concentrations of
rottlerin and LY-294002 on TNF--mediated inhibition of caspase-3 activity was examined to determine whether PI 3-kinase and PKC- are
components of the same signaling pathway. As shown in Figure 8, both rottlerin and LY-294002 abolish
the inhibitory effects of TNF- on caspase-3 activity in a
dose-dependent manner. The inhibitory effect of TNF- on caspase-3
activity was completely ablated at 5 µM rottlerin and at 10 µM
LY-294002 (P < 0.01, n = 4, Fig. 8).
Neither rottlerin, LY-294002, nor the combination of the inhibitors had
any significant effect on caspase-3 activity in the absence of TNF-
(Figs. 2 and 7, and data not shown). When maximal doses of rottlerin (5 µM) and LY-294002 (10 µM) were used in combination, there was no
additive effect, and caspase-3 activity was not significantly different
from TNF- plus rottlerin or TNF- plus LY-294002 alone
(P = NS, Fig. 8). Furthermore, when submaximal doses of
rottlerin and LY-294002 were used in combination, there were no
additive effects on caspase activity, providing further evidence that
both PI 3-kinase and PKC- are components of the same signaling
pathway.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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TNF- is a unique proinflammatory cytokine whose signaling is
linked to both antiapoptotic and proapoptotic pathways
(3, 10, 12, 23, 39, 42, 54). The cellular response to TNF- is dependent on cytokine levels, length of exposure, and input
from other signaling pathways (i.e., engagement of integrins). PKC-
is an important signaling component in TNF--mediated
proapoptotic signaling (13, 17) and has also been
implicated as an important component in neutrophil apoptosis
(16, 25, 45). The results of the present study demonstrate
that PKC- is also a critical component of TNF--mediated
antiapoptotic signaling in adherent neutrophils, a process that may
be mediated by NFB and involve PI 3-kinase.
In this study, the role of -PKC in TNF--mediated
antiapoptotic signaling was examined in fibronectin-adherent
neutrophils. TNF- significantly delayed apoptosis in
cultured fibronectin-adherent neutrophils as determined by DNA
fragmentation and caspase-3 activation. These results are concordant
with previous studies that demonstrated TNF--mediated
antiapoptotic signaling in neutrophils adherent to other matrixes
(3, 10, 12, 23, 39, 42, 54). Rottlerin, an inhibitor of
PKC-, attenuated both TNF--mediated inhibition of DNA
fragmentation and caspase-3 activation. The effect was specific
for PKC-, since inhibitors of calcium-dependent PKC isotypes
or Ca2+/calmodulin kinase II had no effect on
TNF--mediated antiapoptotic signaling. In adherent neutrophils,
PI 3-kinase is also activated by TNF- and PI 3-kinase is an
activator of PKC- (28, 51). Inhibition of PI 3-kinase
with LY-294002 also attenuated TNF--mediated inhibition of
neutrophil constitutive apoptosis. Furthermore, the inhibitory
effects of the PKC- and PI 3-kinase inhibitors were not additive
when used in combination, indicating that PKC- and PI 3-kinase are
components of the same signaling pathway. Thus PKC- and PI 3-kinase
are essential elements in TNF--mediated antiapoptotic signaling
and block caspase-3 activation.
Long-term incubation is required for TNF--mediated attenuation of
neutrophil apoptosis, suggesting that protein synthesis is a
required element. External factors such as cytokines may regulate
apoptosis through the transient activation of antiapoptotic genes (3). Gene expression can be regulated through
modulation of the activity of transcription factors such as NFB
(55). Concordant with the activation of transcription
factors is the requirement for protein synthesis; our studies
demonstrated that preincubation with the protein synthesis inhibitor
cycloheximide abolished the inhibitory effects of TNF- on
apoptosis in fibronectin-adherent neutrophils.
Modulation of the transcription factor NFB is a possible regulatory
site for TNF--mediated attenuation of neutrophil apoptosis. TNF- activates NFB in numerous cell types including neutrophils (38, 55). A role for NFB has been implicated in the
pathophysiology of sepsis, ARDS, and SIRS through enhanced gene
expression of proinflammatory cytokines, chemokines, adhesion
molecules, and inducible nitric oxide synthase and by the attenuation
of phagocytic cell apoptosis (1, 6, 7, 9, 32, 44).
Enhanced NFB activation has been reported in patients with sepsis,
SIRS, and ARDS and is correlated with poor outcome and increased
mortality (7, 32, 44). Inhibition of NFB enhances
spontaneous neutrophil apoptosis, suggesting that activation of
NFB may be an important regulatory site in control of neutrophil
apoptosis (42, 55). The putative antiapoptotic
genes that are activated by NFB in response to TNF- have yet to
be identified; potential candidates include the zinc finger A20,
manganese superoxide dismutase, interleukin-8, the prosurvival Bcl-Xi,
TRAF-1, and the family of cellular inhibitors of apoptosis
(2, 6, 12, 42).
TNF- is a potent activator of NFB in fibronectin-adherent
neutrophils. TNF- enhanced both p65 NFB translocation to the nucleus and increased NFB-DNA binding activity. Rottlerin inhibited both TNF--triggered p65 NFB translocation and NFB-DNA binding. The inhibitory effect was selective for PKC- because inhibition of
calcium-dependent PKC isotypes had no effect on TNF--triggered NFB activation. Thus PKC- is a required signaling component for
TNF--mediated activation of NFB in adherent neutrophils and acts
at a site before NFB translocation. A recent study also reported
inhibition of TNF--mediated activation of NFB by rottlerin but at
a different regulatory site (52). These studies used nonadherent neutrophils and employed rottlerin at a concentration 10-fold greater than employed in this study (50 vs. 5 µM) and at a
concentration far above the IC50 for PKC- (3-6
µM) (52). The discrepancy in the two studies may be
attributed to different responses to cellular activation by TNF- of
adherent neutrophils, compared with those in suspension. Neutrophils in
suspension lack input from -integrin signaling pathways and
activation of key enzymes such as PI 3-kinase (28). The
results presented here demonstrate that in adherent neutrophils, both
PKC- and PI 3-kinase are important components for TNF--mediated
activation of NFB and the attenuation of neutrophil apoptosis.
Previous studies in other cell types have identified PKC- as having
a critical role in TNF--mediated proapoptotic signaling (13, 17). The results of this study indicate that PKC-
is also essential for antiapoptotic signaling. Because both
antiapoptotic and proapoptotic signaling pathways are mediated
principally through the p60TNFR (4, 20, 21), a possible
site of interaction is at the level of the receptor. TNF- binding to
the p60TNFR leads to the recruitment of TNFR-1-associated death domain
protein (TRADD), which acts as a scaffold protein and subsequently
recruits TNFR-associated factor-2 (TRAF2), receptor-interacting protein (RIP), and Fas-associated death domain (FADD) to form the p60TNFR signaling complex (20, 21). TRAF2 and RIP regulate
antiapoptotic pathways through activation of NFB or p42/p44
extracellular signal-regulated kinase (46, 55). FADD is
essential for TNF--induced apoptosis through its association
with and activation of caspase-8, thereby initiating apoptosis
(4). For PKC- to regulate both anti- and
proapoptotic signaling, a likely site of interaction would be
before bifurcation of the signaling pathways, i.e., at the level of the
p60TNFR or TRADD. In support of this concept, our previous studies have
shown that activation of the p60TNFR by TNF- triggers
phosphorylation of the receptor on both serine and threonine residues
(26). Serine phosphorylation of the receptor is mediated
by PKC- (26). Furthermore, both PI 3-kinase and PKC-
associate with the p60TNFR signaling complex in response to TNF- in
adherent neutrophils (26, 28). PI 3-kinase and its product
PI 3,4,5-trisphosphate can activate several novel and atypical
PKC isotypes including PKC- (51). Thus it is suggested that in adherent neutrophils, both PKC- and PI 3-kinase may act at
the level of the p60TNFR in mediating signaling through regulation of
receptor phosphorylation. These studies do not rule out the possibility
that -PKC and/or PI 3-kinase may be acting at several discrete
locations rather than a single site in the TNF--initiated signaling pathway.
In summary, the results of the present study demonstrate that both
PKC- and PI 3-kinase are essential components of the same pathway
for TNF--mediated antiapoptotic signaling in adherent neutrophils. Furthermore, both PKC- and PI 3-kinase are required for
TNF--mediated activation of the transcription factor NFB, suggesting that PKC- and PI 3-kinase regulate TNF--mediated neutrophil survival through induction of antiapoptotic genes. The
requirement for PKC- for both TNF--mediated anti- and
proapoptotic signaling suggests that PKC- is acting at a site
before bifurcation of these signaling pathways, possibly at the level
of the p60TNFR.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Allergy and Infectious Diseases Grants AI-24840 (to H. M. Korchak) and AI-44127 (to K. E. Sullivan).
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. E. Kilpatrick, Immunology Section, Rm. 1207J, Abramson Building, Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104 (E-mail: kilpatrick{at}emailchop.edu).
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.
First published February 6, 2002;10.1152/ajpcell.00385.2001
Received 9 August 2001; accepted in final form 1 February 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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