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INTRODUCTION |
Neutrophils (polymorphonuclear leukocytes) are short-lived
terminally differentiated blood cells that play a vital role in the
inflammatory response; they are one of the first cells recruited to the
site of injury or infection (1, 2). In addition to their phagocytic and
killing properties, neutrophils synthesize numerous proinflammatory
cytokines and chemokines, including
TNF
,1 interleukin
(IL)-1
and IL-1
, IL-8, and macrophage inflammatory protein
,
that may amplify the inflammatory process (3-8). Expression of many of
these proinflammatory proteins is regulated at the level of gene
transcription by transcription factor NF-
B (9, 10).
Since the knowledge that neutrophils are an important source of
cytokines is relatively new, the molecular mechanisms regulating cytokine expression in these cells have only begun to be investigated (11, 12). We have previously shown that NF-
B activity in human
neutrophils consists of p50/50 homodimers and p50/65 heterodimers, and
that their activation in TNF
-stimulated neutrophils is inhibited by
dexamethasone, an anti-inflammatory drug (13). Using
pharmacological inhibitors Nick et al. (12) demonstrated
that lipopolysaccharide (LPS)-induced activation of NF-
B in
neutrophils is mediated by p38
mitogen-activated protein
kinase. However, the signaling pathways leading to NF-
B
activation in response to neutrophil stimulation with TNF
are undefined.
Interestingly, a recent study demonstrated that NF-
B also regulates
both constitutive and TNF
-induced apoptosis in human neutrophils
(14). This neutrophil apoptosis has been recently shown to be mediated
by a novel isoform of protein kinase C (PKC), PKC
(15, 16).
Therefore, we sought to investigate involvement of PKC
in
TNF
-induced activation of NF-
B in human neutrophils. PKC
is
selectively inhibited by rottlerin (15, 17-19), and as any other novel
PKC, it is activated in a Ca2+-independent manner by
diacylglycerol (DAG), which is produced by activated phospholipase C
(PLC) (20).
In this study, we show that inhibition of phosphatidylinositol-specific
phospholipase C (PI-PLC) and PKC
blocks activation of NF-
B in
TNF
-stimulated human neutrophils by inhibiting degradation of
I
B
. The regulation of NF-
B activation by PKC
is specific only for TNF
signaling, since LPS- or IL-1
-induced activation of
NF-
B and degradation of I
B
are not inhibited by rottlerin. In
addition, we show that in human neutrophils, but not monocytes, I
B
localizes in significant amounts in the nucleus of resting unstimulated cells. The NF-
B DNA binding in the neutrophil does not
correlate with nuclear translocation of NF-
B subunits, as is the
case in most mammalian cells (21-23), but rather with the amount of
I
B
in the nucleus, as well as in the cytoplasm.
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EXPERIMENTAL PROCEDURES |
Materials--
Ficoll-Paque PLUS, dextran T-500, T4
polynucleotide kinase, poly(dI-dC), and Sephadex G25 spin columns were
purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Hank's
balanced salt solution, RPMI 1640 medium, and endotoxin tested,
heat-inactivated fetal calf serum (FCS) were obtained from Life
Technologies (Grand Island, NY). Escherichia coli expressed
purified recombinant human TNF
and IL-1
were purchased from R & D
Systems (Minneapolis, MN). [32P]ATP was purchased from
PerkinElmer Life Sciences (Boston, MA). Histone H1, U-73122, U-73343,
D-609, Et-18-OCH3, rottlerin, and Ro-31-8425 were purchased from
Calbiochem (La Jolla, CA). Protein A/G Plus-agarose, purified
polyclonal antibodies to human p50 (sc-7178X), I
B
(sc-371),
PKC
(sc-937), and glucocorticoid receptor (GR, sc-1003), and mouse
monoclonal anti-actin antibody (sc-8432) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Polyclonal antibody to p65 (SA-171) was
obtained from Biomol (Plymouth Meeting, PA). Monoclonal mouse
anti-SUMO-1 antibody was from Zymed Laboratories Inc.
(San Francisco, CA), and polyclonal lactate dehydrogenase (LDH)
antibody (20-LG22) was purchased from Fitzgerald Industries
International (Concord, MA). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG secondary antibodies were
from Amersham Pharmacia Biotech (Arlington Heights, IL). All other
reagents were molecular biology grade and were purchased from Sigma.
All reagents and plasticware used throughout the experiments were
pyrogen-free.
Cell Isolation and Culture--
Fresh blood was obtained from
healthy adult human volunteers and collected in heparinized
preservative-free tubes. Neutrophils and monocytes (95-98% purity)
were separated under endotoxin-free conditions using Ficoll-Paque
centrifugation (24), and the neutrophils were subsequently purified by
dextran sedimentation and hypotonic lysis of residual erythrocytes as
described previously (6). Purified cells were resuspended in RPMI 1640 supplemented with 5% low endotoxin fetal calf serum, at a final
concentration of 5 × 106 cells/ml, and incubated at
37 °C in polypropylene tubes with gentle agitation. For the
inhibition experiments, the inhibitors were dissolved in dimethyl
sulfoxide, and the cells were pretreated 15 min with either the
inhibitor or with Me2SO alone, before stimulation with
TNF
. The incubations were terminated by placing cells on ice and
rapid centrifugation (1 min, 5,000 × g, 4 °C).
Preparation of Cytoplasmic and Nuclear Extracts--
Nuclear and
cytoplasmic extracts were prepared from 5 × 106 cells
as described previously (13). Briefly, the pelleted cells were
resuspended in 300 µl of hypotonic buffer (buffer A: 10 mM Hepes, pH 7.5, 10 mM KCl, 3 mM
NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM dithiothreitol) containing
the following protease and phosphatase inhibitors: 2 mM
phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor, 1 mM benzamidine, 2 mM levamisole, 1 mM Na3VO4, 10 mM NaF,
20 mM glycerophosphate, and protease inhibitor mixture from
Sigma (P-8340), used at concentration 60 µl/5 × 106
cells). After 15 min incubation on ice, 0.05 volumes of 10% Nonidet P-40 were added, the cells were vortexed (10 s) and immediately centrifuged at 500 × g for 10 min at 4 °C. The
supernatants were collected, designated as cytoplasmic extracts,
aliquoted, and stored at
80 °C.
The nuclear pellets were washed in 200 µl of buffer A containing the
protease inhibitors, and re-centrifuged. The pelleted nuclei were
resuspended in 50 µl of ice-cold nuclear buffer (NE buffer: 20 mM Hepes, pH 7.5, 25% glycerol, 0.8 M KCl, 1 mM MgCl2, 1% Nonidet P-40, 0.5 mM
EDTA, 2 mM dithiothreitol) containing the protease and
phosphatase inhibitors as described above. Following a 20-min
incubation on ice (with occasional mixing), the samples were
centrifuged (14,000 × g, 15 min, 4 °C), and the
resulting supernatants (nuclear extracts) were aliquoted and stored at
80 °C. Protein concentration was measured using the Pierce
Coomassie Plus protein assay kit (Pierce, Rockford, IL).
Contamination of nuclear and cytoplasmic fractions by cytoplasmic and
nuclear proteins, respectively, was determined by Western analysis
using LDH and SUMO-1 as specific markers.
Electrophoretic Mobility Shift Assay (EMSA)--
The
oligonucleotide used as a probe for EMSA was a 42-base pair
double-stranded construct
(5'-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3') containing two tandemly repeated NF-
B-binding sites (underlined). Mutant oligonucleotide used for competition studies was
5'-TTGTTACAATCTCACTTTCCGCTTCTCACTTTCCAGGGAGGC-3'. End labeling was accomplished by treatment with T4 kinase in the presence of [
-32P]ATP, and the labeled oligonucleotide
was purified on a Sephadex G-25 column, as described elsewhere
(25).
Nuclear extracts (containing 4-6 µg of protein in 5-7 µl) were
incubated (20 min at room temperature) with 5-10 fmol of radiolabeled oligonucleotide (~70,000 cpm) in 20 µl of binding buffer (20 mM Tris-Cl, pH 7.5, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet
P-40, 6% glycerol) supplemented with 20 µg of acetylated bovine
serum albumin and 2 µg of poly(dI-dC). For competition or supershift
experiments, binding reactions were performed in the presence of 30 M excess of unlabeled oligonucleotide or 1 µg of specific
polyclonal antibody, respectively, and incubated 15 min at room
temperature before adding 32P-labeled oligonucleotide. The
resulting complexes were resolved on 5% nondenaturing polyacrylamide
gels that had been pre-run at 100 V for 30 min in 0.5 × TBE
buffer. Electrophoresis was conducted at 180 V for 2.5 h. After
electrophoresis, gels were transferred to Whatman DE-81 paper,
dried, and exposed to autoradiographic film (Kodak BioMax MS) with
intensifier screen at
80 °C.
Immunoprecipitation PKC
Assay--
PKC
enzymatic activity
was assayed in whole cell lysates immunoprecipitated by PKC
specific
polyclonal antibody as follows. Neutrophils (5 × 106)
were lysed in 0.3 ml of lysis buffer (50 mM Tris-Cl, pH
8.0, 250 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 2 mM
dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor; 1 mM benzamidine, 2 mM levamisole, 1 mM
Na3VO4, 20 mM glycerophosphate, 10 mM NaF and protease inhibitor mixture from Sigma (P-8340),
used at concentration 60 µl/5 × 106 cells). Soluble
proteins were pre-cleared by a 1-h incubation (4 °C) with 10 µl of
Protein A/G Plus-agarose. The precleared supernatants were incubated
with 1 µg of anti-PKC-
or control anti-GR antibody (2 h, 4 °C),
and immunoprecipitated with 10 µl of Protein A/G Plus-agarose for an
additional 1 h. The immune complexes were washed 5 times with
lysis buffer and 1 time with kinase buffer (20 mM Hepes, pH
7.5, 10 mM MgCl2, 2 mM
MnCl2, 20 µM ATP), and resuspended in 20 µl
of kinase buffer. Five µl of 5 × reaction buffer (1 mg/ml
histone H1, 20 µM 1,2-dioleoyl-sn-glycerol, and 0.25 mg/ml L-
-phosphatidyl-L-serine) and
5 µCi of [
-32P]ATP were added, and the samples were
incubated for 5 min at 30 °C. Reactions were stopped by the addition
of 8 µl of 5 × sample buffer, the samples were boiled and
resolved on a 12% SDS-polyacrylamide gel. The gels were stained with
Coomassie, and the extent of histone H1 phosphorylation was determined
by both autoradiography and scintillation counting of the excised
Coomassie-stained histone polypeptide bands. In experiments examining
the effect of rottlerin on PKC
activity in vitro,
rottlerin was added to the PKC
immunoprecipitates in concentrations
given in the text before the addition of 1 µM ATP.
Western Blotting--
Denatured proteins were separated on 12%
denaturing polyacrylamide gels and transferred to nitrocellulose
membrane (Hybond C; Amersham Pharmacia Biotech). Membranes were blocked
overnight with a 5% (w/v) nonfat dry milk solution containing 10 mM Tris-Cl, pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, and 0.1% Tween 20 (TBSTM) before incubating with primary antibodies (1 h for I
B
, p65, p50, SUMO-1, and LDH antibodies, and overnight for actin antibody). Primary antibodies were diluted in TBSTM (1:250 for I
B
and actin, 1:700 for p65, 1:300 for p50, and 1:200 for LDH and SUMO-1). After washing, the membranes were incubated 1 h with horseradish
peroxidase-labeled secondary antibody diluted 1:2000 in TBSTM, and the
labeled proteins were detected using enhanced chemiluminescence (ECL)
reagents as described by the manufacturer (Amersham Pharmacia Biotech).
To confirm equivalent amounts of loaded proteins, or to re-probe the
membrane with another antibody, the membranes were stripped with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM
Tris-Cl (pH 6.7) for 30 min at 50 °C, and incubated with the
appropriate primary antibody diluted in TBSTM. The signal was developed
using secondary IgG-horseradish peroxidase and ECL detection as
described above.
Data Analysis--
Data presented here represent a minimum of
three experiments, and, where appropriate, data are expressed as
mean ± S.E. Statistical significance was evaluated by using ANOVA.
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RESULTS |
Specific Inhibitors of PI-PLC and PKC
Block Activation of
NF-
B in TNF
-stimulated Neutrophils--
To investigate whether
the TNF
-induced NF-
B activation involves PLC- and
PKC
-dependent pathways, we used inhibitors of phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific PLC,
and PKC
: D-609 (26), U-73122, and Et-18-OCH3 (27-29), and rottlerin
(15, 17-19), respectively. Neutrophils were preincubated 15 min with
or without the corresponding inhibitor, stimulated 30 min with TNF
,
and the NF-
B DNA binding activity was measured in nuclear extracts
by EMSA. As seen in Fig. 1A,
neutrophil stimulation with TNF
induced activation of the p50/65
heterodimer, and to a lower extent also the p50/50 homodimer. The
specificity and identity of these complexes was confirmed using
competition and supershift assay as shown in panel B. Since
the NF-
B form responsible for induction of inflammatory and
apoptotic genes is the p50/65 heterodimer, whereas the cellular
function of the p50/50 homodimer is not fully understood (30), we
focused on DNA binding activity of the p50/65 NF-
B heterodimer.

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Fig. 1.
The PI-PLC and PKC
specific inhibitors abolish TNF -induced
NF- B activation in human neutrophils.
A, neutrophils were preincubated 15 min with U-73122 (5 µM), D-609 (50 µM), Et-18-OCH3 (50 µM), and rottlerin (50 µM), and then
stimulated with TNF (10 ng/ml) for 30 min. NF- B DNA binding was
measured by EMSA in nuclear extracts. The result is representative of
three separate experiments. B, competition and supershift
analysis of NF- B DNA binding complexes in nuclear extracts from
TNF -stimulated human neutrophils. Nuclear extracts were incubated
with 32P-labeled NF- B specific DNA probe alone
(lane 1) or in the presence (+) of 30 M excess
of unlabeled wild type (wt) NF- B oligonucleotide (lane
2), or a mutant oligonucleotide (lane 3). Antibodies
used in supershift assay included antibodies to p50 (lane 4)
and p65 (lane 5). This experiment is representative of
four.
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The p50/65 NF-
B DNA binding activity was inhibited by U-73122 (5 µM) and Et-18-OCH3 (50 µM), inhibitors of
PI-PLC, and by PKC
inhibitor rottlerin (50 µM). In
contrast, inhibitor of PC-PLC, D-609 at 50 µM
concentration previously shown to be selectively effective to inhibit
PC-PLC activity (26, 31) did not reduce NF-
B DNA binding (Fig.
1A). The inhibition of NF-
B DNA binding by U-73122 was
dose dependent (Fig. 2A). The
complete inhibition of p50/65 NF-
B was achieved at 5 µM U-73122 concentration, and the IC50 was
~2 µM. This IC50 value is consistent with
the previously reported IC50 for PLC specific inhibition by
U-73122 in the neutrophil (27, 28). The inactive structural analogue of
U-73122, U-73343, in the range of 0.1-5 µM
concentrations, had no effect on NF-
B DNA binding (data not
shown).

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Fig. 2.
U-73122 and rottlerin inhibit
TNF -induced activation of
NF- B in human neutrophils in a
dose-dependent manner. A, autoradiography
showing dose response of U-73122 on TNF -induced NF- B activation.
Neutrophils were preincubated with varying concentrations of U-73122
for 15 min, and stimulated with TNF (10 ng/ml) for 30 min. NF- B
DNA binding was measured in nuclear extracts by EMSA. B,
autoradiograph showing dose response of rottlerin on TNF -induced
NF- B activation. Neutrophils were preincubated with varying
concentrations of rottlerin for 15 min, and stimulated with TNF (10 ng/ml) for 30 min. Both autoradiographs are representative of three
experiments.
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Fig. 2B shows a dose response of the PKC
-specific
inhibitor rottlerin on NF-
B DNA binding in TNF
-stimulated
neutrophils. The complete inhibition of the p50/65 NF-
B heterodimer
was achieved by rottlerin concentrations of 50 µM, and
the IC50 was ~10 µM. These values correlate
well with the previously reported rottlerin IC50 for PKC
inhibition 3-6 µM, whereas the IC50 values
for other PKC isoforms were 40-100 µM (17-19). In
contrast, neutrophil pretreatment with Ro-31-8425 (100 nM), which inhibits the classical isoforms of PKC but not
PKC
(32), had no inhibitory effect on TNF
-induced NF-
B DNA
binding even at concentration 10 times higher than the reported
IC50 (data not shown). Importantly, the inhibitory effect of rottlerin on NF-
B DNA binding was specific only for TNF
induction, since LPS, as well as IL-1
-induced NF-
B activation was
not inhibited by 50 µM rottlerin (Fig.
3, panel A). These results
demonstrate that the rottlerin effect is specific only for the TNF
signaling pathway, and indicate that the NF-
B activation in response
to neutrophil stimulation with TNF
is mediated by PI-PLC and PKC
dependent pathways.

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Fig. 3.
Rottlerin inhibition of
NF- B activation in human neutrophils is
specific for TNF -signaling pathway.
A, autoradiograph of NF- B DNA binding in TNF -, LPS-,
and IL-1 -stimulated neutrophils preincubated with rottlerin.
Neutrophils were preincubated 15 min with rottlerin (50 µM), and then stimulated 30 min with TNF (10 ng/ml),
LPS (10 µg/ml), or IL-1 (10 ng/ml). NF- B DNA binding was
measured by EMSA. B, Western blot analysis of cytoplasmic
extracts prepared from neutrophils preincubated 15 min with rottlerin
(50 µM), and stimulated 30 min with TNF (10 ng/ml),
LPS (10 µg/ml), or IL-1 (10 ng/ml). Expression of I B was
visualized using I B -specific polyclonal antibody (top
panel). To confirm equal protein loading and transfer to
nitrocellulose, the membrane was stripped and reprobed with actin
antibody (lower panel). Both panels are representative of
three experiments.
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Rottlerin Directly Inhibits PKC
Kinase Activity in
Vitro--
Rottlerin was originally reported to inhibit PKC
by
competing for ATP binding (17). To confirm that the same rottlerin concentrations inhibiting NF-
B activation in TNF
-stimulated neutrophils can also inhibit activity of PKC
, the PKC
was
immunoprecipitated from whole cell lysates using PKC
specific
polyclonal antibody, and PKC
kinase activity was measured using
histone H1 as a substrate. For comparative purposes,
immunoprecipitation using irrelevant glucocorticoid receptor (GR)
antibody was performed as a control. As seen in Fig.
4A, while no histone
phosphorylation was detected in lysates prepared from TNF
-stimulated
neutrophils and immunoprecipitated with GR antibody (lane
1), immunoprecipitation with PKC
antibody resulted in strong
phosphorylation of histone H1 (lanes 2-5), demonstrating
that the immunoprecipitation of PKC
was specific.

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Fig. 4.
Rottlerin concentrations inhibiting
NF- B activation in
TNF -stimulated neutrophils inhibit in
vitro kinase activity of PKC .
A, autoradiograph of PKC activity in TNF -stimulated
neutrophils preincubated with rottlerin in vivo. PKC
activity was measured as incorporation of 32P into histone
H1 in immunoprecipitates prepared from TNF -stimulated (10 ng/ml, 15 min) neutrophils preincubated 15 min with varying concentrations of
rottlerin (top panel). The lower panel shows
Coomassie staining of the corresponding histone bands. Lane
1 represents immunoprecipitation from TNF -stimulated
neutrophils using irrelevant GR antibody. B, autoradiograph
of PKC activity immunoprecipitated from TNF -stimulated (10 ng/ml,
15 min) neutrophils, measured with varying concentrations of rottlerin
added to the kinase reaction in vitro, before the addition
of ATP. The top panel shows 32P incorporation
into histone H1, while the lower panel represents Coomassie
staining of the corresponding histone bands. The results are
representative of three experiments.
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To determine whether rottlerin inhibits activity of PKC
directly, or whether it inhibits events upstream of PKC
, we
performed two types of experiments. In the first set of experiments,
PKC
was immunoprecipitated from TNF
-stimulated neutrophils and
incubated with rottlerin in vitro (Fig. 4B).
Rottlerin inhibited PKC
activity in a dose-dependent
manner, the IC50 being about 10 µM, which is
consistent with the rottlerin inhibition of NF-
B activation demonstrated above (Fig. 2B).
In the second set of experiments, neutrophils were preincubated with
varying concentrations of rottlerin in vivo, prior to stimulation with TNF
, and PKC
was immunoprecipitated from
corresponding cell lysates and assayed for histone phosphorylation
(panel A). Since it is very likely that the extensive
washing of the immunoprecipitates efficiently removes rottlerin from
PKC
, this experiment differentiates between the direct and indirect
effect of rottlerin on PKC
activity. If rottlerin targets protein(s)
(for example, another protein kinase(s)) upstream of PKC
, then
neutrophil preincubation with rottlerin in vivo would result
in a reduced activity of the immunoprecipitated PKC
. However, as
seen in Fig. 4A, neutrophil preincubation with varying
concentrations of rottlerin in vivo did not significantly inhibit histone phosphorylation by the immunoprecipitated PKC
. These
results demonstrate that the effect of rottlerin on PKC
is direct,
and further suggest that the activity of PKC
is required for NF-
B
activation in response to neutrophil stimulation with TNF
.
Inhibition of NF-
B Activation by Rottlerin Is Mediated through
Increased Stability of I
B
--
To determine whether PKC
activates NF-
B through regulating cellular pools of the I
B
inhibitor, neutrophils were stimulated with TNF
(15 min, 10 ng/ml)
in the presence of varying concentrations of rottlerin, and cytoplasmic
extracts were analyzed by Western blotting using I
B
specific
polyclonal antibody (Fig. 5A).
Consistent with a previous report (11), neutrophil stimulation with
TNF
substantially reduced the cytosolic pool of I
B
(lane
2). Importantly, neutrophil pretreatment with rottlerin inhibited,
in a dose-dependent manner, the TNF
-induced depletion of
cytosolic I
B
(Fig. 5A). The lower lane
shows reprobing the membrane with control anti-actin antibody,
demonstrating equal protein loading and transfer to nitrocellulose.

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Fig. 5.
Inhibition of PKC
suppresses degradation of
I B .
A, Western blot analysis of cytoplasmic extracts prepared
from TNF -stimulated (10 ng/ml, 15 min) neutrophils pretreated 15 min
with varying concentrations of rottlerin. Expression of I B was
visualized using I B -specific polyclonal antibody (top
panel). To confirm equal protein loading, the membrane was
stripped and reprobed with actin antibody (lower panel).
B, Western analysis of cytoplasmic I B expression in
TNF -stimulated (10 ng/ml, 30 min) neutrophils preincubated with
rottlerin (50 µM, 15 min) with and without prior
pretreatment with cycloheximide (100 µg/ml, 10 min). C,
autoradiograph of EMSA of NF- B DNA binding activity measured in
nuclear extracts from TNF -stimulated neutrophils incubated with and
without rottlerin and cycloheximide as in panel B. Each
panel is representative of three experiments.
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To determine whether the increased cytoplasmic pools of I
B
by
rottlerin resulted from new protein synthesis or increased protein
stability, neutrophils were pretreated with cycloheximide (100 µg/ml,
10 min) prior to incubation with rottlerin (50 µM, 15 min) and stimulation with TNF
(10 ng/ml, 30 min). As seen in Fig. 5,
panels B and C, no new protein synthesis was
required for the rottlerin up-regulation of I
B
and inhibition of
NF-
B DNA binding, respectively. These results indicate that PKC
is involved in the activation of NF-
B in response to neutrophil stimulation with TNF
by activating pathway(s) leading to degradation of I
B
.
To confirm that the PKC
involvement in NF-
B activation is
specific for TNF
signaling (Fig. 3), neutrophils were preincubated with rottlerin (50 µM, 15 min) and stimulated 30 min with
TNF
, LPS, or IL-1
, and cytoplasmic extracts were analyzed for
I
B
expression (Fig. 3B). Consistent with NF-
B DNA
binding (Fig. 3A), the rottlerin effect was specific only
for TNF
, since the LPS- and IL-1
-induced I
B
degradation
were not inhibited (Fig. 3B).
NF-
B Activation in the Neutrophil Is Not Regulated by Nuclear
Translocation of NF-
B Subunits but Correlates with Nuclear Pools of
I
B
--
In most mammalian cells, activation of NF-
B has been
shown to be controlled at the level of nuclear translocation of NF-
B proteins through their tightly regulated association with I
B
anchored in the cytoplasm (21-23). Therefore, we sought to determine whether the inhibition of PKC
-dependent activation of
NF-
B in response to neutrophil stimulation with TNF
is mediated
by cytoplasmic retention of p50 and p65 NF-
B subunits. Neutrophils
were stimulated with TNF
with and without pretreatment with
rottlerin, and the cytoplasmic and nuclear fractions were analyzed by
Western blotting using p50- and p65-specific antibodies. Surprisingly,
both the cytoplasmic and the nuclear levels of p50 and p65 NF-
B
subunits were not significantly affected by neutrophil stimulation with TNF
or pretreatment with rottlerin (Fig.
6). Moreover, both NF-
B proteins were
present in significant amounts in the nucleus even under conditions
when the NF-
B DNA binding is inhibited: in the control unstimulated
neutrophils and in the TNF
-stimulated neutrophils pretreated with
rottlerin (Fig. 5). To exclude any possible cross-contamination of the
cytoplasmic and nuclear fractions, the membrane was stripped and
reprobed with antibodies specific for cytoplasmic (LDH) and nuclear
(small ubiquitin-related modifier, SUMO-1) proteins. That LDH was
detected only in the cytoplasmic fraction, and SUMO-1 in the nuclear
fraction (Fig. 6), demonstrates that both fractions were reasonably
exempt from cross-contamination.

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Fig. 6.
NF- B activation in
the neutrophil is not regulated by nuclear translocation of
NF- B subunits but correlates with the amount
of I B in the nucleus,
as well as in the cytoplasm. Neutrophils were stimulated with
TNF (10 ng/ml, 15 min) with and without preincubation with rottlerin
(50 µM, 15 min). Cytoplasmic and nuclear extracts were
fractionated and analyzed by Western blotting using polyclonal
antibodies specific for p65 and p50 NF- B, and I B . To evaluate
the presence of cytoplasmic proteins in nuclear fractions, the membrane
was stripped and reprobed with LDH antibody. Nuclear contamination in
cytoplasmic fraction was assessed using SUMO-1 specific antibody. The
blot is representative of three experiments.
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The presence of p50 and p65 NF-
B proteins in the nuclear fraction in
the absence of NF-
B DNA binding prompted us to investigate the
subcellular distribution of I
B
. As we hypothesized, significant amounts of I
B
were present in the nuclear fraction of resting, unstimulated neutrophils, and neutrophil pretreatment with rottlerin inhibited TNF
-induced degradation of I
B
in both the nuclear and cytoplasmic compartment (Fig. 6). These results demonstrate that
the extent of NF-
B activation in the neutrophil is not regulated by
the nuclear translocation of its subunits but correlates with nuclear
pools of I
B
.
I
B
Nuclear Localization in Neutrophils Versus
Monocytes--
Since the presence of I
B
in the nuclear fraction
of resting unstimulated neutrophils challenges the current model of
NF-
B activation, it was important to determine whether this is
specific for neutrophils, or whether I
B
localizes in the nucleus
of other inflammatory cells as well. To address this point, neutrophils and peripheral blood monocytes, other type of inflammatory cells, were
analyzed for I
B
expression in the nuclear and cytoplasmic fractions of control unstimulated cells, cells stimulated with TNF
,
and cells pretreated with proteasome inhibitor MG-132 before TNF
stimulation. As shown in Fig.
7A, in contrast to
neutrophils, in monocytes I
B
is predominantly cytoplasmic, with
the I
B
amount in the nucleus being barely detectable. To evaluate
the nucleocytoplasmic distribution of I
B
in the neutrophils and monocytes more quantitatively, the cytoplasmic and nuclear fractions prepared from resting unstimulated cells were serially diluted and the
amount of I
B
was determined by Western blots (Fig.
7B). In neutrophils, about 65% of total cellular I
B
localizes in the nucleus, while in monocytes it is only about 3%
(n = 4, p < 0.001), confirming that
the nuclear localization of I
B
is specific for the
neutrophils.

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Fig. 7.
Nucleocytoplasmic distribution of
I B in neutrophils and
monocytes. A, Western analysis of I B in nuclear
(N) and cytoplasmic (C) fractions in neutrophils
and monocytes. Lanes 1, unstimulated resting cells;
2, TNF -stimulated (15 min, 10 ng/ml) cells; 3,
TNF -stimulated cells preincubated 30 min with MG-132 (200 µM), inhibitor of proteasome. Each lane contains 5 × 105 cells. Blot is representative of three independent
experiments. B, quantitative determination of the total
amount of I B in the nucleus and cytoplasm in resting unstimulated
neutrophils and monocytes. Nuclear (N) and cytoplasmic
(C) fractions were serially diluted and analyzed by Western
blotting and densitometry. Only intensities of bands that fell within
the linear response of the film were used for calculation. Results are
representative of four independent experiments measured in
duplicate.
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DISCUSSION |
A large body of research has focused on understanding the
regulation of NF-
B activation in immune and inflammatory cells, but
very little is known about regulation of NF-
B activity in the
neutrophil. In addition, the signaling pathways leading to NF-
B
activation in response to neutrophil stimulation with TNF
have not
been delineated. The results of the present study lead to two important
conclusions: first, that NF-
B activation in human neutrophils
stimulated with TNF
, but not with IL-1
or LPS, is mediated
through PKC
-dependent degradation of I
B
; and second, NF-
B activation in human neutrophils is not regulated by
nuclear translocation of NF-
B subunits as is the case in most mammalian cells (21-23), but correlates with nuclear pools of
I
B
.
PKC
is a novel type of PKC that is activated by DAG but is
unresponsive to Ca2+ (20). In addition to PKC
,
neutrophils have been shown to contain the classical PKCs
and
,
which are both DAG and Ca2+ dependent, and the atypical
PKC
, which does not require either DAG or Ca2+ for
activation, but may be regulated by 3-phosphorylated inositides produced by phosphoinositide 3-kinase (33-35). Among these PKC isoforms, only PKC
is specifically inhibited by rottlerin at concentrations lower than 40 µM (15, 17-19). PKC
has
been shown to be involved in regulation of apoptosis, and inhibition of
PKC
by rottlerin blocked all parameters of apoptosis in human
neutrophils (15, 16). Since the NF-
B activation has been recently
implicated in the regulation of TNF
-induced apoptosis in human
neutrophils (14), we have utilized rottlerin to determine whether
NF-
B activation in response to neutrophil stimulation with TNF
involves the PKC
-dependent pathway. We demonstrate that
the same concentrations of rottlerin specifically inhibiting the
in vitro PKC
kinase activity from immunoprecipitated
neutrophilic lysates (Fig. 4B), also inhibit TNF
-induced
NF-
B activation in the neutrophil (Fig. 2B). Our data
indicate that PKC
regulates degradation of I
B
, since
inhibition of PKC
resulted in a dose-dependent increased cellular levels of I
B
, independent of new protein synthesis (Fig.
5). The rottlerin inhibition of NF-
B activation is specific only for
TNF
induction, since rottlerin has no effect on NF-
B DNA binding
or I
B
degradation in LPS- and IL-1
-stimulated neutrophils (Fig. 3), suggesting that PKC
is involved in I
B
degradation only in TNF
signaling.
PKC
is activated by DAG generated in vivo by PLC (20). In
this study we demonstrate that in human neutrophils, PI-PLC, and not
PC-PLC is involved in regulation of NF-
B, and is likely to be the
upstream activator of PKC
. With the exception of study by Nick
et al. (12) demonstrating that the LPS-induced activation of
NF-
B involves activation of mitogen-activated protein kinase, the
signaling pathways regulating NF-
B in human neutrophils have not
been delineated. Therefore at present, we do not know the downstream
events regulated by PKC
and leading to I
B
degradation and
NF-
B activation in TNF
-induced neutrophils. One of the critical regulatory steps dictating degradation of I
B
are I
B kinase (IKK), which consists of the catalytic subunits
and
and the regulatory subunit
, and NF-
B inducing kinase (NIK) (36). Recent
studies demonstrated that in T lymphocytes, NF-
B is activated by
PKC
through stimulation of IKK
(37-39). PKC
is another member of the novel PKC family that is selectively expressed in skeletal muscle and T lymphocytes, and plays a vital role in T cell stimulation (37). Neutrophils do not possess PKC
, and PKC
is the only novel
PKC isoform expressed in the neutrophil (20). Therefore, it seems
likely that PKC
stimulates degradation of I
B
in the TNF
-stimulated neutrophil by activating IKK
/
, and the exact molecular mechanism is currently under investigation. Although PKC
has been implicated in the regulation of transcription factors AP1/Jun
(40) and Stat 3 (41), to our knowledge, this is the first report
demonstrating involvement of PKC
in the TNF
-induced NF-
B activation.
According to current models of NF-
B activation, the biological
activity of NF-
B is controlled through the nuclear translocation of
NF-
B subunits (21-23). In the present study we demonstrate that in
human neutrophils, the nuclear pools of I
B
, and not the NF-
B
subunits, correlate with NF-
B DNA binding activity. While the
nuclear and cytoplasmic pools of p50 and p65 NF-
B proteins were not
significantly affected after neutrophil stimulation or inhibition of
NF-
B DNA binding (Fig. 6), it was the amount of I
B
in the
nucleus (and cytoplasm) that reflected the state of NF-
B activation.
Importantly, the nuclear localization of I
B
was specific for the
neutrophil, since in the peripheral blood monocytes, I
B
was
mainly cytoplasmic. Nuclear localization of I
B
has been recently
demonstrated also in cells overexpressing I
B
(42, 43) and in
stimulated cells (44-46), since activated NF-
B can stimulate
neotranscription and neosynthesis of I
B
(47, 48). This newly
synthesized I
B
can then enter the nucleus, remove NF-
B from
gene promoters, and transport it back to the cytoplasm (49-52). In
these models, nuclear localization of I
B
is induced by stimuli
inducing NF-
B activity and can be considered as a cellular mechanism
terminating the NF-
B-dependent transcription. In
contrast, nuclear presence of I
B
in resting unstimulated neutrophils suggests its protective role against induction of NF-
B
activation. In this respect it is important to point out that I
B
has been detected in the nuclear fraction of unstimulated cells also in
peripheral blood T lymphocytes, however, it was resistant to
stimulus-induced degradation, and its levels did not correlate with
NF-
B DNA binding (53). Studies are currently in progress to
determine whether the nuclear retention of I
B
in resting
neutrophils results from its post-translational modification (phosphorylation) and/or whether it is a consequence of its association with other regulatory protein(s).
Neutrophil exposure to TNF
resulted in substantial reduction of both
cytoplasmic and nuclear I
B
, allowing induction of NF-
B DNA
binding (Figs. 5-7). Whether this signal-dependent
reduction of nuclear I
B
content results from nuclear-cytoplasmic
shuttling of I
B
and its degradation in the cytoplasm, or whether
the nuclear I
B
can be phosphorylated and degraded in
situ, remains to be clarified. While further studies are required
to delineate the signaling pathways leading to NF-
B activation in
human neutrophils, this is the first report characterizing the
signaling events resulting in NF-
B activation in response to
neutrophil stimulation with TNF
. Our results suggest that the
TNF
-induced, but not LPS or IL-1
-induced activation of NF-
B in
the neutrophil is mediated by PKC
-dependent degradation
of I
B
. We have shown that NF-
B activation in the neutrophil is
not regulated by nuclear translocation of NF-
B p50 and p65 subunits,
but correlates with nuclear, as well as cytoplasmic, pools of I
B
.
These findings are biologically relevant since they suggest that in the
neutrophil, the presence of I
B
in the nucleus may function as a
safeguard against initiation of NF-
B-dependent
transcription of proinflammatory and anti-apoptotic genes. It will be
important to determine whether the exaggerated expression of
inflammatory genes seen in the neutrophil-mediated diseases (1, 2)
results from de-regulated activation of NF-
B caused by the reduced
I
B
levels in the nucleus. Identification of the key molecular
events regulating nuclear retention of I
B
in human neutrophils
may have major therapeutic implications.