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B Regulation in Human Neutrophils by Nuclear IB
: Correlation to Apoptosis1
Division of Neonatal-Perinatal Medicine, Schneider Children’s Hospital, Long Island Jewish Medical Center-The Long Island Campus, Albert Einstein College of Medicine, and North Shore-Long Island Jewish Research Institute, New Hyde Park, NY 11040
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B plays a key
role in the inflammatory response, regulating the expression of
proinflammatory and anti-apoptotic genes. Recently we have shown
that human neutrophils contain a significant amount of NF-B
inhibitor, IB
, in the nucleus of unstimulated cells. The present
objective was to examine the mechanisms controlling the nuclear content
of IB in human neutrophils and to determine whether increased
accumulation of IB in the nucleus is associated with increased
neutrophil apoptosis. We show for the first time that neutrophil
stimulation with pro-inflammatory signals results in degradation of
IB that occurs in both cytoplasm and nucleus. Prolonged (2-h)
stimulation with TNF and LPS induces resynthesis of IB that is
again translocated to the nucleus in human neutrophils, but not in
monocytic cells. Leptomycin B, a specific inhibitor of nuclear export,
increases nuclear accumulation of IB in stimulated neutrophils by
blocking the IB nuclear export, and this is associated with
inhibition of NF-B activity, induction of caspase-3 activation, and
apoptosis. Based on our data we present a new model of NF-B
regulation in human neutrophils by nuclear IB. Our results
demonstrate that the NF-B activity in human neutrophils is regulated
by mechanisms clearly different from those in monocytes and other human
cells and suggest that the increased nuclear content of IB in
human neutrophils might represent one of the underlying mechanisms for
the increased apoptosis in these cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(11, 12, 13).
NF-B comprises a family of transcription factors that serve as
important regulators of the genes involved in host immune and
inflammatory responses, apoptosis, proliferation, and differentiation
(14, 15, 16, 17, 18, 19). The most abundant and best characterized of the
NF-B dimers is the inducible p50/65 NF-B heterodimer
(20). In the classical model of NF-B activation,
NF-B p50/65 exists in the cytoplasm of unstimulated cells in an
inactive form associated with the inhibitory protein IB
(20, 21). Following cell stimulation by extracellular
stimuli, IB is phosphorylated through a cascade of inducible
protein kinases, ubiquitinated, and selectively degraded in cytoplasm
by the proteasome (21, 22). This results in unmasking of
the nuclear localization sequence (NLS)3
of the NF-B 50/65 heterodimers, which then translocate to the
nucleus and stimulate transcription of inflammatory and
anti-apoptotic genes. One of the first genes induced following
NF-B activation is IB itself, since the IB promoter also
contains the NF-B binding region. This newly synthesized IB
can then enter the nucleus, remove NF-B from gene promoters, and
transport it back to the cytoplasm, representing an important feedback
regulatory mechanism, also called postinduction repression (23, 24). Thus, in this classical model of NF-B activation,
NF-B activity is regulated by cytoplasmic degradation of IB
and nuclear translocation of NF-B dimers, and it is terminated by
nuclear entry of the newly synthesized IB.
Importantly, the NF-B regulation appears to be highly tissue and
cell specific (20). In human neutrophils, NF-B was
first identified by McDonald et al. (25). Interestingly,
the extent of NF-B activation in neutrophils was lower than that in
peripheral blood monocytes (25). We have recently shown
that resting human neutrophils, but not monocytes, contain a
significant amount of IB in the nucleus, thus differing from most
of the human cells described to date (26).
In this study we characterized the mechanisms controlling the nuclear
accumulation of IB in human neutrophils. We show for the first
time that neutrophil stimulation with pro-inflammatory signals induces
the degradation of IB that occurs in both cytoplasm and nucleus.
Furthermore, we demonstrate that sustained stimulation with TNF and LPS
induces resynthesis of IB that is translocated to the nucleus in
human neutrophils, but not in monocytic cells. Our results indicate
that the increased accumulation of IB in the nucleus of human
neutrophils is associated with the inhibition of NF-B activity,
caspase-3 activation, and increased apoptosis in these cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal Abs to human IB (sc-371) and
p21CIP1(sc-469) and mouse monoclonal
anti-actin Ab (sc-8432) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse anti-SUMO-1 mAb was obtained
from Zymed (San Francisco, CA), and polyclonal lactate dehydrogenase
(LDH) Ab (20-LG22) was purchased from Fitzgerald Industries
International (Concord, MA). HRP-conjugated anti-rabbit,
anti-mouse, and anti-goat IgG secondary Abs were obtained from
Amersham (Arlington Heights, IL).
Cell culture
Fresh blood was obtained from healthy adult human volunteers and collected in heparinized preservative-free tubes. Neutrophils and monocytic cells were purified under endotoxin-free conditions as described previously (26, 27). Purified cells were resuspended in RPMI 1640 supplemented with 10% low endotoxin FCS at a final concentration of 5 x 106 cells/ml and incubated at 37°C in polypropylene tubes with gentle agitation. For the inhibition experiments the cells were incubated with leptomycin B (LMB) or the vehicle solution (1/1000 volume of ethanol) for 45 min before stimulation.
Preparation of cytoplasmic and nuclear extracts
Nuclear and cytoplasmic extracts were prepared from 5 x 106 cells as described previously (26). Protein concentration was measured using 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 (26).
EMSA
The oligonucleotide used as a probe for EMSA was a 42-bp
double-stranded construct
(5'-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3')
containing two tandemly repeated NF-B binding sites (underlined).
EMSA was performed using nuclear extracts (containing 4–6 µg protein
in 5–7 µl) and the above NF-B oligonucleotide as described
previously (26, 28).
Western blotting
Denatured proteins were separated on 12% denaturing polyacrylamide gels and transferred to nitrocellulose membrane as described previously (26). To confirm equal amounts of loaded proteins, the membranes were stripped and reprobed with actin Ab. The signal was developed using secondary IgG-HRP and ECL detection.
Assessment of neutrophil apoptosis
Two methods were used for the assessment of neutrophil apoptosis. For morphological assessment, neutrophils were cytocentrifuged, air-dried, stained with a commercial May-Grunwald Giemsa stain (Diff-Quick, Baxter Healthcare, Deerfield, IL), and counted using oil immersion microscopy (x100 objective). Neutrophils (minimum of 200 cells/slide) counted in randomly selected fields were scored as apoptotic and nonapoptotic based on their nuclear morphology (8, 11). The results are expressed as the mean percent apoptosis ± SEM.
For measurement of caspase-3 activity, we used a colorimetric assay kit
from R&D Systems (Minneapolis, MN). Briefly, cells (2.5 x
106) were lysed in 63 µl lysis buffer (R&D
Systems) on ice (15 min), and whole cell lysates were centrifuged
(14,000 x g for 10 min). Fifty microliters of whole
cell lysates (containing 
50 µg protein) were then diluted to 100
µl with reaction buffer (R&D Systems) and incubated with 5 µl Asp
Glu Val Asp-p-nitroanaline substrate (R&D Systems) for
2 h at 37°C. Absorbance was determined at 410 nm. Blanks in the
absence of cell lysate were conducted to determine background
absorbance.
Data analysis
The data presented here represent a minimum of three experiments and, where appropriate, are expressed as the mean ± SEM. Data were analyzed using an InStat software package (GraphPad, San Diego, CA). Statistical significance was evaluated by Mann-Whitney test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B accumulates in the nucleus of
TNF-stimulated neutrophils, but not monocytic cells
First we analyzed whether IB induced by postinduction
repression translocates to the nucleus in TNF-stimulated human
neutrophils and monocytic cells. Cells were stimulated with TNF for 0,
15, 60, and 120 min, and cytoplasmic and nuclear extracts were prepared
and analyzed by Western blotting using IB Ab. The purity of
cytoplasmic and nuclear fractions was monitored using LDH and SUMO-1 as
specific markers, respectively (26). In neutrophils, TNF
stimulation leads to rapid (15-min) depletion of IB in both
cytoplasmic and nuclear fractions (Fig. 1
), and the newly synthesized IB
induced after prolonged neutrophil stimulation with TNF (60 and 120
min) translocates without delay to the nucleus.
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B was detected in the nuclear fraction at
any time point (Fig. 1
), indicating that in these cells neither basal
nor newly synthesized IB can accumulate in the nucleus. However,
since in monocytic cells the kinetics of IB degradation in the
cytoplasm were slower than those in the neutrophils, the following
experiments were performed to distinguish between basal and newly
synthesized IB. Both cell types were stimulated with TNF as
described above in the absence and the presence of prior pretreatment
with cycloheximide (CHX), an inhibitor of protein synthesis. This CHX
experiment confirmed that while in neutrophils IB is degraded
almost completely within 15 min after TNF stimulation, in monocytic
cells a significant amount of basal IB is present in the
cytoplasm even after 2-h stimulation with TNF, due to its slower
degradation.
Neutrophil stimulation with TNF induces nuclear shuttling of
IB
To further characterize the nuclear transport of IB in the
neutrophils, we used leptomycin B (LMB), a specific inhibitor of
nuclear export that interferes with the interaction between nuclear
export sequences (NES), and CRM1, a NES receptor, belonging to the
-importin family (29, 30). As shown in Fig. 2A, blocking the nuclear
export with LMB resulted in substantially increased nuclear
accumulation of IB in TNF-stimulated (2 h) neutrophils
(lanes 7 and 8), while the effect on the
nuclear accumulation of IB in unstimulated cells was much less
pronounced (lanes 3 and 4). In the
TNF-stimulated neutrophils two different scenarios could account for
the LMB-induced nuclear accumulation of IB. In the first model
the newly synthesized IB shuttles between nucleus and cytoplasm
of TNF-stimulated neutrophils, and blocking the nuclear export results
in IB accumulation in the nucleus. Alternatively, this IB
protein accumulated in the nucleus of TNF-stimulated neutrophils in the
presence of LMB could be the original, basal IB, escaping
TNF-induced degradation, provided that this degradation occurs only in
the cytoplasm.
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B). As expected, no IB was detected in the
cytoplasmic fraction in neutrophils stimulated 2 h with TNF in the
presence of CHX, independently of LMB treatment (Fig. 2B,
lanes 3 and 4). In the nucleus, the TNF-induced
resynthesis of IB that is increased by blocking the nuclear
export with LMB (lanes 6 and 7) is
prevented by pretreatment with CHX (lanes 6 and
8). These results imply that the IB protein
accumulating in the nucleus of stimulated neutrophils treated with LMB
is the newly synthesized IB, induced by postinduction repression,
and that neutrophil stimulation with pro-inflammatory signals induces
continuous nuclear-cytoplasmic shuttling of IB. Surprisingly, no
IB was detected in the nucleus of TNF-stimulated neutrophils in
the presence of CHX and LMB (lane 9). The effect of
CHX alone on IB expression and stability was minimal
(lanes 5 and 10) and was comparable to
that of TNF (2 h) alone (lanes 1 and 6).
Together, these results suggested that the TNF-induced degradation of
IB might occur also in the nucleus.
Neutrophil stimulation induces IB degradation that occurs in
the cytoplasm as well as in the nucleus
In Fig. 1 we have shown that neutrophil stimulation with TNF
results in rapid depletion of IB in both cytoplasm and nucleus.
However, it was not clear whether IB is degraded independently in
both compartments or whether it is degraded exclusively in the
cytoplasm after TNF induces its nuclear shuttling. To distinguish
between these two models, neutrophils were stimulated 15 min with TNF
with and without prior incubation with LMB and/or CHX (Fig. 3). As expected, in the cytoplasm 15-min
stimulation with TNF led to degradation of IB (lanes
1 and 3), independently of LMB or CHX
(lanes 4–6). In the nucleus, inhibition of nuclear
export with LMB resulted in a partial, TNF-induced (15-min) degradation
of IB (lanes 2 and 4), independent
of new protein synthesis (lane 6). This result
together with the data shown in Fig. 2B (lane
9) indicated that IB can be degraded in the nucleus as
well.
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B, neutrophils
were stimulated with TNF in the presence of CHX and LMB, and nuclear
IB degradation was measured at 0–60 min. As shown in Fig. 4, blocking the nuclear IB export
with LMB did not prevent time-dependent degradation of IB in the
nucleus. The lower panel shows analysis of
another nuclear protein, p21CIP1
(28), under these conditions, demonstrating the
specificity of TNF-induced nuclear degradation for IB. Together,
these results demonstrate that in stimulated human neutrophils,
IB is degraded in response to stimulation with proinflammatory
signals in the nucleus as well as in the cytoplasm.
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B in stimulated
neutrophils, but not monocytic cells, and this is associated with
NF-B inhibition and increased apoptosis
Since apoptosis in neutrophils as well as other cell types has
been shown to be mediated by the inhibition of NF-B (11, 31), we investigated whether LMB-induced increased nuclear
accumulation of IB in stimulated neutrophils is associated with
NF-B inhibition and increased apoptosis.
Although in our initial studies of the regulation of nuclear
accumulation of IB we used TNF for neutrophil stimulation, to
investigate the role of nuclear IB in the induction of apoptosis
we used LPS. Like TNF, LPS evokes activation of NF-B in human
neutrophils (25, 26, 32). However, unlike TNF, which
inhibits apoptosis after 20-h incubation, while it accelerates
neutrophil apoptosis in a subpopulation of cells at earlier (2–8
h) times (33), LPS promotes neutrophil survival (34, 35).
Similarly as in TNF-stimulated neutrophils, in neutrophils stimulated
with LPS, LMB significantly increased the nuclear accumulation of
IB (Fig. 5), again demonstrating
that the newly synthesized IB induced by sustained neutrophil
stimulation with pro-inflammatory signals continuously shuttles between
nucleus and cytoplasm. The LMB-induced increased nuclear accumulation
of IB was accompanied by inhibition of NF-B, measured as the
extent of NF-B DNA binding in neutrophils stimulated with LPS as
well as TNF (Fig. 6). This correlates
well with the previously reported in vitro inhibition of NF-B DNA
binding by exogenously added purified recombinant IB protein
(36).
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B in the nucleus of human neutrophils
is associated with caspase-3 activation. As shown in Fig. 7A, neutrophil preincubation
with LMB increased caspase-3 activity in neutrophils stimulated with
LPS, and this was most prominent after 3-h incubation with LPS
(p < 0.01; n = 6). To confirm the
above results by an independent method, we determined apoptosis in
LMB-treated neutrophils by morphological evaluation (8, 11). Consistent with caspase-3 activity, LMB accelerated the
induction of apoptosis in LPS-stimulated neutrophils (Fig. 7B), and as with caspase-3 activity, this was most
significant after 3-h stimulation with LPS (p <
0.01; n = 5).
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B in the inhibition of
NF-B activity and the induction of apoptosis in human neutrophils
and to exclude the possibility that LMB-mediated inhibition of NF-B
and induction of neutrophil apoptosis were due to the cytotoxic effect
of LMB, we evaluated the effect of LMB on IB nuclear-cytoplasmic
shuttling, NF-B activation, and caspase-3 activity in human
monocytic cells. As shown in Fig. 8A, LMB did not induce nuclear
translocation of IB in LPS-stimulated monocytic cells, confirming
that in these cells IB is not imported to the nucleus and remains
localized in the cytoplasm. Importantly, in stimulated monocytic cells,
LMB did not inhibit DNA binding of the inducible NF-B 50/65
heterodimer (Fig. 8B). Furthermore, LMB treatment of
LPS-stimulated monocytic cells did not significantly induce caspase-3
activity (p > 0.3; n = 6; Fig. 8C).
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B, LMB does not significantly affect NF-B
and caspase-3 activities suggests that the LMB-mediated inhibition of
NF-B and increased apoptosis in the neutrophils are caused by the
increased amount of IB in the nucleus, rather than by LMB itself.
Together, these results indicate that the increased IB in the
nucleus of human neutrophils inhibits NF-B DNA binding activity and
suggest that this may represent one of the underlying mechanisms for
the increased rate of neutrophil apoptosis. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity in human neutrophils
is regulated by mechanisms clearly different from those in monocytes
and other human cells. The current model of NF-B regulation in human
neutrophils by nuclear IB is presented in Fig. 9. First, we demonstrate that sustained
stimulation with proinflammatory signals induces resynthesis of
IB that is translocated to the nucleus in neutrophils, but not
monocytic cells. Second, we show, for the first time, that neutrophil
stimulation results in rapid IB degradation that occurs in both
cytoplasm and nucleus. Third, our results indicate that the increased
nuclear accumulation of IB in human neutrophils is associated
with the inhibition of NF-B activity and the induction of apoptosis
in these cells.
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B protein contains both NLS activity (24, 39) and
NESs (40, 41, 42, 43). Recent studies have shown that although in
unstimulated epithelial cells, IB is localized almost exclusively
in the cytoplasm, blocking the CRM1-specific export with LMB induces
nuclear localization of IB, indicating nuclear-cytoplasmic
trafficking of basal IB in unstimulated epithelial cells
(40, 41). Moreover, these studies implied that cytoplasmic
localization of IB in epithelial as well as other human cells is
established by a potent CRM1-dependent nuclear export process that is
dominant over the relatively weaker NLS-dependent nuclear import
(41). In contrast, in human neutrophils blocking the
CRM1-dependent export did not substantially increase nuclear
accumulation of basal IB in unstimulated cells (Fig. 2A). This implies that in unstimulated neutrophils, IB
nuclear import is dominant over its export, inhibition of which has
little effect on IB nuclear accumulation.
The lack of significant nuclear trafficking of IB in resting
neutrophils contrasts with its continuous shuttling in neutrophils
stimulated with proinflammatory signals. Since blocking the IB
nuclear export in stimulated neutrophils results in IB nuclear
accumulation (Figs. 2 and 5) and inhibition of NF-B activity (Fig. 6), mechanisms controlling IB nuclear trafficking in human
neutrophils may represent a new potential target for the regulation of
NF-B-driven gene expression in these cells. We and others (44, 45) have previously demonstrated that the rate of nuclear
transport and the extent of nuclear accumulation of nuclear proteins
are regulated by phosphorylation. In this context, IB has been
shown to be phosphorylated, in addition to the IB kinase (IKK) that
phosphorylates it at serine residues 32 and 36 (46, 47),
by casein kinase II (48), protein kinase C
(49), tyrosine kinase (50), and DNA-dependent
protein kinase (51). However, only phosphorylation by IKK
leads to the signal-induced proteolytic degradation of IB.
Phosphorylations by casein kinase II, DNA-dependent protein kinase, and
tyrosine kinase were not demonstrated to result in IB
degradation, albeit they regulate NF-B activity. Therefore, it seems
plausible that one of the mechanisms by which they control NF-B
activation is by regulating the nuclear trafficking of
IB. Studies are currently in progress to determine the
phosphorylation sites and corresponding protein kinases involved in the
regulation of nuclear transport of IB in resting and stimulated
human neutrophils.
Originally, IB has been thought to be phosphorylated by the IKK
complex, ubiquitinated and degraded by the proteasome exclusively in
the cytoplasm (52). However, recent studies suggested that
a functional ubiquitin-proteasome system might be operative within the
nucleus as well. In this study we have established that in human
neutrophils, nuclear export is not required for the stimulus-induced
degradation of IB. This is consistent with recent studies using
epithelial cells, demonstrating ubiquitin-dependent nuclear degradation
of IB and MyoD, another transcription factor (53, 54). In addition, a recent study by Birbach et al.
(55) demonstrated continuous nuclear-cytoplasmic shuttling
of signaling kinases upstream of IB, IB kinase, and
NF-B-inducing kinase. Although the specific role and importance of
nuclear protein degradation are not known at present, it is clear that
the ubiquitin-mediated protein degradation is regulated by
compartmentalization within the cell. In this respect it is interesting
to point out that while in neutrophils, IB is degraded in the
nucleus as well as in the cytoplasm, in monocytic cells IB is
degraded exclusively in the cytoplasm (Fig. 1). Thus, the nuclear
degradation of IB may represent a new additional level of NF-B
regulation in response to neutrophil stimulation with pro-inflammatory
signals.
Our results show that LMB causes nuclear entrapment of IB in
stimulated human neutrophils, but not in monocytic cells (Figs. 5 and 8A), confirming that in the monocytic cells, IB is not
imported to the nucleus. The LMB-induced increased nuclear accumulation
of IB in the neutrophils was accompanied by NF-B inhibition
(Fig. 6) and increased caspase-3 activation and apoptosis (Fig. 7).
Since LMB was shown to decrease cell viability after long term exposure
(2–8 days) in BCR-ABL tyrosine kinase-transformed cells
(56), it cannot be completely excluded that the
LMB-induced NF-B inhibition and induction of neutrophil apoptosis
are caused by a more general effect that is unrelated to the increased
amount of IB in the nucleus. However, this seems unlikely,
because in monocytic cells, where IB remains exclusively
cytoplasmic regardless of LMB treatment, LMB did not significantly
affect NF-B and caspase-3 activities. Furthermore, recent studies
showed that in human prostate cancer cells and lymphocytes, short term
LMB treatment (
24 h) was not sufficient to induce apoptotic changes
(57, 58).
In conclusion, in this study we present a new model of NF-B
regulation in human neutrophils by nuclear IB. Our results
indicate that the increased accumulation of IB in the nucleus of
human neutrophils might represent one of the underlying
mechanisms for the increased apoptosis in these cells. Identification
of the key molecular mechanisms regulating nuclear accumulation of
IB in neutrophils will not only expand our understanding of
NF-B regulation in these cells, but might also provide a new class
of drug targets to selectively modify neutrophil survival and
pro-inflammatory gene expression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ivana Vancurova, Long Island Jewish Medical Center, Research Building B-49, 270-05 76th Avenue, New Hyde Park, New York 11040. E-mail address: vancurov{at}lij.edu
3 Abbreviations used in this paper: NLS, nuclear localization sequence; CHX, cycloheximide; IKK, IB kinase; LDH, lactate dehydrogenase; LMB, leptomycin B; NES, nuclear export signal.
Received for publication March 19, 2002. Accepted for publication July 29, 2002.
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