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Blood, 15 May 2002, Vol. 99, No. 10, pp. 3683-3691
IMMUNOBIOLOGY
Constitutive nuclear factor- B activity preserves homeostasis
of quiescent mature lymphocytes and granulocytes by controlling
the expression of distinct Bcl-2 family proteins
Fabrice Bureau,
Alain Vanderplasschen,
Fabrice Jaspar,
Frédéric Minner,
Paul-Pierre Pastoret,
Marie-Paule Merville,
Vincent Bours, and
Pierre Lekeux
From the Department of Veterinary Physiology,
Department of Parasitic and Infectious Diseases, and Department of
Medical Chemistry and Medical Oncology, University of Liège,
Belgium.
 |
Abstract |
Constitutive nuclear factor kappaB (NF- B) activity protects
quiescent mature immune cells from spontaneous apoptosis. Here, we
examined whether NF-B exerts its antiapoptotic function in these
cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-B were used to achieve total NF-B inactivation in quiescent human blood lymphocytes, granulocytes, and
monocytes. NF-B inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte
apoptosis. T- and B-cell apoptosis was slow and associated with
a gradual down-regulation of the prosurvival Bcl-2 family proteins
Bcl-xL and Bcl-2, respectively. By contrast, granulocyte
apoptosis was fast and accompanied by a rapid cellular accumulation of
Bcl-xS, the proapoptotic Bcl-x isoform that is generated
from alternative splicing of the bcl-x pre-mRNA. Finally,
antisense bcl-xL and bcl-2
knockdown in T and B cells, respectively, and induction of
Bcl-xS expression in granulocytes through antisense
oligonucleotide-mediated redirection of bcl-x pre-mRNA
splicing were sufficient to induce significant apoptosis in these
cells. Taken together, these results reveal that basal NF-B activity
preserves homeostasis of quiescent mature lymphocytes and granulocytes
through regulation of distinct members of the Bcl-2 family. This study
sheds light on the constitutive mechanisms by which NF-B maintains
defense integrity.
(Blood. 2002;99:3683-3691)
© 2002 by The American Society of Hematology.
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Introduction |
Quiescent mature immune cells (QMICs) form a
reservoir that may be rapidly engaged in innate or adaptive immune
responses. In normal conditions, the number of QMICs remains
constant,1 thus ensuring the maintenance of an optimal
defense potential. This suggests that homeostatic mechanisms tightly
control both the emergence of newly formed QMICs and the further
survival of these cells. Although crucial, the molecular basis of QMIC
survival remains elusive. However, convergent studies have shown that
the constitutive presence of transcriptionally active nuclear factor kappaB (NF-B) complexes in resting lymphocytes and phagocytes allows
these cells to escape spontaneous apoptosis, thus contributing to the
maintenance of the size of the QMIC pool.2-5
NF-B is present in an inducible form in virtually all cell
types6 and in a constitutively activated form in most
immune cells and certain neurons.2,4,7,8 The NF-B
family is composed of 5 structurally related DNA-binding proteins,
designated NF-B1/p50, NF-B2/p52, RelA/p65, Rel/c-Rel, and
RelB.9 RelA, Rel, and RelB contain a C-terminal
transactivation domain, whereas p50 and p52 lack a transactivating
domain and are not transcriptionally active. Although the most common
NF-B complex is a heterodimer of the RelA and p50 subunits, the
different family members can associate in various homo- or heterodimer
combinations. Inactive NF-B complexes are sequestered in the cytosol
by inhibitory proteins of the IB family. Following various stimuli,
IB proteins are phosphorylated, ubiquinated, and degraded by the
proteasome, allowing NF-B nuclear translocation and transcriptional
initiation of NF-B-dependent genes.
NF-B-dependent genes are involved in development, immunity, and
cell proliferation and survival.10 NF-B target genes
implicated in cell death prevention include those encoding the tumor
necrosis factor (TNF) receptor-associated factors TRAF1 and TRAF2, the inhibitor of apoptosis proteins c-IAP1, c-IAP2, and X-IAP, the zinc
finger protein A20, the immediate-early response protein IEX-1L, and
the manganese superoxide dismutase.11 Recently, the genes
encoding Bcl-xL and Bfl-1/A1, which are antiapoptotic proteins of the Bcl-2 family, have also been identified as
NF-B-dependent transcriptional targets.12-15 The Bcl-2
family members are essential regulators of cell survival that exhibit
either antiapoptotic (Bcl-2, Bcl-xL, Bfl-1/A1, etc) or
proapoptotic (Bax, Bcl-xS, Bad, Bid, etc)
activities.16,17
To date, only few studies have been devoted to the role of
NF-B-dependent expression of antiapoptotic Bcl-2 family proteins in
promoting survival of primary immune cells. However, Grossmann and
colleagues18 have recently established that NF-B
activation serves a key antiapoptotic function during the later stages
of B-cell maturation through the induction of prosurvival Bcl-2
homologues. In addition, NF-B activation and subsequent Bfl-1
expression protect mature B cells from antigen receptor
ligation-induced apoptosis,13 and T-cell activation via
the T-cell receptor (TCR) enhances survival via a pathway
involving the serine/threonine kinase protein kinase B , NF-B, and
Bcl-xL.19 Although these observations reveal
an essential role for NF-B-induced expression of prosurvival Bcl-2
homologues in preventing the apoptosis of immune cells during
maturation and activation, the question of whether this mechanism is
also crucial for QMIC survival has not been addressed, except in
resting macrophages, where constitutive NF-B activity is required to
maintain Bfl-1 expression and mitochondrial homeostasis.3
Here, we demonstrate using specific pharmacologic inhibitors of NF-B
that basal NF-B activity protects quiescent mature lymphocytes and
granulocytes from spontaneous apoptosis through the regulation of
distinct members of the Bcl-2 family. This study is the first to shed
light on the constitutive mechanisms by which NF-B maintains
peripheral lymphocyte and granulocyte homeostasis, thereby preserving
defense integrity.
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Materials and methods |
Cell sorting, culture, and treatment
Human blood lymphocytes, monocytes, and granulocytes were
obtained from buffy coats (Transfusion Center, Liege, Belgium). Mononuclear cells were separated from granulocytes by density centrifugation (Histopaque; Sigma, Bornem, Belgium). Contaminating erythrocytes were removed from the granulocyte fraction by hypotonic lysis. Granulocyte purity, as determined by counting of cytospin preparations stained with Diff-Quick (Dade Behring, Dudingen, Germany),
was always more than 95%. T cells, B cells, and monocytes were
purified by negative magnetic selection using microbeads coated with
antibodies directed against unwanted cells (Pan T-cell isolation kit,
B-cell isolation kit, and monocyte isolation kit; Miltenyi Biotec,
Paris, France). This procedure yielded T-cell, B-cell, and monocyte
populations that were more than 98% positive for the CD3, CD19, and
CD14 markers, respectively, as determined by flow cytometry analyses
(FACStar Plus; Becton Dickinson, San Jose, CA). Cells were cultured at
2 × 106/mL in RPMI 1640 medium supplemented with 1%
glutamine, 10% fetal calf serum, 50 µg/mL streptomycin, and 50 IU/mL
penicillin (all from Gibco BRL, Merelbeke, Belgium). Cells were
cultured in the presence or absence of gliotoxin (GTX; Sigma), its
inactive analogue methylthiogliotoxin (mGTX; Sigma), cyclopentenone
prostaglandin A1 (PGA1; Cayman Chemical, Ann Arbor, MI), or
oligonucleotides (see below) for different times before analysis.
Antibodies
Fluorescein isothiocyanate (FITC)-conjugated anti-CD3
(MCA463F), anti-CD14 (MCA596F), and anti-CD19 (MCA1111F) monoclonal antibodies were purchased from Serotec (Oxford, United Kingdom). The
antibodies specific for p50 (sc-114 X), p52 (sc-298 X), RelA (sc-372
X), c-Rel (sc-70 X), RelB (sc-226 X), Bfl-1 (sc-8351), Bcl-2 (sc-7382),
and -tubulin (sc-8035) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). The monoclonal antibody directed against Bax (Ab-3)
was purchased from Oncogene Research Products (Darmstadt, Germany), and
the monoclonal antibody recognizing Bcl-xL/S (B61220) was
obtained from Transduction Laboratories (Lexington, KY). The
specificity of the anti-Bfl-1 and -Bcl-2 antibodies was verified
using recombinant Bcl-2, Bcl-xL, Bak, and Bax of human
origin, and protein extracts from TNF--stimulated A549 cells, which
contain high amounts of Bfl-1. Recombinant proteins were obtained from
Santa Cruz Biotechnology, except the recombinant Bax, which was
produced in our laboratory.
Apoptosis assays
Apoptosis was assessed by staining with annexin V and propidium
iodide (PI) using the Annexin-V-FLUOS staining kit (Roche, Mannheim,
Germany) following the recommendations of the manufacturer. Flow
cytometry analyses were performed with a FACStar Plus (Becton Dickinson).
Nuclear protein extraction
Nuclear protein extracts were prepared as previously
described.20 Cytoplasmic buffer contained 10 mM HEPES, pH
7.9, 10 mM KCl, 2 mM MgCl2, 0.1 mM
ethylenediaminetetraacetic acid (EDTA), 0.2% (vol/vol) NP-40,
1.6 mg/mL protease inhibitors (Complete, Roche), and 3 mM of the serine
protease inhibitor diisopropyl fluorophosphate (DFP; Sigma). Pelleted
nuclei were resuspended in 20 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.63 M NaCl, 25% (vol/vol) glycerol,
1.6 mg/mL protease inhibitors, and 3 mM DFP (nuclear buffer), incubated
for 20 minutes at 4°C and centrifuged for 30 minutes at 14 000 rpm.
Protein amounts were quantified with the Micro BCA protein assay
reagent kit (Pierce, Rockford, IL).
Electrophoretic mobility shift assays
Binding reactions were performed for 30 minutes at room
temperature with 5 µg nuclear proteins in 20 mM Hepes, pH 7.9, 10 mM
KCl, 0.2 mM EDTA, 20% (vol/vol) glycerol, 1% (wt/vol) acetylated bovine serum albumin, 3 µg poly(dI-dC) (Amersham Pharmacia Biotech, Aylesbury, United Kingdom), 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 100 000 cpm of
[32P]-labeled double-stranded oligonucleotide probes.
Probes were prepared by annealing the appropriate single-stranded
oligonucleotides (Eurogentec, Liege, Belgium) at 65°C for 10 minutes
in 10 mM Tris, 1 mM EDTA, and 10 mM NaCl, followed by slow cooling to
room temperature. The probes were then labeled by end-filling with the
Klenow fragment of Escherichia coli DNA polymerase I
(Roche), with [-32P]-dATP and
[-32P]-dCTP (Dupont-New England Nuclear [NEN] Life
Science Products, Les Ulis, France). Labeled probes were purified by
spin chromatography on Sephadex G-25 columns (Roche). DNA-protein
complexes were separated from unbound probe on 4% native
polyacrylamide gels at 150 V in 0.25 M Tris, 0.25 M sodium borate, and
0.5 mM EDTA, pH 8.0. Gels were vacuum-dried and exposed to Fuji x-ray
film at  80°C for 12 hours. To confirm specificity, competition
assays were performed with a 50-fold excess of unlabeled wild-type
probes and with mutated probes. For supershifting experiments, 1.5 µL
of each antibody was incubated with the extracts for 30 minutes before
addition of the radiolabeled probe. The sequences of the
oligonucleotides used in this work were as follows: wild-type
palindromic B probe, 5'-TTG GCA ACG GCA GGG GAA TTC CCC TCT CCT TAG
GTT-3'; mutated palindromic B probe, 5'-TTG GCA ACG GCA GAT CTA TTC
CCC TCT CCT TAG GTT-3'.
Immunoblots
Whole-cell extracts (10 µg) were added to a loading buffer (10 mM Tris-HCl, pH 6.8, 1% [wt/vol] sodium dodecyl sulfate, 25% [vol/vol] glycerol, 0.1 mM  -mercaptoethanol, 0.03% [wt/vol]
bromophenol blue), boiled, and run on a 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After
electro-transfer to polyvinylidene difluoride membranes (Roche) and
blocking overnight at 4°C with 20 mM Tris, pH 7.5, 500 mM NaCl, 0.2 (vol/vol) Tween 20 (Tris/HCl/Tween) plus 5% (wt/vol) dry milk, the
membranes were incubated for 1 hour with the first antibody (1:200
dilution), washed, and then incubated for 45 minutes with
peroxidase-conjugated goat anti-rabbit IgG for Bfl-1 (1:5000 dilution;
Kirkegaard & Perry Laboratories, Gaithersburg, MD) and
peroxidase-conjugated rabbit anti-mouse IgG for Bax, Bcl-2, and
Bcl-xL/S (1:1000 dilution; Dako, Glostrup, Denmark). The
reaction was revealed using the enhanced chemiluminescence detection
method (ECL kit, Amersham Pharmacia Biotech). Equal loading of proteins
on the gel was confirmed by probing the blots for -tubulin (data
not shown).
Reverse transcription-polymerase chain reactions
Total RNA was extracted from cells using the Rneasy Mini kit
according to manufacturer's instructions (Qiagen, Hilden, Germany). Poly(A) RNA was primed with oligo(dT) (Roche) and reverse transcribed with AMV reverse transcriptase (Roche) for 1 hour at 42°C. cDNA products were amplified by polymerase chain reaction (PCR) using primers specific for bcl-2 (5' primer GAT GTC CAG CCA GCT
GCA CCT G; 3' primer CAC AAA GGC ATC CCA GCC TCC),
bcl-xL/S (5' primer ATG GCA GCA GTA AAG CAA G;
3'primer GCT GCA TTG TTC CCA TAG A), and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) as a control (5' primer ACT GGC ATG GCC TTC CGT
GT; 3' primer TTA CTC CTT GGA GGC CAT GT). The
bcl-xL/S primers allowed simultaneous
amplification of bcl-xL (353 base pair [bp])
and bcl-xS (164 bp). All primers were purchased
from Eurogentec. A 50 µL PCR reaction was set up containing 5 µL
cDNA, 10 mM Tris-HCl, 25 pmol of each primer, 1.5 mM MgCl2,
0.2 mM dNTP, and 2.5 U of AmpliTaq DNA polymerase (Perkin Elmer,
Boston, MA). Amplification consisted of 30 (bcl-2) or 35 (bcl-xL/S) cycles of denaturation at 94°C for
20 seconds, annealing at 60°C (bcl-2) or 56°C
(bcl-xL/S) for 30 seconds, and extension at
72°C for 1 minute. Amplification products were electrophoresed on
1.2% agarose gels, and PCR product quantities were assessed by
densitometry (Gel Doc 2000; Bio-Rad, Hercules, CA).
Antisense knockdown of bcl-2 and bcl-x
T and B cells were treated with antisense (AS)
oligodeoxyribonucleotides (ODNs) sequences complementary to
bcl-x and bcl-2 mRNA, respectively.
Phosphorothioate backbone AS ODNs targeting bcl-x and
bcl-2 were synthesized by Eurogentec. Their sequences and
those of scrambled (SC) ODNs, which were used as negative controls,
were as follows: bcl-x AS ODN, 5'-TGT ATC CTT TCT GGG AAA
GC-3'; bcl-x SC ODN, 5'-TAA GTT CCG ATG CGA CTT GT-3';
bcl-2 AS ODN, 5'-TCT CCC AGC GTG CGC CAT-3';
bcl-2 SC ODN, 5'-TAC CGC GTG CGA CCC TCT-3'. The ODNs were
delivered to the cells in the form of complexes with a liposome
formulation of the cationic lipid
1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
(DMRIE) and cholesterol (DMRIE-C Reagent; Gibco BRL). A quantity of 1 mL OPTI-MEM I Reduced Serum Medium (Gibco BRL) containing 24 µL
DMRIE-C and 1 mL OPTI-MEM I containing 8 µg DNA was mixed and allowed
to complex for 45 minutes at room temperature. A quantity of
2 × 106 freshly isolated cells in 0.25 mL of serum-free
medium were then added to the transfection medium and incubated for 4 hours at 37°C in a CO2 incubator. Afterward, 2 mL of
growth medium containing 20% fetal calf serum was added, and the cells
were cultured for another 20 hours.
Oligoribonucleotide-mediated redirection of bcl-x
pre-mRNA splicing
Expression of Bcl-xS was induced in granulocytes
through oligoribonucleotide (ORN)-mediated redirection of
bcl-x pre-mRNA splicing, as recently described by Taylor and
coworkers,21 except that a 20-mer phosphorothioate AS ORN
containing uniform 2'-O-methyl (2'-OMe) modifications was used. Its
sequence was as follows: 5'-CUG GAU CCA AGG CUC UAG GU-3'. An ORN
containing 5 mismatched (MM) bp was used as a control (5'-CUG GUU ACA
CGA CUC CAG GU-3'). The ORNs were synthesized by Eurogentec.
Transfection was performed as described above, except that granulocytes
were left in the transfection medium for 8 hours before analysis.
| |
Results |
Total inhibition of constitutive NF-B activity results in late
lymphocyte and early granulocyte apoptosis
The fungal metabolite gliotoxin (GTX) and the cyclopentenone
prostaglandin A1 (PGA1), 2 specific pharmacologic inhibitors of
NF-B,22-24 were used to obtain total NF-B
inactivation in QMICs. Quiescent human blood T and B cells,
granulocytes, and monocytes were isolated from buffy coats by density
centrifugation and negative magnetic selection and cultured in the
presence or absence of various doses of GTX, its inactive analogue
methylthiogliotoxin (mGTX), or PGA1 before analysis of NF-B
DNA-binding by electrophoretic mobility shift assays (EMSAs).
Quantities of 1.5 µM GTX and 48 µM PGA1 were sufficient to obtain
total NF-B inhibition in lymphocytes and granulocytes, whereas 5 µM GTX and 96 µM PGA1 were required to completely inhibit NF-B
activity in monocytes (Figure 1A-D). These concentrations were used throughout the study. Total inhibition of NF-B activity was always observed 90 minutes after treatment with
GTX or PGA1. mGTX did not affect NF-B activity in any cell type
(Figure 1A-D).
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| Figure 1.
Inhibition of constitutive NF-B activity by GTX and
PGA1 induces QMIC apoptosis.
Human blood T cells (A), B cells (B), granulocytes (C), and monocytes
(D) were isolated from buffy coats by density centrifugation and
negative magnetic selection and cultured for 90 minutes in the presence
or absence of 1.5 µM mGTX, 1.5 µM GTX, or 48 µM PGA1, except for
monocytes, which were treated with 5 µM mGTX, 5 µM GTX, or 96 µM
PGA1. Nuclear extracts were then prepared and analyzed for
NF-B-binding activity by EMSAs. The arrows indicate specific
NF-B complexes. EMSAs are representative of at least 3 comparable
assays. Untreated and treated cells were also cultured for 6 hours and
24 hours (mononuclear cells) or 2 hours, 4 hours, and 6 hours
(granulocytes) before conducting apoptosis assays using a dual-color
annexin-V-FITC/PI staining and flow cytometry analyses. Data are
presented as means ± standard deviations (n = 6).
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To characterize the NF-B complexes present in QMICs, supershift
experiments were performed with antibodies directed against the various
members of the NF-B family. These experiments identified RelA/p50,
c-Rel/p50, and p50/p50 dimers in lymphocytes and granulocytes, whereas
only p50 homodimers were present in monocytes (data not shown).
DNA-binding competition experiments using 50-fold excess of unlabeled
wild-type and mutated palindromic B probes confirmed the specificity
of NF-B binding in all cell types (data not shown).
To determine the effects of total NF-B inhibition on lymphocyte,
granulocyte, and monocyte longevity, purified cells were cultured for
different times in the presence or absence of mGTX, GTX, or PGA1 and
assayed for apoptosis and necrosis using dual- color annexin-V-FITC/PI
staining and flow cytometry analyses. NF-B inhibition caused a
drastic induction of lymphocyte death (Figure 1A-B). Nearly all dead
lymphocytes were annexin-V-FITC-positive and PI-negative,
demonstrating that loss of viability was mainly due to apoptosis rather
than necrosis (data not shown). Although increased lymphocyte apoptosis
was already detectable at 6 hours, most cells died later. At 24 hours,
when the rate of spontaneous apoptosis was 5.3% ± 0.4% in T cells
and 26.2% ± 9.7% in B cells, NF-B inhibition caused more
than 70% and more than 90% apoptosis, respectively. Both GTX and PGA1
induced early and massive granulocyte apoptosis (Figure 1C). Indeed,
apoptosis of GTX- and PGA1-treated granulocytes reached 80% at 6 hours, while the rate of spontaneous apoptosis was less than 30%. The
effects of complete NF-B inhibition were less pronounced in
monocytes (Figure 1D). After 24 hours of GTX and PGA1 treatment, the
number of apoptotic monocytes was only increased by 25% to 30%
compared with untreated controls. mGTX had no significant effect on the
spontaneous rate of lymphocyte, granulocyte, and monocyte apoptosis
(Figure 1A-D). To ascertain that apoptosis of GTX- and PGA1-treated
lymphocytes and granulocytes specifically resulted from NF-B
inhibition rather than from nonspecific cytotoxicity of these drugs,
A549 cells, which do not display constitutive NF-B activity, have
been treated with 10 µM GTX or 100 µM PGA1. These treatments did
not affect A549 cell viability over a period of 24 hours, as determined
by dual-color annexin-V-FITC/PI staining (data not shown), confirming
that the effects of GTX and PGA1 were specific.
These data (1) demonstrate that constitutive NF-B activity in QMICs
may be totally inhibited by GTX and PGA1; (2) confirm that NF-B is
essential for the survival of these cells, especially lymphocytes and
granulocytes; and (3) show that complete NF-B deactivation is
associated with slow lymphocyte and fast granulocyte apoptosis.
Complete NF-B inactivation induces Bcl-xL or Bcl-2
down-regulation in lymphocytes and Bcl-xS expression in
granulocytes
NF-B has been demonstrated to induce the expression of
Bcl-xL and Bfl-1, 2 antiapoptotic proteins of the Bcl-2
family.12-15 To address the question of whether
constitutive NF-B activity protects QMICs from apoptosis through the
control of Bcl-2 family proteins, the expression levels of 5 representative members of this family, namely Bax, Bcl-2, Bfl-1, and
Bcl-xL/S, were assessed by immunoblots and reverse
transcription (RT)-PCRs in untreated and in GTX-, mGTX-, and
PGA1-treated QMICs.
Bfl-1 and Bcl-xS were undetectable in freshly isolated
blood T cells. Conversely, T cells contained Bax, Bcl-2, and
Bcl-xL proteins (Figure 2A).
Bax, Bcl-2, and Bcl-xL protein levels remained constant
over a period of at least 24 hours in untreated and mGTX-treated T
cells. Neither Bax nor Bcl-2 protein levels were modified following GTX
or PGA1 treatment. By contrast, the amount of Bcl-xL
protein in T cells began to decrease 6 hours after NF-B inactivation reaching nearly undetectable levels at 24 hours (Figure 2A). To confirm
bcl-xL down-regulation in GTX- and PGA1-treated
T cells, RT-PCR analyses were performed (Figure 2B; data not shown).
bcl-xL mRNA levels were dramatically reduced 6 hours after GTX or PGA1 treatment and remained low for at least 18 hours. Conversely, bcl-xL mRNA levels remained
constant throughout the procedure in untreated and mGTX-treated T
cells.
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| Figure 2.
Complete NF-B inactivation induces Bcl-xL
down-regulation in quiescent human blood T cells.
(A) Blood T cells were isolated from buffy coats by negative magnetic
selection and cultured for 6 hours and 24 hours in the presence or
absence of 1.5 µM mGTX, 1.5 µM GTX, or 48 µM PGA1. Whole-cell
extracts were then prepared and analyzed by immunoblotting for Bax,
Bcl-2, Bfl1, and Bcl-xL/S expression. (B) RNA was prepared
from blood T cells cultured for 6 hours and 18 hours in the presence or
absence of 1.5 µM mGTX or 1.5 µM GTX and analyzed by RT-PCR for
expression of bcl-xL. As a control for
quantification, glyceraldehyde phosphate dehydrogenase (GAPDH) was also
amplified. These results are representative of at least 3 comparable
experiments.
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Freshly purified blood B cells expressed only Bax and Bcl-2
proteins (Figure 3A). Untreated and
mGTX-treated B cells maintained high levels of Bax and Bcl-2 proteins
for at least 24 hours. By contrast, the amount of Bcl-2 protein
gradually decreased following NF-B inhibition reaching very low
levels at 24 hours (Figure 3A). At 6 hours and 18 hours,
bcl-2 mRNA was drastically reduced in GTX- and PGA1-treated
B cells compared with untreated and mGTX-treated controls (Figure 3B;
data not shown), confirming bcl-2 down-regulation in
NF-B-inactivated B cells.
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| Figure 3.
Complete NF-B inhibition results in Bcl-2
down-regulation in resting human blood B cells.
(A) Blood B cells were isolated from buffy coats by negative magnetic
selection and cultured for 6 hours and 24 hours in the presence or
absence of 1.5 µM mGTX, 1.5 µM GTX, or 48 µM PGA1. Whole-cell
extracts were analyzed by immunoblotting for Bax and Bcl-2 expression.
(B) RNA prepared from blood B cells cultured for 6 hours and 18 hours
in the presence or absence of 1.5 µM mGTX or 1.5 µM GTX was
analyzed by RT-PCR for expression of bcl-2. To control for
quantification, GAPDH was amplified. These results represent at least 3 comparable experiments.
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As shown in Figure 4A, blood
granulocytes contained large amounts of the proapoptotic protein Bax
but weakly expressed the antiapoptotic proteins Bcl-2, Bfl-1, and
Bcl-xL. Bcl-xS was barely detectable in freshly
isolated granulocytes. The levels of Bax, Bcl-2, Bfl-1, and
Bcl-xL proteins remained unmodified for at least 6 hours
after treatment with GTX or PGA1. Conversely, a rapid accumulation of
Bcl-xS was observed in GTX- and PGA1-treated granulocytes but not in untreated and mGTX-treated controls (Figure 4A). Maximal levels of Bcl-xS protein were recorded as soon as 4 hours
after NF-B inhibition. Since alternate splicing of bcl-x
pre-mRNA gives rise to 2 transcripts encoding either Bcl-xL
or Bcl-xS,25 the results provided by
immunoblots suggested that redirection of bcl-x pre-mRNA
splicing from bcl-xL toward
bcl-xS occurred in NF-B-inactivated
granulocytes. To confirm increased expression of the variant
bcl-xS transcript in GTX- and PGA1-treated
granulocytes, bcl-xL and
bcl-xS mRNA isoforms were simultaneously
amplified by RT-PCRs using primers designed to hybridize to common
regions of bcl-xL and
bcl-xS. Both bcl-xL and
bcl-xS mRNAs were present in freshly purified
granulocytes (Figure 4B). However, the levels of
bcl-xL mRNA were much higher than those of
bcl-xS mRNA. NF-B inhibition led to a
concomitant decrease in bcl-xL mRNA and increase in bcl-xS mRNA. This process was rapid: equal
amounts of bcl-xL and bcl-xS
transcripts were found in granulocytes as early as 2 hours after
GTX or PGA1 treatment (Figure 4B; data not shown). At 4 hours, the
bcl-xS/bcl-xL mRNA ratio was
completely inverted relative to pretreatment conditions, as
demonstrated by densitometry analyses (Figure 4B). At 4 hours,
bcl-xS mRNA was indeed abundant, whereas
bcl-xL mRNA was nearly undetectable.
Densitometry analyses also showed that total bcl-x mRNA
decreased with time in NF-B-inactivated granulocytes (Figure 4B).
Sequencing of PCR products confirmed that the amplified transcripts
were identical to published bcl-xL and
bcl-xS mRNA sequences25 (data not
shown). To ascertain that the changes in the
Bcl-xS/Bcl-xL ratio was NF-B-dependent,
A549 cells have been transiently transfected with the empty pCDNA3 plasmid or with a pCDNA3 plasmid encoding the superinhibitory form of
IB- (ie, IB- with serine 32 and serine 36 mutated to alanine). Twenty-four hours after transfection, the cells were stimulated with TNF- for 6 hours before being assayed for the expression of Bcl-xL and Bcl-xS using
immunoblots. A549 cells transfected with the superinhibitory form of
IB- before stimulation with TNF- expressed Bcl-xS,
whereas the controls did not (data not shown), demonstrating that
Bcl-xS expression may result from specific NF-B
inhibition. To ensure that the superinhibitory form of IB- was
functionally expressed in transfected cells, immunoblots and EMSAs were
performed. These experiments showed that the superinhibitory form was
expressed at high levels and strongly inhibited TNF--induced
NF-B activation in transfected A549 cells (data not shown).
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| Figure 4.
Complete NF-B inactivation induces rapid accumulation
of Bcl-xS in quiescent human blood granulocytes.
(A) Blood granulocytes were isolated from buffy coats by density
centrifugation and cultured for 2 hours, 4 hours, and 6 hours in the
presence or absence of 1.5 µM mGTX, 1.5 µM GTX, or 48 µM PGA1.
Whole-cell extracts were prepared and analyzed by immunoblotting for
Bax, Bcl-2, Bfl-1, and Bcl-xL/S expression (B). RNA was
prepared from blood granulocytes cultured for 2 hours and 4 hours in
the presence or absence of 1.5 µM mGTX or 1.5 µM GTX and analyzed
by RT-PCR for expression of bcl-xL/S. To control
for quantification, GAPDH was also amplified. Filled columns show the
amount of total bcl-x mRNA and the ratio between
bcl-xS and bcl-xL mRNAs,
as determined by densitometry analyses. These results represent at
least 3 comparable experiments.
|
|
Bfl-1 was not detectable in monocytes (data not shown). The
expression of Bax, Bcl-2, and Bcl-xL/S was not modified
in monocytes cultured for 24 hours in the presence of GTX or
PGA1 when compared with untreated and mGTX-treated controls (Figure
5).
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| Figure 5.
Complete inhibition of NF-B does not affect Bax,
Bcl-2, and Bcl-xL/S expression in human monocytes.
(A) Monocytes were isolated from buffy coats by negative magnetic
selection and cultured for 24 hours with or without 5 µM mGTX, 5 µM
GTX, or 96 µM PGA1. Whole-cell extracts were analyzed by
immunoblotting for Bax, Bcl-2, and Bcl-xL/S expression.
Comparable results were obtained in at least 3 experiments.
|
|
These results suggest that (1) gradual reduction in Bcl-xL
and Bcl-2 expression may be responsible for inducing late apoptosis in
NF-B-inactivated mature T and B cells, respectively, and (2) rapid
cellular accumulation of Bcl-xS following NF-B
inhibition may be the cause of the early granulocyte apoptosis.
Changes in Bcl-2 family protein expression are sufficient to
explain the drastic induction of lymphocyte and granulocyte
apoptosis following NF-B inhibition
To confirm whether the alterations in Bcl-2 family protein
expression observed in NF-B-inactivated lymphocytes and
granulocytes were sufficient to explain apoptosis induction in these
cells, we determined (1) the effects of selectively decreasing the
levels of Bcl-xL and Bcl-2 expression on T- and B-cell
survival, respectively, and (2) the effects of selectively increasing
the levels of Bcl-xS expression on granulocyte longevity.
In these experiments, we used various oligonucleotides, which were
delivered to the cells in the form of complexes with a liposome
formulation of cationic DMRIE and DMRIE-C.
Exposure of freshly purified blood T cells to an optimal dose of
DMRIE-C/bcl-x AS ODN complexes for 24 hours induced a
significant decrease in Bcl-xL protein expression, whereas
DMRIE-C alone had no effect (Figure
6A). SC control bcl-x ODNs
only weakly decreased the levels of Bcl-xL protein. None of
these treatments affected the level of Bcl-2 expression used as a
control. The ability of bcl-x AS ODNs to specifically
down-regulate Bcl-xL allowed exploration of its role in
T-cell survival. Apoptosis assays using dual-color annexin-V-FITC/PI
staining and flow cytometry analyses were performed at 24 hours on T
cells treated with DMRIE-C alone or in combination with
bcl-x SC ODNs or bcl-x AS ODNs (Figure 6B).
bcl-x AS ODNs consistently increased T-cell apoptosis by
40% ± 4%, whereas bcl-x SC ODNs increased apoptosis
only by 10% ± 5%.
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| Figure 6.
Antisense knockdown of
bcl-xL induces human blood T cell apoptosis.
Blood T cells were isolated from buffy coats by negative magnetic
selection, cultured, and treated for 24 hours with DMRIE-C alone (24 µL per 2 × 106 cells) or DMRIE-C combined with
bcl-x AS or SC ODNs (8 µg per 2 × 106
cells). (A) Whole-cell extracts were prepared and analyzed by
immunoblotting for Bcl-xL and Bcl-2. (B) Alternatively,
apoptosis assays using dual-color annexin-V-FITC/PI staining and flow
cytometry analyses were performed on treated T cells. The percentage of
single- or double-positive cells in the individual quadrants is shown.
These results are similar to at least 3 comparable experiments.
|
|
Blood B cells cultured for 24 hours in the presence of bcl-2
AS ODNs exhibited reduced levels of Bcl-2 protein, whereas neither bcl-2 SC ODNs nor DMRIE-C alone was able to alter Bcl-2
protein expression (Figure 7A). None of
these treatments affected the level of Bax expression used as a
control. Figure 7B shows that decreased Bcl-2 expression was associated
with drastic induction of apoptosis in B cells treated with
bcl-2 AS ODNs. Indeed, the percentage of apoptotic B cells
averaged 85% ± 3% after AS treatment, whereas only 10% ± 2%
apoptotic B cells were observed after SC treatment.
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[in a new window]
| Figure 7.
Antisense knockdown of bcl-2 leads to
drastic human blood B-cell apoptosis.
Blood B cells were isolated from buffy coats by negative magnetic
selection, cultured, and treated for 24 hours with DMRIE-C alone (24 µL per 2 × 106 cells) or DMRIE-C combined with
bcl-2 AS or SC ODNs (8 µg per 2 × 106
cells). (A) Whole-cell extracts were analyzed by
immunoblotting for Bcl-2 and Bax. (B) Alternatively,
apoptosis assays using dual-color annexin-V-FITC/PI staining and flow
cytometry analyses were performed on treated B cells. The percentage of
single- or double-positive cells in the individual quadrants is shown.
These results were representative of at least 3 comparable
experiments.
|
|
AS ORNs containing uniform 2'-OMe modifications were used to induce
Bcl-xS expression in resting blood granulocytes. These AS
ORNs were designed to inhibit the use of the 5' splice site in exon II
of bcl-x RNA, thereby redirecting the splicing machinery to
the 5' bcl-xS splice site.21 AS
ORNs efficiently induced Bcl-xS expression in granulocytes,
whereas 5-bp mismatched MM ORNs and DMRIE-C alone did not affect
Bcl-xS protein expression (Figure
8A). None of these treatments affected
the level of Bcl-2 expression used as a control. Immunoblots revealed
that Bcl-xS was clearly present in AS-treated granulocytes
8 hours after treatment (Figure 8A). At this time point, the percentage
of apoptotic granulocytes averaged 60% ± 6% after AS treatment,
whereas 38% ± 3% and 35% ± 4% apoptotic granulocytes were
found after treatment with MM ORNs and with DMRIE-C alone, respectively
(Figure 8B).
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[in a new window]
| Figure 8.
Antisense ON-mediated induction of Bcl-xS
expression in human granulocytes leads to apoptosis.
Blood granulocytes were isolated from buffy coats by density
centrifugation and treated for 8 hours with DMRIE-C alone (24 µL per
2 × 106 cells) or DMRIE-C combined with AS (or MM) ONs
designed to redirect bcl-x pre-mRNA splicing toward
bcl-xS (8 µg per 2 × 106
cells). (A) Whole-cell extracts were analyzed by immunoblotting for
Bcl-xL/S and Bcl-2. (B) Apoptosis assays were performed on
treated granulocytes using dual-color annexin-V-FITC/PI staining and
flow cytometry analyses. The percentage of single- or double-positive
cells in the individual quadrants is shown. These results were
representative of at least 3 comparable experiments.
|
|
Altogether, these results demonstrate that the alterations
in Bcl-2 family protein expression induced by total NF-B inhibition are sufficient to trigger massive lymphocyte and granulocyte apoptosis.
| |
Discussion |
In the present report we have demonstrated that NF-B exerts its
antiapoptotic function in quiescent mature lymphocytes and granulocytes
by controlling the expression of distinct Bcl-2 family proteins.
Quiescent blood T cells contained relatively high levels of Bax, Bcl-2,
and Bcl-xL proteins. Whether Bcl-xL is
expressed in resting mature T cells is a matter of debate. Indeed,
Bcl-xL was first reported to be confined to immature DP
thymocytes and activated mature T cells,25-28 whereas
further study demonstrated substantial amounts of Bcl-xL in
resting mature T cells.19 In the present report, we
confirm the presence of Bcl-xL in quiescent peripheral T
cells and demonstrate that NF-B-dependent expression of
Bcl-xL plays a key role in maintaining the viability of
these cells.
Mice deficient for nfb-1 or
rel29-30 and irradiated SCID mice engrafted with
rela / fetal liver cells31
exhibit no intrinsic defect in the establishment of normal mature
T-cell populations, findings consistent with the potential for
functional redundancy among NF-B family members. Conversely,
lethally irradiated mice engrafted with
rela//rel/ fetal liver
hematopoietic progenitors,32 transgenic mice expressing trans-dominant forms of IB- in the T
lineage,33-35 and bcl-x double-knockout
chimeric mice36 exhibit impaired T-cell maturation. These
findings, coupled with the present results, indicate that suppression
of NF-B activity has similar effects as loss of
bcl-xL expression on the T lineage, raising the
possibility that these 2 major antiapoptotic factors are involved in a
common pathway required for both T-cell maturation and further
survival. However, a small number of mature
bcl-x/ and NF-B-inactivated T cells
exhibiting a normal life span may be found in mouse
models,32-36 a finding consistent with our observation that total NF-B inhibition in resting mature T cells induces drastic
but incomplete apoptosis. Indeed, approximately 30%
NF-B-inactivated mature T cells were still alive 24 hours after GTX
or PGA1 treatment. These findings suggest that a subset of T cells is
less dependent on NF-B-induced Bcl-xL expression for
protection against apoptosis. Because trans-dominant
IB- transgene expression in T cells predominantly blocks NF-B
activity in CD4+ cells but leads to a preferential
reduction in CD8+ cell numbers in the thymus and
periphery,33-35 it is plausible that NF-B-mediated
Bcl-xL expression is dispensable to maintain survival in a
subset of CD4+ cells.
An intriguing question concerns the mechanisms responsible for
continuous induction of NF-B-mediated Bcl-xL expression
in quiescent mature T cells. Thymus-positive selection requires the simultaneous recognition of peptides and major histocompatibility complex (MHC) molecules.1 Similarly, survival of resting
mature T cells requires continuous TCR engagement by MHC
molecules.1 Recent studies devoted to T-cell development
have demonstrated that pre-TCR signaling causes NF-B activation,
which selectively protects pre-TCR-positive cells from
apoptosis.37 Moreover, CD28 costimulation promotes
peripheral T-cell survival through the activation of NF-B and
subsequent Bcl-xL expression.26,38 These
findings, combined with our results, support a model in which
continuous TCR-MHC interaction, and most likely CD28 costimulation, maintains NF-B activation and Bcl-xL expression in
quiescent mature T cells, which thereby avoid spontaneous apoptosis.
Total NF-B inhibition in mature B cells was associated with Bcl-2
down-regulation, and loss of Bcl-2 was sufficient to induce massive
B-cell death. Although recent studies have clearly shown that the
expression of Bcl-xL and Bfl-1 is under NF-B
control,12-15 many investigations failed to demonstrate a
role for NF-B in Bcl-2 expression. However, a few studies provided
evidence for a relation between NF-B activation and induction of
Bcl-2 expression in immature B cells, hippocampal cells, and some
epithelial cancer cell lines,18,39,40 indicating that
regulation of Bcl-2 expression by NF-B is highly restricted to some
cell types, and is of particular importance in the B lineage. In
epithelial cancer cell lines, NF-B likely regulates Bcl-2 expression
through both indirect mechanisms and direct binding to the P2
bcl-2 promoter (P. Viatour, personal communication, October
2001). However, the question of whether NF-B directly or
indirectly controls bcl-2 expression in B cells remains to
be answered.
Lymphoid cells develop normally for a short time after birth in
bcl-2/ mice, indicating that Bcl-2 is
dispensable for lymphocyte maturation but is required for maintaining
immune homeostasis.41 However, further studies have
provided conflicting results. Indeed, transplantation of hematopoietic
stem cells from bcl-2/ mouse bone marrow
into irradiated normal recipient mice results in long-term
reconstitution of nonlymphoid cells but is associated with both the
absence of T lymphopoiesis and severely impaired B-cell
development.42 Similarly, mice engrafted with
rel//rela/ fetal liver
hematopoietic stem cells lack mature IgMloIgDhi
B cells and exhibit increased apoptosis in immature
IgMhiIgDlo and
IgMhiIgDhi B-cell populations.18
Moreover, this decreased survival of double-knockout B cells coincides
with reduced expression of bcl-2 and
bfl-1 and is abolished by enforced
expression of a bcl-2 transgene.18 These
observations, coupled with our results, indicate that NF-B-induced Bcl-2 expression contributes to the maintenance of both immature and
mature B-cell survival. However, NF-B induces coexpression of Bcl-2
and Bfl-1 in immature B cells,18 whereas it induces Bcl-2
expression only in quiescent mature B cells, which require stimulation
to express additional prosurvival Bcl-2 homologues, such as Bfl-1 and
Bcl-xL.13,14,43 This finding suggests
that NF-B-mediated Bcl-2 expression is particularly crucial for
promoting resting mature B-cell survival, a hypothesis that is further
supported by our finding that specific bcl-2 knockdown
results in drastic induction of apoptosis in these cells.
In the present study, we confirm that constitutive NF-B
activity is essential for resting mature granulocyte
survival4 and demonstrate that NF-B inactivation
triggers granulocyte apoptosis through the induction of
Bcl-xS expression. Bcl-xL is the most abundant
Bcl-x isoform found in vivo.44 However, Bcl-xS
may be overexpressed in some circumstances, such as during thymic selection, mammary gland involution, withdrawal of progesterone from
the endometrium, and brain ischemia.44 The mechanisms that regulate alternative bcl-x pre-mRNA splicing are totally
unknown. Our study is the first to provide evidence for a role of
NF-B in controlling the Bcl-xS/Bcl-xL ratio
through the direct or indirect regulation of bcl-x pre-mRNA splicing.
Complete NF-B inhibition had only a weak effect on monocyte survival
and did not affect Bcl-2 family protein expression. Since monocytes
appeared to mainly contain inactive p50 homodimers, a finding
consistent with previous reports,45,46 the inability of
NF-B inactivation to alter Bcl-2 family protein expression and to
substantially induce monocyte apoptosis is not surprising. Monocytes
have a short life span and their transit time in the circulation is
between 1 to 2 days, after which time they extravasate and
differentiate into macrophages. Monocyte differentiation is associated
with RelA expression and subsequent NF-B-dependent Bfl-1
expression, which are critical events in the generation of long-lived
macrophages.3,45 These findings are consistent with
increased apoptosis of maturing
rel//rela/
macrophages32 and collectively show that transcriptionnaly active NF-B complexes are dispensable for monocyte survival until they begin differentiation.
In summary, our study establishes that constitutive NF-B activity
rescues QMICs from spontaneous apoptosis through the regulation of
various Bcl-2 family proteins. Indeed, our results unambiguously demonstrate that constitutive NF-B activity induces directly or
indirectly the expression of prosurvival proteins of the Bcl-2 family
in lymphocytes and prevents the expression of the proapoptotic Bcl-2
homologue Bcl-xS in granulocytes. Furthermore, we show that the transcriptional control of these Bcl-2 family proteins by NF-B
is required to preserve homeostasis of resting lymphocytes and
granulocytes. These findings provide novel insights into the molecular
mechanisms responsible for the maintenance of a sufficient defense reserve.
| |
Acknowledgments |
The authors thank Drs Pierre Chatelain, Bruno Fuks, Jacques Gielen,
and Roy Massingham for advice, and Muriel Chapelier, Martine Leblond,
Miguel Lopez, Ilham Sbaï, and Andrée Villers for
excellent technical and secretarial assistance.
| |
Footnotes |
Submitted June 11, 2001; accepted January 5, 2002.
Supported in part by UCB Pharma (Belgium) and the "Ministère de
la Région Wallonne" (Belgium). F.B. is a Research Assistant, M.-P.M. and A.V. are Research Associates, and V.B. is a Senior Research
Associate at the National Fund for Scientific Research (FNRS, Belgium).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Fabrice Bureau, Department of Physiology, Faculty
of Veterinary Medicine, University of Liège, Boulevard de
Colonster, Bâtiment B42, Sart-Tilman, B-4000, Liège,
Belgium; e-mail: fabrice.bureau{at}ulg.ac.be.
| |
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Top
Abstract
Introduction