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B
-Deficient Mice: Reduction of One T Cell Precursor Subspecies and Enhanced Ig Isotype Switching and Cytokine Synthesis1
*
Unité de Biologie Moléculaire de l’Expression Génique, Centre National de la Recherche Scientifique Unité de Recherche Associée 1773,
Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U277, and
Unité de Pathogénie Microbienne Moléculaire, Institut National de la Santé et de la Recherche Médicale U389, Institut Pasteur, Paris, France; and
§
Institut National de la Santé et de la Recherche Médicale U411, Faculté de Médecine Necker, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel family of transcription
factors, IB
, IBß, and IB
, have been described. To
examine the in vivo role of the most recently discovered member of the
IB family, IB, we generated a null allele of the murine
IB gene by replacement of all coding sequences
with nlslacZ. Unlike IB nullizygous mice, mice
lacking IB are viable, fertile, and indistinguishable from
wild-type animals in appearance and histology. Analysis of
ß-galactosidase expression pattern revealed that IB is mainly
expressed in T cells in the thymus, spleen, and lymph nodes. Flow
cytometric analysis of immune cell populations from the bone marrow,
thymus, spleen, and lymph nodes did not show any specific differences
between the wild-type and the mutant mice, with the exception of a
reproducible 50% reduction of the CD44-CD25+
T cell subspecies. The IB-null mice present constitutive
up-regulation of IgM and IgG1 Ig isotypes together with a further
increased synthesis of these two isotypes after immunization against T
cell-dependent or independent Ags. The failure of observable
augmentation of constitutive nuclear NF-B/Rel-binding activity is
probably due to compensatory mechanisms involving IB and
IBß, which are up-regulated in several organs. RNase-mapping
analysis indicated that IL-1, IL-1ß, IL-1Ra, and IL-6 mRNA levels
are constitutively elevated in thioglycolate-elicited IB-null
macrophages in contrast to GM-CSF, G-CSF, and IFN-
, which remain
undetectable. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B family of transcription factors controls the expression of a
wide array of genes, such as genes encoding cytokines (IL-1 and ß,
IL-2, IL-6, IL-8, TNF-, GM-CSF, etc), cytokine receptors, adhesion
proteins, immunoregulatory molecules (MHC class I, etc), antiapoptotic
and acute-phase response proteins, in addition to viral promoters, like
CMV or HIV (1, 2, 3), and thus plays a key role in immune and
inflammatory processes, as evidenced by recent knockout analyses of
NF-B genes in mice (4, 5, 6, 7). NF-B molecules consist of
homo- and heterodimeric combinations of proteins of the same family,
which comprises five members in mammals, p50, p52, p65 or RelA, c-Rel,
and RelB (1, 2, 6). These proteins share a 300-aa
NH2-terminal Rel homology domain, involved in DNA
binding, dimerization, and nuclear localization (8). In
contrast to p65, c-Rel, or RelB, which contain a C-terminal
transactivation domain, p50 and p52 are synthesized as precursor
molecules, p105 and p100, respectively, which are solely cytoplasmic
and do not bind DNA. NF-B/Rel complexes are constitutively active in
the nuclei of mature T and B lymphocytes as well as in neurons of
certain regions of the brain (8, 9). In the vast majority
of cells, however, NF-B/Rel complexes are sequestered in the
cytoplasm by interaction with inhibitory molecules, called
IBs,5 which mask
the NF-B nuclear localization and DNA binding domains (6, 8, 10). Multiple stimuli, as varied as exposure to proinflammatory
cytokines like TNF or IL-1, viral infection, or chemical agents such as
phorbol esters, UV irradiation, and phosphatase inhibitors, converge
via distinct signaling pathways to activate NF-B by phosphorylation
of IBs, which leads to IB ubiquitination and subsequent
proteolytic degradation by the proteasome (2). Free
NF-B/Rel complexes then migrate to the nucleus and activate
transcription of their target genes.
The IB family comprises three major members, IB, IBß, and
IB, defined by the presence of six ankyrin repeats, a 33-aa motif
that mediates protein-protein interactions (10). IB
and IBß were originally purified from cytosolic fractions as
NF-B-associated inhibitory activities (11, 12) and
their genes were cloned soon after (13, 14). IB was
recently identified in yeast two-hybrid screens on the basis of
protein-protein interactions with p52 (15), p50
(16), or p65 (17). Despite their extensive
structural similarities, IB, IBß, and IB behave
differently in response to external stimuli. Generally, IB is
rapidly degraded and resynthesized, whereas IBß and IB are
degraded with a slower kinetics to gradually reappear (15, 18). Newly synthesized IB has been shown to be responsible
for postinduction repression of NF-B activity by entering the
nucleus and removing NF-B/Rel complexes from target promoters
(19). The nuclear export sequence of IB, which is
exposed upon binding to NF-B, then drives NF-B/IB complexes
out of the nucleus (20). Newly synthesized,
hypophosphorylated forms of IBß interact with the NF-B/Rel
complexes bound on target promoters, without disrupting the DNA-binding
interaction, thereby leading to a sustained NF-B response by
protecting the NF-B molecules from inhibition by nuclear IB
(21). IB is exclusively cytoplasmic and has been
found specifically associated with c-Rel, p65 homodimers, or p65/c-Rel
heterodimers in cell extracts (15, 16). It is unclear
whether different IB family members have distinct roles or redundant
functions in vivo. To address this question, knockout and knockin
studies at the IB locus together with gain of function
analysis of IB and IBß, under the control of T cell-specific
promoters, have recently been undertaken (22, 23, 24, 25, 26).
IB knockout mice die by 7 to 8 days after birth and exhibit
extensive granulopoiesis, acute runting, and abnormal skin formation
(22, 23). Interestingly, the absence of IB induces
the up-regulation of IB (15) and renders mouse
embryonic fibroblasts (MEFs) unable to terminate the NF-B activation
after TNF treatment (22, 23). These results confirm the
essential role of IB in postinduction repression and demonstrate
that this function cannot be substituted for by IBß or IB.
Mice containing a knockin of the IBß coding region at
the IB locus are, on the contrary, totally viable and
show no difference in constitutive or induced NF-B response compared
with wild-type mice (24). These data indicate that
IB and IBß share enough biochemical properties to substitute
one for another and that their specific in vivo roles arise from their
distinct expression patterns.
To gain insight into the physiological role of IB, we created
IB null-mutant mice by targeted IB gene
replacement with nlslacZ. Such mice develop normally and do
not exhibit morphological defects. Analysis of ß-galactosidase
activity in these mice reveals that, within the immune system,
IB is expressed mainly in thymocytes.
IB-deficient animals have normal mature hemopoietic cells, in
spite of a 50% reduction in the number of
CD44-CD25+ precursor T
cells. They respond normally to mitogenic stimuli and bacterial
challenge, but have an altered basal and Ag-specific Ig production. The
expression of a number of cytokine genes is specifically up-regulated
in IB-null mice, but remarkably not those known to be regulated
by p65 and/or c-Rel dimers, such as GM-CSF. The mild phenotype observed
for IB-deficient mice is probably the result of compensatory
mechanisms involving other members of the IB family, since
overexpression of IB and IBß is observed in several
tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 
DASHII library carrying DNA fragments from a partial
Sau3A digest of mouse male 129/SV genomic DNA was
screened with the entire human IB cDNA
(15). Five bacteriophages spanning a 17.5-kb region
encompassing the whole IB gene were isolated, and
their inserts were subcloned into the BamHI site of
Bluescript SK+ (Stratagene, La Jolla, CA).
Restriction mapping and partial sequencing were used to localize
IB encoding regions. A 2.5-kb
SacI-SmaI fragment containing the 5' region of
the murine IB gene was subcloned into a derivative of
p2
nlslacZ (kind gift of S. Tajbakhsh, Institut Pasteur, Paris,
France), containing a XbaI site inserted upstream of the
NcoI site, giving rise to p5'2nlslacZ, a plasmid in which
the 5' region of IB drives the expression of the
lacZ gene with an associated nuclear localization sequence
(nlslacZ). A 1.8-kb
Asp718-HindIII fragment isolated from
pPNT (27), which included the HSV-thymidine kinase gene
(HSV-tk) under the control of the phosphoglycerate kinase
(pgk) promoter together with the pgk poly(A), was introduced upstream
of the 5' region of IB, to generate pPN5'2nlslacZ.
A 6.6-kb BamHI fragment enclosing the 3' region of the
murine IB gene was placed upstream of the tk
promoter-enhancer driving expression of the diphtheria toxin A-fragment
gene (DTA) devoid of poly(A) sequences (ptkDaTA, kind gift
of S. Tajbakhsh), generating p3'tkDaTA. A 5.5-kb NheI
filled-in XhoI fragment from plZ1N (kind gift of S.
Tajbakhsh, Institut Pasteur, Paris, France), containing
nlslacZ, followed by the encephalomyocarditis virus internal
ribosome entry site (IRES) upstream of the neomycin-resistance gene
(neo), was integrated into the
XhoI-EcoRV site of p3'tkDaTA to create
plZ1N3'tkDaTA. Finally, an 11.1-kb XbaI fragment isolated
from pPN5'2wnlslacZ, containing hsvtk, the 5' region of
IB and nlslacZ, was subcloned into the
XbaI site of plZ1N3'tkDaTA upstream of the IRES, to
constitute the final targeting construct.
ES cell transfection and selection
HM1 ES cells (courtesy of David Melton, Edinburgh, U.K.) were grown on primary fibroblast feeder layers in the presence of LIF (104 U/ml). A total of 107 cells were electroporated with 10 µg of NotI-linearized targeting construct in 4-mm cuvettes with a Eurogentec Cellject electroporator at 200 V, 1500 µF, and infinite resistance. Cells were diluted into 40 ml of prewarmed complete medium, plated, and left to recover for 48 h before addition of 0.3 mg/ml G418 (Life Technologies, Cergy, France) with or without gancyclovir at 5 µM. Once established, clones were maintained in complete medium without selection.
Generation and characterization of knockout mice
Two independently targeted ES cell clones were injected into
C57BL/6 blastocysts, which were subsequently reimplanted into
pseudopregnant C57BL/6 x CBA females. Resulting chimeras were mated to
C57BL/6 x DBA/2 mice, and heterozygous offspring for the targeted
allele were interbred. The two independent IB-null mouse lines
generated were found to be identical in all subsequent experiments.
Mice were kept in clean housing conditions in a level 2 barrier animal
house facility. When unspecified, mice were analyzed between 6 and 12
wk of age.
For Southern blot analysis, genomic DNA was isolated from tail biopsies according to Laird et al. (28). A total of 10 µg of DNA was digested with XbaI and BamHI, separated by electrophoresis on 0.9% agarose gel, transferred onto nylon membrane (Amersham Pharmacia Biotech, Amersham, U.K.), and hybridized with a random-primed 32P-labeled 5' external probe (29).
For genotyping by PCR, tail biopsies were lysed in a buffer containing
50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2,
0.1 mg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 1 mg/ml
proteinase K (Boehringer Mannheim, Meylan, France) at 55°C for at
least 3 h. After centrifugation at 14,000 rpm, the supernatant was
recovered, heat denaturated at 100°C for 10 min, and used directly in
PCR reaction. The forward primer is contained within the 5' external
probe used for Southern analysis (5'-cccgctggacccctggacccc-3'). Two
reverse primers were used: one within the first exon of the
IB gene for primer 1
(5'-ccgctcgctctccatccgcatcttctttct-3'), which amplifies the wild-type
allele, and a second within the lacZ gene
(5'-ccgtaaccgacccagcgcccgttgcaccac-3'), which amplifies the mutant
allele. PCR reaction mix included 0.25 µM of each of the three
primers, 1 mM MgCl2, 0.2 mM dNTP, and 1 U of
Taq DNA polymerase (Promega, Charbonnières, France).
Denaturated samples were subjected to 35 cycles of amplification in a
PTC-200 thermal cycler (MJ Research, Cambridge, MA), cycling parameters
being the following: 94°C denaturation for 40 s, followed by a
combined annealing and polymerization step at 72°C for 4 min. After
an extra step at 72°C for 10 min, 10 µl of the reaction was
analyzed on a 2.4% agarose gel (containing one-third of NuSieve GTG
agarose (FMC) and two-thirds of standard agarose from Life
Technologies). Amplified products were detected by ethidium bromide
staining.
RNA isolation and Northern blot analysis
Total RNA was isolated from organs or cells using the TRIzol
Reagent (Life Technologies), according to the manufacturer’s
instructions. The RNA pellet was resuspended in nuclease-free
H2O. A total of 30 µg of total RNA was
separated by electrophoresis on 1% denaturing formaldehyde agarose
gel, transferred onto nylon membrane (Amersham Pharmacia Biotech), and
hybridized with a 32P-radiolabeled probe
corresponding to full-length human IB cDNA.
Western blot analysis and Abs
Whole-cell extracts or cytoplasmic extracts were prepared as
previously described (9), and proteins were separated by
SDS-PAGE. Proteins were then transferred to nitrocellulose (Schleicher
& Schuell, Dasel, Germany) or Immobilon membranes (Millipore, Bedford,
MA). Immunoblots were incubated with rabbit polyclonal Abs at 1/1000
dilution (or 1/200 dilution for commercial Abs) and revealed with the
Pierce (Rockford, IL) enhanced chemiluminescence system, as recommended
by the manufacturer. Sera raised against murine p50 (1263), p65 (1226),
p52 (1267), and c-rel (1051) were kind gifts of N. Rice
(Frederick, MD); serum against RelB was kindly provided by R. Bravo
(Princeton, NJ); serum against IBß was a generous gift of R. Weil
(Institut Pasteur, Paris, France); and sera against IB (C-21) and
IB (M-121) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
Cell culture and treatments
Single-cell suspensions of splenocytes or T cells, isolated from thymic suspensions by purification on MACS CD90 (Thy-1.2) MicroBeads (Miltenyi Biotec., Auburn, CA), were cultured at 37°C, 5% CO2 in RPMI 1640 medium containing 10% heat-inactivated FCS, 100 U/ml penicillin/streptomycin, 2 mM glutamine, and 55 µM 2-ME (all from Life Technologies). Peritoneal macrophages were cultured under the same conditions, but in the absence of 2-ME. LPS from Salmonella abortus equi (Sigma, Saint Quentin Fallavier, France), ALLN (Sigma), and murine TNF (R&D Systems, Abingdon, U.K.) were used at 15 µg/ml, 100 µM, and 10 ng/ml, respectively.
Histological analysis and ß-galactosidase in situ staining
Organs were fixed for 20–30 min in 4% paraformaldehyde in PBS, incubated in 15% sucrose, 1x PBS for 15 h at 4°C, frozen, and embedded in Tissue-Tek OCT compound (Miles-Bayer). Sections (20 µm) were cut using a cryostat (Leica) and collected on SuperFrost Plus slides (Menzel-Glaser, Germany). Sections were X-gal stained, as previously described (9), and counterstained with eosin. Dehydrated sections were mounted using EUKITT and photographed on a Nikon microscope equipped with a camera, using Kodak Ektachrome 64T film.
Flow cytometric analysis
Single-cell suspensions from thymus, bone marrow, spleen, and
lymph nodes were surface stained either with mAbs coupled to
fluorescein or PE, or with biotinylated mAbs (PharMingen, San Diego,
CA; Caltag, San Francisco, CA), followed by tricolor-streptavidin
(Caltag). Viable cells (propidium iodide negative) were then analyzed
using a FACScan fluorocytometer (Becton Dickinson, Mountain View, CA).
The mAbs used were anti-CD3 (145-2C11), anti-CD4 (L3T4),
anti-CD8 (Ly-3.2 and Ly-2/53-6.7), anti-CD11b/Mac1 (M1/70),
anti-CD11c (HL3), anti-CD19 (1D3), anti-CD24/HSA (M1/69),
anti-CD25 (7D4), anti-CD43 (S7), anti-CD44 (Ly-24/Pgp-1),
anti-CD45R/B220 (RA3-6B2), anti-GR1 (Ly-6G/RB6-8C5),
anti-BP-1 (Ly-51/6C3), Ter 119, and anti-IgM (R6-60.2).
For identification of ß-galactosidase-expressing cells, lymphocytes isolated by gradient centrifugation through Ficoll-Isopaque (Amersham Pharmacia Biotech) were first stained with a vital fluorogenic substrate, fluorescein-di-ß-D-galactopyranoside (FDG; Molecular Probes, Interchim, Montluçon, France), according to Fiering et al. (30). Cells were then washed once before additional immunolabeling and analysis by flow cytometry. Fluorescein background was determined by performing the ß-galactosidase assay on lymphocytes isolated in parallel from a wild-type control littermate mouse.
Proliferation assay
Splenocytes (2 x 105/ml) were
incubated in the presence or absence of various concentrations of
anti-CD3 (145-2C11), anti-IgM Abs (PharMingen), or LPS.
Cells were pulsed with [3H]thymidine (1
µCi/ml; Amersham Pharmacia Biotech) and transferred after 72-h
culture onto glass filter mats, and radioactive incorporation was
measured with a ß-scintillation counter.
Electrophoretic mobility-shift assay (EMSA)
Nuclear extracts from whole organs or T cells, purified by MACS
CD90 (Thy-1.2) MicroBeads, were prepared and bandshift assays were
performed as previously described (9), using the B site
derived from the promoter of the MHC class I H-2
Kb gene as a probe.
Ig isotype analysis
Sera were prepared from 5- to 6 wk old
IB-/- and control wild-type sex-matched
littermates. Ig isotypes were quantitatively determined against isotype
standards using a sandwich ELISA with a pan-specific capture Ab
(Southern Biotechnology Associates, Birmingham, AL) and
isotype-specific Abs conjugated to alkaline-phosphatase (Southern
Biotechnology Associates).
Immunization and T cell-dependent and independent humoral immune responses
Sex-matched animals 5 wk old were immunized by i.p. injection of either 100 µg of KLH coupled to DNP precipitated in alum (T cell-dependent responses) or 50 µg of LPS coupled to DNP (T cell-independent responses). Serum samples were collected from the mice before immunization and at 7-day intervals after immunization for a period of 3 wk. Levels of DNP-specific Ig isotype were determined by ELISA using DNP-BSA (17:1) as a capture agent and goat anti-mouse isotype-specific Abs directly conjugated to alkaline-phosphatase (Southern Biotechnology Associates).
RNase protection assay
Animals 12 wk old were injected i.p. with 2 ml of
resazurin-thioglycolate (Sanofi Diagnostics Pasteur,
Marnes-la-Coquette, France). After 5 days, peritoneal macrophages were
collected from pools of five to six mice and incubated for 8 h at
37°C with or without LPS. Total RNA was then isolated from adherent
macrophage cells, as described above, and ribonuclease protection assay
was performed with 6 µg of total RNA and the RiboQuant In Vitro
Transcription and RPA kits (PharMingen). The murine cytokine sets mCK-2
and mCK-4 (PharMingen) were used to obtain radiolabeled antisense RNA
probes for IL-1, IL-1ß, IL-1Ra, IL-3, IL-6, IL-7, IL-11, IL-12p35,
IL-12p40, IL-10, IFN-, LIF, macrophage migration inhibitory factor,
G-CSF, GM-CSF, M-CSF, stem cell factor, L32, and GAPDH. The RNA
duplexes were analyzed by electrophoresis on 5% polyacrylamide/8 M
urea gels, which were then dried and subjected to autoradiography.
Bands were quantified on a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA).
Pathogen challenges
Mice 15 wk old were challenged via intranasal injection under ethyl ether anesthesia to 20 µl (1 x 108 bacteria) of a suspension of Shigella flexneri strain M90T-GFP, established by M. Rathman (unpublished) and grown as described in Phalipon et al. (31). Mice 12 wk old were inoculated i.v. with 5 x 104 bacteria from Listeria monocytogenes EGD strain (32).
At the indicated time after inoculation, lungs were collected from S. flexneri-infected mice, or spleen, liver, and brain were removed from L. monocytogenes-infected mice. Each organ was homogenized with an Ultra-turrax blender in sterile saline solution. For each organ, bacteria were titrated by serial dilutions plated on Congo red agar (infection by S. flexneri) or BHI agar (infection by L. monocytogenes) and incubated at 37°C. Results were expressed as the number of CFU per gram of lung tissue for S. flexneri and as the logarithm of CFU per organ for L. monocytogenes infection.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-deficient mice
To inactivate the murine IB gene by homologous
recombination in ES cells, we cloned a 17.5-kb genomic region
encompassing the IB gene from a 129/SV library, using
the human IB cDNA as a probe (15). The
targeting construct was designed (Fig. 1
A) to replace the entire
coding sequence of IB, starting from the ATG, by
nlslacZ to follow the expression pattern of
IB in vivo (33). The construct contains a
promoterless neo gene (34), positioned
downstream of nlslacZ, and whose translation is dependent on
the presence of an upstream IRES (35). Hsv-tk
and DTA were placed at each end of the construct to achieve
a double-negative selection (36, 37). We checked that
IB mRNA was detectable in ES cells, even if it was at
very low levels compared with those of IBß or
IB, this latter constituting the major IB species
in these cells (data not shown). By transient transfection experiments
into ES cells, we also verified that the 2.5-kb 5' fragment used in the
targeting construct was unable to drive nlslacZ gene
expression by itself (data not shown). Following electroporation of the
targeting vector into HM1 ES cells, G418-resistant colonies obtained in
the presence or absence of gancyclovir were selected and screened by
Southern blot analysis. Of 42 recombinant ES clones examined, 9 were
positive for homologous recombination and 1 of these 9 clones
harbored an additional insertion in the genome, as evidenced by
probing with a lacZ fragment (data not shown). Selection in
the presence or absence of gancyclovir yielded the same percentage of
homologous recombinant clones, indicating that DTA alone was
sufficient for negative selection. Two correctly targeted clones from
two independent electroporations were injected into C57BL/6 blastocysts
and gave rise to chimeric mice that displayed 100% color chimerism and
that all transmitted the recombinant allele to their progeny (with one
exhibiting 50% germline transmission). Two independent IB mutant
mouse lines were thus established, analyzed in parallel, and found to
be indistinguishable in all subsequent experiments. Intercrossing
heterozygous animals, which displayed no obvious phenotype, generated
IB+/+, IB+/-,
and IB-/- mice, identified by Southern
blot and PCR analysis (Fig. 1, B and C). IB
expression was abolished in homozygous mutants, as determined by
Northern blotting with total RNA purified from several organs (Fig. 1D) and Western blot analysis of spleen and thymus total
protein extracts (Fig. 1E). Intermediate levels of IB
mRNA or protein were observed in every heterozygote organ examined
(Fig. 1D, lanes 4, 8, and
11, and Fig. 1E, lanes 2 and
3, spleen; and lanes 2 and 4, thymus),
indicating that both IB alleles are functional in
wild-type mice. IB-/- mice were born at
the expected Mendelian segregation ratios and grew normally. No evident
morphological or behavorial abnormalities were detected. IB-null
mice did not show any gross macroscopic alterations either, as
determined by detailed histopathological analysis, and their skeleton
appeared indistinguishable from that of wild-type mice by x-ray
irradiation and Alcian blue/Alizarin red staining (data not shown).
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B-deficient mice
Examination of tissue sections from thymus, spleen, or lymph nodes
of wild-type (+/+) or homozygous mutant (-/-) mice did not display
any significant differences (Fig. 2). In
situ staining of these sections by X-gal, a colorimetric substrate for
ß-galactosidase, was used to elucidate the expression pattern of
IB in these organs, the nlslacZ gene having
been knocked in to the IB locus and thus placed under
the control of the endogenous IB regulatory sequences.
X-gal staining revealed that the major site of IB
expression is the lymph node, and especially the medulla.
LacZ expression is scattered throughout the thymus and
restricted to a few cells in the spleen (Fig. 2). Further
characterization of the ß-galactosidase-expressing cells was
performed by flow cytometry with simultaneous detection of surface Ags
and fluorescent staining of ß-galactosidase using the FDG fluorogenic
vital substrate (38). Results presented in Table I show that the highest number of
FDG+ cells is located within the lymph node
(50%) and decreasingly in the thymus (30%) and the spleen (5%),
corroborating the in situ staining data. Interestingly, in each organ,
T cells constitute the major FDG+ species. Some B
cells (between 4 and 15% of FDG+ lymphocytes)
also exhibit ß-galactosidase activity in the two secondary lymphoid
organs examined. Immunolabeling of lymph node cells with Mac1, GR1, or
CD11c did not reveal any specific FDG+
population, the number of immunolabeled and FDG+
cells being 1.5%, 4%, and 1%, respectively (data not shown).
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B-deficient mice
We next investigated whether hemopoietic cell populations were
normal in IB-null mice. Results from flow cytometric analyses of
bone marrow, thymic, and splenic cell populations are presented in Fig. 3. No difference between the wild-type
(+/+) and the homozygous mutant (-/-) mice was observed in the
expression of B cell Ags (B220, IgM), erythroid cell Ag (Ter119),
granulocyte Ag (GR1), and macrophage Ag (Mac1) in the bone marrow
(A), or B cell Ags (B220, IgM) and T cell markers (CD3) in
the spleen (C). We also tested surface markers for precursor
B cells according to the classification of Hardy (39),
such as CD43, HSA, CD19, BP-1 in the bone marrow, markers for dendritic
cells like CD11c in the spleen, and markers for granulocytes, GR1, and
macrophages, Mac1, in the lymph nodes (data not shown). No
significant differences were detected between wild-type and mutant
animals. In the thymus (B), the percentage of CD4, CD8
double or simple positive cells are identical in both types of mice.
Remarkably, when we examined the expression of CD44 and CD25 in the
CD4-/CD8- population, we
observed a 50% fold reduction in the percentage of
CD44-/CD25+ cells in
IB-null mice compared with control wild-type littermates, the
other populations being normal. Peripheral blood analysis of
IB-deficient mice revealed a standard cell count for
every cell population, other hemogram parameters being average (data
not shown). Overall, these results establish the presence of normal
numbers of mature B cells, T cells, erythroid cells, dendritic cells,
granulocytes, and macrophages in IB mutants even though one
precursor T cell subspecies is diminished.
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B-deficient mice in
response to mitogenic stimuli
We next investigated whether splenocytes from wild-type (+/+),
heterozygote (+/-), or IB-null (-/-) mice responded normally to
various mitogens known to induce B and/or T cell proliferation via
distinct signaling pathways. Results presented in Fig. 4 reveal no significant difference in the
proliferative response of the splenocytes derived from these three
genotypes to anti-CD3 (A), LPS (B), or
anti-IgM (C), albeit large variations were observed
among individuals.
|
B-deficient mice could be due to compensatory mechanisms
involving up-regulation of other IB family members. We thus assessed
the levels of other IB molecules in the spleen of IB-null
(-/-) mice. A representative Western blot analysis of total splenic
extracts of wild-type (+/+) and IB-null (-/-) mice is presented
in Fig. 5 (n = 4). In
IB-deficient mice, increased levels of IB and IBß were
observed, whereas expression of p105 and p100 was not significantly
affected (A). As expected, IB, found in wild-type
cells, was absent in the homozygous mutant cells (A,
upper panel). Since the synthesis of three members of the
NF-B family, p105, p100, and c-Rel, is directly controlled by
NF-B itself (2, 6), we also looked at the expression of
p50, p65, c-Rel, and RelB (B). The steady state levels of
all these molecules were similar in IB-deficient and wild-type
mice. This latter result together with the observed up-regulation of
IB and IBß suggests that constitutive nuclear NF-B
activity may be unchanged in IB-deficient mice. This was shown to
be the case when we performed bandshift assays with nuclear extracts
from the spleen or resident macrophages of homozygous mutant or control
wild-type littermate mice (data not shown).
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B-deficient mice
Since IB is preferentially expressed in T cells
(see above), we examined how mutant T cells responded to TNF treatment.
Thymocytes purified from wild-type (+/+) (lanes
1–5), heterozygote (+/-) (lanes 6–10), or
IB-deficient (-/-) (lanes 11–15) mice were
incubated with TNF for various periods of time in the absence
(lanes 1–4, 6–9, and 11–14)
or in the presence of ALLN (lanes 5, 10,
and 15) (Fig. 6). Analysis of
nuclear extracts by EMSA (A) showed a similar overall
response of the cells isolated from the three categories of mice: we
observed no modification of constitutive NF-B DNA-binding activity
nor of the kinetics of activation after TNF stimulation. ALLN treatment
through inhibition of the proteasome prevented induction in a
comparable fashion in all three genotypes. The use of specific Abs
directed against individual members of the NF-B family allowed us to
define complex II as p50/p50 homodimers and complex I as p50/p65
heterodimers (data not shown). Analysis of cytoplasmic extracts by
Western blotting (B) confirmed the absence of IB in T
cells of homozygous mutant mice (bottom panel). No increase
of p105 and IBß was detected in mutant thymocytes (upper
panels). IBß was not degraded after TNF treatment in either
wild-type or IB-deficient T cells. IB was up-regulated in
resting mutant thymocytes, and rapidly degraded and resynthesized after
TNF treatment. Its degradation was inhibited by ALLN in both mutant and
wild-type or heterozygous control cells. Stimulation of T cells with
IL-1 again did not discriminate between mutant and control cells (data
not shown).
|
B-deficient mice
To assess the humoral immunity of IB-null mice, we measured
basal Ig levels of naive mutant mice or control wild-type littermates
(Fig. 7A). IgG2a, IgG2b, and
IgG3 levels are equivalent in both types of mice. A slight increase of
IgM levels (2-fold) is detected in IB-deficient mice compared
with in control mice. The main difference between these mice lies in
the augmentation (3-fold, with very small dispersion of the values
obtained with the mutant mice, p < 0.001) of IgG1 seen
in IB-deficient mice compared with wild-type mice.
|
B-null mice or control
wild-type littermates to specific Ab challenge. Mice were injected with
LPS coupled to DNP to induce a T cell-independent response
(B) or with KLH coupled to DNP precipitated in alum to
induce a T cell-dependent response (C) and bled before and
7, 14, and 21 days after immunization. DNP-specific Abs in unchallenged
mice were very low and not significantly different in wild-type and
mutant mice. For each Ag used, only DNP-specific IgG1 and IgM isotype
productions were affected in IB-null mice; IgG2a, IgG2b, and IgG3
values were not statistically different from the wild-type values using
the Student’s test. Considering the T cell-independent response (B), DNP-specific IgG1 production in mutant mice increased by 3-fold at day 7, and this elevation persisted until day 21. DNP-specific IgM synthesis in the mutant mice was 2-fold higher at day 7 and 1.3-fold at day 14, and returned to levels similar to wild type at day 21.
For the T cell-dependent response (C), DNP-specific IgG1
secretion in IB-deficient mice differed from wild type only by a
peak (1.5-fold increase) at day 7. DNP-specific IgM production rose by
2.5-fold at day 7 and remained elevated until day 21.
Constitutive up-regulation of cytokines in IB-deficient mice
Analysis of IB-associated proteins in cells, together with
the finding that some NF-B-responsive genes were activated solely by
c-rel and/or p65 dimers, led to the hypothesis that the role
of IB in vivo might be to regulate this specific subset of genes,
which includes IL-8, tissue factor, and GM-CSF (15, 17).
If this assumption true, these genes should have elevated basal
expression levels in IB-deficient mice. Since some of these genes
are inducible in activated macrophages (40), we examined
the expression of an array of cytokines in thioglycolate-elicited
macrophages (Fig. 8) in the absence
(lanes 1, 2, 3, 7,
8, and 9) or presence of LPS for 8 h
(lanes 4, 5, 6, 10,
11, and 12). Total RNA was purified and subjected
to RNase mapping for the expression of different chemokine genes. The
expression of IL-1, IL-1ß, IL-1Ra, and to a lesser extent IL-6 was
constitutively increased in IB-deficient mice (A,
lanes 3 and B, lane 9), while their
mRNA levels were below detection (for IL-1ß and IL-6) or very low
(for IL-1 and IL-1Ra) in wild-type and heterozygote mutant mice
(n = 5) (A, lanes 1 and
2, and B, lanes 7 and 8).
Remarkably, GM-CSF expression remained unaffected and undetectable in
the mutant mice (B, lane 9). Upon LPS
stimulation, comparable fold induction of IL-1, IL-1ß, IL-1R,
IL-6, IFN-, GM-CSF, and G-CSF expression was observed in mutant and
control mice (A, lanes 4, 5, and
6, and B, lanes 10, 11, and
12).
|
B-deficient mice
It has been previously shown that IL-8, monocyte chemoattractant
protein-1, GM-CSF, TNF, and IL-6 were specifically up-regulated upon
bacterial invasion of human colonic epithelial cells (41, 42), and that the induction of expression of IL-8 and monocyte
chemoattractant protein-1 in HUVEC following infection with L.
monocytogenes was directly correlated with the activation of
NF-B (43). If GM-CSF or the functional equivalent of
IL-8 in the mouse was constitutively overexpressed in
IB-deficient mice, one should expect, in a simplistic view, a
quicker clearance of pathogenic bacteria. We thus asked how
IB-null mice responded to challenge with a Gram-negative,
S. flexneri, or with a Gram-positive, L.
monocytogenes, bacteria. We used a previously described pulmonary
model of Shigella infection by the nasal route, which mimics
the lesions occurring in natural intestinal infection
(31). As shown in Fig. 9A, high bacterial
multiplication was observed in the lungs of wild-type mice 6 h
after inoculation, the bacterial load decreasing after 24 h. An
overall similar pattern of response to S. flexneri was
obtained with heterozygote or homozygous IB mutant mice, the high
SD between individual animals observed at 24 h ruling out any
significant effect in the mutant mice. We next challenged the mice with
L. monocytogenes. Results presented in Fig. 9B
show that for every organ analyzed (liver, spleen, and brain),
IB-null mice responded to the infection identically in intensity
and kinetics to wild-type or heterozygote controls. This absence of
bias between these three populations of mice persisted when we looked
at the spleen 6 days after inoculation when bacterial loads were
considerably diminished (data not shown). By using two distinct
bacterial pathogens, one which led to localized bronchial epithelium
invasion, and a second, which caused a systemic infection that ended in
CNS invasion, we have been unable to establish any discrimination
between the mutant and the wild-type control mice.
|
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bs raises the question of their
individual role and putative functional redundancy in vivo. The
analysis of IB-deficient mice provides some clues about the
function of IB in a whole organism. IB-null mice share none
of the hallmarks of IB-deficient mice (23, 44): they
survive to adulthood and show no increase in NF-B DNA-binding
activity in all organs and cell types analyzed (spleen, thymus,
purified T cells, resident macrophages, or MEFs (data not shown)). They
possess normal hemopoietic cell subsets, in particular displaying
average granulocyte and macrophage numbers together with unaltered skin
structure.
We have shown in this study that IB mRNA is expressed at high
levels in the thymus, spleen, and to a lesser extent in lung and ovary,
in accordance with observations in humans (16). Targeted
replacement of the entire IB coding region by the
lacZ gene carrying a nuclear localization sequence allowed
us to visualize the overall expression pattern of IB in the
immune system with cellular resolution. Analysis of ß-galactosidase
expression by in situ X-gal staining or by FACS analysis of FDG-labeled
cells confirmed our Northern blotting data and revealed that
IB-expressing cells represent one-half of the cells in the lymph
node, one-third in the thymus, and only 5% in the spleen.
ß-galactosidase-positive cells are mostly T cells, in every primary
or secondary lymphoid organ examined. A small fraction of macrophages
(1.5%), granulocytes (4%), dendritic cells (1%), and B cells
(1–2%, depending on the organ) expresses IB. These results
indicate that data from cell lines of the myeloid lineage, like THP1 or
differentiated HL-60 cells, which display high endogenous levels of
IB (15), cannot be extrapolated to primary cells.
Considering the high sensitivity of FDG staining, which has been
reported to detect as little as five molecules of ß-galactosidase per
cell (38, 45), and the fact that two independently
generated knockin lines gave the same pattern of expression, it is
quite unlikely that we overlooked any site of expression of IB in
the lymphoid system.
Up-regulation of IB has been observed in several organs and cell
types (i.e., thymus, spleen, purified T cells) isolated from
IB-mutant mice. IBß was found overexpressed in the spleen,
but not in purified T cells. These results suggest that the relative
contributions of IB and IBß to the regulation of NF-B are
cell type dependent. The prominent role of IB is most likely due
to its preferential expression in hemopoietic cells compared with
IBß (44) and also to its autoregulation by NF-B
(46, 47, 48, 49, 50). IB expression is controlled by NF-B,
and IB protein levels are increased in primary IB-null MEFs
(15). Up-regulation of other IB species is therefore
not a characteristic unique to IB-null mice. However,
up-regulation of IB and IBß in the spleen of
IB-nullizygous mice is intriguing since only 5% of
FDG+ cells were detected in this organ. It may
suggest that overexpression of IB family members could also take
place in cells that do not normally express IB.
No significant increase in NF-B-binding activity was detected in any
IB-deficient organs examined or in purified T cells, of which
80% express IB. This suggests that cytoplasmic retention of
NF-B in IB-deficient mice is achieved by overexpression of
IB and/or IBß. This functional compensation by IB
and/or IBß for the lack of IB is most likely responsible for
the discrete phenotype of IB-null mice. Nevertheless,
substitution by these IBs is not complete and IB-deficient
mice harbor a number of specific features, which might reflect specific
functions of IB.
We observed a 50% decrease in the number of precursor
CD44-CD25+ cells in spite
of the presence of normal mature hemopoietic cell populations.
Differentiation of thymocytes has been correlated with CD25 and CD44
expression during early steps of cortical maturation (51),
and CD25 (or IL-2R-chain) expression in thymocytes is associated
with TCR rearrangement. It is interesting to note that young mice
lacking CD25 exhibit phenotypically normal T cell development
(52). Lack of IB may impair expression of target
genes involved into the proliferation of the
CD44-CD25+ cell population
and thus account for its diminution. Whereas CD25 gene expression was
unaffected in IB-null mice (23, 44) and in
homozygous mice deficient for the different members of the NF-B/Rel
family (53, 54, 55, 56, 57, 58, 59), a similar reduction in the frequency of
CD25+ cells was detected in transgenic mice
expressing constitutive trans-dominant form of IB
(26). Reduction of the CD25+ cell
population in IB-deficient mice might therefore result from an
increase of IB, instead of being a direct consequence of IB
loss. In this case, the up-regulation of IB should be assumed to
be strictly limited to the CD25+ cell subset,
since in IB transgenic mice this phenotype is associated with
severe defects in T cell development and proliferation that are not
observed in IB-null mice.
Another characteristic of IB-deficient mice is their increase in
IgM and especially IgG1 basal levels. Recent reports have implicated
NF-B in the regulation of isotype switching. B binding sites
within the 3' IgH enhancer (60, 61) and the germline
CH promoters, which regulate class switching to
IgG1 and IgE (62, 63), were shown to bind specific NF-B
complexes, and analysis of the knockouts of the various NF-B genes
led to the observation that isotype switching was frequently affected
in these mice. For instance, p50 mice displayed a significant reduction
in all isotypes except for IgM, which was slightly elevated
(55). A pronounced reduction of IgG1 and IgA was seen in
p65-deficient lymphocytes (56). RelB-deficient mice show
increases in IgM, IgE, and IgG1 together with slight decreases in
IgG2a, IgG2b, IgG3, and IgA (64). IgG1 and IgG2 were
drastically reduced in c-Rel-null mice, IgM, IgG2b, and IgG3 being only
slightly diminished (53). IgG1 and IgM levels appear to be
frequently altered in NF-B-deficient mice, and their increase in
IB-null mice might reflect modification of the balance of various
NF-B members in B cell subsets. Ab response to specific Ags was also
modified in IB-deficient mice. T cell-dependent and T
cell-independent immunizations led to increased synthesis of IgM and
IgG1. Impaired Ag-specific Ig isotype switching has also been observed
in several NF-B-deficient mice. C-Rel and RelB knockout mice exhibit
defective T-dependent and T-independent Ab responses (53, 64), while an inability to generate Abs to T-dependent Ags has
been observed in p52-null mice (58, 59). The increase in
IgM and IgG1 levels before stimulation and after T-independent or
T-dependent challenge in IB-nullizygous mice is, however, very
modest compared with the perturbation observed for knockouts of the
NF-B gene family. Moreover, we have shown that only 1–2% of B
cells express IB. Alteration of isotype switching in
IB-deficient mice possibly reflects the contribution of these few
IB-expressing B cells. Alternatively, it is possible that this
modification is indirectly due to IB via the perturbation of
other pathways such as cytokine synthesis.
IB was proposed to be a specific regulator of genes controlled by
p65 and/or c-Rel complexes, since it specifically interacts with p65
and/or c-Rel-containing complexes (15), and inhibits
transcription of the human IL-8 gene, which is only activated by p65
homodimers (17). Analysis of basal cytokine mRNA levels in
thioglycolate-elicited macrophages revealed that expression of
B-responsive genes, such as IL-1, IL-1R, IL-6, or IL-1ß,
which binds p50/p65 dimers (65, 66), was up-regulated in
IB-deficient mice. On the contrary, expression of the GM-CSF gene
that binds only p65/c-Rel heterodimers in vitro (67)
remains undetectable, as does IFN- and G-CSF expression. mRNA levels
of G-CSF, macrophage-inflammatory protein-2 (a mouse IL-8 homologue),
and VCAM-1 were elevated in IB-deficient thymocytes, in constrast
to GM-CSF, IL-2, IL-2R, and IL-6 (44). These data
indicate that different cytokine genes may be controlled by distinct
IBs, and that IB may not function in vivo as a specific
inhibitor of genes specifically regulated by p65 and/or c-Rel
complexes. This assumption is supported by normal responses of
IB-deficient mice to pathogen challenge involving IL-8 and GM-CSF
at the onset of infection (41, 42, 43).
Functional redundancy and up-regulation of other IB members mask the
full importance of IB in vivo and explain the mild phenotype of
IB-deficient mice. Analysis of this mutant nonetheless provides
evidence for specific roles of IB in the regulation of cytokine
synthesis and maybe Ig switching, which cannot be counterbalanced by
other IBs. These studies also illustrate the role of individual
members of the IB family in the tight control of NF-B activity.
Evaluation of the defects present in mice with a knockin of the
IB gene at the IB locus or mice with
simultaneous disruption of multiple IBs should help to unravel the
relative functions and biochemical differences betwen these molecules
and to understand how NF-B activity is controlled in vivo by
individual IBs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sylvie Mémet, Unité de Biologie Moléculaire de l’Expression Génique, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cédex 15, France. E-mail address:
3 Current address: Biology Department, Boston University, 5 Cummington St., Boston, MA 02215.
4 Current address: Hybrigenics SA, Campus Pasteur, 25 rue du Dr Roux, 75724 Paris Cédex 15, France.
5 Abbreviations used in this paper: IB, inhibitory protein that dissociates from NF-B; ALLN, N-acetyl-leu-leu-norleucinal; ES, embryonic stem; FDG, fluorescein-di-ß-D-galactopyranoside; IL-1Ra, IL-1R antagonist; IRES, internal ribosome entry site; KLH, keyhole limpet hemocyanin; MEF, mouse embryonic fibroblast; X-gal, 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside.
Received for publication June 14, 1999. Accepted for publication September 15, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-B and rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:1.[Medline]
B. Immunol. Today 19:80.[Medline]
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B/IB function in mice. Semin. Cancer Biol. 8:93.[Medline]
B transcription factors during lymphocyte activation. Curr. Opin. Immunol. 10:353.[Medline]
B and rel proteins: evolutionary conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B/rel transcription factors. J. Exp. Med. 187:143.B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
B activity in transgenic mice: developmental regulation and tissue specificity. Development 122:2117.[Abstract]
B proteins: structure, function and regulation. Semin. Cancer Biol. 8:75.[Medline]
B by phosphorylation of its inhibitor IB. Nature 344:678.[Medline]
B can rapidly dissociate the complex of the NF-B transcription factor with its cognate DNA. Cell 61:255.[Medline]
B-like activity. Cell 65:1281.[Medline]
Bß regulates the persistent response in a biphasic activation of NF-B. Cell 80:573.[Medline]
B, a novel member of the IB family, controls relA and c-rel NF-B activity. EMBO J. 16:1413.[Medline]
B protein family, IB, inhibits relA (p65)-mediated NF-B transcription. Mol. Cell. Biol. 17:6184.[Abstract]
B. Proc. Natl. Acad. Sci. USA 94:14372.B in response to inducers of NF-B activity. Mol. Cell. Biol. 15:5339.[Abstract]
B negatively regulates DNA-binding and transcriptional activities of NF-B. Mol. Cell. Biol. 15:2689.[Abstract]
B promotes active transport of NF-B from the nucleus to the cytoplasm. J. Cell Sci. 110:369.[Abstract]
Bß in persistent activation of NF-B. Mol. Cell. Biol. 16:5444.[Abstract]
B. Nature 376:167.[Medline]
B deficiency results in a sustained NF-B response and severe widespread dermatitis in mice. Mol. Cell. Biol. 16:2341.[Abstract]
B inhibitors IB and IBß. J. Exp. Med. 188:1055.Bß in T cells of transgenic mice: persistent NF-B activity is required for T-cell immune responses. Mol. Cell. Biol. 18:477.B. J. Exp. Med. 185:1897.B and up-regulation of adhesion molecules and chemokines. Mol. Microbiol. 31:1709.[Medline]
B activation, enhanced granulopoiesis, and neonatal lethality in IB-deficient mice. Genes Dev. 9:2736.B and its inhibitor, IB. Proc. Natl. Acad. Sci. USA 90:2532.B/MAD3 inhibitor of NF-B: positive regulation by members of the rel/NF-B family. EMBO J. 12:5043.[Medline]
B activation by IB. EMBO J. 12:4685.[Medline]
B regulates IB by 2 distinct mechanisms. Genes Dev. 7:1266.B controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway. Science 259:1912. chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
B/Rel family. Cell 80:331.[Medline]
B leads to multifocal defects in immune responses. Cell 80:321.[Medline]
B relA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses. J. Exp. Med. 185:953.B in osteoclast and B-cell development. Genes Dev. 11:3482.B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med. 187:185.B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.B-binding proteins. J. Immunol. 156:2828.[Abstract]
B/Rel by CD40 engagement induces the mouse germ line immunoglobulin C1 promoter. Mol. Cell. Biol. 16:4591.[Abstract]
germline transcripts in B cells via activation of NF-B: synergy with IL-4 induction. J. Immunol. 158:4769.[Abstract]
B site in the human interleukin 1ß promoter: evidence for a positive autoregulatory loop. Mol. Cell. Biol. 13:6231.B regulates IL-1ß transcription through a consensus NF-B binding site and a nonconsensus CRE-like site. J. Immunol. 153:712.[Abstract]
B-like sites that specifically bind c-Rel-p65 heterodimers. J. Biol. Chem. 269:20823.This article has been cited by other articles:
|
|
 |
|
  P. M. Chilton and T. C. Mitchell CD8 T Cells Require Bcl-3 for Maximal Gamma Interferon Production upon Secondary Exposure to Antigen Infect. Immun., July 1, 2006; 74(7): 4180 - 4189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Kearns, S. Basak, S. L. Werner, C. S. Huang, and A. Hoffmann I{kappa}B{varepsilon} provides negative feedback to control NF-{kappa}B oscillations, signaling dynamics, and inflammatory gene expression J. Cell Biol., June 5, 2006; 173(5): 659 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Kaltschmidt, D. Ndiaye, M. Korte, S. Pothion, L. Arbibe, M. Prullage, J. Pfeiffer, A. Lindecke, V. Staiger, A. Israel, et al. NF-{kappa}B Regulates Spatial Memory Formation and Synaptic Plasticity through Protein Kinase A/CREB Signaling Mol. Cell. Biol., April 15, 2006; 26(8): 2936 - 2946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Doerre, K. P. Mesires, K. M. Daley, T. McCarty, S. Knoetig, and R. B. Corley Reductions in I{kappa}B{epsilon} and Changes in NF-{kappa}B Activity during B Lymphocyte Differentiation J. Immunol., January 15, 2005; 174(2): 983 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. I. Samson, S. Memet, C. A. J. Vosshenrich, F. Colucci, O. Richard, D. Ndiaye, A. Israel, and J. P. Di Santo Combined deficiency in I{kappa}B{alpha} and I{kappa}B{epsilon} reveals a critical window of NF-{kappa}B activity in natural killer cell differentiation Blood, June 15, 2004; 103(12): 4573 - 4580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Goudeau, F. Huetz, S. Samson, J. P. Di Santo, A. Cumano, A. Beg, A. Israel, and S. Memet I{kappa}B{alpha}/I{kappa}B{epsilon} deficiency reveals that a critical NF-{kappa}B dosage is required for lymphocyte survival PNAS, December 23, 2003; 100(26): 15800 - 15805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hoffmann, A. Levchenko, M. L. Scott, and D. Baltimore The Ikappa B-NF-kappa B Signaling Module: Temporal Control and Selective Gene Activation Science, November 8, 2002; 298(5596): 1241 - 1245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. N. Suslov, V. G. Kukekov, T. N. Ignatova, and D. A. Steindler Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres PNAS, October 29, 2002; 99(22): 14506 - 14511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-H. Lee and M. Hannink Characterization of the Nuclear Import and Export Functions of Ikappa Bepsilon J. Biol. Chem., June 21, 2002; 277(26): 23358 - 23366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Feuillard, S. Memet, B. Goudeau, A. Lilienbaum, R. Schmidt-Ullrich, M. Raphael, and A. Israel In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-{kappa}B/Rel complexes Int. Immunol., May 1, 2000; 12(5): 613 - 621. [Abstract] [Full Text] [PDF]
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by FACS analysis of FDG-stained and immunolabeled
lymphocytes from I
