By
From the * Department of Oncology and the Department of Experimental Pathology, Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000; and the § Department
of Pathology, Uniformed Services, University of Health Sciences, Bethesda, Maryland 20814
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Abstract |
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The nfkb2 gene is a member of the Rel/NF-B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of
human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas.
To further investigate the function of NF-
B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-
B2-deficient mice
showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph
nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture,
characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired.
Proliferation of NF-
B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were
normal. However, nfkb2 (
/
) animals presented a deficient immunological response to
T cell-dependent and -independent antigens. These findings indicate an important role of NF-
B2 in the maintenance of the peripheral B cell population, humoral responses, and normal
spleen architecture.
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Introduction |
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The nfkb2 gene was originally cloned as a mitogen-inducible cDNA encoding a protein with strong homology to NF-B1 (p105/p50), a member of the Rel/NF-
B family of transcription factors (1). Neri et al. isolated
nfkb2 as the protooncogene involved certain human B cell
lymphomas (4). More recently, several groups have documented that truncations in the COOH-terminal region of
this gene are associated with the development of cutaneous
T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas (5).
The Rel/NF-B family of transcription factors plays a
major role as an early mediator of immune and inflammatory
responses (for reviews see references 9). This group of
proteins contains a highly conserved region named the Rel
homology domain that is responsible for both DNA binding and protein dimerization. In mammalian cells, this protein family can be divided into two classes: one class includes the NF-
B1 (p105/p50) and NF-
B2 (p100/p52) proteins that are synthesized as precursor molecules (p105
and p100, respectively) that remain in the cytoplasm. Upon
proteolytic processing they generate the DNA binding subunits p50 and p52, respectively (1, 15). The second class
of proteins comprises RelA (p65), c-Rel, and RelB. These
proteins do not undergo proteolytic processing, and they harbor transcriptional activation domains. The different biological functions of p50, RelA, RelB, and c-Rel have recently
been addressed by the generation of null mice (18).
In unstimulated cells, the Rel/NF-B dimers associate
with members of the family of inhibitor proteins called I
Bs
and remain as an inactive pool in the cytoplasm. Upon stimulation by different agents, I
B molecules are rapidly phosphorylated and degraded, allowing the NF-
B dimers to
translocate to the nucleus and regulate transcription through
binding to
B sites (9, 12, 23). The in vivo function of
two members of the I
B family, I
B
and Bcl-3, have been
recently analyzed in knockout mice (27).
The biological role of NF-B2 (p100/p52) remains unclear despite the evidence that abnormal NF-
B2 expression correlates with cell transformation (31). Terminally differentiated plasmacytoma and long term LPS-treated pre-B
and B cell lines express p52-containing complexes, implying that p52-c-Rel and p52/-RelB dimers play a role in
the final stages of B cell differentiation (32). Bcl-3, an
oncoprotein overexpressed in human chronic lymphocytic
leukemias, associates with p50 and p52 homodimers (34-
37). Transfection experiments show that these interactions
lead to transactivation of
B-dependent reporter constructs
(8, 35).
To further investigate the function of NF-B2, we have
generated mice carrying a germline mutation of the nfkb2
gene. NF-
B2-null mice have a marked reduction of the B
cell compartment in spleen, bone marrow, and lymph nodes
and are defective in the formation of secondary germinal
centers. B cells from mutant mice have mild proliferative
defects in response to LPS, mIg, and CD40, but have normal maturation and immunoglobulin switching. However,
nfkb2 (
/
) mice have impaired humoral responses to T
cell-independent and T cell-dependent antigens when compared to controls. These findings indicate an important role
for NF-
B2 in the maintenance of the peripheral B cell
population, germinal center formation, and B cell immune
function.
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Materials and Methods |
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Targeting Vector.
To generate the pNF-ES Cell Culture and Generation of Mutant Mice.
CJ7 ES cells were cultured as previously described (40). The electroporation was performed using 25 µg of NotI-linearized pNF-
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Histopathological Analysis and Immunohistochemistry.
Histopathological analysis was performed on a minimum of five animals per age group and genotype. Animals were killed and organs were fixed by immersion in 10% neutral buffered formalin. Tissues were processed by standard methods, embedded in paraffin blocks, sectioned at 4-6 µm, stained with hematoxylin and eosin, and finally examined by light microscopy. For immunohistochemical analysis, frozen tissue sections were stained with anti-IgD (Southern Biotechnology Associates, Birmingham, AL; 1:200 dilution), MOMA-1 (Serotec Ltd., Oxford, UK; 1:100 dilution), peanut agglutinin (PNA)-biotin (Vector Labs., Burlingame, CA; 80 µg/ml) and FDC-M1 (1:100).Cell Labeling and Immunoprecipitation.
Single-cell suspensions from thymus were prepared as previously described (41) and cultured in RPMI 1640 with 10% heat-inactivated fetal calf serum. 2 × 107 cells were labeled for 3 h with 500 µCi/ml of [35S]methionine in methionine-free medium with or without the addition of PMA (10 ng/ml) and PHA (1 µg/ml). Cells were washed with PBS and lysed in 1 ml of radioimmunoprecipitation assay buffer without SDS. Extracts were precleared with preimmune serum (3 µl), immunoprecipitated for 4 h with specific antiserum (3 µl), and then incubated with Protein A-Sepharose CL-4B (Pharmacia Biotech, Piscataway, NJ) for 3 h on a roller at 4°C. The immunocomplexes were washed twice with buffer A (0.2 NP-40, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA); once with buffer B (0.2 NP-40, 10 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 2 mM EDTA); and once with buffer C (10 mM Tris-HCl, pH 7.5). Samples were boiled in 30 µl of Laemmli buffer, and separated on a 12.5% polyacrylamide gel (SDS-PAGE) at 12 milliAmperes for 16 h. Gels were fixed and incubated with Entensify (DuPont-NEN, Boston, MA). After drying, gels were exposed to X-Omat AR film (Eastman Kodak Co., Rochester, NY) atPreparation and Culture of B Cells.
The culture medium and reagents used for the B cell proliferation assays have been previously described (42). Single cell suspensions from spleen were treated with ammonium chloride buffer to lyse red blood cells. Cells were stained with FITC-labeled rat IgG2a anti-mouse B220 mAb (clone RA3-6B2; PharMingen, San Diego, CA) plus PE-labeled hamster anti-mouse CD3e mAb (clone 145-2C11; PharMingen). Small, resting B cells (B220+CD3Electrophoretic Mobility Shift Assays.
Whole cell extracts were prepared as previously described (43). Protein concentrations were determined with the Bio-Rad protein assay kit according to the manufacturer's instructions and were further analyzed by Western blot analysis. Electrophoretic mobility shift assays (EMSAs) were performed using 3 µg of protein extract and 5 × 104 cpm of 32P-labeled probe (Measurement of DNA Synthesis by [3H]thymidine Incorporation.
Purified B cells (105) were cultured for 72 h in a final volume of 0.2 ml in complete RPMI in flat-bottomed 96-well plates. [3H]thymidine was added to the cultures for an additional 18 h. Cultured cells were then harvested onto glass fiber filter paper with a Wallac 1295-001 cell harvester (LKB, Uppsala, Sweden). Specific incorporation of [3H]thymidine was analyzed by scintillation spectroscopy, and results are expressed as the arithmetic mean ± SEM of triplicate cultures.Flow Cytometric Analysis.
Cells were resuspended in PBS containing 1% bovine serum albumin before staining with the following conjugated antibodies: CD3, CD4, CD8, CD25,Quantitation of Secreted Ig Isotype Concentrations in Culture Supernatants.
Purified B cells were stimulated with LPS (20 µg/ml), IL-4 (4.2 ng/ml) + IL-5 (150 U/ml), sCD40L (10 µg/ml), anti- IgD-dextran (3 ng/ml), TGF-Immunization and ELISA Assays.
To induce a T cell-dependent response and analyze the formation of germinal centers in the spleen, five mice of each genotype were immunized by intraperitoneal injection of either sheep red blood cells (SRBCs) or (4-hydroxy-3-nitrophenyl) acetyl (NP-KLH). Each mouse was injected with 250 µl of phosphate-buffered saline containing 10% SRBCs (2.5 × 108 cells) or with 100 µg of alum-precipitated NP-KLH. Animals were killed 10 d after immunization and spleens were frozen in O.C.T. (Tissue-Tek, Miles Inc., Elkhart, IN) for immunohistochemistry. Additional mice were also boosted with a second injection of the same antigens 10 d after the first immunization, and the same assays were performed. The nfkb2-null mice that were used to evaluate the basal immunoglobulin levels and the antigen-specific immunoglobulin production have been previously backcrossed for eight generations to the C57 Bl/6 strain. Basal levels of immunoglobulins were measured in sera collected from control and nfkb2 null mice before immunizations. For T cell-dependent responses, mice were immunized by intraperitoneal injection of 100 µg of alum-precipitated NP-KLH. In the case of the T cell-independent responses, mice were immunized with 10 µg of NP coupled to LPS (20, 44). Serum samples were collected before immunizations and at 7-d intervals during 3 wk. To determine the levels of NP-specific immunoglobulins, we used ELISA plates coated with NP17-BSA for capture, and goat anti-mouse isotype-specific antibodies conjugated to horseradish peroxidase (Southern Biotechnology Associates) as previously described (45). The level of each antigen-specific isotype was determined by comparison with a standard curve. NP-specific Ig levels in unchallenged mice were below detection. One symbol represents one mouse. Statistical analysis was performed using the unpaired Student's t test to calculate second two-tailed P values. ![]() |
Results |
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The strategy to generate the nfkb2-null allele in ES cells involved the introduction of a PGK-neo cassette in exon 4 of the mouse nfkb2 locus in an EcoRI site in nucleotide 54 of the nfkb2 open
reading frame (Fig. 1 A). Heterozygous nfkb2 (+/) animals, containing the 8.0-kb wild-type and 5.3-kb recombinant SpeI fragments, were crossed to obtain homozygous
mice (
/
) (Fig. 1 B). No differences in size, behavior, or
life span were observed between nfkb2-null mice and wild-type littermates. These animals were born at the expected
Mendelian ratios, which demonstrates that NF-
B2 is not required for normal embryo development. The presence of
the NF-
B2 protein was analyzed in thymocytes of control
and mutant mice. While thymic cells from wild-type mice
expressed p100/p52 upon stimulation, neither p100 nor
p52 were present in cells from the mutant mice, demonstrating that the targeted disruption of the nfkb2 locus resulted in a complete loss of both proteins (Fig. 1 C). The
nfkb2 (
/
) mice had no gross developmental deficiencies or health abnormalities while maintained in a pathogen free
facility. No significant difference in protein levels of other
Rel/NF-
B family members was detected in thymus and
spleen whole cell extracts from mutant mice compared to
control littermates by Western blot analysis (data not shown).
EMSAs using whole cell extracts from thymus of control
and nfkb2 (
/
) mice showed no remarkable differences in their DNA binding (Fig. 1 D). In contrast, whole cell
extracts from splenocytes have a decrease DNA binding activity corresponding to p52-containing complexes compared to control cells (Fig. 1 D).
Light microscopic evaluation of all major tissues from nfkb2 (/
)
mice of varying ages (3, 9, 12, 24, and 36 wk) showed that histopathological changes were largely restricted to lymphoid organs. The thymus of the null mice was normal in
size, and corticomedullary ratios were similar to those observed in wild-type age-matched animals (data not shown).
The spleen of the naive mutant mice was grossly similar to
wild-type controls. However, microscopic evaluation of
the spleen of nfkb2 (
/
) mice showed a disruption of the
normal architecture characterized by diffuse, irregular follicular (B cell) areas with the absence of a discrete, perifollicular marginal zone and germinal centers (compare Fig. 2,
A and C). These morphologic abnormalities were substantiated after immunohistochemical detection of B cells. In
contrast to wild-type spleen where numerous B220 positive
(B220+) cells were observed within discrete germinal centers, the spleen from nfkb2 (
/
) mice presented B220+
cells scattered among indiscrete germinal centers (Fig. 2, B and D). No differences were observed in the number of cells
undergoing apoptosis in the red or white pulp of the spleen
of unstimulated control and nfkb2 (
/
) mice as determined by Apoptag (Oncor, Inc., Gaithersburg, MD) histochemical staining (data not shown). However, the number of Apoptag-positive cells in the splenic white pulp of
SRBC-stimulated nfkb2 (
/
) mice was decreased by
~70% as compared to the white pulp of SRBC-stimulated
control mice. The number of cells undergoing apoptosis in
the splenic red pulp of SRBC-stimulated nfkb2 (
/
) and
SRBC-stimulated control mice were similar.
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Morphologic changes similar to those observed in B cell areas in the spleen also occurred in lymph nodes of mutant mice (data not shown).
Reduction of the B Cell Population in the Spleen, Bone Marrow, and Lymph Nodes from Mutant Mice.To further characterize the histopathological changes observed in the nfkb2
(/
) mice, we performed an extensive survey of surface
marker expression on hemopoietic cells from mutant mice. Immunofluorescence staining and flow cytometric analysis
of thymocytes with anti-CD3, -CD4, -CD8, -CD25, and
-Thy 1.2 showed no remarkable changes between control
and mutant mice (data not shown). No difference in the
total cell number was observed in the thymus of null mice with respect to the controls. Consistent with the initial histopathologic and immunohistochemical observations, the
spleen of the naive nfkb2-null mice presented a marked decrease in the B cell population. A 50-80% reduction in the
total number of B cells was detected in the spleen of mutant mice as early as 1 wk after birth (Fig. 3 A). A concomitant increase of the T cell compartment comprising both
CD4 and CD8 single positive cells was detected in the
spleen of mutant mice (Fig. 3 A). A similar decrease in the
number of B cells and increase in the T cell population
were observed in older mice (Fig. 3, B and C). We also examined the stages of B cell differentiation by surface B220,
CD43, HSA, IgM, IgD, and CD19 expression. Both immature and mature B cells were present in the spleen of the
nfkb2 (
/
) mice, indicating that a general reduction of all
the B cell lineages rather than a maturation blockage occurred in the absence of NF-
B2. Similar results were observed in the lymph nodes from mutant mice.
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The bone marrow of the 6-wk-old nfkb2 (/
) mice
have a normal number of B cells (Fig. 4 A). The total number of bone marrow cells recovered from control and nfkb2
(
/
) mice showed no significant differences. However,
mutant mice had a decline of 50% in the absolute number
of B cells between 8 and 12 wk of age in the bone marrow
(Fig. 4 B).
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Normally, B cells
stimulated with LPS, anti-CD40, or anti-IgD-dextran proliferate, and these responses can be further augmented by
IL-4 and/or IL-5 addition. The signal transduction pathways mediating these effects are distinct for each molecule, and
hence could be differentially affected by the absence of
NF-B2 (45). In this context, and in light of the distinctive
roles for p50, c-Rel, and RelB in murine B cell proliferation (20, 21, 42, 45), we determined whether any of these
mitogenic pathways were dependent upon the expression
of NF-
B2. Highly purified resting B cells from naive
nfkb2 (
/
) and control mice were obtained by electronic
cell sorting and stimulated for 72 h with various concentrations of LPS, anti-IgD-dextran, or anti-CD40 mAb in the presence of IL-4 and IL-5. Similar results were obtained in
three independent experiments, which demonstrated only
moderate reduction in the proliferative response of NF-
B2-deficient cells compared to control B cells (Fig. 5, A-C).
At concentrations between 2 and 10 ng/ml of anti-IgD-dextran, NF-
B2-deficient B cells showed a nearly twofold reduction in their proliferative response compared to
controls. These results prompted us to analyze whether the NF-
B2-deficient B cells were more prone to undergo
apoptosis upon stimulation. Splenocytes from control and
nfkb2-deficient mice have a similar number of B cells undergoing apoptosis upon stimulation with anti-IgD-dextran, anti-CD-40 mAb, or TNF-
as detected by Facs® analysis using anti-B220 and anti-Annexin V (data not shown).
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NF-B2 has been shown to be expressed late during B cell
maturation, suggesting that it may regulate B cell maturation to Ig secretion (32, 33). Furthermore, multiple Rel/
NF-
B binding sites have been identified in the Ig heavy
chain locus, including promoters and enhancers involved in
regulating Ig isotype switching (46, 47). Indeed, B cells
lacking NF-
B1 (p105/p50) have distinctive and selective
defects in Ig isotype switching (45). Thus, we tested the
relative ability of B cells lacking NF-
B2 (p100/p52) to
undergo B cell maturation to IgM secretion and switch to
the expression of downstream Ig isotypes in response to
various combinations of B cell activators and cytokines.
IL-4, IFN-, and TGF-
induce class switching to IgG1
and IgE, IgG3 and IgG2a, and IgG2b and IgA, respectively,
in appropriately activated B cells (48). Combinations of
stimuli optimal for induction of class switching to different
sets of isotypes were used to assess switch recombination in
NF-
B2-deficient B cells. In two similar sets of experiments, maturation to IgM secretion was essentially comparable between B cells lacking NF-
B2 and similarly activated control B cells, in response to a variety of distinct
stimuli (Fig. 6). Likewise, induction of all six non-IgM, non-IgD isotypes in NF-
B2-deficient B cells was comparable to that observed for control B cells. The production
of IgM in NF-
B2-deficient B cells was two times higher
than in controls. Thus, in contrast to NF-
B1, NF-
B2 is
not essential for Ig isotype switching.
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Mice rendered deficient for c-Rel, RelB,
NF-B1 (p105/p50), or Bcl-3 have defective basal immunoglobulin levels and antibody production upon antigenic
challenge (20, 21, 29, 30, 49). To evaluate the humoral immunity of the nfkb2-null mice, we measured the basal production of immunoglobulins in naive mutant and control littermates and the statistical analysis was performed using
the Student's t test (Fig. 7 A). While IgG1 and IgG2b
showed similar levels in control and nfkb2 (
/
) mice,
IgM (4.5-fold; P <0.0005) and IgG2a (2-fold; P <0.008)
were increased in null mice. In contrast, a threefold reduction in the levels of IgG3 (P <0.01) and twofold lower levels of IgA (P <0.000004) were detected in the nfkb2-null mice compared to wild-type littermates.
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We next examined the humoral responses of control and
mutant mice to specific antigenic challenges. To assess the
T cell-dependent immune responses, mice were immunized with keyhole limpet hemocyanin coupled to (NP-KLH) and bled 7, 14, and 21 d after immunization. The
production of NP-specific isotype IgG1 by nfkb2 (/
)
mice was reduced fourfold at day 7 (P <0.008), and decrease
to 10-fold (P <0.00001) at 21 d after treatment (Fig. 7 B).
The evaluation of the T cell-independent response was
performed using NP-LPS to immunize control and nfkb2-null mice and the serum levels of NP-specific IgG3 were
measured at 7, 14, and 21 d after immunization (Fig. 7 C).
Under these conditions, the mutant mice had a 10-fold
lower production of specific antibody at day 7 (P <0.01)
and a 30-fold decrease 21 d after immunization (P <0.02).
NP-specific Ig levels in unchallenged mice were below detection. These results demonstrate that NF-B2 is required
for normal levels of antigen-specific immunoglobulins, in
particular, in the absence of T cell help.
The abnormal architecture of the spleen in the
nfkb2 (/
) mice suggests that immune responses dependent on cellular interactions in the follicles may not be fully
functional. To test this hypothesis we immunized mice
with SRBCs and evaluated the formation of germinal centers (GCs) 10 d after immunization. The spleen of wild-type mice had numerous GCs characterized by B cell areas
that bound PNA surrounded by IgD+ cells (Fig. 8, A and
C). In contrast, the spleen of mutant mice had very few cells
stained by PNA and a diffuse perifollicular staining of cells
expressing surface IgD (Fig. 8, B and D). To further evaluate the alterations of spleen follicular structure in the nfkb2
(
/
) mice, tissue sections from the immunized mice were stained with MOMA-1, a specific antibody for metallophilic macrophages (Fig. 8, E and F). While the spleen
of control mice had a ring-like zone of MOMA-1 + cells
demarcating the marginal zone, the spleen of nfkb2 (
/
)
mice contained reduced numbers of loosely organized
MOMA-1-positive cells scattered throughout the white
pulp. MOMA-1 staining in the outer PALS showed no differences between controls and nfkb2 (
/
) mice. The
presence of antigen presenting follicular dendritic cells
(FDCs), that promote B cell maturation, was analyzed by
immunostaining with the FDC-M1 monoclonal antibody
(50, 51). While clusters of FDCs could be clearly seen in
the GCs of the control mice, very few FDCs could be detected in the mutant mice (Fig. 8, G and H).
|
Similar defects in the formation of GCs were observed
when mice received a second immunization boost of SRBC
10 d after the first one. In addition, nfkb2 (/
) mice immunized with NP-KLH also displayed a lack of well defined GC formation.
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Discussion |
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Since the description of the NF-B binding activity
more than 10 y ago (52), an extensive body of information
has been gathered describing the importance of this family
of proteins in the regulation of genes involved in cell growth,
stress, inflammation, and immune responses. An increasing
number of NF-
B target genes have been identified to date,
including cytokines, cytokine receptors, interferons, growth
factors, MHC proteins, and other genes. However, whether or not particular members of the NF-
B family regulate
specific genes in different cell types, and the distinct biological function of each protein, remain to be elucidated. In an
attempt to answer some of these questions, several laboratories have generated mice with mutated genes of the NF-
B
family. The data obtained indicate that the Rel/NF-
B
proteins play an important role in the hematopoietic system. In brief, relA-deficient mouse embryos die at embryonic day 15 following massive apoptosis of hepatocytes in
the liver (18). Recent studies indicate that RelA has a role
in preventing TNF-
-induced apoptosis (53). Secretion of IgG1 and IgA is reduced in RelA-deficient B cells
(56). Also, T and B cells lacking RelA show a marked reduction in their proliferative response. RelB-deficient mice
exhibit a T cell-dependent multiorgan inflammation, myeloid hyperplasia, and splenomegaly associated with extramedullary hemopoiesis (19, 22, 57). RelB-deficient B
cells have defective proliferative responses, although they
undergo normal cell maturation, Ig secretion and isotype
switching (42). On the other hand, c-Rel has been shown
to be required for lymphocyte activation and cytokine expression, as demonstrated by the B and T proliferative defects and IL-2 production by T cell in c-Rel (
/
) mice
(20). The immune responses to T cell-dependent antigens
were defective in these mice.
Based on the studies mentioned above, the NF-B proteins can be divided into two groups according to the role
they play in the hematopoietic system. One of the groups
includes p50 and c-Rel, which regulate genes involved in
rapid immune responses. The other group comprises RelA
and RelB, which control the expression of genes implicated in immune responses and also hemopoietic development and housekeeping functions.
Here, we show that
NF-B2 is required for B cell development and function.
First, nfkb2 (
/
) mice have a dramatic reduction in the
absolute number of B cells in peripheral lymphoid organs. Second, NF-
B2 (
/
) B cells exhibited reduced in vitro
proliferation. Third, the number of B cells in the bone
marrow of the nfkb2 (
/
) mice undergoes a reduction of
50% between 8 and 12 wk of age.
In vitro, NF-B2 (
/
) B cells display normal cell maturation to immunoglobulin secretion and class switching.
The number of splenic T cells is increased although stimulation-induced proliferation is normal in the absence of
NF-
B2. In addition, the nfkb2 (
/
) mice have an abnormal splenic microarchitecture, with diffuse B cell areas,
lack of lymphoid follicles, and a discrete marginal zone, an
area where nfkb2 transcripts have been previously shown (58).
Challenges with T cell-dependent or -independent antigens showed that the nfkb2 (/
) mice are impaired in the
production of antigen-specific antibodies. Consistent with
these defective antibody responses, the spleen of nfkb2 (
/
)
mice lacks well-defined GC structures. The absence of centrocytes and a decreased number of metallophilic macrophages and FDCs are some of the characteristics of the GCs
in the mutant mice. Such centers are the primary sites where T-B cell communication takes place, together with
B cell expansion, somatic hypermutation, isotype switching, and affinity maturation (59).
Our results suggest that either the production or the
lifespan of B cells is defective in nfkb2 (/
) mice. In addition, the migration of the B cells to the peripheral lymphoid organs may be deficient. Moreover, the low number
of splenic B cells in these mutant mice coupled with the
marked reduction of FDCs and the lack of GCs results in
impaired immune responses. Thus, NF-
B2 regulates the
expression of genes involved in B cell production, formation of GCs, and normal levels of antigen-specific antibodies. Although the genes regulated by NF-
B2 are still unknown, we are starting to understand the pathways in
which this protein is involved. Further studies will be necessary to clarify whether nfkb2-null B cells are intrinsically
defective or exhibit abnormalities as a consequence of impaired T lymphocytes, dendritic cells, or the microenvironment of the spleen.
Similar defects in the formation of GCs have been described in mice deficient in different ligands and receptors
including CD40, CD40L, TNF-, LT-
, TNF receptor I,
MHC class II, CD28, the putative chemokine receptor
BLR1, the complement receptor locus Cr II, and OBF-1,
the B cell-specific coactivator of Oct-1 and Oct-2 (63).
Interestingly, CD40, TNF-
, and LT-
signaling induce
NF-
B binding activity, suggesting that p52 may have a role in the signal transduction pathways triggered by these
molecules (73).
Several phenotypic defects are common between bcl-3
(/
) and nfkb2 (
/
) mice (29, 30). Mice rendered deficient for Bcl-3 also have a decreased B cell population,
impaired formation of GCs, and severe defects in the production of antigen specific antibodies. Such strong similarities argue for a synergistic effect of these two proteins in
transcription (35).
The phenotype of the nfkb2-deficient mice is quite
distinct from that of mice rendered deficient in other members of the NF-B family of transcription factors. Although
NF-
B2 (p100) is 52% homologous to NF-
B1 (p105; references 1, 3, 4), their pattern of expression is different. While
nfkb1 is ubiquitously expressed in mouse tissues, nfkb2 expression is more restricted to lymphoid organs, and the
highest mRNA levels are found in thymic medulla, marginal zone, and outer region of the periarterial sheath of the
spleen (58). A comparison between the phenotypic changes displayed by mice lacking NF-
B1 and NF-
B2 reveals
significant differences but also some similarities. The nfkb2-null mice have an abnormal splenic architecture and impaired formation of secondary GCs. In contrast, the nfkb1-null mice do not have any of these deficiencies (21, 30). B
cells from nfkb1-null mice have a very poor proliferative response to LPS, but respond normally to anti-IgD-dextran
and anti-CD40. In the case of nfkb2-null mice, B cells exhibit a moderate decrease in proliferation in the presence of
similar stimuli. These data indicate that NF-
B1/p50 has a key role in the LPS stimulatory pathway, whereas NF-
B2/p52 has a modest effect on proliferation independent
of the pathway. Another important difference in the roles
of NF-
B1/p50 and NF-
B2/p52 is that nfkb1-null B cells
have defects in their maturation and immunoglobulin secretion, germ line CH transcription, and isotype switching
(21, 45), whereas nfkb2 (
/
) B cells do not exhibit any of
these deficiencies. However, the production of antigen-specific antibodies is affected in mice mutant for either NF-
B1 or NF-
B2. T cell-dependent responses are impaired
in nfkb1 mutant mice and reduced in nfkb2-null mice. T
cell-independent immune responses are severely impaired
in nfkb2-null mice.
These phenotypic differences between nfkb1 and nfkb2 mutant mice clearly indicate that p105/p50 and p100/p52 have distinct functions in the B cell lineage. However, we still cannot rule out a functional redundancy for these proteins. Further experiments generating double mutant mice should help to clarify this matter.
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Footnotes |
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Address correspondence to Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000. Phone: 609-252-5744; Fax: 609-252-3307; E-mail: bravo#m#_rodrigo{at}msmail.bms.com Jorge H. Caamaño's present address is Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA, 19104-6008.
Received for publication 23 July 1997 and in revised form 14 November 1997.
1 Abbreviations used in this paper: EMSA, electrophoretic mobility shift assays; ES, embryonic stem; FDC, follicular dendritic cell; GC, germinal center; PNA, peanut agglutinin; SRBC, sheep red blood cell.We are grateful to Mavis Swerdel, Alice Lee, and Sergio Lira in the Transgenic Unit and all the staff in Veterinary Sciences at Bristol-Myers Squibb. We would like to thank Kenneth Class for flow cytometry; Donna Dambach for comments and FDC immunostaining; James K. Loy for photoimaging; and Carol Ryan, Anne Lewin, and Michelle French for excellent technical assistance. We are indebted to Ricardo Attar, Daniel Carrasco, and Falk Weih for technical help and valuable comments of this manuscript. We also thank Albert Bianchi, Anne Gaëlle Borycki, Violetta Iotsova, Heather Macdonald-Bravo, and Rolf-Peter Ryseck for helpful suggestions and comments concerning this manuscript. We are grateful to Marie Kosco-Vilbois for kindly providing the FDC-M1 antibody.
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