By
From the * Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543-4000; the Department of Microbiology, Bristol-Myers Squibb
Pharmaceutical Research Institute,Wallingford, Connecticut 06492; and the § Department of
Pathology, Uniformed Services, University of Health Sciences, Bethesda, Maryland 20814
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Abstract |
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The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-B family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (
c-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in
the c-rel region encoding the COOH terminus of c-Rel (c-rel
CT/
CT) display marked defects
in proliferative and immune functions. c-rel
CT/
CT animals present histopathological alterations
of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary
hematopoiesis, and bone marrow hypoplasia. In older c-rel
CT/
CT mice, lymphoid hyperplasia
was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-rel
CT/
CT mice, the lack
of c-Rel activity is less efficiently compensated by other NF-
B proteins.
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Introduction |
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The c-rel protooncogene encodes a transcription factor
that belongs to the family of Rel/nuclear factor (NF)1-B
proteins that play an important role in the expression of genes involved in immune and inflammatory responses (1).
Rel/NF-
B proteins represent a group of homo- and heterodimeric complexes that are related through a common
NH2-terminal domain known as the Rel homology domain (RHD), which consists of ~300 amino acids and contains sequences important for protein dimerization, DNA binding, nuclear localization, and association with inhibitors of the I
B family. The COOH termini of Rel proteins
have little sequence similarity and have been used to distinguish two classes of Rel proteins. One class includes NF-
B1
(protein [p]105/p50) and NF-
B2 (p100/p52), which, by
proteolytic processing, generates the mature DNA-binding
subunits p50 and p52, respectively. The second class includes c-Rel, RelA (p65), and RelB, which contain transcriptional activation domains in their COOH termini.
The genes of the Rel/NF-
B family are differentially expressed in lymphoid tissues (11) and studies with mice
lacking either p50, RelB, RelA, or c-Rel demonstrate that
individual members of this family have distinct functions in
vivo (for review see reference 13).
Activation NF-B is regulated by posttranslational modification and degradation of I
B proteins that interact with
the Rel/NF-
B complexes and sequester them in the cytoplasm by masking their nuclear localization signal. Members of the I
B family include I
B
, I
B
, I
B
, I
B
,
Bcl-3, p105, and p100, which share conserved ankyrin-like
repeats responsible for interaction with the Rel/NF-
B complexes. In the case of I
B
, phosphorylation and subsequent degradation of the inhibitor releases the active
Rel/NF-
B complexes allowing their nuclear translocation. Degradation of I
B
is mediated by the ubiquitin-
proteasome pathway, and phosphorylation of I
B
involves a ubiquitin-dependent protein kinase (4, 7, 8, 10,
14).
The mammalian c-rel gene was first identified as the cellular homologue of v-rel, the oncogene carried by Rev-T,
an acutely transforming avian retrovirus that induces a variety of neoplastic diseases in chickens. Rearrangements in
the c-rel gene have been associated with human lymphoid
malignancies. Similar to v-rel, the altered c-rel genes lack
sequences encoding the transcriptional activation domain
(17). The in vivo roles of the c-rel gene have been recently addressed by gene targeting. Mice lacking the c-Rel
protein (c-rel/
) exhibit defects in lymphocyte proliferation, humoral immunity, and cytokine production (21).
To understand the in vivo role of c-Rel in greater detail,
we have generated mice lacking only the COOH-terminal
transcriptional activation domain of c-Rel (c-rel
CT/
CT).
This transcriptionally inactive molecule retains an intact RHD, is able to bind DNA, and to interact with other
Rel/NF-
B family members and the I
B family of inhibitory molecules. Therefore, in c-rel
CT/
CT mice, other Rel/
NF-
B family members do not have the possibility of taking the function of c-Rel, as in the case of c-rel
/
mice. In
addition, this approach allows us to address the functional significance of the different transcriptional activation domains present in c-Rel, RelA, and RelB, and the role of
c-Rel COOH-terminal truncations in the generation of
lymphoid malignancies.
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Materials and Methods |
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Targeting Vector and Generation of Mutant Mice.
A genomic library (cloned in lambda DashII; Stratagene Corp., La Jolla, CA) prepared from D3 embryonic stem (ES) cell DNA was screened with the mouse c-rel cDNA probe (24). Two overlapping phages containing a total of 25 kbp of the c-rel gene were isolated and the fragments were subcloned into pBluescript KS+ (Stratagene Corp.). A 0.9-kbp fragment containing the SV40 polyadenylation sequence [p(A)] and a termination codon was prepared by PCR mutagenesis using the pMSG vector (Pharmacia, Piscataway, NJ) and subcloned in the plasmid PGK promoter neomycin thymidine kinase (pPNT) vector (25). A 4.7-kbp c-rel genomic DNA fragment containing exons 7-9 and the first portion of exon 10 (until the XhoI site) was inserted upstream of the stop codon and between the phosphoglycerate kinase (PGK)-neo cassette and the PGK promoter driving the herpes simplex virus thymidine kinase gene (PGK-tk cassette) of a pPNT vector containing the termination codon. A 8.5-kbp fragment from c-rel genomic DNA extending from the XhoI site of exon 10 to the flanking 3' genomic sequences was cloned upstream and in opposite direction to the PGK-neo cassette of the pPNT vector. In this way, the genomic c-rel gene was interrupted by the neo selection marker in exon 10. Introduction of the stop codon 3' to the XhoI restriction site produces a truncated c-rel messenger RNA that lacks the region encoding the truncated c-Rel (
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Histology, Immunofluorescence, Immunohistochemistry, and Flow Cytometry.
Tissues were immersion fixed in 10% buffered formalin and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin. Apoptotic cells were detected with the Apoptag assay and stained with methyl green according to Oncor, Inc. (Gaithersburg, MD). For detection of germinal centers (GCs), mice were immunized with SRBCs, and frozen sections of spleen were prepared and stained as described (27). Flow cytometry analysis was performed as described (26, 28). Anti-mouse macrophage 1 (Mac-1), granulocyte 1 (Gr-1), and total erythroid cells (Ter) 119 were obtained from GIBCO BRL (Gaithersburg, MD).In Vitro Proliferation, Listeria Monocytogenes, and Lymphocytic Choriomeningitis Virus Infections.
B cell proliferation assays were performed as described (29). Lymph node T cells were purified by murine T cell enrichment columns (R&D Systems, Inc., Minneapolis, MN) and (105) cells in 96-well plates were stimulated with IL-2 or with cluster of diffentiation (CD)3 plus CD28 coated antibodies (PharMingen, San Diego, CA) in 200 µl medium for 48 h. Cell proliferation was measured after 12 h of culture by [3H]thymidine incorporation in a scintillation spectroscopy. Results are expressed as the arithmetic mean ± SD of triplicate cultures. Adult mice were injected intraperitoneally with 2,500 CFU of L. monocytogenes and killed after 5 d. Animals were killed after 5 d or on day 4 of infection when moribund. Numbers of viable L. monocytogenes in lung, liver, and spleen of infected animals were determined by plating serial dilutions of organ homogenates in PBS on sheep blood agar. Other mice were injected intraperitoneally with 1.2 × 105 PFU (Armstrong strain) of Lymphocytic Choriomeningitis Virus (LCMV) and killed 3 or 7 d later. Infectious LCMV titers of lung, liver, and spleen (PFU/g tissue) were quantitated by plaque assay using Vero cell monolayers as described (30). Data from six to nine animals per genotype were recorded for each experiment.Thioglycollate-induced Peritonitis, Granuloma Formation, and Nitric Oxide Production.
Peritoneal macrophages were prepared 5 d after intraperitoneal injection with 1.5 ml sterile Brewers thioglycollate broth (3%) as described (31). Three cell preparations from the various genotypes were used in in vitro experiments. Lung granuloma formation was induced by glucan tail injection (32). For nitric oxide synthase (NOS) assays, resting peritoneal macrophages (5 × 105/0.5 ml DMEM in a 24-well plate) were treated for 72 h with LPS (1 µg/ml) alone or in combination with IFN-ELISA.
Purified lymph node T cells (5 × 105/ml) isolated from 6-wk-old mice were incubated with or without coated anti-CD3 and anti-CD28 antibodies for 72 h. Macrophages were stimulated for 2 h with media alone or in the presence of a combination of LPS (1 µg/ml) and IFN-Immunoprecipitation and Electrophoretic Mobility Shift Assays.
Splenocytes from 6-wk-old animals were isolated as described (28) and labeled with 800 µCi/ml of [35S]methionine (Amersham Corp., Arlington Heights, IL) for 6 h. Cells were lysed directly in 1× RIPA buffer, followed by immunoprecipitation as described (34). For electrophoretic mobility shift assays (EMSAs), nuclear extracts were prepared and incubated with a palindromicExpression Vectors, Cell Culture, and Transfections.
Expression vectors for p50, RelA, c-Rel, and ![]() |
Results |
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A targeted disruption of the transcriptional activation domain of c-rel was created by introducing a termination signal at codon 366 followed by SV40 p(A) and a PGK-neo cassette (Fig. 1 A). After electroporation and selection, 200 double-resistant CJ7 ES cell clones were picked and screened by Southern blot analysis (Fig. 1 B). Homologous recombination in 11 clones was identified by the appearance of a 18.5-kbp recombinant band in addition to the 25-kbp wild-type band in XbaI-digested DNA (Fig. 1 B a).
Eight chimeric males transmitted the ES cell-derived agouti coat and the targeted c-rel gene to their offspring (data not shown). Intercrosses between heterozygous animals produced progeny with normal Mendelian transmission of the disrupted c-rel allele according to genotypic PCR analysis (Fig. 1 B b).
Expression of c-Rel Lacking the Transcriptional Activation Domain in Splenocytes.Whole cell lysates from mouse splenocytes labeled with [35S]methionine were immunoprecipitated
with a c-Rel antiserum raised against the RHD. The results
demonstrated that homozygous mutant mice (c-relCT/
CT)
lacked c-Rel, but expressed the truncated c-Rel protein,
c-Rel (Fig. 1 B c). In heterozygous mice (c-rel+/
CT), both
the normal c-Rel and the truncated
c-Rel proteins are detected. We further studied selected protein interactions
of
c-Rel (Fig. 1 C). After immunoprecipitation with c-Rel
antibodies under nondenaturing conditions, the immunocomplexes were dissociated and then sequentially reprecipitated
with p50, RelA, and I
B
antibodies. The immunoprecipitation patterns observed in control (Fig. 1 C, left) and in
c-Rel (Fig. 1 C, right) cells were similar, indicating that
c-Rel, by virtue of its intact RHD, maintains normal interactions with other Rel/NF-
B family members and I
B
.
Young c-relCT/
CTmice, between 3 and 8 wk old, appeared
normal as assessed by habit, weight, posture, and histologic
and flow cytometric analysis of lymphoid cells (data not
shown). However, after 5 to 7 mo of age, an increasing
number of animals began to develop exzematoid skin lesions around the nose, eyes, ears, tail, and foreskin. These
lesions were not related to infection, according to microbiologic testing and serologic analysis (not shown). The disease progressed slowly, without severely compromising the
health status and survival of c-rel
CT/
CT mice if the animals
were kept in microisolators. However, when c-rel
CT/
CT
mice were left in a non-pathogen-free environment, their
survival was reduced compared with control littermates
(data not shown). Apparently, healthy and sick c-rel
CT/
CT
mice were systematically analyzed by histopathology. A constant observation was the presence of enlarged spleens, lymph
nodes, and in 30% of the cases analyzed, the presence of pale
bones and changes in the color and consistency of the stomach, liver, and lung (Fig. 2, a and b, and data not shown).
The TUNEL assay revealed increased numbers of apoptotic nuclei inside of macrophages in medullary areas of the
thymus and within the marginal zone of the spleen in c-
rel
CT/
CT mice (Fig. 2, c and d, and data not shown). This
result is in agreement with recent reports documenting a
role of Rel/NF-
B in preventing apoptosis (39).
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Staining of mutant spleen sections revealed increased
white and red pulp areas when compared with control tissue
(Fig. 2, e and f ). Analysis at higher magnification revealed
mild to moderate increase of normoblast and megakaryocytes and a reduced number of metachromatic macrophages
in red pulp areas of spleens from c-relCT/
CT mice (data not
shown). Reduced cellularity and increased empty spaces were
observed in the bone marrow of c-rel
CT/
CT mice that presented macroscopic alterations in the bones (Fig. 2, g and h).
Lymphoid cellular infiltration was observed in tissues of the
mutant mice with enlarged lymph nodes and macroscopic
alterations in the stomach, liver, and lung (Fig. 2, i-l).
The previous observations suggests an essential role of
c-Rel in normal bone marrow hematopoiesis and lymphoid development. Since these changes were not observed in wild-type and heterozygous c-rel+/CTmice,
c-Rel does not behave as a trans-dominant mutant of c-Rel.
Flow cytometric
analysis of hemopoietic cells from young c-relCT/
CT mice
did not show major alterations in the expression of the surface markers in cells derived from thymus (CD4, CD8, and
TCR-
/
), spleen (CD4, CD8, TCR-
/
, CD25, Mac-1,
Gr-1, B220, IgM, IgK, and Ter 119), and bone marrow
(Ter 119, IgM, IgK, and B220) (data not shown). However, after 5 mo of age, alterations began to be detected in
c-rel
CT/
CT mice (Fig. 3, A and B). For instance, in agreement with the hypocellularity observed in bone marrow at
histopathology, a reduced percentage of macrophages (Fig.
3 A, a and b) and erythroid precursors (Fig. 3 A, c and d)
was detected in bone marrow-derived cells from c-rel
CT/
CT
mice, but not in control littermates. In addition, a concomitant and graded increase in the number of erythroid precursors (Fig. 3 B, a and b) and granulocytes (Fig. 3 B, c and
d) was observed in the enlarged spleens from c-rel
CT/
CT
mice, reflecting the presence of extramedullary hematopoiesis. When B cell markers were used in flow cytometric
analysis, a 1.5-2.5-fold increase in the total number and
percentage of B cells was observed in the enlarged spleens
of c-rel
CT/
CT mice, correlating with the enlarged white
pulp areas (Fig. 3 C, and data not shown). Immunostaining of
control (Fig. 3 C a) and c-rel
CT/
CT (Fig. 3 C b) splenic tissue sections with anti-Mac-1 revealed a dramatic enlargement of the lymphatic follicles with compression and displacement of the red pulp to the periphery of the organ. A
concomitant reduction of Mac-1 positive cells in the red
pulp between the follicles was also observed. Labeling with
anti-B220 antibodies revealed diffusely enlarged white pulp
areas with poorly demarcated white/red pulp boundaries,
marginal zones, and periarterial lymphatic sheaths (Fig. 3 C, c
and d). In addition, a decrease in the intensity of the B220-stained cells was also evident in spleens from c-rel
CT/
CT
mice compared with control littermates. In contrast to the
B cell areas, the T cell areas (periarterial lymphatic sheaths), were not enlarged in spleens from mutant mice, as revealed
by the immunostaining with anti-TCR-
/
monoclonal
antibodies (Fig. 3 C, e and f ).
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Although infectious disease as a primary cause of
the c-relCT/
CT phenotype was ruled out (data not shown),
opportunistic bacterial infections were observed in older
c-rel
CT/
CT mice. To examine whether c-rel
CT/
CT mice
might fail to eliminate bacterial pathogens, we used the L. monocytogenes model (42). Groups of wild-type and homozygous mutant mice were infected intraperitoneally
with L. monocytogenes, killed at day 5, and bacterial CFU
were determined in spleen, liver, and lung. As shown in
Fig. 4 A, c-rel
CT/
CT mice had >20-fold higher listerial titers in all the tissues examined compared with control animals. This result defined an impaired capability of c-rel
CT/
CT
mice in handling bacterial infections and explained the increased susceptibility to bacterial infections of these mice
when kept in a non-pathogen-free environment.
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To investigate whether migration is impaired in c-Rel macrophages, mice were injected with
thioglycollate. The total number of peritoneal inflammatory cells elicited was comparable in control and c-rel
CT/
CT
mice (Fig. 4 B a). However, the differential cell count revealed a twofold reduction in the number of macrophages
and a relative increase in the number of polymorphonuclear
leukocytes in c-rel
CT/
CT mice, indicating that recruitment of macrophages is impaired in the mutant animals
(Fig. 4 B, b and c). The altered clearance of the facultative
intracellular bacterium L. monocytogenes in c-rel
CT/
CT mice
may be due to reduced bactericidal activity of the macrophage, which is known to be mediated by release of
toxic nitrogen intermediates (43). Resident c-rel
CT/
CT
peritoneal macrophages after in vitro stimulation with LPS
and IFN-
had a significantly lower NO production compared with control macrophages (Fig. 4 C).
Cytokines synthesized by activated macrophages include,
among others, IL-1, TNF-
, IL-6, and GM-CSF (44).
The production of these cytokines was compared in nonstimulated and LPS/IFN-
-activated control and
c-Rel
resident peritoneal macrophages (Fig. 4 D). Basal levels of
IL-1
(Fig. 4 D a), GM-CSF (Fig. 4 D b), TNF-
(Fig. 4
D c), and IL-6 (Fig. 4 D d) were reduced in nonstimulated
c-Rel resident peritoneal macrophages; however, upon
LPS and IFN-
activation, TNF-
and IL-6 production
was almost completely reestablished. This finding contrasts
with the production of IL-1
and GM-CSF by activated
macrophages, whereas cytokine levels secreted by
c-Rel
macrophages were significantly reduced.
The
intravenous administration of glucan to a variety of experimental animals results in a marked proliferation of macrophages and granuloma formation (32). Control and c-relCT/
CT
mice were injected intravenously with glucan and after 24 h, lung tissue sections were prepared for analysis of granuloma
formation (Fig. 4 E). In control lungs, multiple and massive
angiocentric granulomas were observed (Fig. 4 E a). Analysis at higher magnification revealed that the granulomas were
composed primarily of granulocytes and, to a lesser extent,
by macrophages (Fig. 4 E b). In contrast, in c-rel
CT/
CT
mice, lung compromise was minimal with small scattered
granulomas (Fig. 4 E c), composed primarily of macrophages
and rare granulocytes (Fig. 4 E d). This result indicates that
macrophage proliferation and granulocyte recruitment in
response to glucan injection is impaired in c-rel
CT/
CT
mice. Since we have not detected c-Rel expression in
granulocytes (12), the impairment in granulocyte recruitment observed after glucan injection is most probably due
to defective cytokine production by alveolar macrophages.
Collectively, these results define an essential need of a transcriptionally active c-Rel in macrophages.
To evaluate the humoral immunity of c-relCT/
CT mice, we measured the basal production of immunoglobulins in naive
animals. Serum isotype levels from nonimmunized c-rel
CT/
CT
mice and control littermates are shown in Fig. 5 A. Although the levels of IgG1 (threefold), IgG2b (twofold), and
IgG3 (fourfold) in c-rel
CT/
CT mice were reduced compared with wild-type littermates (Fig. 5 A a), those of IgG2a
(twofold), IgM (threefold), and IgA (twofold) were increased
(Fig. 5 A b).
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Splenic B cells from control and c-relCT/
CT mice were
stimulated with different agents over a 72-h period and
proliferation was monitored by [3H]thymidine incorporation. Control B cells stimulated with
-IgD-dex, LPS, ligand
for the mouse CD40 (mCD40L), or
-CD40 showed proliferation, and these responses were further augmented by
IL-4 and IL-5 addition.
c-Rel B cells showed only minimal response to LPS and mCD40L and IL-4 plus IL-5, but
did not proliferate under other stimuli (Fig. 5 B). However,
when unpurified
c-Rel B cells were used for in vitro proliferation assays, significant levels of [3H]thymidine incorporation were observed (data not shown). These results indicate that there are two different pathways leading to B
cell proliferation, one intrinsic and c-Rel dependent, and another extrinsic, c-Rel independent, which requires additional exogenous stimuli.
The abnormal architecture of the spleen in c-relCT/
CT
mice suggests that the immune responses dependent on cellular interactions in the lymphatic follicles may not be fully
functional. Therefore, we immunized mice with SRBCs
and evaluated the formation of GCs 7 d later (Fig. 5 C).
The spleen of control mice had numerous GCs as defined
by central areas of cells that bind peanut agglutinin (PNA;
Fig. 5 C a) surrounded by IgD+ cells (Fig. 5 C b). In contrast, spleens of c-rel
CT/
CT mice had reduced numbers and
poorly defined GCs (Fig. 5 C c and d). These results indicate that clonal expansion of B cells in response to the T
cell-dependent antigen (SRBC) is impaired in c-rel
CT/
CT
mice.
Acute
infection of adult mice with LCMV induces a protective
immunity and it has been shown that virus-specific CTLs
play an essential role in virus elimination from the infected
host (45). To examine whether a transcriptionally active
c-Rel is required to induce a CTL response, control and
c-relCT/
CT mice were infected with LCMV and their capacity to clear virus loads from various organs was determined
at days 3 and 7 after virus inoculation. As shown in Fig. 6 A,
c-rel
CT/
CT and control mice exhibited equivalent viral titers in all organs tested at 3 (Fig. 6 A a) or 7 (Fig. 6 A b) d
after inoculation. This result demonstrates that c-rel
CT/
CT
mice can mount a protective CTL response against LCMV.
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Proliferative response of T cells
from c-relCT/
CT mice was comparable to that seen in control cells (Fig. 6 B). Equivalent levels of [3H]thymidine incorporation were observed in control and mutant T cells
after stimulation of either IL-2 or with a combination of
anti-CD3 plus anti-CD28.
The cytokine secretion profile of normal and c-Rel T
cells in the absence of stimuli or after anti-CD3 plus anti-CD28 stimulation is shown in Fig. 6 C. In the absence of
stimuli, cytokines were undetectable in either control or
c-Rel T cells (Fig. 6 C, a-c). Anti-CD3 plus anti-CD28
stimulation increased production of GM-CSF, TNF-
,
and IL-2 in both control and c-rel
CT/
CT T cells. However,
quantitative differences were observed. For instance, the
levels of GM-CSF and TNF-
were higher in c-rel
CT/
CT
T cells, whereas the levels of IL-2 production were equivalent in control and c-rel
CT/
CT T cells.
The functional alterations observed in some hematopoietic cell compartments but not in others in c-relCT/
CT
mice could be due to differential alterations in the NF-
B activity. Thus, we determined the
B-binding activity of purified in vitro activated control and mutant T and B cells
(Fig. 7 A). Nuclear protein extracts from B cells (Fig. 7 A a)
and T cells (Fig. 7 A b) from control (lanes 1-6) and
c-Rel
(lanes 7-12) cells were analyzed by EMSA using a palindromic
B-binding site. Antibody challenge of the nuclear
extracts demonstrate that the major
B-binding complexes
in control B cells are p50/c-Rel heterodimers and p50/p50 homodimers, whereas in control T cells, p50/c-Rel and p50/
RelA heterodimers together with p50/p50 homodimers
are the major
B-binding complexes (Fig. 7 A b). In
c-Rel B cells, the major
B-binding complex is a p50/
c-Rel heterodimer (Fig. 7 A a), whereas in
c-Rel T cells,
two major complexes containing
c-Rel were identified, RelA/
c-Rel and p50/
c-Rel heterodimers (Fig. 7 A b).
As expected, due to the COOH-terminal deletion present
in
c-Rel, the p50/
c-Rel and RelA/
c-Rel heterodimers migrate faster than p50/c-Rel and RelA/c-Rel
heterodimers, respectively.
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The transcriptional activity of c-Rel complexes was
studied in cotransfected S107 cells (Fig. 7 B). These studies
demonstrate that the activity of the RelA/
c-Rel heterodimer is similar to the wild-type RelA/c-Rel heterodimer (compare lanes 5 and 8), probably due to the
presence of one transcriptional activation domain in RelA.
The p50/
c-Rel heterodimer is much less active than the
wild-type p50/c-Rel heterodimer (compare lanes 7 and 9),
most likely due to the absence of transcriptional activation domains in both p50 and
c-Rel. In summary, these results
indicate that the presence of p50/c-Rel activity is indispensable for normal B cell function, but not for T cell
function.
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Discussion |
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c-Rel was the first member of the Rel/NF-B family of
transcriptional factors demonstrated to be a protooncogene.
v-Rel is the oncogene carried by the retrovirus of the reticuloendotheliosis virus strain T that produces lymphoproliferative disorders in birds (17). The chicken v-Rel has
also been demonstrated to induce multicentric lymphoma/
leukemia in mammalian cells (34). Although all Rel/NF-
B
members contain the highly conserved RHD, they have a
highly divergent COOH-terminal end that in c-Rel, RelA, and RelB confer powerful transcriptional activity
(24, 36, 46, 47). Interestingly, v-Rel represents a mutated
version of c-Rel in which one of the major differences is a
deletion of its COOH-terminal sequence (17). To better understand the functional consequences of the COOH-terminal truncation on the transforming capability of c-Rel
and the functional role of the different transcriptional activation domains among different Rel/NF-
B members, we
have generated mice in which the endogenous c-Rel protein has been replaced by a truncated version lacking the
transcriptional activation domain (c-rel
CT/
CT mice). In this
paper, the initial characterization of the phenotype of
c-rel
CT/
CT mice is described.
In agreement with the broad
expression pattern of c-Rel in hematopoietic cell lineages
(12, 36), a complex spectrum of hematopoietic alterations
was observed in mice lacking the transcriptional activation
domain of c-Rel. c-relCT/
CT mice showed increased susceptibility to bacterial infection, impaired bone marrow
hematopoiesis, and histopathologic alterations in hematopoietic tissues (including B cell hyperplasia). These alterations appear to represent defects in the cross-regulation between various hematopoietic cells, and for this reason, it
is difficult to sort out secondary from primary defects. For
instance, the fact that bone marrow hypoplasia was accompanied by compensatory extramedullary hematopoiesis in
the spleen indicates that there are no primary abnormalities
in hematopoietic precursors. Instead, there is an inappropriate microenvironment in the bone marrow of c-rel
CT/
CT
mice, probably related to altered cytokine production by
stromal cells, macrophages, B cells, or T cells. The defective GC formation in c-rel
CT/
CT mice is likely due to the
structural alterations in the spleen, or due to functional alterations at some level in the network between macrophages, dendritic follicular cells, T cells, and B cells that are
essential for the normal development of GCs (48, 49). Understanding the primary cellular alterations in c-rel
CT/
CT
mice will require specific cellular assays in purified single cell populations. A successful example of this strategy is the recent observation that isolated c-rel
CT/
CT-derived B cells
exhibit selective defects in germline transcription and Ig
class switching (50).
The fact that c-relCT/
CT mice present lymphadenopathy
and lymphoid hyperplasia in stomach, liver, and lungs is
particularly interesting in light of the information that
chromosomal translocations associated with structural alterations of the Rel/NF-
B family of proteins have been documented in several cases of human lymphomas (51). More specifically, c-rel rearrangements in several human
non-Hodgkin's lymphomas have been found. In one cell
line derived from a pre-T diffuse large cell human lymphoma, several abnormal c-rel cDNAs were isolated that
encoded a large portion of the RHD that is fused to cellular sequences of unknown origin (53). The explanation of
lymphoid hyperplasia found in older c-rel
CT/
CT mice is
complex and may be related to a combination of different factors. The fact that in vitro [3H]thymidine incorporation
was decreased in purified c-rel
CT/
CT B cells but not different from control unpurified B cells indicates that the truncated
c-Rel protein does not affect the intrinsic mitogenic activity of B cells. Instead, it may affect the external signals regulating B cell proliferation. Cytokines are potent regulatory molecules secreted by cells of the immune system; deletion of certain cytokines in mouse mutants leads
to immune dysregulation and expansion of some hematopoietic lineages (44). For instance, IL-8-deficient mice exhibit lymphoadenopathy, which results from an increase in
B cells, and splenomegaly, which results from an increased
number of metamyelocytes, band cells, and mature neutrophils (54). GM-CSF null mice present extensive lymphoid
hyperplasia associated with lung airways and blood vessels
(55). Interestingly, disruption of cytokine production and
myeloid hyperplasia has been observed in mice deficient in
RelB (26), and in mice lacking the COOH-terminal
ankyrin domain of NF-
B2 (56).
The fact that B cell hyperplasia appears at later times, and
in older animals, indicates that the lymphoid expansion
may be related to a reduced clearance of aged cells by the
reticuloendothelial system. This possibility is particularly
attractive in light of the observation of reduced total number and functional alterations of macrophages in c-relCT/
CT
mice. It is also possible that a secondary event may lead to an increase in B cell proliferation in older c-rel
CT/
CT
mice. In this sense, it is important to mention that there are some biochemical similarities between
c-Rel in c-rel
CT/
CT
cells and v-Rel in transformed T cells (34). For example, a significant amount of
c-Rel is found in the nucleus (data
not shown) and increased
B-binding activity, mainly
composed of p50/
c-Rel, is observed in
c-Rel cells, as in
v-Rel-transformed T cells. In addition, similar to v-Rel
transgenic mice, lymphoid hyperplasia in c-rel
CT/
CT animals was evident after a long period of latency, suggesting that a secondary event is required for the appearance of this phenotype. We have previously expressed
c-Rel in transgenic thymocytes by using the lck T cell-specific promoter
(34). In this model,
c-Rel did not produce T cell abnormalities, suggesting that the use of a rearranged c-rel gene
under the control of its own regulatory elements, as in
c-rel
CT/
CT mice, is required to recapitulate the situation
found in lymphoproliferative disorders.
The phenotype of c-relCT/
CT mice has quantitative
and qualitative differences from the phenotype described
previously for mice lacking the entire c-Rel molecule (21-
23) with c-rel
CT/
CT mice showing a more severe phenotype. Since the same strain of mice were used to generate
both c-rel
/
and c-rel
CT/
CT mice, these differences must
be attributable to other factors. In c-rel
CT/
CT mice, a transcriptionally inactive molecule has been generated that retains its capability to bind DNA and interact with other Rel/
NF-
B members. The absence of phenotypic alterations in
heterozygous c-rel+/
CT mice indicate that
c-Rel does not
behave as a transdominant mutant protein of c-Rel. We
speculate that by keeping the original niche of c-Rel,
c-Rel may prevent partial compensation of its function by
other Rel/NF-
B members in some cell lineages, which may be the case in c-rel
/
mice. The degree of compensation in c-rel
/
mice depends on the cell type, its physiologic state, and the level of expression of other Rel/NF-
B
members. In the case of
c-Rel, its degree of inactivation
will also depend on similar parameters, because of the possibility of forming either transcriptionally inactive or transcriptionally active heterodimers. For instance, in c-rel
/
,
but not in c-rel
CT/
CT mice, defective proliferation of T
cells and cytokine production by T cells was identified (22).
Band shift assays performed with nuclear protein extracts
from control T cells showed p50/RelA heterodimers as
one of the major
B-binding components, whereas in
c-Rel T cells, a significant amount of complexes containing
c-Rel were detected, including
c-Rel/RelA heterodimers (Fig. 7 A a). Since cotransfection transcriptional
assays demonstrated that
c-Rel/RelA heterodimers are as
active as p50/RelA heterodimers (Fig. 7 B, lanes 6 and 8),
it is possible that T cell proliferation and cytokine production are normal in c-rel
CT/
CT mice due to cellular-specific
compensation by
c-Rel/RelA heterodimers. On the
other hand, in c-rel
/
mice, T cell proliferation is altered
because other Rel family proteins in the
B-binding complexes do not compensate for the loss of c-Rel in T cells
(22). Alteration in B cell function in c-rel
CT/
CT mice is
likely related to the fact that the major
B-binding activity
in normal B cells is composed of p50/c-Rel heterodimers (Fig. 7 A a), which cannot be substituted by the transcriptional inactive p50/
c-Rel heterodimer present in
c-Rel
B cells (Fig. 7 B, lanes 7 and 9). Quantitative differences in
the level of cytokine production in resting macrophages
have also been observed between c-rel
/
and c-rel
CT/
CT
mice. For instance, GM-CSF and IL-6 production are decreased in c-rel
CT/
CT mice, but increased in c-rel
/
mice.
Since p50/c-Rel is the major
B-binding activity in resting macrophages (data not shown and reference 23), replacement with the inactive p50/
c-Rel heterodimer in
c-Rel
macrophages results in decreased expression of GM-CSF
and IL-6 (Fig. 7 B, lanes 7 and 9). In c-rel
/
macrophages,
the p50/c-Rel heterodimer is substituted by the p50/RelA
heterodimer that is a more powerful transcriptional activator (Fig. 7 B, lanes 6 and 7). In this way, the expression of
GM-CSF and IL-6 genes will likely increase, instead of decreasing as in resting c-rel
/
macrophages.
The function of individual Rel/NF-B family members
has been studied by a gene targeting approach (13). The
phenotypic differences between c-rel
/
and c-rel
CT/
CT
mice suggest the existence of partial compensation of c-Rel
function by other Rel/NF-
B members. Molecular compensation has also been suggested in mice deficient for
other members of the Rel/NF-
B family. For instance,
mice lacking only NF-
B1 or NF-
B2 do not show alterations in bone development. However, mice lacking both NF-
B1 and NF-
B2 develop osteopetrosis due to a defect
in osteoclast differentiation (57).
The c-relCT/
CT mice are a useful model system to study in
more detail the role of NF-
B factors in cells of the immune
system. The pathological changes observed in c-rel
CT/
CT
mice may help to understand the pathogenesis of human
immune and lymphoproliferative disorders.
![]() |
Footnotes |
---|
Address correspondence to Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543. Phone: 609-252-5744; Fax: 609-252-6051; E-mail: bravo#m#_rodrigo@msmail.bms.com
Received for publication 28 August 1997 and in revised form 12 January 1998.
1Abbreviations used in this paper:We are grateful to S. Lira, M. Swerdel, and L. Chen for generating mutant mice; R.-P. Ryseck, J. Caamaño, E. Claudio, and J. Cates for critical discussion; C. Reventos and K. Class for FACS® analysis; T. Gridley (The Jackson Laboratory, Bar Harbor, ME) for CJ7 ES cells; and the staff of Veterinary Sciences at Bristol-Myers Squibb (Princeton, NJ) for their excellent support.
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