By
From the * Laboratory of Immunology and the Laboratory of Experimental Pathology, Aichi Cancer
Center Research Institute, Chikusa-ku, Nagoya 464; and the § Research Institute for Biological
Sciences, Science University of Tokyo, Noda 278, Japan
To investigate the function of NF-B RelA (p65), we generated mice deficient in this NF-
B
family member by homologous recombination. Mice lacking RelA showed liver degeneration
and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote
matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the
SCID mice receiving relA
/
fetal liver transplants, similar to the relA+/+ and +/
cases.
T cells were found to mature to Thy-1+/TCR
+/CD3+/CD4+ or CD8+, while B cells had
the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the
secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B
cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con
A, anti-CD3, anti-CD3+anti-CD28, LPS, anti-IgM, and PMA+calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.
The NF- The RelA/p50 heterodimer was the original NF- In the present study, we generated RelA-deficient mice
with a targeting vector expected to yield a null mutation,
different from the vector used in the previous study which
would be expected to produce a truncated form of RelA
(11). Our RelA-deficient mice also died during embryogenesis but in an attempt to explore the role of RelA in lymphoid development, we transplanted the fetal liver cells from
relA Construction of the Targeting Vector.
The mouse relA gene was
isolated from a C57BL/6 (B6)1 mouse genomic library using a
mouse relA cDNA probe (codons 185-277, reference 16). The
targeting vector was constructed in pBluescript as shown in Fig. 1.
It contained 7 kb of the mouse relA gene including exons 1 to 6, PMC1-neo inserted into the first exon of relA at an NcoI site 3 bp
downstream of the translation initiation codon, and the herpes
simplex virus-thymidine kinase gene (HSV-tk) flanking at the 3
Derivation of relA-deficient Mice.
20 µg DNA of the targeting
vector was transfected into 5 × 107 CCE embryonic stem (ES)
cells (kindly provided by Dr. Motoya Katsuki, Institute of Medical Science, University of Tokyo, Tokyo, Japan [17]) by electroporation (T-300; Biotechnologies & Experimental Research Inc.,
San Diego, CA). Transfected cells were cultured for 10 d in positive-negative selection medium (18) containing G418 (400 µg/ml;
Sigma Chem. Co., St. Louis, MO) and Gancyclovir (5 µM, Demosine; F. Hoffmann-La Roche Ltd., Palo Alto, CA, provided by
Tanabe Seiyaku Co. Ltd., Osaka, Japan). Growing colonies were tested for homologous recombination by DNA blot analysis using 5 DNA and RNA Blot Analyses.
DNA and total RNA were
isolated using the proteinase K/SDS and the guanidium thiocyanate/CsCl procedures, respectively (19, 20). DNA was digested by
restriction enzymes and separated by agarose gel electrophoresis
and then transferred to nitrocellulose filters (Schleicher & Schuell,
Dassel, Germany). Total RNA was separated by 2.2 M formaldehyde agarose gel electrophoresis and transferred to nitrocellulose
filters. RNA blots were analyzed by cDNA probes for relA, c-rel
(codons 144-277, reference 21), p50 (codons 391-518, reference
22) and relB cDNA (codons 458-580, reference 23). DNA blots
were analyzed with 5 Immunohistochemistry and Histology.
Whole embryos were fixed
in buffered formalin and embedded in paraffin. Sagittal sections (5 µm) were reacted with rabbit anti-RelA specific antibody (no. sc372; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and binding sites were visualized by an avidin-biotin enzyme complex
(ABC) method (Vectastain Elite ABC kit; Vector Laboratories Inc.,
Burlingame, CA), and then counterstained in hematoxylin. For
histological examination, the sections were stained also with hematoxylin-eosin (HE).
Transplantation of Fetal Liver Cells.
SCID mice between 5-8
wk age were irradiated (2.5 Gy; Hitachi MBR-1520R; Hitachi,
Tokyo) and each injected intravenously with 3 × 106 fetal liver
cells from ED13.5 relA+/+, +/ Serological Analysis.
Two-color analysis of cell surface antigens was performed with a FACScan® (Becton Dickinson and
Co., Mountain View, CA). Thymocytes and spleen cells were
stained with antibodies to H-2Kb (E121.46; Seikagaku Kogyo,
Tokyo, Japan), Thy-1.2 (30-H12; Becton Dickinson and Co.),
TCR ELISA for Measurement of Levels of Serum Ig.
For measurement
of IgM, IgG1, IgG2a, IgG2b, IgA, and IgE, 96-well flat-bottom
plates (Immuno Plate 430341; Nunc, Roskilde, Denmark) were
pre-coated with affinity-purified goat anti-mouse Ig IL-2 Bioassay.
3 × 105 spleen cells from transplanted mice
were plated in 96-well plates (200 µl per well). Con A (2 µg/ml;
Boehringer Mannheim GmbH, Mannheim, Germany), anti-CD28
(1 µg/ml; Caltag Laboratories, South San Francisco, CA), LPS
(20 µg/ml; Sigma), anti-IgM (60 µg/ml; Capel Research Products, Durham, NC), and PMA (10 ng/ml; Sigma) plus calcium
ionophore (100 ng/ml; Sigma) were added to the medium. For
anti-CD3 antibody stimulation, plates were pre-coated with the
antibody (10 µg/ml). After 18 h of culture, the supernatants were
collected for the assay. To measure the levels of IL-2, serially diluted culture supernatants were added to IL-2-dependent NRB cells
(5 × 103, reference 31) in 96-well plates. NRB cells were cultured for 44 h and their proliferation was quantitated using a CellTiter 96TM AQueous Non-Radioactive Cell Proliferation Assay
(Promega, Madison, WI) and a microplate reader (model 3550;
Bio-Rad). Recombinant human IL-2 (Takeda Chemical Industries, Osaka, Japan) was used as a standard.
Proliferation Assay.
Conditions for cell culture and stimulation
were the same as for the IL-2 bioassay. After 48 h of culture, the
proliferation response was measured by uptake of [3H]thymidine
(New England Nuclear, Boston, MA) over a 16-h pulse. To determine the percentage of cells in apoptosis during the course of
the proliferation assays, cells at 0, 24, 48, and 64 h after stimulation were stained by the TdT-mediated dUPT nick labeling (TUNEL) technique (32, 33), using an In Situ Cell Death Detection Kit (Boehringer Mannheim GmbH) and the results assessed
by FACScan®.
Mice heterozygous for a disrupted relA were normal and fertile, but no homozygous relA-deficient mice were
born from heterozygote mating. Sequential DNA blot analysis and histological examination of embryos from timed
matings of heterozygous mice were conducted. Until embryonic day (ED) 13.5, all embryos were apparently normal and homozygous mutants (
During normal embryonic development, hematopoietic stem cells
emerge in the fetal liver on ED9.5 (36). To test whether
the RelA-deficient hematopoietic stem cells can develop in
the fetal liver and also whether they can differentiate to mature lymphocytes, fetal liver cells of ED13.5 embryos were
transplanted into irradiated SCID mice. Transplantation of
fetal liver cells not only from relA+/+ and +/
To
test whether RelA-deficient stem cells can differentiate into
T and B cells, the cell surface markers on lymphocytes of transplanted mice were examined. As shown in Fig. 5, the
relA
As the RelA-deficient B and T cells matured normally, the
role of RelA in lymphocyte function was examined. To assess B cell function, the levels of serum Ig isotypes in SCID
mice transplanted with fetal liver cells were measured (Fig.
6). The results showed that relA
In T cells, the
RelA/p50 heterodimer has been reported to be a potent
transcription factor for the IL-2 gene after stimulation by
various agents (39). The relA Table 1.
Levels of IL-2 Production by Spleen Cells from SCID Mice Transplanted with Fetal Liver Cells in Response to Various Stimuli
B family of transcription factors are conserved
from flies to humans and regulate the expression of a
wide variety of cellular and viral genes (reviewed in reference 1). Biochemical and molecular characteristics of NF-
B
and their activation pathways have been extensively studied,
especially in terms of immune and acute phase responses.
The mammalian NF-
B proteins, RelA (p65), c-Rel, RelB,
p50/p105, and p52/p100, share the "rel" homology domain
which facilitates dimerization, nuclear translocation and DNA
binding. Transactivation domains are also present at the
COOH termini of RelA, c-Rel, and RelB. These NF-
B
proteins form multiple interchangeable heterodimers and
homodimers and their activity is regulated by binding of
I
B inhibitory factors which determine the localization of
NF-
B dimers, either in the cytoplasm or in the nucleus.
Upon activation, NF-
B dimers dissociate from I
B and
translocate to the nucleus and then bind to the
B sites in promoters and enhancers of NF-
B responsive genes, consequently activating their transcription.
B
identified, as a transcription factor for Ig
light chain gene
(2), and has the strongest transactivating capacity among
NF-
B dimers as well as the most widely distributed
B
binding activity (3). In the B cell lineage, RelA/p50 is the
major NF-
B in pre-B cells, while c-Rel/p50 is predominant in mature B cells and RelB/p52 in plasmacytomas and
LPS-activated B cells (4). This suggests that RelA/p50
plays an important role in certain steps of B cell development, although genes regulated by RelA/p50 have yet to be identified. In the T cell lineage, RelA/p50 has been reported to be critical for antigen activation (5) and cytokine
production (3). Studies in vitro have suggested that RelA/
p50 regulates expression of the T cell receptor
chain
gene (6), cytokine genes such as IL-2 (7), IL-6 (8), and
TNF
(9), and the IL-2 receptor
chain gene (10). However, because of the presence of several related proteins and
their pleiotropic effects, the specific roles of RelA in vivo
remain to be elucidated. Studies on the functions of other
NF-
B proteins have faced similar problems. To overcome
this drawback, mutant mice lacking RelA (11), c-Rel (12),
RelB (13), p50 (14), or I
B
(15) have been derived by
homologous recombination to assess specific functions of
individual NF-
B proteins. All except RelA-deficient mice
demonstrate normal birth and development but with certain abnormalities in immune responses. In the case of RelA
deficiency, however, embryonic mortality occurs, concomitant with liver degeneration (11), so that clarification of the
function of this family member in the immune system has
faced difficulties.
/
embryos into SCID mice and found that the
RelA-deficient stem cells could then differentiate to mature T and B cells. To investigate the roles of RelA further, we examined RelA-deficient T and B cells for their functions and their proliferative responses to various stimuli.
end of the relA sequence. We expected that this targeting vector
would generate a null mutant allele by homologous recombination.
Fig. 1.
Structure of the relA targeting vector. The wild-type mouse
relA allele is shown at the top. The targeting vector is in the middle and
the predicted mutant allele is at the bottom. The area predicted to undergo homologous recombination is indicated by the dotted lines. Exons
are indicated by closed boxes. The probes used for diagnostic DNA blot
analysis are also indicated at the top. Restriction enzyme sites: H, HindIII;
N, NcoI; P, PstI; Sm, SmaI; S, SphI.
[View Larger Version of this Image (16K GIF file)]
and 3
flanking region probes. Eight clones with the targeted allele were obtained and injected into B6 blastocysts. The blastocysts were then transferred into the uterus of pseudopregnant Jcl:
MCH(ICR) (MCH) mice. Three clones produced chimeric mice
which transmitted a mutant allele to offspring by mating with B6
mice. Three relA mutant mouse strains, RKO-1, -2, and -3, were
maintained by brother-sister mating of heterozygous mice in our
animal facility. All three strains showed identical phenotypes and
RKO-1 mice were mainly used in this study. B6 and MCH mice
were purchased from Japan SLC Inc. (Hamamatsu, Japan) and
Clea Japan Inc. (Tokyo, Japan), respectively.
and 3
flanking region probes (see Fig. 1).
, or
/
embryos. 4 wk after
the transplantation, these mice were used for experiments. SCID
mice contain very few lymphocytes in the thymus, spleen and lymph nodes (24), due to a defect in the gene coding for DNAdependent protein kinase p350 (25). SCID mice were maintained
in our breeding colony.
(H57-597; provided by Dr. R. T. Kubo, National Jewish Center for Immunology and Respiratory Medicine, Denver,
CO [26]), CD3 (145-2C11; provided by Dr. J. A. Bluestone, The University of Chicago, IL [27]), CD4 (GK1.5; obtained
from Dr. N. Shinohara, Mitsubishi Kasei Institute for Life Science, Machida, Japan, [28]), CD8 (53-6.7; obtained from Dr. N. Shinohara [29]), CD25 (IL-2R
, 7D4; reference 30), CD44
(Pgp-1, NU5-50; Seikagaku Kogyo), B220 (RA3-6B2; PharMingen, San Diego, CA), and IgM (DAKO, Glostrup, Denmark).
(5 µg/ml,
100 µl/well; Bethyl Laboratories Inc., Montgomery, TX). For
Ig
, plates were coated with affinity-purified rabbit anti-mouse
IgG (5 µg/ml, 100 µl/well; Southern Biotechnology Associates, Inc., Birmingham, AL). Diluted serum samples and standard Ig
were added and bound Ig were detected with horseradish peroxidase-labeled affinity-purified goat anti-mouse Ig isotype-specific
antibodies or an anti-
light chain-specific antibody (Southern
Biotechnology). o-Phenylendiamine solution (0.04%; Sigma) was
added to each well as a substrate. The optical density at 490 nm
was measured with a microplate reader (model 3550; Bio-Rad,
Hercules, CA).
Requirement of RelA for Embryonic Development of the
Mouse.
/
) were present in an expected ratio. On ED14.5, the relA
/
embryos were still
present but some showed signs of abnormalities in their
liver (Fig. 2 A). On ED15.5, a portion of the embryos became necrotic and were typed homozygous mutant (
/
),
while normal embryos were all wild type (+/+) or heterozygous (+/
). RNA blot analysis of relA+/+ embryos
from ED8.5 until birth as well as CCE ES cells showed the
presence of relA transcripts, while no relA transcripts could
be detected in relA
/
embryos at any stage (Fig. 2 B).
Positive immunostaining with anti-RelA antibody correlated with the presence of RNA transcripts, showing that
RelA proteins were present in almost all tissues of ED13.5
relA+/+ embryos, while no staining of relA
/
embryos (Fig. 2 C). Although a different vector construct was used
in our study, the generated RelA-deficient mice showed
the same phenotype as those of Beg et al. (11), indicating
that RelA is essential for embryonic development of the
mouse. In contrast to RelA-deficient mice, mice lacking
other NF-
B proteins are known to develop normally at
least until birth (12). The difference may simply reflect the fact that RelA is expressed ubiquitously from an early
stage of development while the others are expressed in restricted tissues from a much later stage (34, 35). Identification of RelA responsive genes in developing embryos,
especially in the liver, should open new avenues for elucidation of the function of NF-
B in embryonic development.
Fig. 2.
Liver degeneration and the absence of relA transcripts and
RelA protein in relA/
embryos. (A) Histological features of livers of
ED14.5 relA+/+ and relA
/
embryos. In the liver of a relA+/+ embryo, hepatocytes with large cell size and light nuclear staining are mixed
with the hematopoietic cells with dark nuclear staining. In the liver of
relA
/
embryos, hepatocytes are disintegrated, while the hematopoietic cells are apparently normal. Magnification is 240-fold. (B) RNA blot
analysis of ED13.5 relA+/+, +/
, and
/
embryos. 10 µg of total
RNA were loaded per lane and analyzed with the relA, c-rel, relB, and p50
cDNA probes. relA transcripts were present in the relA+/+ and +/
embryos, but not in the relA
/
embryos. The relA+/+, +/
and
/
embryos expressed similar amounts of c-rel and p50 transcripts. No relB
transcripts were detected in ED13.5 embryos (data not shown). (C) RelA
protein in ED13.5 relA+/+ and relA
/
embryos. With rabbit antiRelA antibody and the ABC method, RelA protein is ubiquitously detected in the relA+/+ embryo, but is completely absent in the relA
/
embryo. Magnification is eightfold.
[View Larger Version of this Image (40K GIF file)]
/
Fetal Liver Cells.
but also
from relA
/
embryos greatly increased the number of
cells in the thymus, spleen and lymph nodes of SCID mice.
The numbers of cells in spleen and lymph nodes of the
mice transplanted with relA
/
fetal liver cells were similar to those receiving either relA+/+ or +/
fetal liver
cells. The thymus from mice transplanted with relA
/
fetal liver cells contained fewer cells than those with wild-type
or heterozygous fetal liver cells. The origin of the lymphocytes in transplanted mice was determined by testing the
expression of H-2Kb antigen. The fetal liver cells were
from crosses between 129 and B6, both of which express
H-2KbDb, while the recipient SCID themselves express
H-2KdDd. As shown in Fig. 3, >95% of lymphocytes of
mice transplanted with fetal liver cells expressed H-2Kb, indicating that they were definitely of fetal liver origin. The
donor origin of the lymphocytes in transplanted mice with fetal liver cells was further confirmed by the presence of disrupted relA genes and the absence of relA transcripts (Fig. 4).
Thus, these results indicated that RelA-deficient hematopoietic stem cells can indeed develop in the fetal liver and
also proliferate in SCID mice.
Fig. 3.
The fetal liver origin of lymphocytes in transplanted SCID
mice. By transplantation of the fetal liver from relA+/+, +/, or
/
ED 13.5 embryos into irradiated SCID mice, the number of lymphocytes
increased. Average numbers of the cells in the spleen, lymph nodes, and
thymus in groups of three mice receiving transplants are indicated above
the distribution plots. Staining in the presence and absence of mAb to H-2Kb
is indicated by solid and open curves, respectively. Flow cytometric analysis of spleen cells, lymph node cells, and thymocytes showed the expression of H-2Kb, indicating the fetal liver origin.
[View Larger Version of this Image (39K GIF file)]
Fig. 4.
DNA and RNA
blot analyses of the spleen cells of
the transplanted SCID mice. (A)
DNA blot analysis. DNA digested by PstI was analyzed with
a 5 franking probe (see Fig. 1);
the wild-type relA allele yielding
a 4.1-kb fragment, and the mutant allele a 2.2-kb fragment. (B)
RNA blot analysis. Note the lack
of relA transcripts in the spleen
cells of mice transplanted with
relA
/
fetal liver cells. The
relA
/
, +/+, and +/
spleen cells express similar
amounts of c-rel, relB, and p50
transcripts.
[View Larger Version of this Image (27K GIF file)]
/
hematopoietic stem cells differentiated to Thy1+/TCR
+ /CD3+/CD4+ or CD8+ T cells and IgM+/
B220+ B cells in the periphery, similarly to the relA+/+
and +/
stem cells. In addition, testing of the expression
of activation markers of T cells, IL-2R
(CD25) and CD44
(Pgp-1), revealed 6.6 and 2.5% of relA
/
spleen cells to
be positive, respectively, with no significant differences
from the relA+/+ or +/
cases (data not shown). The results with cells in lymph nodes were essentially identical to
those of spleens (data not shown). The relA
/
thymuses with much fewer cells than those of relA+/+ and +/
,
showed reduction of the CD4+/CD8+ immature population, which may have been caused by the absence of RelA.
Altogether, these results suggested that RelA is not necessary for the maturation of lymphocytes or that the loss of RelA function can be compensated for by other members
of the NF-
B family. In this regard, it was interesting to
note that none of the mice deficient in any NF-
B subunit,
whether RelA, c-Rel, RelB, or p50, showed abnormality in
the development of T cells and B cells. There is a vast
amount of evidence indicating the importance of NF-
B
in lymphocyte development (for review see references 1, 3,
37). Thus, it is most likely that the development of T and B
cells proceeds with certain combinations of NF-
B subunits and may not require the presence of specific NF-
B
dimers.
Fig. 5.
Surface antigen profiles of spleen cells and thymocytes of
SCID mice transplanted with ED13.5 fetal liver cells. Cells were examined by two-color staining using various combinations of antibodies. The
expression of H-2Kb by spleen cells confirms their fetal liver origin. As for
T cell markers, Thy-1, TCR, CD3, CD4, and CD8 were examined
and as for B cell makers, IgM and B220 were examined. Even relA
/
fetal liver cells develop normally to mature T and B cells in the transplanted SCID mice.
[View Larger Version of this Image (35K GIF file)]
/
B Cells.
/
B cells were capable
of secreting Ig as well as switching classes of Ig isotypes.
The levels of total Ig and individual classes of IgM, IgG2a,
IgG2b, IgE, and Ig
produced by relA
/
B cells were
comparable to those with relA+/+ or +/
B cells. Although RelA was originally identified, together with p50,
as an enhancer binding protein for an Ig
chain gene, surprisingly the absence of RelA had no effect on the levels of
Ig
production. The RelA-deficient B cells, however, produced 10-fold and 100-fold less IgG1 and IgA, respectively, than the control B cells. Reduced production of
certain Ig classes has been also reported in mice deficient in
p50 (14) and c-Rel (12): IgG1, IgA, and IgE in the former
and IgG1 and IgG2a in the latter. Thus, each NF-
B member is critically involved in the production of certain
Ig isotypes, presumably by regulating the transcription of Ig
genes directly and/or acting on various cytokine genes
which ultimately control Ig class switching and production.
In this regard, it is interesting to note that IgA reduction
has also been reported in IL-6-deficient mice (38) and that
the expression of IL-6 is controlled by RelA/p50 heterodimers (8).
Fig. 6.
The serum Ig levels in SCID mice transplanted with fetal
liver cells. 4 wk after the transplantation, mice with relA/
fetal livers
were able to secrete Ig in their sera, with the total amount being comparable to those with relA+/+ or relA+/
cells, but the IgA and IgG1 levels were found to be significantly lower. SCID mice secrete hardly detectable levels of any Ig isotypes. Open, striped, and closed circles correspond
to mice transplanted with relA+/+, +/
, and
/
fetal liver cells, respectively.
[View Larger Version of this Image (21K GIF file)]
in relA
/
T Cells.
/
spleen cells, however,
produced similar levels of IL-2 to relA+/+ or +/
spleen
cells after stimulation with Con A, anti-CD3, anti-CD3+
anti-CD28, or PMA+calcium ionophore (Table 1). The
results were in contrast to those for c-Rel-deficient mice
which showed ~50-fold reduction in IL-2 production after stimulation by anti-CD3, and anti-CD3+anti-CD28
and threefold reduction by the PMA+calcium ionophore.
Thus, it was strongly suggested that the main component of
the NF-
B transcription factor for the IL-2 gene in vivo is
c-Rel rather than RelA.
relA genotype
of
fetal liver
donor*
Stimuli
Con A
Anti-CD3
Anti-CD28
Anti-CD3 + Anti-CD28
PMA + calcium
ionophore
None
IL-2 levels
U/ml §
+/+
167 ± 16.2
63.7 ± 10.3
5.7 ± 0.84
446 ± 19.8
361 ± 69.8
4.5 ± 1.42
+/
146 ± 18.5
42.8 ± 5.27
5.2 ± 0.82
448 ± 10.9
312 ± 26.1
2.0 ± 0.05
/
118 ± 16.8
60.3 ± 8.11
6.5 ± 1.08
402 ± 88.0
276 ± 42.3
2.4 ± 0.32
None
1.5 ± 0.66
3.4 ± 1.03
2.3 ± 0.62
5.2 ± 1.84
6.5 ± 1.32
1.4 ± 0.32
*
3 × 106 fetal liver cells from relA +/+, +/ , or
/
embryos were transplanted into irradiated SCID mice.
3 × 105 spleen cells were stimulated as described in the Materials and Methods.
§
The levels of IL-2 were measured by bioassay using NRB cells (see the Materials and Methods).
Mean ± SD. The results obtained from three mice were averaged.
RelA has been reported to be involved also in the upregulation of IL-2R expression with stimulation by various
agents (40). Before stimulation, the levels of IL-2R
on
relA
/
, +/+, or +/
lymphocytes were similar to one
another as mentioned above. With stimulation by PMA+
calcium ionophore or Con A, the levels of IL-2R
expression on relA
/
T cells increased and did not significantly differ from relA+/+ or relA+/
T cells (data not shown).
The c-Rel-deficient T cells also showed no reduction in
the basal or induced expression of IL-2R
(41). These observations suggest the following two possibilities: (a) neither
RelA nor c-Rel is required for the expression of IL-2R
,
or (b) both can participate in the control of IL-2R
expression and one works in the absence of the other. Identification of binding subunits to the
B motif of IL-2R
in the
absence of RelA or c-Rel and derivation of mice lacking
both RelA and c-Rel should sort out these possibilities.
To further analyze the role of RelA in lymphocyte activation, spleen cells from mice transplanted with relA/
fetal liver cells were stimulated with various agents. With both
T cell specific stimuli, Con A, anti-CD3 and anti-CD3+
anti-CD28, and B cell-specific stimuli, LPS and anti-IgM,
relA
/
spleen cells showed a much lower [3H]thymidine
uptake than relA+/+ or +/
spleen cells (Table 2). To
test whether this low response of relA
/
spleen cells is
due to reduced cellular proliferation or to increased apoptotic cell death, the percentages of cells in apoptosis during
the course of proliferation assays were determined. As
shown in Fig. 7, the percentages and the actual numbers of
apoptotic cells with relA
/
were not significantly different from the relA+/
case. Although the number of viable
cells may not be as indicative as [3H]thymidine uptake because only a small component of the spleen cells can proliferate in response to certain mitogenic stimuli, relA
/
yielded constantly fewer viable cells than relA+/
. These results indicate that RelA-deficient lymphocytes indeed have
an impaired proliferative response to various mitogens. As
the production of IL-2 and the expression of IL-2R
were
normal in RelA-deficient T cells, the results suggested that
RelA is also involved in yet unidentified critical steps of proliferative responses. Furthermore, RelA-deficient lymphocytes exhibited impaired responses to various stimuli whose
signals are transduced by distinctive pathways (42, 43).
Thus, RelA may be involved in each single pathway or in a
critical merging step downstream of these different pathways. Identification of RelA responsive genes involved in
proliferation should reveal the role of RelA in these responses. T and B cells of c-Rel-deficient mice have also
been found to demonstrate a defective proliferation response to various stimuli, generally with severe reduction
(12). These results indicate that RelA and c-Rel are essential for certain steps of proliferation and that they cannot
compensate for each other. It is interesting to note that
relA
/
lymphocytes showed an impaired proliferative response to PMA+calcium ionophore in this study while
c-rel
/
lymphocytes respond normally to this agent (12).
Presumably, the involvement of RelA in proliferative responses is thus wider. Furthermore, relA
/
embryonic
fibroblasts also showed reduced proliferation after PMA+
calcium ionophore stimulation, down to 30% of the levels
of their relA+/+ or +/
counterparts (data not shown).
As expression of relA is not restricted to lymphocytes, in
contrast to that of c-rel (44), this also suggests a role in a
wider range of biological processes.
|
In conclusion, transplantation of fetal liver cells into
SCID mice in the present investigation allowed light to be
cast on a number of the functions of RelA in the immune
system, despite the fact that the relA/
genotype is lethal
for embryos. Further study with in vitro and in vivo immunization by T-dependent and -independent antigens should
facilitate clarification of RelA roles. In addition, since lymphocytes can be rescued from dying embryos by transplantation of fetal liver cells as described here, mice lacking
multiple NF-
B proteins, such as RelA and c-Rel, or
RelA and RelB, should now be testable for their actions
exerted in concert.
Address correspondence to Yuichi Obata, Laboratory of Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan.
Received for publication 16 October 1996 and in revised form 26 December 1996.
1Abbreviations used in this paper: ABC, avidin-biotin enzyme complex; B6, C57BL/6; ED, embryonic day; ES, embryonic stem; HE, hematoxylineosin; HSV-tk, herpes simplex virus-thymidine kinase; MCH, Jcl: MCH(ICR); TUNEL, TdT-mediated dUTP nick end labeling.We thank Drs. Motoya Katsuki, Hitoshi Niwa, and Kunio Tsujimura for their valuable suggestions. We also thank Mineko Izawa, Yasue Matsudaira, Hitomi Nishiwaki, Hiromi Tamaki, and Satoshi Ozeki for their excellent technical assistance. We are grateful to Dr. Malcolm A. Moore for his editorial assistance.
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grantin-Aid for General Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. This work was also supported in part by grants from the Naito and Imanaga Foundations.
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