By
From the * Department of Oncology, Department of Experimental Pathology, Bristol-Myers Squibb
Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000; and the § Department of
Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
RelB-deficient mice (relB/
) have a complex phenotype including multiorgan inflammation
and hematopoietic abnormalities. To examine whether other NF-
B/Rel family members are
required for the development of this phenotype or have a compensatory role, we have initiated
a program to generate double-mutant mice that are deficient in more than one family member.
Here we report the phenotypic changes in relB
/
mice that also lack the p50 subunit of NF
B (p50
/
). The inflammatory phenotype of p50
/
relB
/
double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death
within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in
contrast to relB
/
single knockouts, B cells were absent from inflammatory infiltrates. Both
p50
/
and heterozygous relB
/+ animals are disease-free. In the absence of the p50, however, relB
/+ mice (p50
/
relB
/+) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased
B-binding
activities of NF-
B/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-
B activity is not
required for the development of the inflammatory phenotype.
Cellular decisions involved in growth, differentiation,
and development require the coordinated expression
of a wide variety of genes. The transcriptional regulation of
genes is mediated by factors that bind singly or in association with other proteins to cis-regulatory sequences found
in promoters, enhancers, and silencers. These transcription
factors frequently form families in which individual members have distinct or similar functions.
The NF- Recent evidence using mice with targeted disruptions
for individual members of the NF- RelB, originally identified as an immediate-early gene in
growth factor-induced fibroblasts (13, 14), is expressed predominantly in lymphoid tissues where RelB heterodimers
represent the major constitutive Mice with a targeted disruption of RelB have pathological changes including inflammatory cell infiltration in several organs, myeloid hyperplasia, and splenomegaly due to
extramedullary hematopoiesis. RelB is also required for the
normal development of thymic medulla and antigen-presenting DC. Besides the pathological changes, RelB-deficient mice have multifocal defects in cellular and humoral
immune responses (21 and 21a). Similarly, a mutation disrupting the relB locus by the random integration of transgene sequences resulted in mice with a syndrome of excess
production of macrophages and granulocytes, reduced populations of thymic dendritic and medullary epithelial cells,
and impaired antigen-presenting cell function (22).
Different NF- In this report we compare the phenotype of p50 Mice.
RelB- and p50-deficient mice were originally established from relB Histopathology and Immunohistochemistry.
Histopathological analyses were performed on a minimum of three animals per age group
and genotype. Tissues were immersion-fixed in 10% buffered formalin, embedded in paraffin blocks, and processed by routine
methods. Sections (4-6 µm thick) were stained with hematoxylin
and eosin, and examined by light microscopy. For immunohistochemical staining of CD4+ and CD8+ T cells, tissues were frozen in O.C.T.-embedding medium (Miles Inc., Elkhart, IN),
sectioned at 8 µm, and immediately fixed in buffered formalin/
acetone. Sections were blocked with avidin D/biotin reagents
(Vector Labs., Inc., Burlingame, CA), followed by 0.5% casein in
PBS, and incubated with purified CD4- or CD8-specific mAb
from PharMingen (San Diego, CA; clone RM4-5 diluted 1:50 and clone 53-6.7 diluted 1:20, respectively) for 180 min at room
temperature. For immunohistochemical staining of B cells, mature macrophages, and neutrophils, formalin-fixed paraffin sections were treated with 0.1% trypsin at 37°C for 20 min, blocked
with avidin D/biotin reagents and normal mixed serum (Shandon
Inc., Pittsburgh, PA), and incubated with purified specific mAbs
(clone RA3-6B2 diluted 1:300 [PharMingen] and clone F4/80 diluted 1:10 and clone 7/4 diluted 1:50 [both from Harlan Bioproducts for Science Inc., Indianapolis, IN]) for 90 min a room
temperature. Endogenous peroxidase activity was quenched in 3%
hydrogen peroxide in PBS for 10 min at room temperature. Incubation with a biotinylated secondary Ab (mouse-absorbed anti-
rat IgG from rabbit, diluted 1:100) was for 30 min, followed by
avidin-linked peroxidase for 30 min, followed by diaminoben- zidine chromogen for 3 min, and hematoxylin counterstain for 1 min. Unless otherwise stated, all reagents were obtained from Vector Labs., Inc. For negative control slides, the primary Ab was
substituted with normal mixed serum.
Flow Cytometry.
Flow cytometry was done using a flow cytometer and cell sorter (Coulter Epics Profile II; Coulter Corp.,
Hialeah, FL). Splenocytes and bone marrow cells were isolated
and mature red blood cells were lysed according to standard procedures (27). Cell suspensions were incubated with mAbs ( Semi-Quantitative Reverse Transcription-PCR Analyses.
Total thymus RNA from 2-wk-old mice was isolated using TRIzol according to the manufacturer's instructions (GIBCO BRL). cDNA was synthesized from 2 µg total RNA (Superscript Preamplification System; GIBCO BRL). Semi-quantitative PCR was performed
under conditions where amplification of the cDNAs was linearly
dependent on the concentration of the corresponding mRNAs
(28, 29). The sequences of the primers were: for nfkb1, 5 Electrophoretic Mobility Shift Assays.
Whole cell extracts from
2-wk-old thymuses were prepared as described (16). The oligodeoxynucleotide comprising a palindromic, Mice with a targeted deletion of p50 are fertile, whereas
the majority of RelB-deficient mice are sterile due to
marked inflammatory infiltrates in the reproductive organs
of males and females older than 40 d (8, 21). Therefore, the
p50 2-3 wk after birth, however, all the p50
The histopathological findings in 20-d-old mice of various genotypes are summarized in Table 1. No changes were observed following
an extensive histopathological examination of lymphoid and
nonlymphoid tissues from several wild-type, relBB/Rel family of transcription factors represents
a group of homodimeric and heterodimeric complexes that
plays an important role in the function of lymphocytes and
other cells of hematopoietic origin. Five members of this
family have been identified in vertebrates: NF-
B1 (encoding the precursor molecule p105 that is proteolytically
processed to p50), NF-
B2 (encoding the precursor p100
and the processed form of p52), RelA (p65), RelB, and
c-Rel (Rel). One hallmark of the family is a highly conserved domain of ~300 amino acids, termed the Rel homology domain, that contains sequences important for
dimerization, DNA binding, and nuclear localization. In most
cell types NF-
B/Rel proteins associate with the inhibitor
molecule I
B forming an inactive cytoplasmic complex that
can be activated by a wide range of stimuli leading to degradation of I
B and nuclear translocation of the NF-
B/
Rel proteins and their binding to so-called
B sequence motifs. Many target genes are involved in immune, inflammatory, acute phase, and stress responses (1).
B/Rel family indicates
that the different proteins play distinct biological roles. For
example, p50-deficient mice appear normal and lymphocyte development is not impaired. However, these animals
have multifocal defects in immune responses and B cells are
defective in mitogenic activation and specific antibody production (8, 9). Mice with a targeted disruption of RelA are
embryonic lethal due to a defect in liver development (10).
Mature B and T cells from mice that lack c-Rel were found to be unresponsive to most mitogenic stimuli (11, 12).
B-binding activity (15).
In thymus, relB transcripts are confined to the medulla and
high levels of RelB are expressed in the nucleus of interdigitating dendritic cells (DC)1. In addition, lower levels of
RelB expression can be detected in macrophages as well as
B and T cells (15, 16, 18).
B/Rel complexes have different sequence
preferences and transcriptional activation properties. However, the DNA binding domain is highly conserved between
NF-
B/Rel family members and p50-RelA, p50-RelB, or
p50-c-Rel heterodimers can bind to similar regulatory
B
sequences and activate transcription of target genes (23).
Although the phenotypic changes in relB
/
mice show that
RelB function is crucial for a normal hematopoietic system
(21, 22), it is possible that other members of the NF-
B/
Rel family may, at least in part, functionally compensate for
the lack of RelB. p50-deficient mice do not have any
pathological changes and the absence of the "classical" p50RelA-NF-
B activity as well as other p50-containing
NF-
B/Rel complexes (8) make this mutant mouse line a
useful model system to address functional redundancy
within the NF-
B/Rel family. In addition, p50
/
relB
/
double-knockout mice may provide an answer to the question of whether p50-RelA-NF-
B complexes are required
in the effector cells that mediate the relB
/
phenotype.
/
and
relB
/
single-mutant, p50
/
relB
/
double-mutant, and
p50
/
relB
/+ mice, and focus on the different cell types
involved in the pathological changes observed in these animals. The results indicate that the lack of RelB is partially
compensated by other p50-containing complexes and that
the p50-RelA-NF-
B activity is not essential for the development of the inflammatory phenotype in RelB-deficient mice.
/+ (129/Sv × C57BL/6J background) and p50
/+
(129/Ola × C57BL/6J background) mice, respectively, and were subsequently maintained by intercrosses (8, 21). Tail DNA was
prepared as described (26). The genotype of RelB-deficient mice
was determined by PCR amplification (1 min at 95°C, 1.5 min at
60°C, and 1 min at 72°C for 33 cycles) using primer pairs recognizing the intact relB gene (5
-GTG GTG CCC GGG AAT
AGG ATT GCT G-3
and 5
-CCA TTT TGC TCT GGG
TCT GTG TCT G-3
) and the targeted relB-neo locus (5
-CAT
CGA CGA ATA CAT TAA GGA GAA CGG-3
and 5
-AAA
TGT GTC AGT TTC ATA GCC TGA AGA ACG-3
). PCR
amplification conditions for p50-deficient mice were 1 min at
95°C and 2 min at 66°C for 30 cycles using primers specific for
the intact nfkb1 gene (5
-GCA AAC CTG GGA ATA CTT
CAT GTG ACT AAG-3
and 5
-ATA GGC AAG GTC AGA
ATG CA CAG AAG TCC-3
) and the targeted nfkb1-neo locus
(5
-AAA TGT GTC AGT TTC ATA GCC TGA AGA ACG3
). All animals were housed and bred within the same room (in
Veterinary Sciences Department of Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). The absence of
pathogens was assessed by extensive, periodic, comprehensive serology, and histopathology of sentinel animals housed within the
same room.
1 µg
each/106 cells) for 30 min on ice, spun, and washed with PBS/2%
fetal calf serum. Conjugated CD4-FITC, CD8-PE, and CD45R/
B220-FITC mAbs were obtained from GIBCO BRL (Gaithersburg, MD); TCR-
/
-FITC, IgM-FITC, and Gr-1-FITC mAb
were from PharMingen; Mac-1-PE mAb was from Boehringer
Mannheim (Indianapolis, IN). An average of 104 cells was recorded in each case.
-GCA
CCG TAA CAG CAG GAC CCA AGG ACA-3
and 5
-CCC
GTC ACA CAT CCT GCT GTT CTG TCC ATT CT-3
; for
nfkb2, 5
-GCC TGG ATG GCA TCC CCG-3
and 5
-CTT CTC ACT GGA GGC ACC T-3
; for relA, 5
-TAG CCT TAC
TAT CAA GTG TCT TCC TCC-3
and 5
-GTT CAG AGC
TAG AAA GAG CAA GAG TCC-3
; for relB, 5
-CCT CTC
TTC CCT GTC ACT AAC GGT CTC-3
and 5
-ACG CTG
CTT TGG CTG CTC TGT GAT G-3
; for c-rel, 5
-TGG
CTG ACT GAC TCA CTG ACT GAC TGA CTC GTG CCT
TGC-3
and 5
-CCA ACT AAA TCA TGA GGA TGA GGC
TTA TAT GGA TCA TTC-3
; for ikba, 5
-CAG GAC TGG GCC ATG GAG GG-3
and 5
-TGG CCG TTG TAG TTG
GTG GC-3
; for ikbb, 5
-CCC TTC CTG GAT TTC CTC
CTG GGC TTT TC-3
and 5
-GTG GGG TCA GCA CCG
GCT TTC AGG AGA-3
; and for
-actin, 5
-AGA GGT ATC
CTG ACC CTG AAG TAC C-3
and 5
-CCA CCA GAC
AAC ACT GTG TTG GCA T-3
.
B-binding site
has been described previously (13, 24). 4 µg of thymus extract
were incubated with 10 fmol of the radiolabeled probe in 10 mM
Tris × HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 5% glycerol, and 0.6 µg herring sperm DNA;
B-binding activity was analyzed in electrophoretic mobility shift assays (EMSAs)
as previously described (18). Extracts were activated by treatment
with 0.6% deoxycholate (DOC) for 15 min on ice followed by
the addition of NP-40 to a final concentration of 1.2%. As a control, binding of unactivated extracts was also performed in the
presence of 1.2% NP-40. Integrity of extracts was checked with
an octamer binding site.
Increased Mortality of p50/
relB
/
Double-knockout Mice.
/
mutation was crossed into a relB
/+ background
generating p50
/
relB
/+ animals that were interbred to obtain p50
/
relB
/
double-mutant mice. The resulting litters were normal in size, all animals appeared healthy at
the time of birth, and p50
/
relB+/+, p50
/
relB
/+, and
p50
/
relB
/
mice were born at expected Mendelian ratios, demonstrating that p50
/
relB
/
double-knockout
mice can develop to birth (data not shown).
/
relB
/
animals were runted, lethargic, had abdominal distension, and
their body weights were reduced 30-50%, compared to
control littermates; relB
/
mice of the same age were less
severely affected (Fig. 1 A). The disease progressed rapidly
in p50
/
relB
/
mice leading to premature death of >95%
of the animals between 2 and 4 wk of age (Fig. 1 B). relB
/
single-knockout mice also had increased mortality, but
only 33% died within the first 4 wk after birth. Heterozygous relB
/+ on a wild-type or p50
/
background did not
have increase mortality, although several p50
/
relB
/+ mice
of 5 mo and older appeared sick and died prematurely (Fig. 1
B and data not shown). At necropsy of 20-d-old mice,
marked thymic atrophy, splenomegaly, and liver and kidney turgidity were observed in all p50
/
relB
/
double
knockouts. Moderate thymic atrophy and splenomegaly
was observed in relB
/
single-knockout animals. Wildtype controls, p50
/
single knockouts, and relB
/+ mice
were nonremarkable (Fig. 1 C and data not shown).
Fig. 1.
(A) Gross appearance of 20-d-old
wild-type (right), relB/
(left), and p50
/
relB
/
(middle) mice. (B) Survival of relB
/+, p50
/
relB
/+, relB
/
, and p50
/
relB
/
mice. Surviving mice expressed as a percentage of the initial number of animals in each genotype group
are plotted. relB
/+ mice (
); p50
/
relB
/+
mice (
); relB
/
mice (
); and p50
/
relB
/
mice (
). (C ) Thymus (Th), spleen (Sp), liver
(Li), and kidney (Ki) from 20-d-old wild-type,
relB
/
, and p50
/
relB
/
animals. Genotypes
are indicated at the bottom.
[View Larger Version of this Image (74K GIF file)]
/
relB
/
Double-knockout Mice.
/+, and
p50
/
animals. A minimal inflammatory phenotype accompanied by a moderate myeloid hyperplasia occurred in
heterozygous relB
/+ mice in the absence of p50 (p50
/
relB
/+). relB
/
single-knockout mice had a moderate
multiorgan infiltrate.
In contrast, p50/
relB
/
double-knockout mice by
day 20 developed an inflammatory infiltrate that was of
greater severity and extent of organ system involvement as
compared to relB
/
single-knockout animals. Interestingly, 8-10-d-old p50
/
relB
/
mice were only slightly
more affected than similarly aged relB
/
animals. Thereafter, the infiltrate in RelB-deficient mice progressed much
more rapidly and aggressively in the absence of the p50 subunit of NF-
B, and was reflected by increased mortality (Fig.
1 B and data not shown). Similar to relB
/
single-knockout animals, the lung and liver were also markedly affected
in p50
/
relB
/
double-knockout mice. In the lung of
double-knockouts, the infiltrate was in a perivascular and
periairway orientation that extended into and effaced the
surrounding parenchyma, whereas in the relB
/
single
knockouts, the infiltrate was predominantly restricted to a
perivascular orientation (Fig. 2 A-D). In the liver of relB
/
and p50
/
relB
/
mice, the infiltrate originated in periportal areas and extended into surrounding hepatocellular
trabeculae and sinusoids. However, the infiltrate in doubleknockout animals was so severe that infarction was commonplace, most likely resulting from infiltrate-induced interference with vascular flow (data not shown). In addition, the inflammatory infiltrate of 20-d-old double-knockout
mice also involved kidney (Fig. 2, E-H), heart (Fig. 2, I-L),
striated musculature, and salivary glands, organs that were
not affected in 3-wk-old relB
/
single-knockout mice.
Myeloid hyperplasia in the bone marrow and spleen was
also more pronounced in double knockouts as compared to
relB
/
single-knockout animals (data not shown).
Role of T Cells, B Cells, Macrophages, and Neutrophils in the Inflammatory Phenotype.
To examine the cellular composition of the inflammatory infiltrates in nonlymphoid tissues
in more detail, lung and liver sections from 20-d-old wildtype controls, relB/
, and p50
/
relB
/
mice were analyzed by immunohistochemical criteria. Only resident leukocyte populations were detected in control tissues stained with the different mAbs (Fig. 3, A and D; and Fig. 4, A, D,
and G). In contrast, abundant staining was observed in pulmonary infiltrates of both relB
/
and p50
/
relB
/
mice
with mAbs specific for CD4+ helper T cells (Fig. 3, B and
C ) and CD8+ cytotoxic T cells, although a smaller number
of CD8+ lymphocytes occurred both quantitatively and
proportionally in the infiltrates of double-knockout mice
(Fig. 3 E and F). Prominent infiltrates, consisting predominantly of CD4+ and CD8+ lymphocytes, were observed in
the lung and liver of these animals as early as 10 d after
birth, suggesting that T cells are crucially involved in the
onset of the inflammatory phenotype (data not shown).
To examine which non-T cell types are involved in the
inflammatory infiltrates, liver sections from 20-d-old wildtype, relB/
, and p50
/
relB
/
mice were stained with
mAbs specific for B cells, macrophages, and neutrophils
(Fig. 4). B cells were readily detected within the periportal
infiltrate in sections from relB
/
mice (Fig. 4 B). In marked
contrast, the inflammatory infiltrate in p50
/
relB
/
liver
contained very few B cells (Fig. 4 C), indicating that B
cells are not required for the development of the inflammatory phenotype in p50
/
relB
/
animals. Kupffer cells,
specialized tissue macrophages residing along hepatic sinusoids, showed strong positive staining in wild-type mice as
compared to relB
/
and p50
/
relB
/
animals (Fig. 4, D-F).
Little F4/80-positive staining was detected within the infiltrates of single- and double-knockout mice (Fig. 4, E and F).
This finding, coupled with the observation that few infiltrative cells had morphological criteria of macrophages, suggest that this cell population is not a prominent component of the inflammatory phenotype of these animals at this
age. Since adult RelB-deficient mice have increased granulopoiesis in bone marrow and spleen (21, 22), we examined
whether polymorphonuclear leukocytes are an important
component of the inflammatory infiltrates. Markedly increased numbers of neutrophils were detected in the periportal infiltrates of p50
/
relB
/
mice compared to relB
/
animals (Fig. 4, G-I). Similar results were obtained when
lung sections were stained for B cells, macrophages, and
neutrophils (data not shown).
Flow cytometric analysis of
3-wk-old p50/
relB
/
double-knockout mice revealed
marked alterations in thymus, spleen, and bone marrow cell
subpopulations. Mice deficient in the p50 subunit of NF
B, like heterozygous relB
/+ mice, do not have any
changes compared to wild-type animals (8, 21; data not
shown) and were used as controls. We also included relB
/+
mice on a p50
/
background (p50
/
relB
/+) since these
animals develop a mild inflammatory phenotype (see Table 1).
CD4+CD8+ double positive (DP) TCRlo thymocytes were
dramatically reduced in double knockouts, whereas CD8+
and, in particular, CD4+ single positive TCRhi T cells were
relatively increased (Fig. 5 A). Mature T cells were present
in p50
/
relB
/
spleen whereas B220+IgM+ B cells were
markedly reduced. In contrast, numbers of myeloid cells
(Mac-1+, Gr-1+, 7/4+) were dramatically increased (Fig. 5
B and data not shown). With respect to the reduced number of B cells, p50
/
relB
/
double-knockout spleens
were characterized by poorly-developed B cell follicles, hyperplastic periarterial lymphatic sheath (T cell) areas, and
indiscrete marginal zones (data not shown). Flow cytometric analysis of bone marrow cells also revealed increased numbers of myeloid cells and markedly reduced B220+
IgM
and B220+IgM+ B cell populations (Fig. 5 C). Interestingly, p50
/
relB
/+ mice also had a moderate reduction
in splenic B cells and increased numbers of myeloid cells in
the bone marrow, but normal T cell subsets in thymus and
spleen (Fig. 5, A-C).
Since the interpretation of the results obtained from 3-4-wk
old animals is hampered by the severe pathological changes
in lymphoid organs of p50/
relB
/
double-knockout
mice, we analyzed 2-wk-old animals by flow cytometry. At
this age, both relB
/
and p50
/
relB
/
mice show a milder
phenotype, and the pathological differences between these
two mutant lines are less pronounced. Wild-type animals
were included as a control; similar results were obtained with p50
/
or relB
/+ mice (data not shown). Similar to
the results shown in Fig. 5, although less severe, 2-wk-old
p50
/
relB
/
mice had decreased numbers of DP thymocytes
and an increased population of thymic CD4+TCRhi T cells
(Fig. 6 A). T cell subsets in double-knockout spleen were
normal, but both B220+IgM
and B220+IgM+ B cell populations were reduced, and the number of myeloid cells was increased (Fig. 6 B). Similarly, the number of Gr-1+
cells was increased in double-knockout bone marrow while
both B cell subpopulations were markedly reduced (Fig. 6
C). In contrast, relB
/
mice of the same age had only
moderate myeloid hyperplasia in spleen and a very mild reduction in B220+IgM+ B cells compared to wild-type littermates (Fig. 6 and data not shown).
Expression and DNA Binding of NF-
To examine whether
there is any compensatory upregulation of other family
members in mice lacking p50, RelB, or both, mRNA levels
of all NF-B/Rel family members and I
B
/
in thymus
were determined by semi-quantitative reverse transcription-PCR. Expression of
-actin was used as a reference
(Fig. 7). Whereas nfkb1 and relB transcripts were absent in
the respective mutant lines, mRNAs specific for relA, c-rel,
ikba, and ikbb were readily detected in all genotypes examined. Expression of these genes was not significantly altered
but nfkb2 mRNA levels were reduced in both single- and double-mutant mice, suggesting that nfkb2 expression may
be regulated by p50-RelB heterodimers. Interestingly, p52
protein levels were only slightly reduced in extracts from
single- or double-knockout mice (8, 21; data not shown).
These results indicate that there is no compensatory upregulation of other NF-
B/Rel family members in the absence of p50, RelB, or both.
To correlate mRNA levels with B-binding activity,
EMSA with thymus extracts from wild-type, p50
/
, relB
/
,
and p50
/
relB
/
mice were performed (Fig. 8). The
Bbinding activity was strongly reduced in both p50
/
and
relB
/
single-mutant mice (8, 21) and almost completely
abolished in p50
/
relB
/
double-mutant thymus (Fig. 8
A, lanes 1-4). Challenge with anti-p52 (lanes 5-8) and
anti-RelA (lanes 9-12) antiserum reduced the respective binding complexes in wild-type or single-mutant mice, and
did not reveal any new homodimers or heterodimers in
mice lacking p50, RelB, or both. The complex in extracts
from p50-deficient single knockouts consisted of p52-
RelB heterodimers since it was reduced in relB
/
mice
(lanes 3 and 4) and in the presence of anti-p52 antiserum (lanes 2 and 6). We were unable to detect significant binding of c-Rel under these conditions, and only the very
weak
B-binding activity that remained in extracts from
double-knockout mice was reduced by antiserum directed
against c-Rel, p52, or RelA (lanes 4, 8, 12, and data not
shown). Integrity of extracts was checked with an octamer
binding site (lanes 13-16). Also, no compensatory complexes could be detected in spleen extracts or with
Bbinding sites derived from the immunoglobulin
light
chain enhancer or the HIV LTR (data not shown).
In most cells, NF-B/Rel activities are retained in an inactivated form in the cytoplasm through their interaction
with the inhibitory proteins I
B
and I
B
or the p100
and p105 precursors. These inactive complexes, however,
can readily be activated by DOC treatment resulting in increased
B-binding activity (30). Therefore, we analyzed
whether any new compensatory complexes can be found in
DOC-treated extracts from single- or double-mutant mice
(Fig. 8 B). Under these conditions, binding of p50 homodimers in wild-type and relB
/
extracts was significantly reduced. In all activated extracts, an additional complex of slower mobility could be observed, although the
signal was clearly reduced in the absence of p50, RelB, or both (compare lanes 1-4 and 5-8). Challenge with antiRelA and anti-c-Rel antiserum showed that this
B-binding activity most likely consisted of c-Rel homodimers and
c-Rel-RelA heterodimers, although RelA homodimers
could not be excluded since the anti-c-Rel antiserum
weakly cross-reacts against RelA (lanes 9-12 and 13-16 and data not shown). In any case, these complexes were
neither increased in single- nor double-mutant mice, indicating the lack of compensatory
B-binding activities in
both single- and double-knockout mice.
More than 10 potential homodimers and heterodimers
can be formed among members of the NF-B/Rel family
of transcription factors that can bind to similar cis-regulatory sites and modulate gene expression. This results in a
high degree of complexity within this family of transcription factors. Several reports of mice with targeted disruptions of individual NF-
B/Rel family members demonstrated that the different proteins play distinct biological roles (6, 7). It is unclear, however, whether functional redundancy also exists within this family that would result in a (partial) compensation of the phenotype in single-knockout animals.
Both thymic atrophy and myeloid hyperplasia in bone marrow and spleen were markedly increased
in p50/
relB
/
double-mutant mice compared to relB
/
single-mutant animals. Also, multiorgan inflammation was
dramatically increased in severity and extent resulting in
premature death of all double-knockout mice. Another notable difference is the penetrance of the pathological changes
in thymus and other organs. The phenotype was markedly
increased in severity in all p50
/
relB
/
double knockouts
examined, whereas there was considerable interanimal variation in relB
/
single-knockout mice. Heterozygous relB
/+
mice that also lacked p50 had moderate pathological
changes that were qualitatively similar to homozygous
relB
/
mice, further lending support to the notion that the
lack of RelB is partially compensated by other p50-containing complexes.
There are examples to support the theory of functional
compensation within a family of related proteins. For instance, embryos that are exposed to retinoic acid during development have a wide spectrum of malformations. Whereas
the disruption of individual retinoic acid receptors did not
result in the expected phenotypes, double-mutant mice have
severe developmental abnormalities (31). Similarly, increased
severity in phenotypic changes was observed in mice with
combined deficiencies of the homeobox genes hoxa-3 and
hoxd-3 (32) or the tyrosine kinases Src, Fyn, and Yes (33). Increased expression of a related family member as a compensatory mechanism has been proposed for the myogenic
transcription factor Myo D. Mice with a targeted disruption of myo D do not have morphological abnormalities in
skeletal muscle, but do have markedly increased levels of
myf-5 mRNA. Simultaneous disruption of both myo D and myf-5, however, results in a more severe phenotype (34).
Our results also suggest functional redundancy within the
NF-B/Rel family and, in contrast to Myo-D-deficient
mice, we were unable to detect a compensatory upregulation of other NF-
B/Rel family members in both single-
and double-knockout animals. Compared to relB
/
single-knockout mice, p50
/
relB
/
double knockouts also
lack p50-p50, p50-p52, p50-RelA, and p50-c-Rel complexes. Thus, additional mouse mutants deficient for different combinations of NF-
B/Rel proteins will be necessary
to further elucidate the network of redundancy and to understand the specific functions of each NF-
B/Rel family
member in more detail.
Several of the
histopathologic features and cellular constituents observed
in the lung of relB/
and p50
/
relB
/
mutant mice are
similar to those observed in certain human interstitial lung
diseases, including idiopathic pulmonary hemosiderosis (35), Goodpasture's syndrome (36), and acute systemic lupus erythematosis with a hemorrhagic component (37, 38). The
inflammatory cell infiltration in p50
/
relB
/
mice, in
particular in the skeletal and cardiac musculature, kidney,
and salivary glands, resembles that seen in certain human autoimmune diseases, including immune-mediated myocarditis and polymyositis, and Sjögren's syndrome (39, 40).
Many experimental models have shown that T cells can be
pathogenic mediators in autoimmune diseases (41, 42). Indeed, by using a transgenic mouse line that lacks T cells, we
have recently demonstrated that both multiorgan inflammation and myeloid hyperplasia in RelB-deficient mice are
T cell-dependent (43). This result correlates with the finding
that the inflammatory infiltrates in 10-20-d-old relB
/
single- and p50
/
relB
/
double-knockout mice predominantly consisted of CD4+ and CD8+ T cells. In thymus,
RelB expression is restricted to medullary DC and epithelial cells (15, 21, 22), cell types that have been implicated in
the process of negative selection (44, 45). Since development of thymic medulla is impaired in the absence of RelB,
a defect in clonal deletion of autoreactive T cells may eventually result in potentially pathogenic T cells promoting the
inflammatory phenotype observed in RelB-deficient mice.
This model is further supported by the finding that relB
/
mice poorly delete autoreactive thymocytes and have splenocytes that generate an autoreactive response (46).
The thymus of p50/
mice is nonremarkable and T cell
development is not impaired in these animals (8). Despite
thymic atrophy and impaired development of a thymic medulla, RelB-deficient mice have normal thymocyte subsets
as defined by CD4, CD8, CD3, CD25, and TCR-
/
surface markers (21). Simultaneous disruption of p50 and
RelB, however, resulted in a markedly increased thymic atrophy with a 10-20-fold reduction in cellularity, the
complete absence of a medullary compartment, and an altered profile of thymocyte subpopulations. Consistent with
the thymocyte profile, mature CD4+ and CD8+ single
positive cells could be detected in peripheral lymphoid organs and inflammatory infiltrates of double-knockout animals, indicating that T cell differentiation is not blocked in
the absence of both p50 and RelB. The basis for the lack of
immature CD4+CD8+ DP thymocytes in 3-wk-old p50
/
relB
/
mice is not clear. Interestingly, very similar changes
in thymus have been observed in mice lacking the protooncogene c-fos (47). The drastic reduction in the number
of CD4+CD8+ DP thymocytes occurs only in 40-50% of
the c-fos
/
mice, and it has been suggested that this defect
could be an indirect consequence of impaired bone marrow function and general stress on these animals (48). Similar mechanisms may be responsible for the dramatic reduction in the number of DP thymocytes in mice lacking both
p50 and RelB.
B cell development in relB/
mice appears normal and B cells lacking RelB undergo normal maturation to Ig secretion and Ig
class switching, but they have decreased proliferative responses (21, 49). Immunohistochemical analysis of tissue sections revealed that B cells are a prominent component of
the inflammatory infiltrates in relB
/
single-mutant animals. Similar to RelB, p50 is not required for normal B cell
development (8). However, purified B cells from p50-deficient mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching
(9). In contrast to relB
/
single mutants, p50
/
relB
/
double-mutant mice did not have significant numbers of B
cells in inflamed tissues despite markedly increased severity
of the phenotype. This result correlates with impaired B
cell development resulting in markedly reduced numbers of
both immature B220+IgM
and mature B220+IgM+ B cells
in bone marrow and spleen of double-knockout mice. Thus, B cells appear not to be required for the development of
the multiorgan inflammation, although we cannot rule out the
possibility that B cells may modulate the inflammatory response in RelB-deficient mice. Further studies, such as in
vitro differentiation and bone marrow reconstitution experiments, are required to understand the developmental potential of T and B cells from p50
/
relB
/
mice in more detail.
We also analyzed the contribution of myeloid cells to the
inflammatory infiltrate using mAbs specific for macrophages
(F4/80) and neutrophils (7/4). In 20-d-old animals, only
very few F4/80+ cells were present in the inflammatory infiltrates of both relB/
single- and p50
/
relB
/
doubleknockout mice. In 5-6-week-old relB
/
single mutants,
however, positive F4/80 staining could be detected in the
inflammatory infiltrates of liver and lung (data not shown), suggesting that macrophages are not playing a major role
during the early stages of the inflammatory phenotype. The
faint F4/80 staining of sinusoidal Kupffer cells is most likely
due to an activated state of these tissue macrophages triggered by the inflammatory infiltrates, since it has been
shown that F4/80 expression is significantly reduced upon
antigen stimulation and activation (50). In contrast to macrophages, mature neutrophils contribute to the inflammatory infiltrate in 20-d-old single- and, in particular, doubleknockouts. This finding correlates with a marked increased of neutrophils in bone marrow and spleen of p50
/
relB
/
mice compared to relB
/
animals. The cause of this prominent neutrophilia is still unclear. One possible explanation
is that polymorphonuclear leukocytes become activated by
cytokines released by autoreactive T cells, a scenario that is
supported by an altered cytokine milieu in mice lacking
RelB and an attenuated myeloid hyperplasia in T cell-deficient relB
/
mice (21a and 43).
The phenotypic changes in p50/
relB
/
doubleknockout and p50
/
relB
/+ mice, compared to the respective single-knockout and relB
/+ animals, indicate that the
lack of RelB is partially compensated by other p50-containing complexes. Our results also show that the classical
p50-RelA-NF-
B activity is not required for the development of the multiorgan inflammatory phenotype. Additional NF-
B/Rel double-knockout mice may give further
insight into both functional redundancy and specificity
within this family.
Address correspondence to Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000. The present address of W.C. Sha is Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200.
Received for publication 21 January 1997.
1Abbreviations used in this paper: DC, dendritic cells; DOC, deoxycholate; DP, double positive; EMSA, electrophoretic mobility shift assays.We gratefully acknowledge Kenneth Class for flow cytometry, Michele French and Sophie Komar for excellent technical assistance, and James Loy for photoimaging. We also thank Violetta Iotsova and Jorge Caamano for valuable comments on this manuscript and all the staff in Veterinary Sciences of Bristol-Myers Squibb.
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