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B-
Develop Normally, But Have Selective Defects in Proliferation and Function1
,
,¶
Departments of
*
Microbiology and Immunology and
Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232;
Howard Hughes Medical Institute, Chevy Chase, MD 20815;
Vanderbilt-Ingram Cancer Center, Nashville, TN 37232; and
¶ Transplantation and Immunobiology Section, Division of Allergy, Immunology, and Infectious Disease, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B has been implicated in the development, activation, and
function of B and T lymphocytes. We have evaluated the in vivo effects
of deletion of IB-
, a major inhibitor of NF-B, on lymphocyte
development, proliferation, and function. To elucidate the long term
role of IB- in lymphocytes, fetal liver cells of 14.5-day-old
IB--/- or wild-type embryos were transplanted into
irradiated recombinase-activating gene-2-deficient mice. Within 4 wk,
the IB--/- fetal liver cells reconstitute mature B
and T cell populations in the recipients comparable to those produced
by wild-type fetal liver cells. However, the proliferative responses of
IB--/- B cells are enhanced, whereas those of
IB--/- T cells are reduced. The levels of IgG1,
IgG2a, IgA, and IgE produced by IB--/- B cells are
elevated relative to those produced by IB-+/+ or
IB-+/-. Moreover, the specific immune
responses to OVA and the generation of germinal centers are impaired in
recipients of IB--/- fetal liver cells. These
results indicate that IB- plays a vital role in signal
transduction pathways regulating lymphocyte proliferation and also in
the production of specific Ig isotypes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, plays a critical role
in a wide variety of cellular functions, including proliferation,
differentiation, and programmed cell death. NF-B complexes are
segregated in the cytoplasm by association with an inhibitor IB. The
current model for the activation of NF-B is that second messenger
cascades activate the IB kinases (IKKs) that phosphorylate IB-
on specific serines. Hyperphosphorylation of IB- leads to
inactivation and destruction of IB by proteasome-mediated
proteolysis, which results in the release of NF-B, translocation
into nucleus, and its binding to specific DNA elements
(1, 2, 3, 4). The NF-B family contains five mammalian members
(RelA (p65), c-Rel, RelB, p52, and p50) and three Drosophila
homologues (dorsal, dif, and relish) (5, 6, 7). These
proteins complex together as a variety of homodimers or heterodimers
(8) that can coexist simultaneously in a given cell type.
The activity of such dimers must be regulated coordinately or
independently. IB, an evolutionarily conserved multigene family, is
comprised of nine members, including IB-, IB-
, Bcl-3, p100,
p105, IB-
, IB-
, and two Drosophila homologues
(cactus and relish). These proteins share three to seven
repeats of a conserved motif known as an ankyrin repeat, which is
required for association with the NF-B complex and inhibition of the
DNA binding activity of the NF-B complex (9). The
different IB forms may differentially regulate distinct NF-B
dimers. IB- associates with and blocks the activity of two major
transcription activators, c-Rel and RelA. Hence, IB- is one of
the primary inhibitors of inducible NF-B binding activity.
Gene disruption has been used to generate mice lacking individual
NF-B members. As predicted, deficiency of Rel/NF-B members
results in defects of various immune functions (reviewed in Ref.
10). However, the variety of heterodimeric combinations
that occur within the NF-B family makes it difficult to interpret
the function of NF-B in the immune system by studying the deletion
of a single NF-B member. In this case, more information might be
gained by altering the genes that control NF-B activity, namely
IBs. It was proposed that gene disruption of IB- would result
in constitutive NF-B activity. Indeed, NF-B DNA binding activity
is elevated in several tissues from IB--deficient mice. They
display granulopoiesis and severe skin disease and typically die within
9 days after birth, although they develop normally until birth
(11, 12). However, lymphoid cells and immune responses in
these mice were not characterized.
The neonatal lethality exhibited by these animals precludes the study
of immune system development and function in adult mice. Therefore, we
have used fetal liver cell adoptive transfer to extend our
characterization to include effects of IB- deficiency on the
development and function of lymphocytes. IB- deficiency results
in alterations in the B and T cell populations consistent with more
differentiated and activated states. Elevated NF-B activity was
observed in several tissues, including hemopoietic cells of
IB--deficient mice, and the expression of several cytokines was
altered. Proliferation of IB--/-
lymphocytes is affected, and alterations in specific isotype Igs are
observed. Our results demonstrate for the first time that
IB--/--deficient lymphocytes display
defects in proliferation and hypermaturation and a profound alteration
in their ability to mount an immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mouse IB- gene, mad3, fragment was cloned
from a mouse 129 genomic library into the gt11 phage. An Acc65I
fragment from phage clone 4 was subcloned into pBluescript (Stratagene,
La Jolla, CA). Plasmid containing both 5' and 3' regions of IB-
cDNA was used to create the targeting construct. NarI was
used to remove a 300-bp region containing 5' untranslated region and
part of exon I region and was replaced with a PGK-Neo cassette to
construct the targeting vector MAD3 PGK-Neo. This single selection
construct, PGK-Neo, was verified by Southern blot using external and
internal probes. To create the double selection construct, an
XbaI fragment containing PGK-Neo was subcloned into a PGK-tk
plasmid to create a PGK-tk Neo IB- construct.
Embryonic stem (ES)3 cell culture and generation of mice
Linearized targeting vector (50 µg), was introduced into TL-1
ES cells (a gift from Trish Labosky, University of Pennsylvania,
Philadelphia, PA). Cells were cultured in the presence of G418 for 7
days, and resistant colonies were selected and cultured as described by
Hogan et al. (13). Genomic DNA isolated from individual
colonies was digested with HindIII and SmaI,
screened by Southern blot analysis using a 500-bp
KpnI/HindIII fragment as an external probe (probe
a) and Neo cassette as internal probe (probe b). Targeted clones were
used for microinjection into C57BL/6 blastocysts. Chimeric males with
>80% agouti coloring were backcrossed to C57BL/6 mice, and
IB-+/- mice were verified by Southern blot
analysis. IB--/- mice were subsequently
generated from crosses of IB-+/- siblings.
To generate IB--/- bearing a luciferase
reporter of NF-B activity, IB-+/-
animals were mated with HLL transgenics, then
IB-+/- HLL+ siblings
were mated. HLL transgenic mice contain a reporter gene encoding
luciferase under the control of HIV long terminal repeat region, which
includes NF-B DNA binding elements. Animals carrying HLL were
identified by PCR or Southern blot analyses as well as luciferase
activity from the brain tissue (F. E. Yull et al., manuscript in
preparation). Mice were kept in a pathogen-free environment, provided
with autoclaved food, water, and cage.
Immunofluorescence staining and flow cytometry
Single-cell populations from thymus, spleen, lymph node, peripheral blood, and bone marrow were isolated and surface stained as previously described (14, 15), with directly fluoresceinated mAbs (PharMingen, San Diego, CA). Stained cells were subjected to analysis by FACS flow cytometer (FACS caliber at Howard Hughes Medical Institute; flow cytometry at Vanderbilt University). The mAbs used were anti-B220 (PharMingen 01128A), anti-IgD (02214D), anti-CD4 (01065A), anti-CD8 (01048A), anti-CD3 (01084D), anti-CD44 (01224D), anti-CD69 (01575A), anti-heat-stable Ag (anti-HSA; 01575A), and anti-IgM (1021-09; Southern Biotechnology Associates, Birmingham, AL). FACS analysis was performed using the CellQuest program (Becton Dickinson, Mountain View, CA).
Preparation of purified splenic B and T cells
Splenocytes from recipients of fetal liver cells were first depleted of erythrocytes by hypotonic shock, followed by incubation at 37°C in complete medium for 1 h to allow macrophages to attach to the bottom of the plate. After macrophage depletion, the suspended cells were gently transferred to 15-ml tubes and counted. Cells (3 x 107) were incubated with 1.5 ml of anti-Thy1.2, anti-CD4 (G.K 1.5) and anti-CD8 (T1B105) hybridoma culture supernatant on ice for 40 min followed by washing twice with fresh cold medium. Cells were then transferred onto plates coated with mouse anti-rat IgG (115-006-075, Jackson ImmunoResearch, West Grove, PA) to deplete the T cell population at 4°C for 1 h. Spleen cells (3 x 107) were incubated in anti-IgM (BioSource, Camarillo, CA)-bound plates at 4°C for 1 h to deplete B cells. After panning out B and T cells, the purity of T and B cell populations was verified by FACS analysis using anti-CD3, anti-B220, and anti-Mac-1 (PharMingen). Normally, 80–85% purity of B or T cells was obtained.
Ex vivo proliferation assays
All primary cells were cultured in complete medium containing RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 µM 2-ME, and 2 mM L-glutamine. Spleen cells were cultured at an initial concentration of 1 x 106 lymphocytes/ml. Purified B and T cells were cultured at concentrations of 5 x 105 cells/ml. For proliferation assays, the purified B or T lymphocytes were dispensed into 96-well microtiter plates at 200 µl/well. B lymphocytes were stimulated with anti-IgM (Jackson ImmunoResearch), anti-CD40 (PharMingen, HM 40-3), or LPS (Sigma, St. Louis, MO) at the indicated concentrations. T lymphocytes were stimulated with Con A (Pharmacia, Piscataway, NJ), anti-CD3 (culture medium from 1452C11 hybridoma), or OVA in the presence of irradiated splenic cells from C57BL/6 at the indicated concentration. After 48 h of stimulation, cells were pulsed with 0.75 mCi of [3H]thymidine (Amersham, Arlington Heights, IL)/well for the next 16 h. Cells were harvested with a cell harvester (Tomtec orange), and uptake of radioactivity was measured with a betaplate recorder (Wallac, Gaithersburg, MD).
Immunization and Ig isotype analysis
Samples representing preimmune sera for analysis of basal levels
of Ig isotypes were collected from 6-, 7-, and 8-wk-old recipients of
fetal liver cells from IB--/- embryos and
control littermates. For analysis of Ig levels, prechallenge serum was
collected at 7 wk after fetal liver cell transfer. The mice were then
immunized with 100 µg/ml OVA emulsified in CFA (Becton Dickinson) via
i.p. injection. Sera were obtained at 7-day intervals after
immunization for a period of 3 wk. The immunized mice were boosted with
OVA emulsified together with IFA at wk 3. Mice were bled 1 wk after
secondary challenge, and sera were collected for Ig isotype analyses.
Lymph nodes and part of the spleen were taken for immunohistochemical
analyses. T cells were isolated from part of the spleen and used for
the proliferative response of OVA stimulation. The level of specific Ig
in each sample was determined by ELISA using goat anti-Ig (H+L)
polyclonal Ab as the capture agent and goat anti-mouse
isotype-specific polyclonal Ab conjugated with HRP (both from Southern
Biotechnology Associates, Birmingham, AL) as detecting agents. The
levels of Ag-specific Ig isotypes in immune sera and serum IgE were
determined by ELISA as described by Singh et al. (16). The
concentrations of Ig isotypes from immunized or immune sera were
determined using purified myeloma proteins as standards (Sigma).
Immunohistochemical analysis
Spleens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 6 µm. Sections were stained with hematoxylin and eosin or used for immunohistochemistry. For immunohistochemistry, rehydrated serial sections were treated for Ag retrieval and quenching of endogenous peroxidase activity as previously described (17). The slides were blocked at room temperature with unchallenged mouse serum for 1 h and then labeled with peroxidase-conjugated peanut agglutinin (PNA; EY Laboratories, San Mateo, CA) or anti-B220 for 1 h at room temperature. Peroxidase-conjugated anti-rat Abs were used as secondary Ab for B220 staining. FDC-M1 (anti-FDC, a gift from Dr. M. H. Kosco-Vibois) Ab staining was performed as described by Schubart et al. (18). After washing, diaminobenzidene was added to form precipitate. Peanut agglutinin-positive germinal centers were counted, and pictures were taken at x100 magnification (FDC-M1 40X).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B- deficiency results in constitutive NF-B
transcriptional activity in multiple tissues
The IB- locus was disrupted by homologous recombination in
ES cells using two alternative targeting vectors, PGK-Neo-IB- and
PGK-tk-Neo-IB-, each replacing the promoter and first exon of the
IB- gene with a PGK-Neo cassette. This strategy is designed to
disrupt IB- transcription and translation. Consistent with other
groups’ findings (11, 12),
IB--/- mice were comparable in size and
behavior to wild-type or heterozygous mice at birth, and their growth
and development were equal to those of their wild-type and heterozygous
littermates for the first 3 days. At this point, IB--deficient
pups stop gaining weight and by 6 days, their weight is approximately
one third that of wild-type pups. The IB-
-/- pups develop a dry, red, flaky skin; suffer
from severe runting; and typically die within 9 days after birth.
Six-day-old IB--/- pups have formed
internal organs. The spleens of IB- null mice appear
smaller than those of wild-type or heterozygous littermates. Thymic
atrophy is also observed in IB--/- mice,
whereas spleen and liver appear anemic. The cause of the neonatal
lethality in IB--/- mice remains unknown.
In agreement with other groups’ findings, a significant increase in
specific protein:DNA complexes was observed in nuclear extracts
prepared from the thymocytes and splenocytes of
IB--/- compared with those from
IB-+/+ and
IB-+/- animals (data not shown). The
transcriptional levels of several cytokines, including TNF-, IL-6,
macrophage inflammatory protein-1, and GM-CSF, were elevated in the
IB--deficient skin, whereas the transcriptional levels of IL-2
and IFN- remained comparable to those in wild-type animals (data not
shown). To examine the effect of IB- deficiency on NF-B
transcriptional activity in vivo, heterozygous mice were mated with
transgenic mice in which the expression of a luciferase reporter gene
is under the control of the HIV-long terminal repeat region,
which contains NF-B-responsive elements. This line of luciferase
reporter animals is termed HLL and is a useful reporter of NF-B
activity as a measure of luciferase activity. The phenotype of
IB--/- HLL+/- mice
is identical with that of IB--/- mice.
Luciferase activity, indicative of NF-B transcriptional activity,
was elevated in the organs tested (Fig. 1
). These observations confirm that lack
of IB- results in elevated NF-B activity. This unregulated
NF-B activity may contribute to the neonatal lethality caused by
IB--/- deficiency (12).
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B activation perturbs T and B cell development
in IB--/- mice
To determine whether constitutive NF-B activation perturbs T
and B cell development, several stage-specific or function-specific
cell surface markers were selected to examine T and B cell development
in IB--/- mice. Consistent with the
observed thymic atrophy in IB--/- mice,
the total cell number of thymocytes was dramatically decreased in
IB- deficient mice.
An increased apoptotic rate could result in a reduced total cell number of thymocytes. Therefore, the percentage of apoptotic cells was measured in stimulated wild-type and null T and B cell populations. T cells (2 x 106) were stimulated with either bound anti-CD3 (10 µg/ml) or Con A (2.5 µg/ml). After 48 h, TUNEL assays were performed, and the percentage of apoptotic cells in CD4 and CD8 populations was determined by FACS analysis. No significant differences between null and wild-type cells were detected (data not shown).
Staining of thymic cells with anti-CD4 or anti-CD8 revealed an
alteration in the T cell population of
IB--/- mice (Fig. 2A). The population of
single-positive (CD4+ or
CD8+) cells was increased in
IB--/- mice, whereas the population of
double-positive (CD4+/CD8+)
cells was decreased, relative to those in littermate controls.
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B- null T cells,
samples were stained with CD3 and HSA. During the maturation of
single-positive cells the TCR level increased, while the level of
expression of HSA normally decreased. By comparison to control
littermates, FACS analysis showed that CD8 single-positive T cells from
IB--deficient thymus expressed higher levels of CD3 but lower
levels of HSA than T cells from control littermates (Fig. 2
B, gated on CD8+). However, CD4
single-positive T cells expressed levels of CD3 and HSA in
IB--/- pups comparable to those observed
in control littermates (data not shown). These results indicate that
constitutive NF-B activation accelerates T cell development toward a
single-positive state and, in particular, toward a more mature CD8
single-positive state.
The functional status of IB--/- T cells
was determined by staining with anti-CD69 Abs, a surface marker
indicating transient activation of T cells. Interestingly, FACS
analysis revealed that 59% of IB--/-
CD8+ T cells express a high level of CD69
compared with 6% in wild-type T cells. The level of CD69 expression
was comparable in CD4 single-positive cells. These results indicate
that although there are fewer T cells present, constitutive NF-B
activity produces a higher proportion of mature, activated
CD8+ cells (Fig. 2B, gated on
CD8).
To determine whether constitutive NF-B activity perturbs the
differentiation of B cells, spleen cells were isolated from 5-day-old
pups and stained with fluorescence-conjugated B220 and IgM Abs. A
larger population of B cells expressed high levels of IgM in
IB--/- spleen compared with the wild-type
spleen (Fig. 2, C and D). However, fewer total
splenic B cells were harvested from
IB--/- mice, consistent with the
reduction in the size of the spleen of
IB--/- mice compared with wild-type mice.
To investigate whether the reduction in total B cell numbers was due to
increased apoptosis, B cells (2 x 106) were
stimulated with either anti-IGM (2 µg/ml) or LPS (10 µg/ml).
After 48 h TUNEL assays were performed, and the percentage of
apoptotic cells in B220 populations was determined by FACS analysis. No
significant differences between null and wild-type cells were detected
(data not shown).
The results from the FACS analysis suggest that although fewer B cells
are present in IB--/- mice, these B cells
are more differentiated than their wild-type counterparts. Together,
our results lead us to propose that hyperactivation of NF-B is
sufficient for accelerating differentiation of T cells and activating T
cells (as assessed by certain cell surface markers). B cell maturation
is affected by the removal of IB-, which suggests that NF-B
may be involved in distinct mechanisms of differentiation in T and B
cells.
IB--/- fetal liver cells can develop into adult
lymphocytes
Although the lack of both p50 and p65 resulted in the blockade of
B and T cell development, normal lymphocyte development occurred when
p50/65-deficient fetal liver cells were adoptively transferred together
with wild-type bone marrow cells (19). This observation
suggests that some effects of NF-B activity on B and T cell
development are extrinsic. To extend our investigation of the
development of lymphocytes from IB--/-
pups into adult mice and to examine whether the effects are intrinsic
or extrinsic, adoptive transfers of fetal liver cells were performed.
The same number (5 x 106) of fetal liver
cells isolated from 14.5-day-old embryos from a heterozygous mating
were transferred into lethally irradiated 12-wk-old
recombinase-activating gene-2-deficient mice. Reconstitution of B and T
cells by IB--/- fetal liver cells was
confirmed by FACS analysis, using selected markers 5 wk after transfer.
No severe phenotype was observed in the recipients, and the thymus and
spleen appeared to develop normally upon gross morphologic and
histologic examination. Mice sacrificed at 6, 7, and 8 wk were used to
investigate the development of B and T cells collected from bone
marrow, thymus, spleen, and peripheral blood. Total numbers of
reconstituted B and T cells in the spleen were comparable between
recipients of wild-type and IB--/- cells
(Fig. 3A). Single-positive T
cell populations in thymus, spleen, or peripheral blood were normal in
IB--/- recipients (Fig. 3B). Populations
of mature B cells were normal in bone marrow, spleen, and peripheral
blood (Fig. 3C). Several surface markers, including HSA,
CD3, CD69, and CD44, indicative of differentiation and activation
status were examined, and no abnormalities were observed (data not
shown). To investigate the population of granulocytes, bone marrow
cells and peripheral cells were stained with Mac-1. The results reveal
slightly elevated granulocytosis (data not shown). Our results suggest
that the observed defects in lymphocyte development in
IB--/- newborns are not cell intrinsic.
We hypothesize that factors extrinsic to the
IB--/- lymphocytes and myeloid cells
(i.e., cytokines, contacting accessory cells, etc.) contribute to the
more pronounced phenotypes in the homozygous animals.
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B--/- lymphocytes display proliferative
defects in response to polyclonal stimuli
To investigate the role of IB- in the regulation of
lymphocytes, we performed ex vivo proliferation assays using spleen
cells from recipients of IB--/- or
wild-type fetal liver cells. Splenic B and T cells were purified, then
equal numbers of enriched B or T cells were treated with specific
activators to stimulate proliferation (14, 20, 21). The
loss of IB- alters the proliferative response of B and T cells
compared with that of wild-type cells. Interestingly, IB-
deficiency affects the proliferative response of B cells differently
than it affects T cells. In response to the T cell stimuli
(anti-CD3 or Con A), IB--/- T cells
exhibit a 50% decreased proliferative response compared with controls
(Fig. 4A). Medium from these
proliferation assays was used for ELISA to investigate potential
differences in levels of cytokines critical for T cell proliferation.
Levels of IL-2, IFN-, and IL-4 were measured, and no significant
differences were detected between medium collected from wild-type or
null cells when stimulated with anti-CD3 or Con A (data not
shown).
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B--/- B cells displayed a 2- to 4-fold
increase in proliferation (Fig. 4B). T and B cell numbers
were comparable between recipients of
IB--/- and wild-type fetal liver
cells.
We interpret these disparate results between the cell types as being
due to intrinsic differences in the ways in which T and B cells use
NF-B to establish proliferation and activation.
Ab production by IB--/- B cells
To examine further the role of IB- in B cell function, the
levels of serum Ig isotypes in recipients of fetal liver cells were
mea- sured. The results indicate that
IB--/- B cells were capable of Ig
secretion and class switching of Ig isotypes. The basal serum levels of
IgM, IgG2b, IgG3 (Fig. 5A),
and Ig (data not shown) in the recipients of
IB--/- fetal liver cells were comparable
to those in wild-type cells. However, there was a 10-fold increase in
the levels of IgG2a, IgA, and IgE and a 3-fold increase in IgG1 in the
unchallenged mice (Fig. 5A). Levels of Ig
were also
elevated (data not shown). These data suggest that NF-B is involved
in Ig production and in switching of Ig isotypes, activities that are
critical for B cell function.
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B--/- B
cells
To test the effects of IB--/-
deficiency on Ag-specific immune responses, we challenged recipients of
IB--/- or wild-type fetal liver cells
with the T cell-dependent Ag, OVA (emulsified in CFA), or the T
cell-independent Ag, DNP-Ficoll. The Ags were administered 7 wk after
fetal liver cell transfers, then readministered 3 wk later for OVA.
Sera collected at 1-wk intervals were used for measuring the specific
Ig isotypes by ELISA (Fig. 5, B and C). The
OVA-specific anti-IgM levels produced by
IB--/- B cells are comparable to those
produced by IB-+/- or
IB-+/+ B cells. This result indicates that
OVA-specific B cells are present in the
IB--/- fetal liver cell recipients.
However, the levels of IgG1, IgG2a, and IgG2b specific for OVA in
IB--/- recipients were significantly
reduced even after the secondary challenge, whereas the levels of these
Abs produced by IB-+/- or
IB-+/+ B cells were significantly elevated
(Fig. 5B). To explore the function of T cells in
response to challenge, T cells were purified from OVA-challenged mice
and stimulated with OVA ex vivo. The proliferative response of
IB--/- T cells was comparable to that of
IB-+/+ T cells (Fig. 5C). This
result suggests that the response of
IB--/- T cells to OVA challenge is
normal. Therefore, the observed alteration in B cell production of
IgG1, IgG2a, and IgG2b in response to T cell-dependent OVA is not due
to a defect in T cell priming. To test the B cell function, recipients
were challenged with a T cell-independent Ag, DNP-Ficoll. The
production of IgG3, specific for DNP, is lower by
IB--/- B cells than by
IB-+/+ or
IB-+/- B cells (Fig. 5D). This
result indicates that the impaired production of Ig may partly be due
to defects in IB--/- B cell
function. Our results suggest that the deficiency of IB- has a
more profound impact on B cell differentiation than on T cell
differentiation.
Loss of splenic germinal centers in the IB--/-
fetal liver recipients
Germinal centers are prominent histological structures found
within the secondary lymphoid organs that contain activated B cells
undergoing proliferation, differentiation, and programmed cell death
(22). Activated germinal center B cells undergoing
maturation, hypermutation, and class switching express binding sites
for PNA. Since Ag-specific Ab responses are lower in
IB--/- fetal liver cell recipients, we
investigated the formation of germinal centers in the spleens of
recipients of IB--/- fetal liver cells by
histological analysis and by histochemical staining for PNA. Although
FACS analysis shows that the population of splenic B and T cells
remains comparable between the recipients of
IB--/- and wild-type fetal liver cells
after challenge (Fig. 6A),
results from PNA staining show lack of germinal center formation in
IB--/- recipients (Fig. 6B).
The follicular dendritic cell (FDC) clusters, which are normally
associated with germinal centers, were comparable between mutant and
wild-type control animals (Fig. 6B, FDC-M1). This suggests
that although mature IB--/- B cells are
present, IB- deficiency interferes with germinal center
formation. These results are qualitatively similar to findings for
other related knockout animals in which the lack of Bcl-3 or CD40 has
been demonstrated to result in the disruption of germinal centers
(15, 23). Our results indicate that IB- deficiency
results in the impairment of B cell differentiation to PNA-positive
germinal center B cells.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B family has varied
effects on regulation of the immune response (reviewed in Ref.
10). A deficiency of an individual family member has minor
effects on the development of lymphocytes, which may be due to a
significant level of functional redundancy within the family. To
overcome this, transgenic mice expressing a transdominant negative
inhibitor of NF-B, IB-
N, in T or B cell lineage were
generated (5, 24, 25) (F. E. Yull et al., manuscript
in preparation). These studies showed the important role of NF-B in
B and T cell development. Our results suggest that lack of IB-
has an extrinsic rather than an intrinsic effect on development. Our
data show that the population of single-positive cells in the thymus of
IB--/- pups is elevated relative to that
in control littermates. However, this elevation may be due to the
reduced total cell number in IB--/-
thymus specifically in
CD4+CD8+ double-positive
cells. In addition, the surface markers on the CD8 single-positive
cells as well as on B cells indicate that the majority of these cells
are at an advanced stage of maturation (Fig. 3). We cannot rule out the
possibility that the increased level of CD69 on
IB--/- T cells represents committed
double-positive T cells; however, the changes in measured levels of CD3
and HSA suggest that this effect is due to the transient activation of
T cells in IB--/- pups. While the
findings from our IB--/- pups also reveal
perturbation of lymphocyte populations, the
IB--/- fetal liver transfers successfully
reconstituted normal lymphocyte populations. Considering the data from
fetal liver recipients, we hypothesize that the effects of IB-
deficiency on lymphocyte development are due to extracellular factors,
such as cytokines or cell:cell communications, which are absent in
IB--deficient animals. Other studies in which there is failure of
lymphopoiesis after adoptive transfer of p50/p65 double-deficient fetal
liver into irradiated SCID mice also support this hypothesis
(19). These p50/p65 double-deficient fetal liver cells can
develop into normal lymphocytes when cotransferred with wild-type bone
marrow cells. This suggests that NF-B mediates the development or
survival of early lymphocyte precursors through regulation of
extracellular factors.
Lymphocytes lacking either of the two major inducible NF-B
transcription activators of the NF-B family, c-Rel and RelA, have B
and T cell proliferative defects in response to certain stimuli
(20, 21). From these previous data the assumption would be
that IB--/--deficient lymphocytes would
have a hyperproliferative response to B and T cell mitogens. Our data
show that IB--/- B cells indeed have
hyperproliferative responses to the B cell-specific stimuli.
Intriguingly, T cells exhibit impaired proliferative responses. These
observations are consistent with studies of p105-deficient lymphocytes
(26). Data accumulating from studies using the
transdominant inhibitor, mutant IB-, have shown that blocking
NF-B can enhance the number of activated T or B cells undergoing
apoptosis in response to stimulation (27). However, we
have not detected any difference in the number of activated
IB--/- T cells or B cells undergoing
apoptosis (data not shown). This finding is consistent with the studies
on RelA-deficient lymphocytes, which indicate that the actual numbers
of RelA-/- apoptotic cells are not
significantly different from those of RelA+/-
cells(21). Although we did not detect an effect of
IB--/- deficiency on apoptosis, we cannot
rule out the possibility that NF-B activity can prevent apoptosis in
activated T and B cells, and that this was not detected due to
functional redundancy within the NF-B/IB family. An alternative
explanation for the proliferative response of
IB--/- lymphocytes is the involvement of
NF-B in cell cycle control, a hypothesis that is supported by a
growing body of evidence (28). Thus, we speculate that
disruption of the IB- gene may lead to persistent nuclear NF-B
activity, which results in unregulated entry into the cell cycle in a
tissue-dependent manner.
Our data show that the basal serum levels of IgG1, IgG2a, IgA, and IgE
produced by IB--/- B cells were
significantly elevated, whereas the levels of IgM and light chain
were comparable to those produced by wild-type lymphocytes. These
results are consistent with the measured serum Ig levels in c-Rel-,
p50-, and RelA-deficient mice. The production of IgG1 and IgG2a in
c-Rel-/- mice; IgG1, IgG2a, and IgE in
p50-/- mice; and IgG1 and IgA in the irradiated
recipient of p65-/- fetal liver cells is
impaired. In addition, we have observed that serum levels of
light chain produced by IB--/- B cells is
elevated (data not shown). This finding is consistent with studies
showing that blocking NF-B activity leads to reduced expression of
light chain (29).
It is surprising that the levels of Ag-specific Ig produced by
IB--/- B cells are significantly reduced.
A lack of PNA-positive germinal centers correlates with the poor
production of specific Abs in the IB--/-
recipients. We hypothesize a lack of hypersomatic mutation and specific
Ig clonal expansion as the reason why Ag-specific Igs are significantly
reduced. It is unlikely that this result is due to the priming defects
of T cells, since the proliferative response of
IB--/- T cells from OVA-challenged mice
is comparable to that of wild-type T cells (Fig. 6D).
However, defects in the interactions between B cells and T cells cannot
be ruled out a contributing as factor, since Ig production by B cells
is affected in response to OVA, a T cell-dependent Ag. The observation
of impaired production of Ig by IB--/- B
cells is very similar to the findings in Bcl-3-deficient mice. The
proto-oncogene bcl-3 is another member of the IB family
that may regulate different genes or different physiological processes
than IB- (30, 31). In contrast with usual inhibitory
functions of the other known members of the IB family, it has been
postulated that Bcl-3 facilitates trans-activation
(32, 33, 34). However, the available data suggest that it
probably retains some biological functions similar to those of the
other family members in the proliferation or differentiation of B cells
(35).
In addition to results in Bcl-3-/- mice, the
role of NF-B/IB in the formation of germinal centers was also
shown in p52-deficient mice (36), p52/p50 double-deficient
mice (37), and transgenic mice expressing IB-N in
B cell lineages (25). It is interesting that NF-B
activity seems to be capable of opposing roles (agonist or antagonist)
in the generation of germinal centers. The molecular mechanism of
generation of germinal centers remains largely unknown. Impaired
germinal center formation results in poor production of specific Igs.
Thus, it is reasonable to speculate that differentiation of B cells and
formation of germinal centers may share a common etiology. Studies on
the signal transduction pathways have revealed that several molecules,
including TNF-, lymphotoxin-, CD40, and CD40 ligand, are
important for the generation of germinal centers (38, 39).
Not surprisingly, the NF-B/IB family is involved in the signal
transduction pathways of these molecules (27). The role of
CD40 signaling in B cell terminal differentiation remains
controversial. Animals deficient in either CD40 or its ligands are
unable to form germinal centers or make class-switched Abs after
immunization with T cell-dependent Ags (23, 40). Several
in vitro experiments indicate that CD40 signaling promotes B cell
differentiation (41, 42, 43). However, other experiments have
indirectly suggested that cross-linking of CD40 may actively inhibit B
cells from differentiating into Ab-secreting cells (23, 44, 45). Taking CD40 as an example, we speculate that NF-B
activity may be necessary for B cell differentiation to a certain
stage, but after this stage unregulated NF-B activity may prevent
the terminal differentiation of B cells. Although our histochemical
analysis of spleen from IB--/- fetal
liver recipients did not show a defect in the formation of the network
of FDCs, our current data do not rule out the possibility that
NF-B/IB activity may play an important role in the function of
FDCs, and loss of IB--/- may alter FDCs
in such a manner as to interfere with germinal center formation. Our
current model provides a good system in which to explore the effects of
unregulated NF-B activity on the formation of germinal centers and
Ag-specific B cell differentiation.
|
Acknowledgments |
|---|
B probes for EMSA. We also thank Trish Labosky and Bridgid Hogan
for providing ES cells, and David Martin for technical advice. We are
grateful to Dr. M. H. Kosco-Vibois for generously providing
anti-FDC-M1 Ab, and to Drs. Singh and Rodey for the training in the
technique of fetal liver cell transfer. Special thanks to members of
the Kerr laboratory and to Drs. Earl Ruley, Luc Van Kaer, Terry
Dermody, Barney Graham, Roland Stein, Gene Oltz, and Mark Boothby at
Vanderbilt University for helpful comments on the manuscript. Finally,
we acknowledge the Vanderbilt Transgenic Mouse/ES Cell Shared Resource
for generating the animals used in this study. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Fiona E. Yull, Vanderbilt University School of Medicine, Department of Microbiology and Immunology, MCN A4314, 1161 21st Avenue South, Nashville, TN 37232.
3 Abbreviations used in this paper: ES, embryonic stem; PNA, peanut agglutinin; HSA, heat-stable Ag; FDC, follicular dendritic cell.
Received for publication April 10, 2000. Accepted for publication August 14, 2000.
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