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B Kinase 
Is Critical for B Cell Proliferation and Antibody Response
Division of Hematology-Oncology, Department of Medicine, Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390
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Abstract |
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B proteins are critical in the regulation of the immune
and inflammatory response. Stimulation of the NF-B pathway leads to
increases in I-B kinase 
(IKK) kinase activity to result in
the enhanced phosphorylation and degradation of I-B and the
translocation of the NF-B proteins from the cytoplasm to the
nucleus. In this study, a dominant-negative IKK mutant expressed
from the IgH promoter was used to generate transgenic mice to address
the role of IKK on B cell function. Although these transgenic mice
were defective in activating the NF-B pathway in B cells, they
exhibited no defects in B lymphocyte development or basal Ig levels.
However, they exhibited defects in the cell cycle progression and
proliferation of B cells in response to treatment with LPS,
anti-CD40, and anti-IgM. Furthermore, selective defects in the
production of specific Ig subclasses in response to both T-dependent
and T-independent Ags were noted. These results suggest that IKK is
critical for the proliferation of B cells and the control of some
aspects of the humoral response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B comprises a family of transcription factors that play a
critical role in the control of immune and inflammatory responses
(1, 2). Members of the NF-B family including p50, p52,
p65, RelB, and c-Rel are present predominantly in the cytoplasm, where
they are bound to a group of inhibitory proteins known as I-B
(1, 2, 3, 4). In response to a variety of stimuli, including the
cytokines TNF-
and IL-1, the I-B proteins are phosphorylated,
leading to their ubiquitination and degradation by the 26S proteasome
(4). This process results in the nuclear translocation of
NF-B and the binding of these proteins to promoter elements in a
variety of genes and activation of their expression.
One of the major steps involved in the control of the NF-B pathway
is the phosphorylation of the I-B proteins by the I-B kinases
(5, 6, 7, 8, 9, 10). The I-B kinase complex consists of three
proteins: I- kinase
(IKK)2 (5, 8, 9), IKK (5, 6, 8), and IKK
/NF-
essential modulator (NEMO) (11, 12, 13, 14). IKK and IKK are
kinases that are each capable of phosphorylating I-B
(5, 6, 7, 8, 9), whereas IKK/NEMO is a scaffold protein that is
critical for IKK and IKK kinase activity (11, 12, 13, 14).
Treatment of B cells with a variety of agents, including cytokines and
LPS in addition to the CD40 ligand on the surface of T cells, increases
IKK kinase activity, resulting in the phosphorylation and subsequent
degradation of I-B and NF-B nuclear translocation
(10).
IKK and IKK have a high degree of amino acid homology and a
similar domain organization, which includes an N-terminal kinase
domain, a leucine zipper that facilitates their heterodimerization and
homodimerization, and a C-terminal helix-loop-helix domain (5, 6, 7, 8, 9, 15). However, IKK and IKK appear to have different
functions. Homozygous disruption of the IKK gene results in marked
decreases in NF-B activation and embryonic lethality in mice
(16, 17, 18). These mice die of severe apoptosis of the liver
due to their failure to activate NF-B-responsive genes that help to
prevent apoptosis. In contrast, IKK disruption plays only an
auxiliary role on activation of the NF-B pathway. Mice carrying
homozygous deletions of this gene die shortly after birth due to severe
skin and skeletal abnormalities (19, 20, 21). The ability of
IKK to regulate epidermal proliferation suggests that this kinase
can most likely activate signal transduction pathways other than those
involved in activating NF-B (22). Finally, homozygous
disruption of IKK/NEMO leads to embryonic lethality due to hepatic
apoptosis much like that seen in the IKK-deficient mice
(23, 24, 25). These results indicate that the IKK function is
critical for a variety of biologic processes.
The NF-B proteins are critical for the regulation of immune function
(1, 2, 10). For example, they regulate the expression of a
variety of genes encoding cytokines and cytokine receptors, chemokines,
cell adhesion molecules, and cell surface receptors that are critical
for T and B lymphocyte function (26). Targeted
inactivation of genes encoding individual NF-B subunits demonstrates
the importance of these proteins in regulating the immune system
(27). Gene disruption of single NF-B subunits in mice,
including p105/p50 (28, 29), p100/p52 (30, 31), c-Rel (32), RelA (33), and RelB
(34), leads to reduced B and T cell proliferation and
immune defects, but no major defects in the maturation of T and B
cells. However, mice lacking multiple NF-B subunits, including
p105/p50 and p100/p52 (35), p105/p50 and RelB (36, 37), RelA and c-Rel (38), and p105/p50 and RelA
(39), have more severe defects in B and T cell development
than do mice with mutations of single NF-B subunits. These results
indicate that the NF-B pathway is critical for the function of both
B and T lymphocytes.
Because IKK-deficient mice die in utero, the role of IKK on B
cell development and function has not been addressed
(16, 17, 18). Transgenic mice containing dominant-negative
(DN) mutants in NF-B regulatory proteins such as I-B have
previously been useful in defining the role of the NF-B pathway in
the immune system (27). To investigate the role of a
DNIKK mutant on regulating B cell development and differentiation in
transgenic mice, we inserted this mutant downstream of the IgH promoter
and enhancer and characterized transgenic mice expressing this protein.
In this study, we demonstrate that inhibiting IKK function does not
affect B cell development, but results in marked defects in cell cycle
progression, B cell proliferation, and in the humoral response to both
T-independent and T-dependent Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mutant in B
cells
An expression vector containing the B cell-specific Ig H chain
(IgH) promoter and enhancer has been described (40). The
rat insulin intron A and an SV40 polyadenylation signal were also
inserted into this vector. A human IKK cDNA containing substitutions
of serine residues 177 and 181 with alanine and an amino-terminal Flag
epitope (8) was inserted into a NotI site
between the intron and the poly(A) elements. The linearized transgene
IgH/DNIKK was microinjected into the pronuclei of ICR (CD-1) strain
in the Transgenic Core Facility at University of Texas Southwestern
Medical Center, and mice were maintained in a specific pathogen-free
colony. The presence of the transgene was confirmed by PCR and Southern
blot analysis.
Flow cytometry analysis
Splenocytes from either wild-type or transgenic littermates were
placed in RPMI media, washed twice, and resuspended in buffer
containing PBS with 1% FBS. Approximately 5 x
105 cells were incubated with fluorescent Abs: PE
B220, FITC Thy-1.2, FITC IgM, FITC Ig, FITC IgD,
biotin IgM, biotin B220, FITC CD21, PE CD23, APC
streptavidin, and PE streptavidin (BD PharMingen, San Diego, CA).
Fluorescence analysis was performed using a FACSCalibur flow cytometer
(BD Biosciences, San Diego, CA).
Purification of B cells from mouse spleen
To purify B cells from the spleens of 8- to 12-wk-old mice, the MACS system (Miltenyi Biotec, Auburn, CA) was utilized. The splenocytes were incubated with anti-CD43 microbeads and separated into CD43 positive (non-B cells) and CD43 negative (B cells), according to the manufacturer’s instructions. Purified B cells were either stimulated with B cell mitogens or used to make whole cell extract. To purify small resting B cells from the spleen, T cells in the unfractionated splenocytes were depleted by cytotoxic elimination using anti-CD90 Ab and Low-Tox-M rabbit complement (Cedarlane, Hornby, Ontario, Canada), and the remaining cells were fractionated through Percoll gradients (41). Small resting B cells which sediments between 75% and 100% Percoll were collected and used in in vitro cultures.
Immunoprecipitation and Western blot analysis
To prepare whole cell extracts from either unfractionated
splenocytes, purified B cells, or non-B cells, the cells were lysed in
TNE buffer (1% Triton X-100, 10 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM
EDTA) containing a protease inhibitor mixture (Roche, Somerville, NJ).
Cell lysates containing 10 µg protein were incubated with
anti-Flag Ab (Sigma, St. Louis, MO), followed by incubation with
protein G-Sepharose beads (Sigma) and immunoblotting using an IKK
rabbit polyclonal Ab (sc-7607; Santa Cruz Biotechnology, Santa Cruz,
CA). The B cells were also analyzed by immunoblotting using a rabbit
polyclonal Ab directed against either p100/p52 (sc-298), p105/p50
(sc-7178), or p65 (sc-372) obtained from Santa Cruz.
RT-PCR analysis of IKK mRNA isolated from the B cells of
wild-type and transgenic mice
B cells were purified from the splenocytes of both transgenic
and wild-type mice using the MACS magnetic sorting system. Total RNA
was extracted from these cells and analyzed by semiquantitative RT-PCR
analysis. The oligonucleotide primers used to amplify a 341-bp
homologous fragment from both mouse IKK and human DNIKK included
the sense primer, 5'-gtgtcagctgtatccttc-3' and the antisense primer,
5'-gctccacagcctgctcc-3' with the sense primer end labeled with
[-32P]ATP. Oligonucleotide primers for
amplifying GAPDH mRNA have been described previously (42).
The PCR products were analyzed by digestion with EcoRI,
which cuts the cDNA fragment amplified from mice expressing human
DNIKK to generate two fragments of 176 and 165 bp. Following gel
electrophoresis and autoradiography, the intensity of the species was
measured by PhosphorImager analysis (Cyclone; Packard,
Meriden, CT) and compared with that of the 341-bp mouse fragment.
Stimulation of primary B cells and EMSAs
Magnetically purified B cells were incubated with either RPMI alone or RPMI containing the F(ab')2 fragment of anti-IgM (10 µg/ml; Jackson ImmunoResearch, West Grove, PA), LPS (10 µg/ml; Difco, Detroit, MI), or PMA (50 ng/ml; Sigma) and ionomycin (200 ng/ml; Sigma) for 45 min to 2 h, respectively. The cytoplasmic and nuclear extracts of the nonstimulated and stimulated B cells were prepared and analyzed according to published methods (43, 44).
To detect NF-B binding, a 32P-labeled
oligonucleotide probe containing the class I MHC B site
(45) or NF-Y binding site was incubated with the nuclear
extracts. The binding reaction contained 60,000 cpm of the radiolabeled
probe, 4–5 µg nuclear extract, 500 ng poly(dI-dC) (Pharmacia,
Piscataway, NJ), 10 µg BSA, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1%
Nonidet P-40, 5% glycerol, and 5 mM DTT in a final volume of 20 µl.
Reactions were incubated at room temperature for 30 min and subjected
to electrophoresis on a 5% native gel in 0.5x Tris-buffered
EDTA. For supershift assays, 5 µg goat polyclonal Ab directed
against p65 or normal goat sera was added to the binding reactions and
incubated for 30 min on ice before the samples were subjected to gel
electrophoresis. The gels were dried and exposed to x-ray film and
quantified by PhosphorImager analysis.
In vitro proliferation assay and Ig production of primary B cells
Small resting B cells were prepared from the spleens of two
transgenic mice and two wild-type littermates. The cells were pooled
and resuspended in RPMI and plated in 96-well plates in quadruplicate
at 105 cells/well for each condition. LPS (10
µg/ml), anti-IgM F(ab')2 (10 µg/ml),
anti-CD40 (5 µg/ml), IL-4 (100 U/ml), IL-5 (0.1%)
(46), and IFN- (10 ng/ml) (R&D Systems, Minneapolis,
MN) were added to the cells in each well, cultured for 3 days, and
pulsed with [3H]thymidine (1 mCi/well)
overnight. The [3H]thymidine incorporated was
quantified by scintillation counting, and the secreted Ig in the
culture supernatant in day 6 cultures were measured by ELISA using
class-specific antisera.
Cell cycle analysis
Cell cycle analysis of B cells was performed using a BrdU (5-bromo-2-deoxyuridine) Flow Kit (PharMingen). Resting B cells were cultured in RPMI media supplemented with 10% FBS in the presence of LPS (10 µg/ml), anti-IgM F(ab')2 (10 µg/ml), or anti-CD40 (5 µg/ml) for 60 h. Cells were then pulsed with BrdU for 40 min and processed for BrdU and 7-amino actinomycin D (7-AAD) staining, according to the manufacturer’s instructions. Flow cytometry analysis was performed using a FACSCalibur (Becton Dickinson).
Apoptosis in quiescent and activated B cells
Resting B cells were cultured in RPMI containing 10% FBS or in the presence of either LPS (10 µg/ml), anti-IgM F(ab')2 (10 µg/ml), or anti-CD40 (5 µg/ml). Cells were harvested at 24 and 48 h. Apoptotic cells were quantified using an Annexin VFITC Apoptosis Detection kit (PharMingen), according to the manufacturer’s instructions.
Semiquantitative RT-PCR analysis
To compare the relative mRNA levels of µM, µS, 3, and
2a in stimulated B cells from mutant and normal mice,
semiquantitative RT-PCR analysis was performed as described
(47). A total of 1 x 106
resting B cells was stimulated with either LPS (10 µg/ml) alone or
both LPS and IFN- (10 ng/ml) for a period of 3–4 days. Total RNA
was prepared from the cells using an RNeasy Kit (Qiagen, Chatsworth,
CA). Equal portions of total RNA from each sample were reverse
transcribed, and titrations were performed so that PCR products
corresponding to the µM, µS, 3, and 2a transcripts were
within linear range. These same dilutions of the cDNA samples were used
as templates in the RT-PCR reactions. For µM, the sense primer was
5'-ggtatgcaaaatccactacggaggc-3', and the antisense primer was
5'-gataaaagctggagggcaac-3'; for µS, the same sense primer was used
and the antisense primer specific to the µS exon,
5'-gacatgatcagggagacattgtac-3', was used; for 2a and 3, a
sense primer with the sequence 5'-tatggactactggggtcaag-3' was used,
while the antisense primer for 2a was 5'-ggccaggtgctcgaggtt-3', and
the antisense primer for 3 was 5'-aatagaacccagactgcagga-3'. One of
the primers was end labeled with [-32P]ATP,
and the PCR products were subjected to electrophoresis on a 1% agarose
gel and quantified by PhosphorImager analysis.
In vivo response to T-independent and T-dependent Ags
Immunization of mice with T-independent and T-dependent Ags was performed as previously described (48). Briefly, littermates of 9- to 11-wk-old mice were injected i.p. with the type 2 T-independent Ag trinitrophenyl (TNP)-Ficoll (Biosearch Technologies, Noveto, CA) at 40 µg/mouse or the type 1 T-independent Ag TNP-LPS (Sigma) at 20 µg/mouse in 100 µl PBS. Sera were collected from the tail vein at day 0 (before injection) and day 14. TNP-specific IgM, IgG3, and IgG2a were measured by ELISA. To determine the Ab response to the T-dependent Ag, TNP-keyhole limpet hemocyanin (KLH) (48), 100 µg of this Ag was dissolved in 100 µl PBS with Ribi adjuvant (Corixa, Seattle, WA) and was injected i.p. into each littermate. Sera were collected at days 0 (before injection) and 14, and TNP-specific IgM, IgG1, and IgG2a were measured by ELISA.
Enzyme-linked immunosorbent assay
ELISA was performed as described previously (48). To determine the concentrations of total IgM, IgG3, IgG2a, and IgG1 in the culture supernatant or the sera of naive mice, 96-well MicroTest III flexible plates (Becton Dickinson) were coated with AffiniPure goat anti-mouse IgG and IgM Abs (Jackson ImmunoResearch). TNP-BSA (48) was used to detect TNP-specific Ig in the sera of immunized mice. Ig levels in the mouse sera were determined at three dilutions of each sera in duplicate. The baseline level of TNP-specific Ig in the sera from mice before immunization was subtracted from the levels in the TNP-immunized mice to determine TNP-specific responses. Isotype standards (IgM, IgG1, IgG3, and IgG2a), HRP-conjugated goat anti-mouse IgM, IgG3, IgG2a, IgG1, and Ig(H+L) Abs, and the substrate ABTS were obtained from Southern Biotechnology Associates (Birmingham, AL), and the plates were read on a Universal Microplate Reader (Bio-Tek Instruments, Burlington, VT) at 405 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein in B
lymphocytes
In an attempt to inhibit inducible NF-B activity specifically
in B lymphocytes, we generated transgenic mice that expressed a DN form
of human IKK (A176/181) (8) that was inserted between
the B cell-specific Ig heavy (IgH) promoter and enhancer (40, 49) (Fig. 1
A). The
IKK cDNA contained an amino-terminal Flag epitope to facilitate its
detection in murine B cells. Previous studies have demonstrated that an
IKK protein in which serine residues 177 and 181 in the
mitogen-activated protein 3 kinase activation loop were substituted
with alanine has a DN phenotype that inhibits NF-B activation in
response to treatment with proinflammatory cytokines such as TNF-
and IL-1 (5, 6, 8, 50, 51). Since mouse and human IKK
have greater than 90% amino acid identity, we assumed that this
DNIKK mutant should be able to inhibit the function of endogenous
mouse IKK and thus alter NF-B activation in mouse B
lymphocytes.
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DN construct shown in Fig. 1
A was
microinjected into pronuclei of the murine CD-1 strain. Southern blot
analysis indicated that three founders designated A, B, and C had an
integrated transgene, although only founder C transmitted the transgene
(Fig. 1B). Next we addressed whether the DNIKK protein
was expressed in splenocytes isolated from transgenic mice derived from
founder C. These cells were either unfractionated or fractionated into
either B lymphocytes or non-B cells that contained predominantly T
cells, macrophages, and myeloid cells. Western blot analysis performed
with a mAb directed against the Flag epitope indicated that there was
more DNIKK protein present in B lymphocytes from the transgenic mice
(Fig. 1C, lane 3) than in the same number of
cells from unfractionated splenocytes (Fig. 1C, lane
2). There was no detectable DNIKK expression in non-B cells
isolated from this transgenic mouse (Fig. 1C, lane
4).
In an attempt to compare the expression level of DNIKK with that of
the endogenous IKK in the B cells of the transgenic mice, we used
RT-PCR analysis to assay the levels of IKK mRNA (Fig. 1D). We chose to amplify a 341-bp fragment that includes
sequences encoding the leucine zipper motif of IKK and that contains
an EcoRI site in the human, but not the mouse sequence. The
176-bp fragment generated by EcoRI digestion of the
32P-labeled PCR product represents the mRNA level
of DNIKK expressed in transgenic B cells. Thus, we could compare the
relative levels of the expression of DNIKK and the endogenous murine
IKK gene. PhosphorImager analysis demonstrated that there was 1.5-
to 2-fold more of the fragment from the human DNIKK than from the
endogenous murine IKK (Fig. 1D, lane 8).
Residual DNIKK mRNA was also detected in the splenocytes of the
transgenic mice in which the majority of B cells have been removed, and
this is most likely due to residual B cell contamination in this
fraction of cells (Fig. 1D, lane 6). The
transgenic mice that expressed the DNIKK protein were housed in a
specific pathogen-free colony and used in the phenotypic studies
described below.
Reduced NF-B DNA binding in B lymphocytes isolated from DNIKK
transgenic mice
Next we addressed whether DNIKK expression altered NF-B
activation in response to various agents that are known to stimulate
this pathway. Previous studies have demonstrated that LPS,
anti-IgM, and PMA and ionomycin potently stimulate NF-B
DNA-binding properties in B lymphocytes (52, 53, 54). To
analyze the effects of DNIKK expression on NF-B DNA binding, we
performed EMSAs using nuclear extracts prepared from both nonstimulated
and stimulated B lymphocytes isolated from wild-type and DNIKK
transgenic mice. In B cells isolated from wild-type mice, treatment
with the F(ab')2 fragment of either anti-IgM,
LPS, or PMA and ionomycin strongly induced NF-B DNA-binding activity
(Fig. 2, A and B).
In contrast, there was significantly less NF-B DNA binding in B
cells isolated from the transgenic mice (Fig. 2, A and
B). There was only modest inhibition of NF-B DNA binding
in anti-CD40-treated B cells isolated from DNIKK mice (data not
shown). NF-B binding in untreated wild-type and transgenic B cells
was similar in four different experiments. There was little difference
in the DNA-binding properties of nuclear extracts prepared from B
lymphocytes isolated from the wild-type and transgenic mice using the
control NF-Y probe (Fig. 2, A and B). The p65 Ab,
but not the normal goat IgG, resulted in a supershift of the NF-B
DNA-binding protein complex, indicating the presence of p65 in this
complex (Fig. 2C). These results indicated that NF-B
activation was significantly inhibited in B lymphocytes isolated from
the DNIKK transgenic mice.
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may prevent
activation of the NF-B pathway is by inhibiting IKK function.
Recently, IKK was demonstrated to be important in the processing of
the p100 NF-B subunit to generate the p52 subunit (55).
When IKK is absent from B cells, this processing is markedly
reduced, leading to increases in p100 and corresponding decreases in
p52. The deficiency in p52 results in marked defects in the development
and function of B cells. Thus, it was important to address whether
there was a reduced level of p52 in B cells isolated from DNIKK
transgenic mice. Western blot analysis of extracts prepared from B
cells isolated from wild-type and DNIKK mice revealed no differences
in the levels of the NF-B subunits p100, p52, p105, p50, and p65
(Fig. 2D). Thus, DNIKK did not appear to interrupt IKK
function to result in the decreased processing of p100 nor alter the
level of other NF-B subunits.
Normal B cell development in DNIKK transgenic mice
Since the DNIKK inhibited NF-B activation in the B cells of
the transgenic mice, we asked whether the expression of DNIKK
altered B cell development in the transgenic mice. The DNIKK
transgenic mice have spleens of normal size and normal architecture, as
demonstrated by H&E staining (data not shown). To confirm that B
lymphocyte development in these mice was normal, the surface markers on
the splenocytes isolated from these mice were analyzed by flow
cytometry. A representative analysis is shown in Fig. 3. The DNIKK transgenic mice have
similar percentages of B and T cells as compared with wild-type mice,
as reflected by similar B220 and Thy-1.2 surface expression (Fig. 3A). The percentage of B cells expressing surface IgM and
Ig was also similar in the transgenic and wild-type mice (Fig. 3A). Since surface IgD expression reflects the state of
maturation and activation of B cells, we also compared the surface
expression of IgM and IgD (Fig. 3B). There were
comparable percentages of
IgDhigh/IgMhigh
(upper right) and
IgDlow/IgMhigh (upper
left) populations of cells in transgenic and wild-type mice,
indicating that the DNIKK protein does not significantly alter the
maturation and activation of B cells.
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function in B cells from the transgenic
mice affected the accumulation of this subset of B cells. As shown in
Fig. 3C, the B cell population defined by staining with CD21
and CD23
(B220+CD21highCD23low)
in our transgenic mice was similar to that of wild-type mice in three
different experiments, indicating that inhibition of the NF-B
pathway in B cells does not affect the development of marginal zone B
cells. In summary, our results are consistent with previous studies of
c-rel and RelA-deficient mice, demonstrating that inhibition
of NF-B function does not cause marked defects in B lymphocyte
development (28, 32, 33).
B cells from DNIKK mice exhibit proliferative defects
Although B cell development is normal in the DNIKK transgenic
mice, previous data demonstrating that NF-B is involved in
regulating B cell function prompted us to analyze the B cell
proliferative responses in these mice. Resting B cells isolated from
the splenocytes of wild-type and transgenic mice were stimulated over a
72-h period with either LPS, anti-CD40, or anti-IgM, and their
proliferation was monitored by [3H]thymidine
incorporation. A representative experiment is shown in Fig. 4. Stimulation of transgenic B cells with
LPS resulted in only 
30% of the proliferation seen in the wild-type
B cells (Fig. 4A). In contrast, B cell proliferation induced
by either anti-CD40 or anti-IgM was less significantly
compromised in the DNIKK transgenic mice, with a
[3H]thymidine incorporation of 70% and 55% of
normal levels, respectively (Fig. 4B). Furthermore, the
proliferation defects seen with anti-CD40 and anti-IgM were
partially compensated by the addition of IL-4, which further enhances B
cell proliferation (Fig. 4B). These results, which were
repeated three times, indicated that IKK is involved in the B cell
proliferative responses induced by LPS as well as anti-CD40 and
anti-IgM.
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It was important to address whether the decreased
[3H]thymidine incorporation in LPS-,
anti-IgM-, and anti-CD40-treated B lymphocytes isolated from
the transgenic mice was due to either defective cell cycle progression
or increased cell death. B cells were isolated from transgenic and
wild-type splenocytes and cultured with either LPS, anti-IgM, or
anti-CD40 for 60 h and pulsed with bromodeoxyuridine (BrdU)
for 40 min. Flow cytometry analysis was then performed to determine the
amount of BrdU incorporation into newly synthesized DNA, and 7-AAD was
used to detect total cellular DNA content. Wild-type mice exhibited a
marked increase in the number of cells present in the S phase in
response to treatment with either LPS, anti-IgM, or anti-CD40
(Fig. 5). The percentage of cells in the
S phase is shown on the top line within each panel and represents the
cells shown in the R2 grid (Fig. 5). In contrast, there was a reduction
in the number of transgenic B cells in the S phase following treatment
with these agents (Fig. 5). This decrease in the percentage of B cells
in the transgenic mice was associated with a corresponding increase in
the percentage of B lymphocytes in the
G0/G1 phase of the cell
cycle as compared with wild-type mice. These results that were repeated
three times are consistent with the
[3H]thymidine incorporation assays, which
indicate that B cell proliferation induced by LPS is more dependent on
NF-B than that induced by anti-IgM and anti-CD40.
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B subunit
have a higher rate of apoptosis (58, 59). Next we
determined the percentage of apoptosis in B cells isolated from
wild-type and DNIKK mice either when untreated or when treated with
LPS, anti-IgM, or anti-CD40 (Table I). Annexin V staining of these B cells
indicated that there were similar percentages of apoptotic B cells
isolated from wild-type and transgenic mice. These results indicated
that the decreased proliferation of transgenic B cells in response to a
variety of different mitogens was not due to an increased amount of
apoptosis.
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transgenic mice exhibit defects in in vitro
Ig secretion
Next we determined whether inhibition of IKK activity altered
in vitro Ig secretion and class switching of B cells. Resting B cells
isolated from wild-type or DNIKK transgenic mice splenocytes were
cultured in the presence of either LPS; LPS and IFN-; or
anti-CD40, IL-4, and IL-5 over a 6- to 7-day period. IgM (Fig. 6A), IgG2a and IgG1 (Fig. 6B), and IgG3 (Fig. 6C) levels in the culture
supernatants were measured by ELISA. Total IgM and IgG3 secretion was
reduced approximately 50% in B cells from the transgenic mice as
compared with wild-type mice in the LPS-stimulated culture (Fig. 6, A and C), while IgG2a secretion was reduced to
20% of that seen in wild-type B cells in the presence of LPS and
IFN- (Fig. 6B). The synthesis of IgM and IgG1 in response
to treatment with anti-CD40 and both IL-4 and IL-5 was also assayed
in transgenic and wild-type B cells. In the presence of IL-4 and IL-5,
anti-CD40 stimulation induced similar levels of IgM (Fig. 6A) as well as IgG1 (Fig. 6B), indicating that
signaling through CD40 and switching to IgG1 are much less dependent on
NF-B activation than is LPS signaling.
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The decreased amounts of IgM, IgG3, and IgG2a in the cultures of
the DNIKK B cells in response to LPS treatment could be due to a
decreased number of Ig-secreting cells or to a decreased capacity for
Ig secretion from individual cells. When B cells were counted following
stimulation with either LPS or LPS and IFN-, there were 2.9 times
more wild-type cells than transgenic cells following LPS stimulation
and 4.6 times more wild-type cells than transgenic cells in the
presence of LPS and IFN-. These results suggested that proliferative
defects were largely responsible for the decreased Ig levels present in
the transgenic mice.
In an attempt to further distinguish between these two
possibilities, we performed semiquantitative RT-PCR to compare the
synthesis of mRNA for the secretory forms of µ, 3, and 2a heavy
chain from the B cells of wild-type and the DNIKK mice (Fig. 7). The levels of mRNA from each Ig
isotype should correlate with the level of secreted Ig protein. First,
it was important to demonstrate that the RT-PCR was performed in
the linear range relative to RNA abundance for µM, µS,
2a, and 3 (Fig. 7A). Next we analyzed RNA prepared
from resting and LPS-stimulated B lymphocytes isolated from wild-type
and DNIKK transgenic mice (Fig. 7B). Since surface IgM
(µM) expression is relatively constant after B cell stimulation and
roughly correlates with the cell number (46, 60), the
abundance of mRNA corresponding to each of the secreted Ig in the B
cells can be adjusted for cell number by estimating the ratio of its
intensity to that of µM. This analysis demonstrated that there were
similar ratios of µS/µM, 3/µM, and 2a/µM mRNAs in the LPS
and LPS and IFN--stimulated B lymphocytes isolated from the
transgenic and normal mice (Fig. 7C). These results indicate
that when corrected for differences in cellular proliferation, the
synthesis of IgM, IgG3, and IgG2a from transgenic B cells is similar to
that from wild-type B cells. Thus, the signals for isotype switching to
3 and 2a are not reduced in B cells isolated from transgenic mice
that are activated by polyclonal stimulators such as LPS.
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transgenic mice display normal basal Ab production, but
impaired Ab production in response to specific Ags
The restriction of the DNIKK defect to B lymphocytes allowed us
to determine whether in vivo B cell responses were compromised in the
presence of T cells that had intact NF-B function. To determine
whether inhibition of the NF-B pathway in B cells plays a role in
regulating basal Ab production, we analyzed serum Ig levels in naive
mice. Although there was a somewhat lower level of serum IgM in the
transgenic mice, the levels of IgG2a, IgG3, and IgG1 in the serum of
naive wild-type and transgenic mice were similar (Fig. 8). These results indicate that blocking
the activation of the NF-B pathway by introduction of the DNIKK
protein into B cells does not substantially affect basal Ig
production.
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function altered the
responsiveness of mature B cells to specific antigenic challenge.
DNIKK transgenic mice and their wild-type littermates were immunized
with both a type 1 T-independent Ag (TNP-LPS) and a type 2
T-independent Ag (TNP-Ficoll) as well as a T-dependent Ag (TNP-KLH).
The levels of specific Abs against TNP were tested by ELISA, and the
results are summarized in Fig. 9.
TNP-specific IgM was only slightly lower in the transgenic mice as
compared with wild-type littermates in mice immunized with TNP-LPS,
whereas TNP-specific IgG2a and IgG3 levels were similar (Fig. 9A). These results indicate that in vivo class switching of
TNP-specific Abs to IgG2a and IgG3, which depends on the mitogenic
properties of LPS, is not affected following immunization with a type 1
T-independent Ag.
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B). These
results indicate that the response to the type 2 T-independent Ag
TNP-Ficoll requires NF-B activity.
Surprisingly, the TNP-specific IgG1 and IgG2a, but not the IgM
levels were significantly lower in the transgenic mice immunized with
the T-dependent Ag TNP-KLH as compared with the wild-type mice (Fig. 9C). This result indicates that class switching to 1 and
2a in response to in vivo immunization with a T-dependent Ag is
altered in the DNIKK transgenic mice. Taken together, these results
indicate that introduction of a DNIKK mutant does not change the
basal Ab production, but does play a role in the humoral response to a
type 2 T-independent Ag. In addition, switching to downstream isotypes
in response to both type 2 T-independent and T-dependent Ags may be
affected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway is critical for the regulation of immune
function (1, 2, 3). Since disruption of the IKK and IKK
genes results in early lethality of mice, we studied the role of a
DNIKK mutant expressed exclusively in B cells while maintaining
intact T cell function. The use of a DNIKK mutant does not totally
inhibit IKK activity, but instead results in competition with the
wild-type IKK protein. There are several potential steps at which
the DNIKK protein can inhibit the NF-B pathway. First, the
DNIKK protein can interact with wild-type IKK to form inactive
homodimers. Second, it may bind to IKK/NEMO and prevent its
interaction with wild-type IKK/IKK. Alternatively, DNIKK may
bind to IKK to inactivate its function in generating p52
(55). However, our results demonstrate intact p52
processing in DNIKK mice, suggesting that this aspect of IKK
function is intact. Finally, DNIKK can form a complex with IKK
and IKK/NEMO to bind the I-B proteins, and thus prevent IKK
phosphorylation of this substrate (61). It is likely that
the interaction of DNIKK with multiple components of the IKK complex
is involved in the inhibition of the NF-B pathway. Although the
mechanism of DNIKK inhibition of the NF-B pathway remains to be
elucidated, this mutant allowed us to address the role of IKK on B
cell development and function under conditions in which the function of
the wild-type protein is partially blocked.
The results of our study indicate that complete activation of the
NF-B pathway by IKK is not essential for the development of B
cell subsets. Since there is most likely residual IKK function in
these transgenic mice, the complete loss of this kinase may result in
more profound effects on B cell development. In addition, we did not
detect abnormalities in lymph node or splenic architecture as has
previously been noted with disruption of specific NF-B subunits
(data not shown). Consistent with these findings, basal Ig levels were
also not altered. Although previous data suggest that blocking specific
components of the NF-B pathway can result in enhanced apoptosis in B
cells (59, 62, 63), we did not detect increased apoptosis
of B cells when cells were either unstimulated or treated with LPS,
anti-CD40, or anti-IgM. Our failure to detect increased
apoptosis when IKK function is altered in B cells may be due to
residual NF-B function or the fact that B cell apoptosis is less
dependent on NF-B than apoptosis in either hepatocytes or T cells,
both of which undergo enhanced apoptosis in IKK knockout animals
(6, 7, 8).
However, our results indicate that IKK is critical for B cell
proliferation in response to various mitogens and in vivo Ig production
in response to both T-dependent and T-independent Ags. B cells isolated
from the transgenic mice expressing the DNIKK exhibited
proliferative defects in response to treatment with LPS, anti-IgM,
or CD40 ligand. The DNIKK protein interfered with the proliferation
of B cells induced by LPS more than that induced by anti-IgM and
anti-CD40. This result is consistent with those obtained from p50
knockout mice, which demonstrate that proliferation of B cells from
these mice could be induced by Ag receptor engagement more efficiently
than following treatment with LPS (28). Since NF-B
activation is not completely inhibited by the DNIKK, it is possible
that the remaining NF-B activity induced by anti-IgM and
anti-CD40, but not LPS, is sufficient to transactivate genes
involved in B cell proliferation. It is also possible that B cell
proliferation induced by anti-IgM and anti-CD40 can partially
bypass the NF-B pathway.
The defects in B cell proliferation in response to LPS, anti-IgM,
and anti-CD40 are associated with reduced cell cycle progression
from the G1 to S phases of the cell cycle, as
determined by BrdU labeling. Similar defects have been noted in B cells
isolated from mice in which other NF-B subunits such as p50 were
disrupted (58). These results indicate that the NF-B
pathway is involved in the expression of genes that are critical for
cell cycle progression following mitogenic stimulation of B
lymphocytes. For example, NF-B has been shown to be involved in the
expression of cyclin D1, which, in combination with CDK4, is critical
for the phosphorylation of the retinoblastoma protein, leading to the
progression of cells from the G1 to the S phase
(64). Additional studies are underway to identify factors
regulated by NF-B that are involved in the cell cycle progression of
B cells.
Activation of the NF-B pathway in B lymphocytes can be induced by
engagement of either the B cell receptor (BCR), CD40, or stimulation
with LPS. The engagement of the BCR initiates signaling pathways
mediated through nonreceptor protein tyrosine kinases, including Fyn,
Lyn, Syk, and Bruton’s tyrosine kinase (BTK) (65). Both
BCR-dependent and independent pathways can lead to NF-B activation,
and there is most likely cross talk between these pathways. For
example, LPS stimulation of B cell proliferation is mediated by the
Toll-like receptor 4 in addition to other related receptors
(66). Mice with mutations in these receptors exhibit
defects in LPS-induced cell cycle progression and proliferation, which
are most likely due at least in part to decreased activation of BTK and
other downstream kinases (67, 68). Although the in vitro
proliferation of transgenic B cells following LPS treatment is
defective, the in vivo response of transgenic mice following
immunization with the type 1 T-independent Ag TNP-LPS is intact. Thus,
TNP-LPS may induce signals through both Toll-like receptor 4 and the
BCR that are sufficient to override the inhibitory effects of the
DNIKK mutant.
In contrast to the results with TNP-LPS, the transgenic mice exhibited
marked defects in the B cell response to the type 2 T-independent Ag
TNP-Ficoll. B cell responses to TNP-Ficoll are most likely due to
signaling through the BCR to activate BTK with subsequent activation of
the NF-B pathway (69). Marginal zone B cells have been
shown to be critical for both the proliferative response to LPS and the
humoral response to type 2 T-independent Ags (56, 57, 70).
Mice deficient in B cell surface receptors such as transmembrane
activator and calcium-modulating ligand interactor
(71) or downstream signaling molecules such as
Pyk-2 (57), BTK (72), and phospholipase C2
(73) are also defective in development of marginal zone B
cell as well as their response to type 2 T-independent Ags. We did not
find changes in the development of these B cells in the transgenic mice
despite the dramatic defects in response to type 2 T-independent Ags.
Thus, inhibiting IKK function most likely prevents efficient
signaling induced by the BCR that normally activates the NF-B
pathway in the marginal zone B cells.
The transgenic mice also exhibited a defective humoral response to the
T-dependent Ag TNP-KLH that is most likely due to effects on class
switching. Since B cell activation in response to TNP-Ficoll is
defective in the DNIKK mice, as indicated by the low IgM levels, it
is not possible to determine whether switching to downstream isotypes
is compromised in response to this Ag. In contrast, IgM production in
response to TNP-KLH is not affected in the transgenic mice, indicating
that the initial stimulation of B cells following immunization with
this T-dependent Ag does not require high levels of NF-B. In
contrast, switching to IgG1 and IgG2a is markedly reduced. It is
interesting to note that neither activation by anti-CD40 nor
isotype switching in response to cytokines in vitro is affected in the
transgenic B cells. Therefore, there must be significant differences in
the level of NF-B that are required for initial activation of B
cells in response to CD40 and/or other T cell costimulatory signals in
the context of BCR signaling vs the level of NF-B required for the
induction of class switch recombination.
Recent studies utilizing bone marrow chimeras have investigated the
role of IKK on B cell development (55). Control and
IKK-deficient fetal liver cells were transferred into RAG2-deficient
irradiated mice (55, 63). These mice exhibited reduced
numbers of mature B cells with decreased formation of secondary
lymphoid organs and impaired Ag-specific immune responses. The B cells
also showed decreased survival and reduced proliferation to mitogens,
indicating a role for IKK in preventing apoptosis and in
facilitating the functional development of mature B cells most likely
due to IKK-mediated processing of p52 (55). A similar
analysis utilizing IKK-deficient fetal liver cells demonstrated that
these cells could completely reconstitute T cell development, although
they exhibited a marked increase in TNF--induced apoptosis
(74). Thus, this study and our analysis suggest that
IKK is critical for both B and T cell function. The in vivo and in
vitro analysis presented in this work have allowed us to gain
additional insights into the mechanism of B cell activation that may
not have been readily apparent in mice with specific disruption of
NF-B genes that affect additional cell types. Furthermore, the
partial disruption of the NF-B pathway in the transgenic B cells has
permitted us to better define the signals transmitted via various B
cell response elements that lead to activation of the NF-B pathway
and to establish a role for IKK in modulating B cell function.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: IKK, I-B kinase; 7-AAD, 7-aminoactinomycin D; BCR, B cell receptor; BrdU, 5-bromo-2-deoxyuridine; DN, dominant-negative; KLH, keyhole limpet hemocyanin; NEMO, NF-B essential modulator; TNP, trinitrophenyl.
Received for publication September 11, 2001. Accepted for publication November 16, 2001.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B. Annu. Rev. Cell Biol. 10:405.
B/IB family: intimate tales of association and dissociation. Genes Dev. 9:2723.B kinase. Cell 90:373.[Medline]
B kinase complex (IKK) contains two kinase subunits, IKK and IKK, necessary for IB phosphorylation and NF-B activation. Cell 91:243.[Medline]
B kinase-: NF-B activation and complex formation with IB kinase- and NIK. Science 278:866.B kinases essential for NF-B activation. Science 278:860.B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B activity. Annu. Rev. Immunol. 18:621.[Medline]
B activation. Cell 93:1231.[Medline]
B kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex. Mol. Cell. Biol. 19:1526. is an essential regulatory subunit of the IB kinase complex. Nature 395:297.[Medline]
B activity and as a target of an adenovirus inhibitor of tumor necrosis factor -induced apoptosis. Proc. Natl. Acad. Sci. USA 96:1042.B kinase activity through IKK subunit phosphorylation. Science 284:309.
