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Molecular and Cellular Biology, September 1998, p. 5523-5532, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Ability of CD40L, but Not Lipopolysaccharide, To Initiate
Immunoglobulin Switching to Immunoglobulin G1 Is Explained by
Differential Induction of NF-
B/Rel Proteins
Shih-Chang
Lin,1
Henry H.
Wortis,2 and
Janet
Stavnezer1,*
Department of Molecular Genetics and
Microbiology and Program in Immunology and Virology, University of
Massachusetts Medical School, Worcester, Massachusetts
01655-0122,1 and
Department of
Pathology, Tufts University School of Medicine, Boston,
Massachusetts 02111-18002
Received 14 November 1997/Returned for modification 13 January
1998/Accepted 25 June 1998
 |
ABSTRACT |
Antibodies of the immunoglobulin G1 class are induced in mice by
T-cell-dependent antigens but not by lipopolysaccharide
(LPS). CD40 engagement contributes to this preferential isotype
production by activating NF-
B/Rel to induce germ line
1
transcripts, which are essential for class switch recombination.
Although LPS also activates NF-
B, it poorly induces germ line
1
transcripts. Western blot analyses show that CD40 ligand (CD40L)
induces all NF-
B/Rel proteins, whereas LPS activates predominantly
p50 and c-Rel. Electrophoretic mobility shift assays show
that in CD40L-treated cells, p50-RelA and p50-RelB dimers are the major
NF-
B complexes binding to the germ line
1 promoter, whereas
in LPS-treated cells, p50-c-Rel and p50-p50 dimers are the major
binding complexes. Transfection of expression plasmids for NF-
B/Rel
fusion proteins (forced dimers) indicates that p50-RelA and p50-RelB
dimers activate the germ line
1 promoter and that p50-c-Rel and
p50-p50 dimers inhibit this activation by competitively binding to the
promoter without activating the promoter. Therefore, germ line
1
transcription depends on the composition of NF-
B/Rel proteins.
 |
INTRODUCTION |
After activation by immunization or
infection, naive resting B cells expressing IgM and IgD switch to
expression of IgG, IgE, and IgA isotypes. Isotype, or class, switching
is mediated by a DNA recombination event called class switch
recombination. Recombination occurs between two switch regions, one
located 5' to the Cµ gene and the other located 5' to one of the
downstream heavy chain constant region (CH) genes. Class
switching does not alter the antigen specificity of the antibody
isotype but does alter its effector function, e.g., the ability to bind
to complement, to transcytose across epithelial cells, or to mediate an
allergic reaction (41, 44).
Cytokines, B-cell mitogens, and the nature of antigen determine the
choice of isotype. For example, the IgG1 and IgG3/IgG2b antibody
classes are preferentially induced in mice by T-cell-dependent and
-independent antigens, e.g., proteins and bacterial LPS, respectively (31, 40).
An initiating event in class switching is the induction of
transcription of the unrearranged, or GL, CH gene to which
the cell will switch. Transcription from the GL CH
gene initiates at exon I, upstream of the switch region of
each CH gene (44). Subsequent RNA splicing to
produce mature GL transcripts, also called switch transcripts, appears
to be required for switch recombination (26). The effect of
targeted disruption of the promoter and exon I of GL CH
gene transcripts provides solid evidence for the requirement for GL
transcripts in class switching (4, 20, 54).
Since GL transcripts direct switch recombination, an understanding of
the mechanisms of regulation of GL transcripts is necessary for
understanding the regulation of class switching. Expression of GL
transcripts is regulated at the transcriptional level by cytokines,
such as IL-4, gamma interferon, and transforming growth factor
, and
by B-cell activators, such as LPS, CD40L, and stimuli that induce
signaling through surface Ig (19, 27, 37, 44, 45, 48, 53).
For example, IL-4 and CD40L induce GL
1 transcripts, whereas LPS, in
the absence of IL-4, induces GL
2b and
3 transcripts (27,
37, 48).
We previously reported that CD40L, but not LPS, induces the activity of
the GL
1 promoter in a reporter plasmid (25). This induction by CD40L is mediated by activating NF-
B/Rel proteins to bind to three tandem
B sites located in the CD40RR of the GL
1
promoter, which is located just downstream of an IL-4-responsive Stat6
binding site (3). Overexpression of NF-
B/p50 together with RelA or RelB is sufficient to transactivate the GL
1 promoter or the CD40RR inserted upstream of a minimal c-fos promoter,
whereas overexpression of NF-
B/p50 together with c-Rel fails to
induce even though this heterodimer binds to the GL
1 promoter.
These observations suggest that NF-
B/Rel proteins are important
for initiating class switching to IgG1 in response to T-dependent antigens.
Although LPS is an activator of NF-
B, LPS is a poor inducer of GL
1 transcripts. In this study, we have addressed the reason for the
differential effects of LPS and CD40L on induction of GL
1 promoter
and transcripts. We have tested the hypothesis that GL
1
transcription depends on the composition of the induced NF-
B/Rel
dimers.
 |
MATERIALS AND METHODS |
Abbreviations.
Abbreviations used in this paper are as
follows: Ig, immunoglobulin; GL, germ line; CD40L, CD40 ligand; CD40RR,
CD40 responsive region; LPS, lipopolysaccharide; IL, interleukin; PDTC,
pyrrolidine dithiocarbamate; TLCK,
N
-p-tosyl-L-lysine chloromethyl
ketone; DMSO, dimethyl sulfoxide; CHX, cycloheximide; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol
acetyltransferase; RT, reverse transcription; EMSA,
electrophoretic mobility shift assay(s); GM-CSF,
granulocyte-macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; TNF-
, tumor necrosis factor alpha.
Splenic B cells and cell lines.
Splenic B cells from BALB/c
mice were prepared by T-cell depletion by adding monoclonal antibodies
for Thy1 (J1j.10), CD4 (GK1.5), and CD8 (3.168.3), followed by addition
of anti-rat
chain monoclonal antibody (MAR 18.5) and guinea pig
complement. Elutriation was used to obtain small resting B cells. The
1B4.B6 cell line was obtained by immortalizing LPS-stimulated BALB/cByJ splenic B cells by in vitro transfection with the J-2 retrovirus expressing avian v-raf and v-myc, as described
previously (6, 32). 1B4.B6 cells are CD45(B220), IgM, and
IgD positive (data not shown). 1B4.B6, M12.4.1 (15), and
splenic B cells were maintained in a 5% CO2 incubator in
RPMI 1640 medium with 10% fetal bovine serum, 50 mM 2-mercaptoethanol,
0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine,
100 U of penicillin per ml, 100 µg of streptomycin per ml, and 0.1 mg
of kanamycin sulfate (GIBCO, Grand Island, N.Y.) per ml. HEK 293 cells,
used for overexpression of p50-RelA, p50-c-Rel, p50-RelB, and p50-p50
fusion proteins (see below), were cultured in Dulbecco modified Eagle
medium with 10% fetal calf serum.
Soluble CD40L-CD8
fusion protein, recombinant IL-4, LPS,
NF-
B inhibitors, and okadaic acid.
Cell culture supernatant
containing soluble CD40L-CD8
fusion protein was prepared from J558L
mouse myeloma cells stably transfected with the CD40L-CD8
fusion
gene as described previously (24, 25). Cell culture
supernatant from untransfected J558L cells was prepared as a control.
Recombinant mouse IL-4 was a gift of W. E. Paul, National
Institutes of Health. LPS (Escherichia coli serotype
O55:B5), NF-
B inhibitors PDTC and TLCK, and an NF-
B activator
(okadaic acid) were purchased from Sigma. TLCK and okadaic acid were
dissolved in DMSO at 75 mM and at 125 µg/ml, respectively, and were
diluted 1,000-fold when added to cells. LPS and PDTC were dissolved in
RPMI medium.
RNA isolation.
Total RNA was prepared from 1B4.B6 and
splenic B cells by the hot phenol method (33).
Semiquantitative RT-PCR. (i) RT.
RT was performed with the
following specific primers: 5'-CTGAGCTGCTCAGAGTGTA-3'
(positions 300 to 282, GenBank no. V00793) (18) for GL
1 transcripts and 5'-TCACAAACATGGGGGCATC-3'
(positions 437 to 419, GenBank no. M32599) (35) for
GAPDH transcripts. These two specific primers were mixed with 4 µg of
total RNA from splenic B cells or 30 µg of total RNA from 1B4.B6
cells. The mixture was heated at 65°C for 10 min and cooled down to
room temperature. To the mixture were added the following reagents: 6 µl of 5× RT buffer (containing 250 mM Tris-HCl [pH 8.3], 375 mM
KCl, 50 mM dithiothreitol, and 15 mM MgCl2), 2.5 µl of
2.5 mM deoxynucleoside triphosphate, 10 U of RNasin (Promega Corp.,
Madison, Wis.) and 200 U of Moloney murine leukemia virus reverse
transcriptase (Promega). Diethyl pyrocarbonate-treated water was added
to obtain a 30-µl volume. The mixture was incubated at 39°C for
1 h to synthesize the cDNAs of GL
1 and GAPDH transcripts.
(ii) PCR.
Primers used to amplify the GL
1 cDNA were
5'-ACAGCCTGGTGTCAACTAG-3' (top strand positions 1772 to
1790, GenBank no. M12389) (28) and the C
1-specific RT
primer. The primers used to amplify the GAPDH cDNA were
5'-CAAATTCAACGGCACAGTC-3' (positions 202 to 220, GenBank no.
M32599) (35) and the GAPDH-specific RT primer. For PCR
amplification of cDNAs from 1B4.B6 cells, the 50-µl reaction volume
contained 75 pmol of each primer, 5 µl of RT product, 1× PCR buffer
(5 mM Tris-HCl [pH 8.3], 42.5 mM KCl, and 0.1% Triton X-100), and
MgCl2 (1.4 mM for GL
1 RNA and 3 mM for GAPDH RNA). The
cDNA of GL
1 transcripts was amplified for 40 cycles (1.5 min at
94°C, 2 min at 62°C, and 2 min at 72°C), and the cDNA of GAPDH
transcripts was amplified for 20 cycles (1.5 min at 94°C, 2 min at
55°C and 2 min at 72°C). Similar PCR conditions were used for PCR
amplification of cDNA from splenic B cells, except that 15 µl of RT
product and 1× PCR buffer (27.5 mM KCl and 0.1% Triton X-100) were
used for amplification of GL
1 cDNA for 44 cycles and GAPDH cDNA was
amplified for 28 cycles. PCR product (30 µl) was loaded onto an
8% polyacrylamide gel, which was subsequently stained with ethidium
bromide to visualize the bands. The specificity of the amplified GL
1 fragment from 1B4.B6 and splenic B cells was determined in the
early experiments by digestion with three restriction enzymes and also
by DNA sequencing.
Plasmids. (i) Plasmids for expression of p50-RelA, p50-c-Rel,
p50-RelB, and p50-p50 fusion proteins.
(a) The eukaryotic
expression plasmid p50-RelA(pEF-1
) was generated for expression of a
p50-RelA fusion protein. The relA gene was excised from the
pcDNA-I vector (39) with NdeI and XbaI
digestion, and the p50 gene was excised from pcDNA-I (25) with EcoRV and HindIII digestion. Both gene
fragments were cloned into the pEF1
-neo vector (23)
between the EcoRI and XbaI sites with the p50
coding region located 5' to the RelA coding region. A 19-amino-acid
linker between the two gene products was obtained by insertion of the
HindIII/EcoRV segment from the multiple
cloning region of the pcDNA-3 vector. (b) To generate the
p50-c-Rel(pEF-1
) plasmid for expression of a p50-c-Rel fusion
protein, the c-rel gene in pcDNA-I (25) was
excised with NcoI and XbaI and used to replace
the relA gene between the NdeI and
XbaI sites of the p50-RelA(pEF-1
) plasmid. (c) For
generation of the expression plasmid for the p50-RelB fusion protein,
the p50 and relB genes were cloned into the pEF1
-neo
vector by the following procedure. The HindIII fragment
from pcDNA-I containing the p50 coding region (25) was
inserted into the HindIII site of the pcDNA-3 vector (Invitrogen, San Diego, Calif.). Then, p50 cDNA was excised from pcDNA-3 with EcoRV and XbaI digestion and cloned
into pEF1
-neo vector digested with EcoRI and
XbaI. The EcoRI/SacII fragment at the
5' end of relB cDNA in pcDNA-I (25) was replaced
with a PCR fragment produced by using primers
5'-GCTGGAATTCTGCAGATAATGCCGAGTCGCCGCGCT-3' and
5'-CACTCGTAGCGGAAGCGCAT-3' and relB cDNA in
pcDNA-I (25) as the template. The relB gene was
then excised from this plasmid by EcoRI and inserted into
pEF1
-neo containing the p50 gene at the EcoRI site. (d)
To generate p50(pEF-1
), p50 cDNA, excised from pcDNA-I by
EcoRV and XbaI digestion, was inserted into
pcDNA-3 digested with EcoRV and XbaI. The p50
cDNA in pcDNA-3 was then excised with EcoRI and
XbaI digestion and cloned into pEF1
-neo vector digested
with EcoRI and XbaI. To generate
p50-p50(pEF-1
), the eukaryotic expression plasmid for the
p50-p50 fusion protein, p50 cDNA in p50-RelA(pEF-1
) was excised with
EcoRI and inserted into p50(pEF-1
) at the
EcoRI site.
All plasmids for expression of NF-
B/Rel fusion protein contain p50
cDNA located upstream of the other NF-
B/Rel gene. The upstream p50
gene is terminated at an internal HindIII site,
resulting in a 27-amino-acid deletion at the C terminus of p50 protein
and deletion of the stop codon. p50 lacking the C-terminal 27 residues has been found to be able to dimerize with other NF-
B/Rel
proteins (13). The amino acid sequence of the linker between
two proteins (GTELGSTSNGRQCAGILQI), which was confirmed by DNA
sequencing, is the same for all fusion proteins except that the p50-p50
fusion protein contains 3 additional amino acids (SMA) at the C
terminus of the linker. The plasmids were transfected into M12.4.1 or
HEK 293 cells, and expression of fusion proteins was detected with anti-RelA, anti-c-Rel, or anti-RelB antibody.
(ii) Other plasmids.
The
954WT luciferase reporter gene
plasmid contains the GL
1 promoter fragment from nucleotides
954
to +202 relative to the first RNA initiation site (51). The
pCD40FL-F plasmid has the CD40RR from the GL
1 promoter inserted
upstream of a mouse minimal (
71) c-fos promoter segment in
a luciferase reporter plasmid (25). The pFosCAT plasmid,
containing a minimal (
71) c-fos promoter segment ligated
upstream of a CAT reporter gene, was used for internal control of
transfection efficiency (14). The previously generated RelA
expression plasmid (25) was cotransfected with p50(pEF-1
)
plasmid into HEK 293 cells. The
-162Luc plasmid contains the mouse
GL
promoter fragment, as described previously (7).
DNA transfections.
Electroporation was performed as
described previously for transient transfection of M12.4.1 cells
(25). Cells (2 × 107) were washed once
with serum-free RPMI medium and were resuspended in 1 ml of serum-free
RPMI medium. The cell suspension was mixed with plasmid DNA (a total of
about 95 µg, varied to maintain equimolar amounts) and electroporated
at 1,250 µF and 750 V/cm. After transfection, cells were left at room
temperature for 10 min and then cultured in 10 ml of complete RPMI
medium in six-well plates.
The calcium phosphate method (
21) was used for expression of
NF-

B fusion proteins in HEK 293 cells by transient transfection.
Cells (2 × 10
6) were transfected with 5 µg of
plasmid and harvested 48 h after
transfection. Nuclear extracts
were obtained for EMSA.
Luciferase and CAT assays.
Luciferase (5) and CAT
(29) assays were performed and analyzed as described
previously (25).
Cytoplasmic and nuclear extracts.
The modified small-scale
method for extraction of cytoplasmic and nuclear proteins was described
previously (25, 36).
Western blot analysis.
Two micrograms of nuclear extracts
from splenic B cells or 10 µg of nuclear extracts from M12.4.1 or
1B4.B6 cells was fractionated on 10% reducing sodium dodecyl
sulfate-polyacrylamide gels and transferred to Immobilon-P membranes
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions. The membrane was first incubated with antibody against
specific NF-
B/Rel family members in TBS-T buffer (20 mM Tris-HCl
[pH 7.6], 137 mM NaCl, and 0.5% Tween 20) in the presence of 5%
powdered skim milk overnight. After a wash with TBS-T buffer, the blot
was incubated with goat anti-rabbit peroxidase conjugate (Santa Cruz
Biotechnology, San Diego, Calif.) for 5 h. The immunoreactive
bands were visualized on films by using the ECL system (Amersham
Corp.). To remove antibodies so that the blot could be used for further
detection of other NF-
B/Rel family members, the blot was incubated
with antibody-stripping buffer (62.5 mM Tris-HCl and 100 mM
2-mercaptoethanol) at 50°C for 30 min.
EMSA.
Nuclear extract was combined with 1 µg of annealed
poly(dI-dC) · poly(dI-dC) (Sigma) and 32P-labeled
CD40RR fragment in a reaction mixture containing 10% glycerol, 17.5 mM
HEPES (pH 7.5), 5 mM KCl, 103 mM NaCl, 0.35 mM EDTA, 0.25 mM EGTA, and
1 mM dithiothreitol. The mixture was incubated at room temperature for
30 min, and samples were loaded onto a 5% nondenaturing polyacrylamide
gel (acrylamide/bisacrylamide ratio, 37.5:1) and electrophoresed in
0.5× Tris-borate-EDTA buffer at 150 V. Gels were dried and subjected
to autoradiography. For EMSA with antibody supershift, antibodies were
premixed with nuclear extracts for 15 min before labeled probe was
added. Probe was labeled as previously described (25).
Compared to the reaction conditions reported previously
(
25), a higher, more physiological salt concentration (total
of
126 mM) in the reaction mixture was used in EMSA in this study,
as
formation of DNA-protein complexes is sensitive to salt concentration.
The p50-RelA, p50-c-Rel, and p50-p50 complexes in EMSA with nuclear
extracts from M12.4.1 cells were barely detected in our previous
study
(
25), in which 30 mM salt was used, including 5 mM KCl
(not
50 mM, as in reference
25, a typographic error),
whereas
these complexes can be detected under the higher salt
conditions,
as indicated in Fig.
4A. In addition, under the low-salt
conditions
previously used, an additional RelB-containing complex was
detected
and supershifted by anti-RelB but not by other NF-

B
antibodies,
although it can be competed away by an oligonucleotide with
two

B sites. Under the higher salt conditions, all complexes can
be
supershifted by antibody to p50 (see Fig.
4A; also data not
shown).
Antibodies against NF-
B/Rel and I
B proteins.
Antibodies against p50 (sc-114), c-Rel (sc-071X), RelB (sc-226X),
I
B
(sc-371), and I
B
(sc-945) for Western blot analyses were
purchased from Santa Cruz Biotechnology. Antiserum against RelA
(34) for Western blot analyses was a gift from N. Rice (National Cancer Institute, Bethesda, Md.). Antibodies to specific NF-
B/Rel family members, including anti-RelA (sc-109X), and antibody against ATF-2 as the control antibody for EMSA were all from Santa Cruz
Biotechnology.
 |
RESULTS |
GL
1 transcripts can be induced by CD40L but are only poorly
induced by LPS.
Using luciferase reporter assays in M12.4.1 B
lymphoma cells, we found previously that CD40L, but not LPS, induces
the GL
1 promoter. The different effects of CD40L and LPS on the GL
1 promoter are consistent with previous observations that
T-dependent antigens, but not T-independent antigens or LPS, induce
IgG1 production in mice (31, 40). To determine if CD40L and
LPS show a differential ability to induce GL
1 transcripts in normal
B cells, splenic B cells were stimulated with CD40L or LPS for 6 h, and the levels of GL
1 transcripts were assayed by RT-PCR. Figure
1 shows that CD40L, but not LPS, induces
GL
1 transcripts in splenic B cells. CD40L-induced GL
1
transcripts persist for at least 18 h, whereas they are still not
induced by LPS at this time (data not shown). The results are
consistent with a previous report showing that treatment for 18 h
with CD40L expressed on Sf9 insect cells induces GL
1 transcripts in
splenic B cells (48).

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FIG. 1.
RT-PCR analysis to demonstrate induction of GL 1
transcripts in splenic B cells. Resting splenic B cells were left
untreated or were treated with LPS (50 µg/ml), control supernatant
(sup.) (20%), or CD40L supernatant (20%) for 6 h in the presence
or absence of the NF- B inhibitor PDTC (50 µM) or TLCK (75 µM). Expression of GL 1 transcripts was assayed by RT-PCR with
amplification of GAPDH transcripts as the internal control.
|
|
To obtain a system that could be more readily manipulated, we examined
the effects of CD40L and LPS on GL

1 transcripts in
the B-cell line
1B4.B6. This B-cell line was derived by transformation
of
LPS-stimulated splenic B cells with the J-2 retrovirus expressing
avian
v-Raf and v-Myc. This B-cell line has been shown to secrete
IgG1 upon
stimulation with Th2 cells or with CD40L plus IL-4 (
50a).
1B4.B6 cells were treated with CD40L or LPS for various times,
and the
levels of GL

1 transcripts were assayed by RT-PCR. As
shown in Fig.
2A, CD40L induces GL

1 transcripts
within 2 h and
the RNA levels gradually increase for up to 24 h. In contrast,
LPS only poorly induces GL

1 transcripts. Since
M12.4.1 cells
do not contain GL C

1 genes (
24a),
regulation of expression of
GL

1 transcripts could not be studied in
this cell line.

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FIG. 2.
RT-PCR analysis of induction of GL 1 transcripts in
1B4.B6 B cells by LPS, CD40L, and okadaic acid. (A) 1B4.B6 cells were
left untreated or were treated with LPS, control supernatant (sup.), or
CD40L supernatant, and cells were harvested at the time points
indicated for detection of GL 1 transcripts by RT-PCR. (B)
Expression of GL 1 transcripts was analyzed in 1B4.B6 cells,
untreated or treated with control supernatant CD40L supernatant, or
IL-4 (1,000 U/ml) for 6 h in the presence or absence of the
NF- B inhibitor PDTC or TLCK. Induction of transcripts by okadaic
acid (125 ng/ml), an NF- B activator, was evaluated at 6 h.
|
|
Induction of GL
1 transcripts by CD40L is mediated by NF-
B
activation.
We found previously that induction of the GL
1
promoter by CD40L in M12.4.1 cells is mediated by activation of
NF-
B/Rel proteins that bind three tandem
B sites in the CD40RR
(
99 to
43, relative to the first RNA initiation site) of the
promoter (25). To determine whether induction of GL
1
transcripts by CD40L in 1B4.B6 and splenic B cells is also mediated by
activation of NF-
B/Rel proteins, we tested the effects of addition
of NF-
B inhibitors on induction of the GL
1 transcripts. Two
inhibitors which each inhibit NF-
B activation by different
mechanisms were used, the anti-oxidant PDTC and the protease inhibitor
TLCK. 1B4.B6 or splenic B cells, pretreated with PDTC at 50 µM or
TLCK at 75 µM for 30 min, were incubated with CD40L in the presence
of PDTC or TLCK for 6 h. At this time, greater than 90% of PDTC-
or TLCK-treated 1B4.B6 cells and TLCK-treated splenic B cells and about
70% of PDTC-treated splenic B cells were viable (data not shown). The levels of GL
1 transcripts were assayed by RT-PCR. As shown in Fig. 1 and 2B, induction of GL
1 transcripts by CD40L in splenic B
cells and in 1B4.B6 cells is inhibited by PDTC and by TLCK. The
inhibition of induction of GL
1 transcripts by blocking of NF-
B
activation appears to be specific to CD40L treatment, because TLCK has
no effect on induction of GL
1 transcripts by IL-4 in 1B4.B6 cells
(Fig. 2B).
To determine if the inhibitors did indeed eliminate NF-

B
activation by CD40L, an aliquot of the inhibitor-treated 1B4.B6
or splenic B cells used to assay the levels of GL

1
transcripts
were used for preparation of nuclear extracts for EMSA or
for
Western blot analyses, respectively. We determined previously
that
all complexes that bind to the CD40RR in EMSA contain NF-

B/Rel
proteins (
25). The results confirmed that NF-

B DNA
binding
activity induced by CD40L was abolished by treatment of 1B4.B6
cells with PDTC or TLCK and that NF-

B activation in splenic B
cells was inhibited by PDTC and TLCK treatment (data not shown).
In
conclusion, induction of GL

1 transcripts by CD40L, but not
by IL-4,
requires NF-

B activation.
To examine whether NF-

B activation is sufficient to induce GL

1 transcripts, we tested whether GL

1 transcripts can be
induced
by treatment with okadaic acid, which activates NF-

B
by
stimulating activity of an I

B kinase, resulting in degradation
of
I

B proteins (
8). As expected, okadaic acid treatment for
6 h was found to activate NF-

B binding activity in 1B4.B6
cells
(data not shown) and to induce GL

1 transcripts (Fig.
2B). In
addition, we found that the GL

1 promoter in the

954WT reporter
plasmid is induced threefold by okadaic acid in M12.4.1 cells
(data not
shown). The effect of okadaic acid treatment on GL

1
treatment in
splenic B cells cannot be examined due to induction
of cell death. In
conclusion, experiments using okadaic acid together
with previously
reported experiments showing that overexpression
of NF-

B/Rel
proteins induces GL

1 promoter activity (
25) demonstrate
that NF-

B activation is sufficient for induction of GL

1
transcripts.
NF-
B activation by CD40L and LPS.
Since CD40L-activated
NF-
B/Rel proteins induce the GL
1 promoter, we might expect
that LPS, a known NF-
B activator, would also induce the
promoter. However, LPS has very little effect on the GL
1 promoter
(25) or on the levels of GL
1 transcripts (Fig. 1 and
2A), although LPS activates NF-
B/Rel proteins in M12.4.1,
1B4.B6, and splenic B cells (see below). Three possible mechanisms
could explain this discrepancy. The first is that CD40L, but not LPS,
induces a coactivator protein(s) which is required for promoter
activation by NF-
B/Rel proteins. This possibility is not
supported by the finding that overexpression of p50-RelA or p50-RelB is
sufficient to transactivate the GL
1 promoter and a reporter gene
driven by a minimal c-fos promoter and the CD40RR (pCD40FL-F
plasmid) (25). The second possibility is that LPS induces a
repressor protein(s) which interacts with NF-
B/Rel proteins and
suppresses their transactivation activity. The LPS-induced repression
would act at the CD40RR, because LPS cannot induce expression of the
pCD40FL-F plasmid. Therefore, the LPS-induced repressor protein(s) must
bind to the CD40RR or associate with NF-
B/Rel proteins bound to
the CD40RR. However, CD40L and LPS induce similar protein complexes,
all of which contain NF-
B/Rel proteins, to bind the CD40RR in
EMSA, suggesting that no additional repressor protein is induced by LPS
(see Fig. 4A, lanes 3 and 11, and further description below).
The third possibility is that CD40L and LPS might activate different
profiles of NF-

B/Rel proteins. We have shown that overexpression
of p50 alone or together with c-Rel cannot transactivate the GL

1
promoter, but overexpression of p50 together with RelA or RelB
strongly
transactivates the promoter. Therefore, it is possible
that LPS induces
more p50 and c-Rel proteins and/or fails to induce
RelA and RelB
proteins. To test this possibility, we compared
the patterns and
kinetics of NF-

B activation induced by CD40L
and LPS. The
nuclear level of each NF-

B/Rel protein was determined
by Western
blot analyses of nuclear extracts from M12.4.1, 1B4.B6,
and splenic B
cells, and cells from the same culture were used
for analyzing
induction of promoter activity by a reporter gene
assay or induction of
GL

1 RNA by RT-PCR. Two or three independent
experiments were
performed with nearly identical results for each
cell type.
As shown in Fig.
3A and B, CD40L induces
greater nuclear accumulation of NF-

B/Rel proteins than does LPS
in both splenic
B cells and M12.4.1 cells, with a greater difference
between CD40L
and LPS treatments in splenic B cells and at later time
points
in M12.4.1 cells. All NF-

B/Rel proteins can be induced by
CD40L
for at least 12 h in M12.4.1 cells and for at least 24 h in splenic
B cells, although RelA levels are slightly reduced at
longer time
points. In contrast, the induction by LPS of RelA and RelB
declines
to levels lower than those induced by CD40L at 12 h.
However,
c-Rel is more persistently induced than RelA and RelB during
LPS
stimulation.

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FIG. 3.
Western blot analyses of NF- B activation by CD40L
and LPS treatment. Nuclear extracts from splenic B cells (A) and from
the mouse B-cell lines M12.4.1 (B) and 1B4.B6 (C), left untreated or
treated with LPS, control supernatant (sup.), or CD40L supernatant for
the indicated times, were analyzed. (D) Western blot analysis with
nuclear extracts from M12.4.1 B cells untreated or treated for 6 h
with control supernatant, CD40L supernatant, or LPS in the presence or
absence of CHX (5 µg/ml). Each Western blot was probed sequentially
with four antibodies against different NF- B/Rel proteins. These
experiments were performed two or three times with nearly identical
results.
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|
In 1B4.B6 cells, LPS activates NF-

B better than does CD40L and
induces significant amounts of RelA and RelB (Fig.
3C). Therefore,
poor
induction by LPS of GL

1 transcripts in 1B4.B6 cells is
not due to
its inability to induce nuclear translocation of the
transactivating
NF-

B/Rel proteins RelA and RelB. The induction
of
NF-

B/Rel proteins by CD40L in 1B4.B6 cells occurs with slow
kinetics and is still increasing 24 h after addition of CD40L
(Fig.
3C). These data are consistent with the gradual increase
in GL

1 transcripts in CD40L-treated 1B4.B6 cells (Fig.
2A).
Most
importantly, LPS, but not CD40L, stably induces abundant
nuclear c-Rel
and p50 in this cell line.
Taken together, the Western blot results from the two B-cell lines and
splenic B cells indicate that CD40L and LPS induce
different patterns
of NF-

B activation. Three conclusions can
be drawn. (i) LPS
induces more c-Rel, and in 1B4.B6 cells also
preferentially induces
p50, than other NF-

B/Rel proteins, suggesting
that p50-p50
and/or p50-c-Rel dimers may be the predominant NF-

B/Rel
complexes accumulating in the nucleus in response to LPS treatment.
This conclusion is subject to the caveat that the NF-

B
antibodies
used may have different sensitivities, but data presented in
the
next section, with EMSA used to quantitate individual
NF-

B/Rel
proteins, support this conclusion. (ii) CD40L induces
greater
amounts of RelA and RelB, in comparison to LPS, and also
induces
p50 and c-Rel. (iii) The kinetics of NF-

B activation
show that
nuclear translocations of individual NF-

B/Rel proteins
are regulated
differently by different stimuli.
The p50-c-Rel heterodimer and p50 homodimer are the main
NF-
B/Rel dimers bound to the GL
1 promoter in cells treated
with LPS.
To determine if the different pattern of induction of
NF-
B/Rel proteins by CD40L in comparison to LPS results in
different amounts of individual NF-
B/Rel complexes binding to
the GL
1 promoter, EMSA were performed to examine NF-
B/Rel
complexes formed with the CD40RR segment of the GL
1 promoter and
nuclear extracts from M12.4.1, 1B4.B6, and splenic B cells treated with
CD40L or LPS for 12 h. Nuclear extracts from CD40L-treated cells
form three major complexes with the CD40RR probe, as do nuclear
extracts from LPS-treated cells (Fig. 4A,
lanes 3 and 11). As indicated in Fig. 4A, complex I contains
p50-RelB and complex II consists of p50-c-Rel, p50-RelA, and p50-RelB
dimers. Complex II can be entirely supershifted if the amount of
anti-p50 antibody is increased to 3 µl (data not shown). Since the
CD40RR has three
B sites, it is possible that complex I
contains CD40RR DNA fragments with two or three
B sites
occupied. Complex III consists of p50-p50 homodimers, as it can be
supershifted by anti-p50 but not by antibodies against other
NF-
B/Rel family members (Fig. 4A) (25).

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FIG. 4.
EMSA and densitometry analyses to compare NF- B
activation by LPS and CD40L. (A) Labeled CD40RR probe was incubated
with nuclear extracts (NE) from M12.4.1, 1B4.B6, and splenic B cells
treated with CD40L or LPS for 12 h to determine binding activity
of each NF- B/Rel dimer. The three major DNA-protein complexes
formed are indicated. To supershift or deplete the specific
NF- B/Rel dimers, nuclear extracts were preincubated with
antibody against a specific NF- B/Rel protein, a control serum,
or the indicated combinations of antibodies before incubation with
labeled CD40RR probe. The total amount of antibody was kept constant at
3 µl by adding control antibody. (B) Densitometry analyses of the
NF- B/Rel complexes from EMSA results shown in panel A. The
quantity of binding activity of complex I, containing p50-RelB, was
obtained by subtracting the signals at the position of complex I in
lanes 8 and 16 from the signals of complex I in lanes 3 and 11, respectively. The quantity of complex III, containing p50-p50, was
obtained by subtracting the signals at the position of complex III in
lanes 4 and 12 from the signals of complex III in lanes 3 and 11, respectively. The quantity of p50-c-Rel in complex II was obtained by
subtracting the signals of complex II in lanes 5 and 13 from the
signals of complex II in lanes 3 and 11, respectively. The quantity of
p50-RelA in complex II was obtained by subtracting the signals of
complex II in lanes 6 and 14 from the signals of complex II in lanes 5 and 13, respectively. The quantity of p50-RelB in complex II was
obtained by subtracting the signals at the position of complex II in
lanes 7 and 15 from the signals of complex II in lanes 6 and 14, respectively.
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To discern the contribution of the individual NF-

B/Rel dimers to
the binding activity, we depleted or supershifted the specific
NF-

B/Rel dimers by antibodies and used densitometry analyses
to
determine the amounts of individual NF-

B/Rel dimers binding
to
the promoter (Fig.
4B). As shown in Fig.
4A, anti-c-Rel antibody
eliminates the majority of binding activity of complex II from
nuclear
extracts of LPS-treated cells (lanes 3 and 5). Addition
of anti-c-Rel
and anti-RelA to these nuclear extracts almost completely
abolishes
binding activity of complex II (lane 6). These results
suggest that
p50-c-Rel, predominantly induced by LPS, is the major
NF-

B/Rel
dimer in complex II and that some p50-RelA is also induced.
Little or
no RelB is detected in complex II (lane 6 versus lane
7 and lane 3 versus lane 8), consistent with the poor induction
of complex I (lane
3). Also, LPS induces complex III in 1B4.B6
cells. We also examined
earlier time points (1 and 6 h) after
treatment of 1B4.B6 cells
with LPS and again found that p50-c-Rel
is the predominant complex
(data not shown).
Densitometry analyses of these EMSA (Fig.
4B) show that a greater
amount of p50-c-Rel plus p50-p50 binding activity than p50-RelA
plus
p50-RelB binding activity is detected in nuclear extracts
from
LPS-treated cells. This conclusion is consistent with the
Western blot
results in Fig.
3 except for the finding that p50-RelB
binding activity
is barely detectable in nuclear extracts from
LPS-treated cells. This
inconsistency suggests that most of the
nuclear RelB may be unavailable
for binding the CD40RR. A similar
finding has recently been reported
for RelB induced by anti-Ig
treatment of splenic B cells
(
11).
When we examined the NF-

B/Rel binding activities in nuclear
extracts from CD40L-treated cells, it was apparent that anti-c-Rel
only
partially depletes (M12.4.1 cell extracts) or barely depletes
(splenic
B and 1B4.B6 cell extracts) the binding activity of complex
II (Fig.
4A, lanes 11 and 13). The remaining binding activity
of complex II can
be greatly reduced by addition of anti-RelA
(lane 14). Depletion of
c-Rel and RelA reveals the p50-RelB component
in complex II, which is
completely eliminated by addition of anti-RelB
(lane 15). These data
indicate that p50-RelA and p50-RelB are
the main components in complex
II. Therefore, in contrast to LPS,
CD40L induces much greater p50-RelB
binding activity; this is
also indicated by the induction of complex I,
which appears to
consist mostly of p50-RelB. Densitometry analyses of
the EMSA
results (Fig.
4B) demonstrate that the combined binding
activities
of p50-RelA and p50-RelB are greater than those of
p50-c-Rel plus
p50-p50 dimers in nuclear extracts from cells treated
with CD40L.
Generation of p50-p50, p50-RelA, p50-c-Rel and p50-RelB fusion
proteins.
The DNA-binding data suggest that the different effects
of CD40L and LPS on the expression of GL
1 transcripts are due to differential induction of NF-
B/Rel dimers binding to the
promoter. These NF-
B/Rel dimers might influence the promoter
activity by competing with each other for binding to the promoter. To
examine this possibility, we tested the effects on the GL
1
promoter of overexpressing specific NF-
B/Rel dimers. The
conventional method for overexpressing NF-
B/Rel dimers is to
cotransfect cells with plasmids for individual NF-
B/Rel
proteins. However, overexpressed NF-
B/Rel subunits will form
various homodimers and heterodimers, as demonstrated by
coexpression of p50 and RelA (Fig. 5,
lane 8); thus, the effect of a specific NF-
B/Rel dimer cannot be
evaluated.

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FIG. 5.
EMSA to demonstrate the ability of NF- B/Rel
fusion proteins to bind the CD40RR. EMSA was performed with labeled
CD40RR fragment as the probe and 2 µg of nuclear extracts (NE) from
HEK 293 cells transiently transfected with empty vector or with
expression plasmids for p50-RelA, p50-c-Rel, p50-RelB, and p50-p50
fusion proteins (lanes 2 to 6). Nuclear extracts from cells transfected
with p50 plasmid alone (lane 7) or both p50 and RelA plasmids (lane 8)
demonstrate formation of multiple NF- B complexes when two
NF- B/Rel subunits are coexpressed. Nuclear extracts from M12.4.1
cells (3 µg) (lane 9) and 1B4.B6 cells (6 µg) (lane 10) treated
with CD40L for 12 h illustrate complexes formed by endogenous
NF- B/Rel dimers.
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To overexpress specific NF-

B/Rel dimers, we created plasmids for
expressing NF-

B/Rel fusion proteins (p50-c-Rel, p50-RelB,
p50-RelA, and p50-p50) by fusing cDNA encoding p50 protein with
cDNA
encoding p50, RelA, c-Rel, or RelB. To evaluate the expression
of the
NF-

B/Rel fusion proteins, Western blot analyses were performed
with nuclear extracts from M12.4.1 cells stably expressing p50-c-Rel,
p50-RelB, p50-RelA, and p50-p50 (Fig.
6).
The results are compared
with nuclear extracts from HEK 293 cells
transiently transfected
with the same plasmids. HEK 293 cells do not
express nuclear NF-

B
proteins, thus allowing a distinction
between endogenous and transfected
NF-

B proteins. As shown in
Fig.
6, intact fusion proteins can
be detected in nuclear extracts of
M12.4.1 cells and of HEK 293
cells. The fusion proteins, except for
p50-p50, can also be detected
in M12.4.1 cytoplasmic extracts (data not
shown). These results
indicate that expressed p50-c-Rel, p50-relB, and
p50-RelA can
be kept in the cytoplasm, presumably by associating with
I

B proteins,
and also that they can be translocated into the
nucleus. Treatment
of the M12.4.1 transfectants with CD40L or LPS
induces nuclear
accumulation of the NF-

B/Rel fusion proteins
(Fig.
6D and data
not shown).

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FIG. 6.
Western blot analyses demonstrate that NF- B/Rel
fusion proteins can be stably expressed and translocated into the
nucleus in M12.4.1 and HEK 293 cells. (A to C) Nuclear extracts were
prepared from M12.4.1 cells (stably) or HEK 293 cells (transiently)
transfected with the p50-c-Rel, p50-p50, or p50-RelB plasmid. The
fusion proteins and endogenous NF- B proteins in the nuclear
extracts were detected by Western blot analyses using anti-c-Rel,
anti-p50, and anti-RelB antibodies. (D) M12.4.1 cells, untransfected or
stably transfected with the p50-RelA expression plasmid, were left
untreated or were treated with LPS, control supernatant (sup.), or
CD40L supernatant for 6 h. p50-RelA and RelA in the nuclear
extracts were detected by using anti-RelA in the Western blot analysis.
Expression of intact p50-RelA in transiently transfected HEK 293 cells
was also detected in the nucleus.
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|
To test whether overexpressed fusion proteins can form DNA-protein
complexes similar to endogenous NF-

B/Rel dimers, we investigated
the ability of p50-p50, p50-RelA, p50-c-Rel, and p50-RelB fusion
proteins to bind to the CD40RR. In order to ensure that the
binding
activity was due to the transfected NF-

B/Rel
dimers, NF-

B/Rel
fusion proteins overexpressed in HEK 293 cells by transient transfection
were examined. As shown by EMSA,
overexpressed p50-RelA, p50-c-Rel,
p50-RelB, and p50-p50 fusion
proteins can bind the CD40RR (Fig.
5, lanes 3 to 6). When the
DNA-protein complexes formed by fusion
proteins are compared to
complexes formed by endogenous dimers
in B-cell nuclear extracts (Fig.
5, lanes 9 and 10), it can be
seen that p50-RelA, p50-c-Rel, and
p50-RelB form complexes which
migrate slightly more rapidly than
complex II and that the complex
formed by the p50-p50 fusion protein
migrates clearly faster than
complex III. This more rapid migration is
probably due to the
27-amino-acid deletion of the C terminus of p50
produced during
creation of the fusion proteins. The C-terminal 27 residues of
p50 have been documented to be not required for NF-

B
dimerization
and DNA-binding activity (
13). In addition,
overexpressed p50-RelB,
and to a lesser extent p50-RelA, form an
additional slowly migrating
complex, presumably corresponding to
complex I. p50-RelA also
forms a rapidly migrating complex, probably
due to protein degradation.
Taken together, these results suggest that
the fusion proteins
have structures similar to that of endogenous
NF-

B/Rel dimers.
Overexpression of p50-p50 or p50-c-Rel protein inhibits
transactivation activity of p50-RelA and p50-RelB proteins.
To
test if p50-RelA, p50-RelB, and p50-c-Rel fusion proteins can
transactivate the GL
1 promoter, the luciferase reporter plasmid
containing the
954WT segment was cotransfected with plasmids expressing NF-
B/Rel fusion proteins into M12.4.1 cells and the promoter activity was determined 9 h after transfection.
Overexpression of p50-RelA or p50-RelB fusion protein transactivates
the promoter activity, whereas overexpression of p50-c-Rel fusion
protein poorly induces the promoter (Fig.
7). These data are consistent with previous results showing that p50 and RelA or p50 and RelB activate the
promoter whereas p50 and c-Rel or p50 alone do not (25).

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FIG. 7.
Reporter gene assays demonstrate that overexpression of
p50-c-Rel or p50-p50 fusion protein suppresses the transactivation
activity of p50-RelA and p50-RelB fusion proteins. The 954WT
luciferase reporter plasmid (20 µg) that contains the GL 1
promoter and plasmid pFosCat (15 µg) as the transfection control were
cotransfected into 2 × 107 M12.4.1 cells with empty
vector or different amounts of expression plasmids for NF- B/Rel
subunits or fusion proteins, as indicated (in picomoles). Empty vector
was added to equalize the amount of DNA in each transfection.
Luciferase activity, representing the promoter activity, was assayed
9 h after transfection and normalized by CAT activity. The fold
induction was calculated by the ratio of the luciferase activity from
cells transfected with NF- B/Rel plasmids to the luciferase
activity from cells transfected with empty vector. The mean and
standard error (SE) of fold induction were calculated from at least
three independent experiments. Similar experiments performed with the
-162Luc plasmid containing the mouse GL promoter fragment as the
reporter plasmid demonstrate that p50-c-Rel is able to transactivate
the mouse GL promoter.
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|
The EMSA data in Fig.
4 show that although LPS predominantly induces
p50-c-Rel binding in all three B-cell lines and induces
p50-p50 in
1B4.B6 cells, it also induces significant binding of
p50-RelA to the
CD40RR in nuclear extracts from M12.4.1 and 1B4.B6
cells. Yet
LPS-induced p50-RelA does not induce GL

1 promoter
activity and
transcripts. Therefore, we determined whether p50-c-Rel
and p50-p50
inhibit transactivation of the promoter by p50-RelA
and p50-RelB dimers
by cotransfecting the

954WT reporter plasmid
and the p50-RelA or
p50-RelB plasmid together with the p50-c-Rel
or p50-p50 plasmid.
The effects of three different doses of p50-c-Rel
or p50-p50 on GL

1 promoter activity are shown in Fig.
7. It
can be seen that
transcription induced by p50-RelA or p50-RelB
is inhibited by
p50-c-Rel or p50-p50. When present at a 2:1 ratio,
p50-c-Rel inhibits
induction by p50-RelA or p50-RelB by 80%. It
appears likely that the
inhibition is due to a competition for
binding to the

B sites in the
GL

1 promoter, since all of these
NF-

B/Rel proteins bind the

1

B sites. The inability of p50-c-Rel
to transactivate the GL

1 promoter appears to be specific to
this promoter, because
p50-c-Rel transactivates the mouse GL
promoter by about 14-fold
(Fig.
7).
These data provide an explanation for the fact that the GL

1
promoter and GL

1 transcripts are significantly induced by
CD40L but
are poorly induced by LPS. LPS induces the binding of
p50-RelA to the
GL

1 promoter but also induces a greater amount
of p50-c-Rel and
p50-p50, which do not activate the GL

1 promoter
and override the
transactivation activity of the p50-RelA dimer.
Although CD40L also
induces binding of c-Rel and p50 to the promoter,
it induces an excess
of transactivating dimers. At early time
points (Fig.
2A), however, the
level of GL

1 transcripts induced
in 1B4.B6 cells by LPS was
approximately the same as the level
induced by CD40L. This appears to
be consistent with the higher
ratio of RelA to c-Rel at early time
points after LPS addition
(Fig.
3C). In contrast to LPS, CD40L induces
persistent binding
activity of the transactivating NF-

B/Rel
dimers p50-RelA and
p50-RelB.
Comparison of the mechanisms of activation of NF-
B/Rel
proteins by LPS and by CD40L.
The mechanisms that mediate more
persistent nuclear accumulation of RelA and RelB induced by CD40L,
compared to LPS, and the mechanisms for maintenance of c-Rel activation
by LPS and CD40L treatment have not been elucidated. The slow kinetics
of RelB induction compared to other NF-
B/Rel proteins (Fig. 3A
to C) suggests that induction of RelB may require protein synthesis. To
examine this possibility, we tested the effect of CHX treatment on the
induction of RelB by LPS or by CD40L. As shown in Fig. 3D, CHX
treatment inhibits induction of RelB by LPS and CD40L but not of p50,
RelA, or c-Rel in M12.4.1 cells. In splenic B cells, CHX treatment
enhances induction of RelA and c-Rel by CD40L but it reduces the
induction of RelB by CD40L (Fig. 3A). These data indicate that
activation of RelB is regulated differently from activation of RelA and
c-Rel and requires protein synthesis.
We next examined whether LPS and CD40L differentially target I

B

and I

B

. The degradation of cytoplasmic I

B

and I

B

proteins
induced by LPS and CD40L in the two B-cell lines and in
splenic
B cells was examined by Western blot analyses. As shown in Fig.
8A to C, both LPS and CD40L cause
degradation of I

B

and I

B
proteins in these B cells, except
that degradation of I

B

and
I

B

induced by CD40L in 1B4.B6
cells is not obvious, consistent
with the slow kinetics of NF-

B
activation induced by CD40L in
this cell line. LPS or CD40L treatment
in the presence of CHX
leads to complete or nearly complete
disappearance of I

B

and
I

B

proteins in M12.4.1 and splenic
B cells (Fig.
8D), indicating
that these I

B proteins are undergoing
synthesis in the activated
B cells.

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FIG. 8.
Western blot analyses demonstrate induction of I B
degradation by CD40L and LPS. Cytoplasmic extracts (15 µg) from
splenic B (A), M12.4.1 (B), and 1B4.B6 (C) cells, left untreated or
treated with LPS, control supernatant (sup.), or CD40L supernatant for
the indicated times, were analyzed by Western blotting. (D) Cytoplasmic
extracts from M12.4.1 and splenic B cells, left untreated or treated
for 6 h with control supernatant, CD40L supernatant, or LPS in the
presence or absence of CHX, were used in Western blot analyses. Each
Western blot was analyzed with antibodies against I B and I B
proteins sequentially.
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|
Several cell-specific differences exist in the degradation patterns.
CD40L appears to induce greater and more persistent degradation
of
I

B

than LPS in splenic B cells, which may explain why CD40L
activates NF-

B/Rel proteins much better than LPS in splenic B
cells. In 1B4.B6 cells, I

B

is greatly reduced for at least
24
h after LPS treatment (Fig.
8C). However, in M12.4.1 cells, LPS
and CD40L have similar effects on both I

B

and I

B

(Fig.
8B),
despite their differential effects on NF-

B activation. These
data suggest that differential NF-

B activation by CD40L and by
LPS may be partly due to targeting of different I

B
proteins,
but other mechanisms also exist.
 |
DISCUSSION |
Role of NF-
B/Rel proteins in IgG1 production during
T-cell-dependent immune responses.
Preferential induction of the
IgG1 antibody isotype during T-cell-dependent immune responses in mice
appears to be mediated by CD40 signaling, as disruption of CD40L-CD40
interaction severely affects production of IgG1 but not other IgG
isotypes (for a review, see reference 16). CD40
engagement contributes to the preferential IgG1 production via
induction of GL
1 transcripts by activating NF-
B/Rel
proteins. NF-
B/Rel proteins have been shown to associate critically with immune responses (1). Targeted disruptions of individual NF-
B/Rel genes in mice have various effects on expression of Ig classes, providing further evidence of the important roles of NF-
B in isotype switching. Data presented in this
report demonstrate that p50, RelA, and RelB are activated by CD40L,
resulting in transactivation of the GL
1 promoter. Thus, IgG1
production in response to T-dependent antigens should be reduced in
mice deficient in p50, RelA, or RelB. It has been shown that mice
deficient in p50 or RelB have impaired IgG1 production in response to
T-dependent antigens (38, 49). The defect in IgG1 production
is more severe in p50
/
mice than in
RelB
/
mice, which can be explained by the observations
that all CD40L-induced NF-
B/Rel dimers binding to the GL
1
promoter contain p50 and that p50-RelA transactivates the promoter.
However, when splenic B cells from p50
/
mice were
tested for the ability to express GL
1 transcripts in vitro, it was
found that CD40L plus IL-4 plus IL-5 induces wild-type levels of
transcripts (43). It is likely that IL-4 circumvents the
defect in NF-
B activation, since the GL
1 promoter can also
be activated by IL-4. In the in vivo situation, the cytokines and
T-cell contact help are probably more limiting and locally delivered;
therefore, each signaling pathway may be important for GL
1 promoter
activation. This may also explain why cultured splenic B cells from
RelB
/
mice produce normal levels of IgG1 in response to
CD40L plus IL-4 plus IL-5 (42). However, it is still
possible that p50 and RelB are involved at additional levels in class
switching in vivo.
Disruption of the
relA gene causes embryonic lethality
(
2). However, SCID mice with B cells reconstituted by
transplantation
of RelA
/
fetal liver cells show a
10-fold decrease in serum IgG1, but
not in all isotypes, compared to
SCID mice receiving normal fetal
liver cells. IgG1 production in
response to T-dependent antigens
was not investigated (
10).
In c-Rel-deficient mice, cytokine production and T-cell functions are
affected, so the effects on antibody production are
probably indirect
(
22). It has recently been reported that B
cells from mice
with a mutated c-
rel gene that contains the DNA-binding
domain but no transactivation domain do not express GL

1 transcripts
in response to LPS and IL-4 (
52). These results may seem to
contradict our data, but it is likely that the c-Rel DNA-binding
domain
competes with the transactivating NF-

B/Rel dimers for
binding to
the GL

1 promoter and thereby inhibits promoter activity.
CD40L and LPS have different patterns of NF-
B
activation.
Induction of nuclear translocation of NF-
B/Rel
proteins by a variety of NF-
B inducers is regulated by
degradation of I
B proteins and can be transient or persistent
(1). LPS has been shown to induce NF-
B binding
activity more persistently than phorbol myristate acetate and
TNF-
(46). However, our data demonstrate that LPS
persistently activates p50 and c-Rel, but not RelA or RelB, in B cells,
indicating that persistent induction can be restricted to certain
NF-
B/Rel proteins and that nuclear accumulation of individual
NF-
B/Rel proteins is regulated differentially. By contrast,
CD40L activates all NF-
B/Rel proteins in splenic B cells and in
the two B-cell lines we examined for more than 24 h (Fig. 3 and
data not shown). Neumann et al. (30) reported similar
results, showing that CD40L expressed on L cells induces RelA for
24 h and p50, c-Rel, and RelB for up to 48 h in splenic B cells.
The mechanisms that mediate more persistent induction of RelA and RelB
by CD40L and the mechanisms for persistent induction
of c-Rel by LPS
and CD40L treatment need to be further investigated.
Analyses of the
degradation of I

B proteins induced by CD40L and
LPS indicate that
targeting of different I

B proteins may contribute
to differential
NF-

B activation. However, CD40L and LPS induce
similar
degradation kinetics of I

B

and I

B

in M12.4.1 cells,
indicating that other mechanisms must regulate differential NF-

B
activation by these inducers. Other I

B proteins that we have
not
examined may be differentially regulated (
1,
50). Another
possibility is that nuclear RelA and RelB induced by CD40L, as
compared
to LPS, may be more stable.
The mechanisms that mediate induction of RelB appear to differ from
those activating RelA and RelB and remain to be elucidated.
Although
CHX treatment inhibits nuclear translocation of RelB,
we found that the
levels of RelB mRNA and cytoplasmic RelB protein
are not changed after
12 h of CD40L treatment in M12.4.1 cells;
thus, it is unlikely
that synthesis of RelB protein mediates RelB
induction at this time
(data not shown). It has been shown, however,
that after 24 h of
CD40L stimulation of splenic B cells, RelB
mRNA levels are induced
(
30). One possible mechanism for the
unique characteristics
of RelB induction is that a newly synthesized
RelB transporter(s)
and/or signaling protein is required for nuclear
translocation and
activation of RelB.
Positive and negative regulatory effects of c-Rel on gene
expression.
Gene expression can be positively or negatively
regulated by c-Rel, depending on the gene and cell type. In
c-Rel-deficient mice, production of IL-3 and GM-CSF by T cells and
production of TNF-
and inducible nitric oxide synthase by
resident peritoneal macrophages are significantly reduced,
indicating that c-Rel can transactivate gene expression (12,
17). In contrast, the expression of GM-CSF, G-CSF, and IL-6 in
resident peritoneal macrophages is increased in c-Rel-deficient
mice (17), indicating that c-Rel also plays a negative role
in controlling gene expression. Further evidence for a negative role is
the finding that expression of c-Rel inhibits RelA-mediated
transactivation of the long terminal repeat of human immunodeficiency
virus (9).
By overexpression of NF-

B/Rel fusion proteins, we found that the
p50-c-Rel dimer has different effects on the mouse GL

1
and

promoters. The p50-c-Rel dimer poorly induces the GL

1
promoter and
suppresses transactivation of the GL

1 promoter
by p50-RelA and
p50-RelB dimers. Since the p50-c-Rel heterodimer
and p50 homodimer
bind well to the CD40RR of the GL

1 promoter,
repression appears to
be mediated by competition with transactivating
NF-

B/Rel
dimers for DNA binding. In contrast, GL

promoter activity
is
efficiently induced by p50-c-Rel fusion protein, albeit still
not as
well as by p50-RelA.
The transactivation activity of c-Rel may be mediated by interaction
with other transcription factors. For example, Wang et
al.
(
47) reported that coexpression of c-Rel together with
NF-ATc
protein, but not c-Rel or NF-ATc alone, strongly induces
IL-2
promoter activity. RelA did not appear to substitute for c-Rel.
Therefore, c-Rel may preferentially activate promoters that also
bind
NF-ATc.
The preferential induction of c-Rel might cause LPS to inhibit
induction of the GL

1 promoter by CD40L. However, we found
that when
added together with CD40L, LPS does not significantly
reduce induction
of promoter activity by CD40L in M12.4.1 cells
(
25).
p50-RelA and p50-RelB were found to be the dominant NF-

B/Rel
dimers binding to the GL

1 promoter in nuclear extracts from
M12.4.1
cells treated with LPS and CD40L (data not shown). Surprisingly,
however, the combination of LPS and CD40L synergistically induces
the
endogenous GL

1 transcripts in 1B4.B6 cells, although p50-c-Rel
is
still the predominant NF-

B/Rel dimer induced by LPS plus CD40L
in these cells (data not shown). The synergistic effect of LPS
and
CD40L might be explained by activation of transcription via
sequences
outside the promoter segment present in the luciferase
reporter gene we
use, perhaps by interaction of the p50-c-Rel
dimer with another
transcription factor(s) induced by LPS plus
CD40L.
In conclusion, the ability of CD40 signaling to induce GL

1
transcription contributes to the preferential production of IgG1
production during T-dependent immune responses. Persistent
activation
of transactivating NF-

B/Rel dimers in excess over
nontransactivating
NF-

B/Rel dimers mediates induction of GL

1
transcripts by CD40L.
In contrast, LPS predominantly activates
nontransactivating NF-

B/Rel
dimers, explaining why LPS in the
absence of T-cell help does
not induce IgG1 production in B cells.
 |
ACKNOWLEDGMENTS |
We thank N. Rice for antisera to RelA and p50. We thank W. E. Paul for recombinant mouse IL-4. We thank C.-H. Shen for the RelA
expression plasmid.
The research was supported by a grant to J.S. from NIH (AI23283) and by
grants to H.H.W. from NIH (AI15803 and AR43773).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Microbiology and Program in Immunology and
Virology, University of Massachusetts Medical School, 55 Lake Ave.
North, Worcester, MA 01655-0122. Phone: (508) 856-4100. Fax: (508)
856-1789. E-mail: janet.stavnezer{at}banyan.ummed.edu.
 |
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