INTRODUCTION

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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 (C_H) 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, C_H gene to which the cell will switch. Transcription from the GL C_H gene initiates at exon I, upstream of the switch region of each C_H 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 C_H 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

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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 C1-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.

(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.

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).

Antibodies against NF-B/Rel and IB proteins. Antibodies against p50 (sc-114), c-Rel (sc-071X), RelB (sc-226X), IB (sc-371), and IB (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

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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).

View larger version (31K): [in this window] [in a new window]   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.

View larger version (48K): [in this window] [in a new window]   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).

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).

View larger version (50K): [in this window] [in a new window]   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.

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).

View larger version (51K): [in this window] [in a new window]   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.

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.

View larger version (69K): [in this window] [in a new window]   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.

View larger version (41K): [in this window] [in a new window]   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.

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).

View larger version (26K): [in this window] [in a new window]   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.

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.

View larger version (36K): [in this window] [in a new window]   FIG. 8.   Western blot analyses demonstrate induction of IB 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 IB and IB proteins sequentially.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

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.

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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 IB 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.

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).