NF-{kappa}B is required for CD38-mediated induction of C{gamma}1 germline transcripts in murine B lymphocytes

Hiroaki Kaku1, Keisuke Horikawa1, Yuichi Obata2, Ichiro Kato3, Hiroshi Okamoto3, Nobuo Sakaguchi4, Steve Gerondakis5 and Kiyoshi Takatsu1

1 Division of Immunology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan 2 Department of Pathology, Aichi Cancer Center, Nagoya 464-8681, Japan 3 Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 4 Department of Immunology, Kumamoto University School of Medicine, Kumamoto 960-8575, Japan 5 Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia

The first two authors contributed equally to this work
Correspondence to: K. Takatsu; E-mail: takatsuk{at}ims.u-tokyo.ac.jp
Transmitting editor: K. Sugamura


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ligation of CD38 on murine B cells with agonistic anti-CD38 mAb induces B cell proliferation, expression of germline {gamma}1 transcripts and enhances IL-5 receptor expression. This leads to Ig class switch recombination from the µ to {gamma}1 heavy chain gene, and high levels of IgM and lgG1 production, particularly in response to anti-CD38 and IL-5 co-stimulation. Although some of the post-receptor signaling events initiated by CD38 ligation have been characterized, signaling pathways involved in CD38-mediated germline {gamma}1 transcript expression in B cells are poorly understood. Here we show that CD38 ligation of murine splenic B cells activates members of the NF-{kappa}B/Rel family of proteins including c-Rel, p65 and p50. The activation patterns and kinetics of NF-{kappa}B-like proteins in CD38-stimulated B cells differ somewhat from those seen in CD40-stimulated B cells. Activation of NF-{kappa}B-like proteins by CD38 ligation is not observed in splenic B cells from Bruton’s tyrosine kinase (Btk)-deficient (Btk–/–) mice, with inhibitors of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase also suppressing NF-{kappa}B activation in CD38-activated B cells. We infer from these results that activation of Btk, PI-3 kinase and PKC play, at least in part, important roles in the induction of NF-{kappa}B in CD38-stimulated murine B cells. Consistent with a role for NF-{kappa}B/Rel signaling in CD38-mediated germline {gamma}1 transcript expression, p50–/– B cells show significant impairment of germline {gamma}1 transcript expression in response to CD38 ligation, whereas the CD40-induced response was not altered. In contrast, c-Rel–/– B cells show a severe impairment of germline {gamma}1 transcript expression in response to CD38 or CD40 ligation. These results indicate an essential role for NF-{kappa}B proteins in the induction of germline {gamma}1 transcripts by CD38-ligated murine B cells giving rise to IL-5-induced IgG1 production.

Keywords: Bruton’s tyrosine kinase, c-Rel, IgH switch recombination, IL-5, phosphatidylinositol-3 kinase, transcription factors


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The NF-{kappa}B/Rel family of transcription factors, which includes p50, p52, p65 (RelA), RelB and c-Rel, forms homodimers and heterodimers that in most cell types are retained as inactive complexes in the cytoplasm by specific inhibitor (I{kappa}B) proteins (17). Cellular activation by a range of stimuli involved in inflammation and immunity, such as BCR cross-linkers, CD40 ligand, cytokines, chemokines and lipopolysaccharide (LPS), leads to the phosphorylation and degradation of the I{kappa}B component of the NF-{kappa}B complex, resulting in the translocation of NF-{kappa}B/Rel complexes to the nucleus (1,2,5,6). Subsequent binding of NF-{kappa}B to decameric {kappa}B sequence motifs within the promoter/enhancer of a wide variety of genes plays a key role in regulating their transcription in a positive manner (8). The subunits of the NF-{kappa}B/Rel family of transcription factors can interact with each other to create different heterodimeric forms with resulting differences in binding specificity, function and interactions with other transcription factors. Pre-B cells express mainly p50 and p65, whereas p50 and c-Rel predominate in mature B cells.

BCR cross-linking leads to the activation of Syk, phosphatidylinositol (PI)-3 kinase, Bruton’s tyrosine kinase (Btk), BLNK, phospholipase C (PLC)-{gamma}2, protein kinase C (PKC) and NF-{kappa}B (9). Btk acts in concert with Syk to phosphorylate and activate PLC-{gamma}2 (10,11), which in turn mediates production of the second messengers inositol 1,4,5-triphosphate and diacylglycerol (12). These second messengers stimulate the activity of PKC and increase intracellular calcium levels, resulting the activation of downstream transcription factors (13,14). It has recently been shown that Btk (15,16), BLNK (17), PLC-{gamma}2 (18) and PKC (19,20) are all involved in BCR-induced NF-{kappa}B activation. In addition, PI-3 kinase gene-targeted (p85–/–) mice reveal a profoundly impaired B cell phenotype (21,22). Whilst PI-3 kinase promotes cell survival by activating Akt kinase, which phosphorylates and inactivates the pro-apoptotic molecule, Bad (23), a connection between Akt and IKK-{gamma} has also been shown to be responsible for targeting I{kappa}B for degradation and NF-{kappa}B activation (24,25).

CD38 is a type II transmembrane glycoprotein that possesses both ADP-ribosyl cyclase and cADP-ribosyl hydrolase activities, and is widely expressed in both hematopoietic and non-hematopoietic cells (2632). Murine CD38 is expressed in follicular B cells, but is down-regulated in germinal center B cells (30,33,34). Stimulation of CD38+ lymphocytes with agonistic anti-CD38 mAb has profound effects on cellular viability, activation, proliferation and differentiation (29,30,35). We previously reported that ligation of splenic B-2 cells with agonistic anti-CD38 (clone CS/2) induces the expression of germline {gamma}1 transcripts, enhances the expression of the IL-5 receptor {alpha} chain (IL-5R{alpha}) and prevents B cell apoptosis (3537). Furthermore, IL-5 stimulation of anti-CD38-activated B-2 cells induces µ–{gamma}1 class switch recombination (CSR) and IgM and IgG1 production in an IL-4-independent manner (3739). Because of its multiple functions, the signaling pathways triggered by CD38 are expected to be diverse. CD38 ligation by anti-CD38 facilitates activation of tyrosine kinases (Lyn and Btk) (36,41,42), PI-3 kinase (43,44) and phosphorylation of c-Cbl (45). Nonetheless, a direct link between specific signaling pathway and the expression of germline {gamma}1 transcripts remains unclear.

B cells lacking p50 (4648), c-Rel (46,49,50), RelB (51) and, to a lesser extent, p52 have proliferative defects that may depend upon the manner by which the B cells become activated. The presence of multiple NF-{kappa}B/Rel binding sites in the Ig heavy chain locus including I regions and the switch regions has further suggested a potential role for NF-{kappa}B/Rel family members in regulating CSR. Interestingly, through studies performed in knockout mice, it became clear that individual members of the NF-{kappa}B family have distinct roles in the immune system. Snapper et al., using B cells from p50/NF-{kappa}B1-deficient mice, demonstrated that p50/NF-{kappa}B plays key selective roles in germline heavy chain RNA expression and CSR (48). They also reported results obtained by using B cells genetically deficient in the c-Rel transactivation domain ({Delta}c-Rel) that {Delta}c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS plus IL-4 (50). These failures of CSR are associated with a corresponding loss of germline {gamma}3, {gamma}1 or {epsilon} RNA. The ability of {Delta}c-Rel B cells to switch to IgG1 can be restored through the action(s) of additional stimuli that were associated with induction of normal levels of germline {gamma}1 RNA expression relative to controls (50). Their data demonstrated a key and selective role for c-Rel in the regulation of CSR, and suggested the distinct differences in the Ig isotype induction profiles of B cells lacking c-Rel compared with those deficient in p50/NF-{kappa}B.

To gain a better understanding of the role of NF-{kappa}B in the cellular activation of CD38-ligated B cells, we analyzed splenic B cells from p50–/– and c-Rel–/– mice for their ability to undergo proliferation, germline {gamma}1 RNA expression and IgG1 production. We found that both the p50 and c-Rel proteins play an important role in CD38-induced germline {gamma}1 gene expression. We will also discuss the roles of PI-3 kinase, Btk and PKC in the CD38-dependent activation of the NF-{kappa}B/Rel family proteins.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female C57BL/6 and BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan) and used at 8–12 weeks of age. CD38–/– mice and c-Rel–/– and (49,52) were backcrossed to C57BL/6 mice for eight generations and used for experimentation at 8–12 weeks of age. The genotype of CD38–/– mice was determined by PCR of ear DNA using the following primers: C30 (5'-GTCAACCCCCTAGAGTAAGCAGCAA-3'), C63 (5'-CAAGGGTCTACACAGGATCACCAAG-3') and L129 (5'-TCTCAGGAGGTATCAGTTCAAACCC-3'). The p50–/– mice and wild-type littermates were purchased from the Jackson Laboratory (Bar Harbor, ME). Btk–/– mice (53), kindly provided by Dr Fred Alt (Harvard University, Boston, MA), were backcrossed to C57BL/6 mice for seven generations and maintained in our animal facility. All mice were housed in microisolator cages under pathogen-free conditions, maintained on a diet of laboratory chow and water available ad libitum in the animal facility of the Institute of Medical Science, University of Tokyo. All experiments were performed according to the guidelines for animal treatment at the Institute of Medical Science, University of Tokyo.

Antibodies and reagents
Agonistic mAb for murine CD38 (clone CS/2, rat IgG1) and CD40 (clone LB429, rat IgG2a) were prepared as previously described (35,54). Affinity-purified F(ab')2 fragments of goat anti-mouse IgM antibody and LPS were purchased from Cappel (Durham, NC) and Difco (Detroit, MI) respectively. Streptavidin-conjugated magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The following mAb were obtained from ATCC (Rockville, MD): RA3-6B2 (anti-B220), 2.4G2 (anti-mouse Fc{gamma}R) and M1/70 (anti-Mac-1). Anti-mIL-5R{alpha} (clone H7, rat IgG1) was prepared as described (55,56). Purified mAb was coupled with biotin (Pierce, Rockford, IL), FITC (Sigma, St Louis, MO) or phycoerythrin (PE; Pierce). Streptavidin–PE was purchased from Life Technologies (Tokyo, Japan). Mouse IL-4 was purified from cultured supernatant of IL-4-producing cells using 11B11 (anti-IL-4 mAb)-coupled beads as previously described (57). IL-5 was purified according to previously described procedures (36,56). PI-3 kinase inhibitor, LY 294002 (Calbiochem, San Diego, CA) and PKC inhibitors, Gö6976 and Gö6983 (Calbiochem), were dissolved in DMSO and further diluted with cell culture medium before use.

B cell culture
Splenic B cells were prepared using MACS column CS (Myltenyi, Stuttgart, Germany) by negatively selecting with anti-CD3–biotin, anti-Mac-1–biotin, and streptavidin–magnetic beads as previously described. This protocol yielded >95% pure population of B cells that were B220+ CD3, as determined by flow cytometry. The B cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY), supplemented with 8% FCS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, penicillin (50 U/ml) and streptomycin (50 µg/ml) in 96-well flat-bottom microtiter plates (Coaster, Pittsburgh, PA) at a concentration of 1 x 105 cells/well in a volume of 200 µl of medium with or without stimulants. To determine proliferative responses, cells were cultured in triplicate for 3 days and pulse-labeled with [3H]thymidine (0.2 µCi/well; 1 µCi = 37 GBq, Amersham Life Science, Little Chalfont, UK) for the last 6 h of the culture. Incorporation of [3H]thymidine was measured according to procedures previously described (38). Results were expressed as the arithmetic mean c.p.m. ± SD of triplicate cultures. To determine Ig secretion, splenic B cells were cultured at a density of 1 x 105 cells/well in 200 µl of media for 7 days as previously described (37). Anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), IL-5 (100 U/ml), IL-4 (50 ng/ml) or a selected combination of those agents was added at the time the cells were plated. Cultures were set up in triplicate. The amounts of total IgM and IgG1 present in the culture supernatants were determined by ELISA. In some cases, cells were pretreated with LY294002 (10 µM), Gö6976 (300 nM) or Gö6983 (500 nM) for 1 h before adding anti-CD38 mAb (final concentration of DMSO did not exceed 0.1%). Each experiment was repeated at least 3 times, and one of the representative results is shown. For FACS analysis and preparation of RNA, B cells were cultured in a six-well plate at a density of 1 x 106 cells/ml.

Flow cytometry
For single-color flow cytometric analysis, cells were stained with FITC- or PE-conjugated mAb on ice for 30 min, and washed with PBS containing 2% FCS and 0.5% NaN3. Stained cells were analyzed on a FACSCalibur instrument (Becton Dickinson, Mountain View, CA) equipped with CellQuest software. 7-Aminoactinomycin D (2 µg/ml) (Sigma) was used to exclude dead cells from the analysis. For each sample, at least 1 x 104 cells were collected and analyzed.

Nuclear protein extract preparation and electrophoretic mobility shift assay (EMSA)
Nuclear protein extracts were prepared according to described procedures (5860). In brief, Cells were disrupted for 10 min on ice by hypotonic lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl and 1.5 mM MgCl2. To extract their nuclear proteins, cells were suspended for 20 min on ice in extraction buffer (20 mM HEPES, pH 7.9, 420 mM KCl, 1.5 mM MgCl2 and 25% glycerol) containing leupeptin (2 µg/ml), pepstatin (2 µg/ml), aprotinin (2 µg/ml), PMSF (0.5 mM), sodium orthovanadate (1 mM) and DTT (1 mM). After incubation, samples were centrifuged, and supernatants were collected as nuclear protein extracts and stored at –70°C. Protein concentration was determined by the Bradford method (Biorad, Hercules, CA) (61).

EMSA was carried out as previously described (58,59) in the following buffer: 10 mM HEPES pH 7.9, 50 mM sodium chloride, 1.5 mM EDTA, 5% glycerol and 0.1% NP-40. Each reaction mixture (25 µl) also contained 3 µg/ml of poly(dI:dC) (Amersham Pharmacia Biotech) and 4 x 104 c.p.m./2 µl of 32P-end-labeled probe. The probe was the following: sense, 5'-AGCTTCAGAGGGGACTTTCCGAGAGG-3'; anti-sense, 5'-TCGACCTCTCGGAAAGTCCCCTCTGA-3'. For EMSA supershift experiments or for antibody inhibition experiments, optimal amounts of antibodies specific for each component of the NF-{kappa}B/Rel family of proteins were incubated with a mixture of nuclear protein extracts and poly(dI:dC) in DNA binding buffer at 4°C for 60 min before the addition of 32P-labeled probe. Reaction mixtures were incubated at room temperature for another 30 min and the DNA–protein complexes were resolved on a native 4% polyacrylamide gel in 0.25x Tris borate–EDTA buffer for electrophoretic analysis at 150 V. Gels were dried and subjected to image analyzer (Fuji Photo Film, Tokyo, Japan). Antibodies for supershift or inhibition experiments against c-Rel, p50, p52, p65, RelB and normal rabbit serum IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Semi-quantitative RT-PCR analysis of germline {gamma}1 transcripts
Total RNA was extracted from splenic B cells before or after culture (2 day culture for germline {gamma}1 transcript assay) using TRIzol (Gibco/BRL, Gaithersburg, MD) according to the manufacturer’s instructions. cDNA synthesis was carried out in 20-µl aliquots of reaction mixture containing 5 µg total RNA and oligo(dT) primer and Superscript II RNase H reverse transcriptase (Gibco/BRL) as described previously (38). For semi-quantitation, serial dilutions of the cDNA templates were subjected to PCR amplification using the following primers: I{gamma}1 and C{gamma}1R for the germline {gamma}1 transcript and HPRT S1 and HPRT AS1, as described previously (38). PCR products were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.

Western blot analysis
Cellular extracts of B cells were prepared as previously described (36) and subjected to SDS–PAGE (7.5% gel). For Akt immunoblotting, equal amounts of whole-cell lysates (equivalent to 4 x 105 cells/sample) were loaded onto each lane of a 7.5% SDS–PAGE gel. Proteins were transferred on a PVDF membrane (Amersham) that was blocked with 5% non-fat dry milk in TBST buffer (10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) for 1 h at room temperature. The membranes were further incubated with optimal concentrations of anti-phospho-Akt antibodies (1:1000) (New England Biolabs, Mississauga, Ontario, Canada). The membranes were then washed 3 times with TBST and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated donkey anti-rabbit IgG (PharMingen, San Diego, CA) in TBST plus 5% BSA. The membrane was washed with TBST 3 times and immunoreactive bands were visualized by ECL detection (Amersham Pharmacia Biotech). The same membrane was re-probed with anti-Akt antibody (New England Biolabs) after removing anti-phospho-Akt.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Activation of NF-{kappa}B upon stimulation of naive B cells with agonistic anti-CD38 antibody
It has been reported that treatment of murine splenic B cells with various mitogenic stimuli induces activation of NF-{kappa}B. Cross-linking of CD38 on the murine B cell surface by agonistic anti-CD38 (CS/2 clone) generates a proliferation signal and the increased expression of germline {gamma}1 transcripts (3539,42). However, it still remains unclear whether CD38 ligation of naive B cells activates NF-{kappa}B. To examine whether CD38 ligation activates NF-{kappa}B, we stimulated purified splenic B cells for various periods of time with anti-CD38. As controls, B cells were stimulated with anti-CD40, anti-IgM or LPS. Nuclear protein extracts of cells from each group of stimulation were prepared and subjected to EMSA using a NF-{kappa}B binding DNA probe. As can be seen in Fig. 1(A), anti-CD38 stimulation of wild-type B cells induced NF-{kappa}B activation at similar levels to that seen in the anti-CD40, anti-IgM or LPS stimulation. As expected, CD38–/– B cells did not show NF-{kappa}B activation upon anti-CD38 stimulation, but showed NF-{kappa}B activation similar to that of wild-type B cells in response to anti-CD40, anti-IgM and LPS stimulation (Fig. 1B). Kinetic analysis revealed that the NF-{kappa}B activation by anti-CD38 stimulation was observed within 3 h, peaked at 6 h and declined by 12 h. The kinetics of anti-CD38-induced NF-{kappa}B activation differed from that of anti-IgM and anti-CD40, which peaked at 1 and 3 h respectively (Fig. 1C and D). These results indicate that CD38 ligation on splenic B cells induces the NF-{kappa}B activation differently from that of CD40 and BCR stimulation.



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Fig. 1. Activation of NF-{kappa}B in splenic B cells by CD38 ligation. Splenic B cells (10 x 106 cells/stimuli) from C57BL/6 or BALB/c (A, C and D) and CD38–/– (B) mice were stimulated with either anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), anti-IgM (10 µg/ml) or LPS (10 µg/ml) for 6 h and then lysed. Nuclear proteins (0.5 µg) prepared from each of the treatments were subjected to EMSA using a {gamma}-32P-radiolabeled DNA probe containing the NF-{kappa}B-binding site. (C and D) Kinetic analysis for NF-{kappa}B activation in anti-CD38-stimulated B cells. Arrows indicate probe–NF-{kappa}B complexes.

 
To address which NF-{kappa}B/Rel proteins are activated by CD38 ligation, we carried out EMSA by using antibodies specific for each NF-{kappa}B/Rel family member. Nuclear extracts isolated from splenic B cells incubated with anti-CD38, anti-CD40, anti-IgM or LPS for 6 h were used for EMSA. Nuclear complexes induced by anti-CD38 were supershifted with antibodies specific for p50, p65 and c-Rel, but not p52 or RelB (Fig. 2). These supershifted patterns were similarly to those observed upon anti-IgM or LPS stimulation, indicating that CD38 ligation of splenic B cells induces p50, p65 and c-Rel activation like BCR cross-linking and LPS stimulation. Interestingly, anti-CD40 stimulation activated RelB in addition to p50, p65 and c-Rel (Fig. 2).



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Fig. 2. NF-{kappa}B family members in B cells activated by CD38 ligation differ from those in CD40-activated B cells. Nuclear proteins (0.5 µg) prepared from each of the treatments were subjected to EMSA using a {gamma}-32P-radiolabeled DNA probe containing the NF-{kappa}B-binding site. Rabbit antibody (1 µg) against p50, p52, p65, RelB or c-Rel was added to a separate set of reactions for 1 h prior to EMSA. As a control, normal rabbit serum (NRS) was added. Smaller arrows indicate probe–NF-{kappa}B complexes and larger arrows indicate supershift complexes with anti-NF-{kappa}B antibodies.

 
Requirement of Btk and downstream signaling molecules for CD38-induced activation of NF-{kappa}B
We have reported that the activation of Btk, Lyn and Fyn is involved in CD38-induced B cell activation (43,44). Prior studies have demonstrated that BCR-induced NF-{kappa}B activation in murine B cells is profoundly impaired in Btk-deficient (Btk–/–) mice (15,16). However, a biochemical link between Btk and NF-{kappa}B has not been well established. Splenic B cells from either wild-type or Btk–/– mice were exposed to various B cell stimuli that induce NF-{kappa}B activation. Nuclear extracts were prepared and analyzed for NF-{kappa}B DNA-binding activity by EMSA. As shown in Fig. 3(A), anti-CD38, anti-CD40, anti-IgM or LPS stimulation of splenic B cells led to a marked increase in NF-{kappa}B activity (Fig. 3A, cf. lanes 1 and 3, 5, 7 and 9). However, anti-CD38- and anti-IgM-mediated induction of NF-{kappa}B was negligible in Btk–/– B cells (Fig. 3A, lanes 4 and 8). Interestingly, anti-CD40 and LPS stimulation led to the activation of NF-{kappa}B (Fig. 3A, cf. lanes 5 versus 6 and 9 versus 10).



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Fig. 3. (A) Impaired NF-{kappa}B activation of Btk–/– B cells in response to CD38 ligation. The {gamma}-32P-labeled probe containing an NF-{kappa}B-binding site was incubated with 0.5 µg of splenic B cell nuclear extract (10 x 106 cells/stimuli) from Btk–/– B cells (10 x 106 cells/stimuli) stimulated with anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), anti-IgM (10 µg/ml) or LPS (10 µg/ml) for 3 and 6 h. EMSA was performed as above. The arrows indicate DNA-binding complexes. (B) Effect of PI-3 kinase inhibitor on anti-CD38-induced NF-{kappa}B activation. Splenic B cells (10 x 106 cells/stimuli) were pre-treated with PI-3 kinase inhibitor, LY294002 (10 µM) for 60 min at 37°C before addition of anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), anti-IgM (10 µg/ml) or LPS (10 µg/ml). The B cells were incubated for another 3 or 6 h. EMSA was performed as above.

 
One of the critical downstream events following activation of Btk is the recruitment of PI-3 kinase to the plasma membrane. Activated Btk, together with Syk, phosphorylates and activates PLC-{gamma}2 (10,11), resulting hydrolysis of phosphatidylinositol 4,5-bisphosphate and production of the second messengers inositol 1,4,5-triphosphate and diacylglycerol (12). These second messengers stimulate the activity of PKC and increase intracellular calcium levels, resulting in activation of downstream transcription factors (13,14). When splenic B cells were stimulated with anti-CD38, tyrosine phosphorylation of Btk was clearly evident (36). However, tyrosine phosphorylation of BLNK, PLC-{gamma}2 or Vav was not observed in anti-CD38-stimulated B cells (data not shown). This was in sharp contrast to anti-IgM stimulation in which significant tyrosine phosphorylation of Vav, BLNK and PLC-{gamma}2 was induced within 2 min (data not shown).

To investigate the relationship between PI-3 kinase and NF-{kappa}B in anti-CD38-induced B cell activation, we stimulated splenic B cells with anti-CD38 in the presence of the well-characterized PI-3 kinase-specific inhibitor LY294002 and monitored NF-{kappa}B activation. LY294002 abrogated the NF-{kappa}B activation induced by anti-CD38 and anti-IgM (Fig. 3B). This was not due to an increased rate of cell death because anti-CD38 stimulation maintained cell viability following treatment with LY294002. Interestingly, LY294002 showed little inhibition, if any, of NF-{kappa}B activation upon stimulation with anti-CD40 and LPS.

Another important protein kinase thought to be involved in NF-{kappa}B activation is PKC. To address whether PKC activation is involved in NF-{kappa}B activation, we added PKC inhibitors Gö6983 and Gö6976 to the B cell culture with various stimuli. Gö6983 and Gö6976 can inhibit the activity of PKC isoforms of {alpha}, ß, {gamma}, {delta} and {zeta}, and {alpha}, ß and µ respectively, all of which are expressed in B cells. Our results revealed that both Gö6983 and Gö6976 inhibited NF-{kappa}B activation induced by anti-CD38, but not by anti-CD40 or LPS (Fig. 4). To elucidate the role of PKC on germline {gamma}1 expression of splenic B cells activated by anti-CD38, we performed RT-PCR using B cells pre-treated with PKC inhibitors. Both Gö6983 and Gö6976 inhibited germline {gamma}1 transcript expression in response to anti-CD38 (Fig. 4B). These data indicate that PKC and NF-{kappa}B activation is involved in CD38-mediated germline {gamma}1 transcript expression.



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Fig. 4. Effect of PKC inhibitor on CD38-induced NF-{kappa}B activation. (A) Splenic B cells were pre-treated with PKC inhibitor Gö6976 or Gö6983 for 60 min at 37°C, and stimulated with anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), anti-IgM (10 µg/ml) or LPS (10 µg/ml) for 3 and 6 h. EMSA was performed as above. (B) Semi-quantitative RT-PCR analysis of germline {gamma}1 transcript expression in B cells activated by CD38 ligation. Splenic B cells (5 x 106 in a 5 ml culture) were pre-treated with PKC inhibitors as Fig. 4(A) and cultured in the presence of anti-CD38 (1.0 µg/ml) for 48 h. Total RNA was prepared from both pre-cultured and cultured cells, and cDNA was prepared. Serial dilutions (4-fold) of the cDNA templates were subjected to PCR analysis using a set of primers amplifying germline {gamma}1 transcript. The HPRT gene was amplified in order to calibrate quantities of cDNA in each sample.

 
Proliferation in p50–/– and c-Rel–/– B cells in response to CD38 activation
There is numerous evidence that NF-{kappa}B/Rel family proteins play distinctive roles in cell growth and differentiation. To address whether NF-{kappa}B family proteins play a role in CD38-induced B cell activation, we stimulated splenic B cells from p50–/– and c-Rel–/– mice with various individual and combinations of stimuli including anti-CD38, LPS, IL-5 and anti-IgM for 72 h. B cell proliferation was monitored by [3H]thymidine incorporation. As a control, splenic B cells of wild-type littermate mice were cultured separately. The proliferative response of p50–/– B cells to anti-CD38 and anti-CD38 plus IL-5 and LPS were significantly lower than that of wild-type B cells (Fig. 5A). The proliferation induced by anti-CD40 and anti-CD40 plus IL-5 was similar to that of wild-type B cells. In contrast, proliferative responses of c-Rel–/– B cells to various stimuli examined were all severely impaired to the same extent (Fig. 5B). These results indicate that c-Rel activation is indispensable for B cell proliferation in response to anti-CD38, anti-CD40, LPS and anti-IgM. In contrast, p50 activation is required for inducing maximum levels of B cell proliferation induced by anti-CD38 and LPS.



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Fig. 5. Proliferative response of splenic B cells from p50–/– and c-Rel–/– mice in response to CD38 ligation. Splenic B cells from either 8-week-old wild-type littermate, p50–/– mice (A) and c-Rel–/– mice (B) were cultured (1 x 105 cells in a 200 µl culture) for 3 days with anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml), anti-IgM (10 µg/ml) or LPS (10 µg/ml). Cells were pulse-labeled with 0.2 µCi of [3H]thymidine for the last 6 h of the culture. The results represent mean c.p.m. + SD of triplicate cultures. We tested four mice for each group.

 
Role of NF-{kappa}B activation in the expression of germline {gamma}1 transcripts
To elucidate a role of NF-{kappa}B activation in the expression of germline {gamma}1 transcripts, we stimulated splenic B cells from wild-type, p50–/– or c-Rel–/– mice with anti-CD38, anti-CD40 and IL-4 for 2-days, and the expression of germline {gamma}1 transcript was monitored by RT-PCR. In wild-type B cells, anti-CD38 stimulation induced a significant expression of the germline {gamma}1 transcripts (Fig. 6A and B). However, in p50–/– B cells, the anti-CD38-induced expression of germline {gamma}1 transcripts was significantly decreased (Fig. 6A). The stimulation of p50–/– B cells with anti-CD40 or IL-4 induced the expression of germline {gamma}1 transcripts to a similar extent to that of wild-type B cells. In contrast, c-Rel–/– B cells showed markedly reduced expression of germline {gamma}1 transcripts in response to various stimuli including anti-CD38 (Fig. 6B). In particular, anti-CD40-induced expression of {gamma}1 transcripts was not induced in c-Rel–/– B cells. These results imply that activation of both p50 and c-Rel is indispensable for anti-CD38-induced germline {gamma}1 transcript expression. Further more, c-Rel plays a more important role than p50/NF-{kappa}B in CD40-induced germline {gamma}1 RNA expression.



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Fig. 6. Semi-quantitative RT-PCR analysis of germline {gamma}1 transcript expression in B cells activated by CD38 ligation. Splenic B cells (5 x 106 in a 5 ml culture) from either wild-type littermate, p50–/– mice (A) or c-Rel–/– mice (B) were cultured in the presence of anti-CD38 (1.0 µg/ml), anti-CD40 (1.0 µg/ml) or IL-5 (50 ng/ml). Total RNA was prepared from both pre-cultured and cultured cells 48 h after plating, and cDNA was prepared. Serial dilutions (4-fold) of the cDNA templates were subjected to PCR analysis using sets of primers amplifying germline {gamma}1 transcript. The HPRT gene was amplified in order to calibrate quantities of cDNA in each sample.

 
IgM and IgG1 production by p50–/– and c-Rel–/– B cells stimulated with anti-CD38 and IL-5
We cultured splenic B cells from wild-type, p50–/– and c-Rel–/– mice with anti-CD38, anti-CD40, IL-4, IL-5 or combinations of these stimuli for 7 days after which the concentration of IgM and IgG1 in the culture supernatants were then measured. Stimulation of splenic B cells from wild-type mice with IL-5, anti-CD38 plus IL-5 and anti-CD40 plus IL-5 induced significant IgM production, while p50–/– and c-Rel–/– B cells showed impaired IgM production (p50–/– B cells in Table 1 and c-Rel–/– B cells in Table 2). Stimulation of the wild-type B cells with anti-CD38 plus IL-5 and anti-CD40 plus IL-5 induced significant IgG1 production (Tables 1 and 2).


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Table 1. IgM and IgG1 production by wild-type and p50–/– B cells in response to various B cell mitogen and cytokinea
 

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Table 2. IgM and IgG1 production by wild-type and c-Rel–/– B cells in response to various B cell mitogen and cytokinea
 
Neither anti-CD38 plus IL-5 nor anti-CD40 plus IL-5 induced IgG1 production in splenic B cells from p50–/– mice (Table 1) and c-Rel–/– mice (Table 2). Stimulation of p50–/– and c-Rel–/– B cells with anti-CD40 plus IL-4 did not induce significant IgG1 production (data not shown). These results indicate that both p50 and c-Rel are indispensable for IgG1 production by B cells stimulated with either anti-CD38 or anti-CD40 in conjunction with IL-5 or IL-4.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
As we reported, anti-CD38 and IL-5 can induce a high level of IgM and IgG1 production in addition to promoting switching from IgM to IgG1 in naive (sIgD+) splenic B cells (37). In this system, ligation of splenic B cells with anti-CD38 induces the expression of germline {gamma}1 transcripts and enhances the expression of the IL-5R{alpha} (36,37). Furthermore, IL-5 stimulation of the anti-CD38-activated B cells induces µ–{gamma}1 class switch recombination and IgM and IgG1 production (3739). Accumulating data suggest that activation of NF-{kappa}B family proteins is indispensable for CSR. Thus, we examined the activation of NF-{kappa}B by anti-CD38 stimulation in murine B cells. The data presented in this paper demonstrated that stimulation of naive B cells with anti-CD38 alone activates NF-{kappa}B family proteins, resulting in the expression of germline {gamma}1 transcripts.

CD38 ligation of splenic B cells activates NF-{kappa}B/Rel
Our studies with murine splenic B cells indicated that CD38 ligation induced the NF-{kappa}B activation (Figs 1 and 2). Analysis of B cells from mice deficient for CD38 or individual NF-{kappa}B family members demonstrated that like CD40- and BCR-mediated B cell activation and proliferation (62,63), NF-{kappa}B is important in CD38-mediated B cell activation and proliferation (Figs 1, 6 and 7). Both p50 and c-Rel together with p65 were activated in B cells by anti-CD38 ligation (Fig. 2). Kinetic analysis revealed that the time-course of p50 and c-Rel activation induced by anti-CD38 differed from anti-CD40 stimulation (Fig. 1), suggesting that anti-CD38 stimulation activates NF-{kappa}B differently from anti-CD40 stimulation. IL-5 by itself did not activate NF-{kappa}B family proteins (data not shown). While this is the first report which has highlighted a link between NF-{kappa}B activation and CD38 ligation of B cells, the underlying molecular mechanisms by which they are linked are still obscure. It would be important in future to examine whether the NF-{kappa}B activation is primary response or separate response requiring de novo protein synthesis.

Btk is essential for CD38-induced NF-{kappa}B activation
Btk is critical in early B cell development as well as in mature B cell activation and survival. Genetic defects in Btk cause human X-linked agammaglobulinemia, which is characterized by reduced numbers of peripheral B lymphocytes, low concentrations of serum Ig and varying degrees of bacterial infections. Likewise, xid mice, as well as Btk–/– mice, show impaired B cell development and function. NF-{kappa}B activation is one of the major downstream effects of Btk.

The NF-{kappa}B/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Nuclear NF-{kappa}B is induced in B cells by engagement of either the BCR, CD40 cross-linking or stimulation with LPS. Two groups independently reported diminished IgM-, but not CD40-mediated NF-{kappa}B/Rel nuclear translocation and DNA binding in B cells from xid as well as Btk–/– mice (15,16). As shown in Fig. 3(A), NF-{kappa}B activation was severely impaired in Btk–/– B cells upon anti-CD38 or anti-IgM stimulation. Interestingly, in the absence of Btk, NF-{kappa}B was activated by CD40 ligation and LPS. These results are in agreement with Bajpai’s report (15).

PI-3 kinase and PKC are involved in CD38-mediated NF-{kappa}B activation
The pleckstrin homology (PH) domain is the best characterized domain of Btk. Molecules binding to the PH domain include phosphoinositides, several isoforms of PKC, TFII-I transcription factor (also known as BAP-135), F-actin, STAT3 and Fas (64). Mutation of the conserved arginine residue at position 28 (R28) in the PH domain of Btk in X-linked agammaglobulinemia patients markedly reduces the affinity between Btk and phosphoinositides, and highlights the importance of this domain in Btk signal transduction.

Despite the crucial role of PI-3 kinase in cell growth and differentiation, the downstream signaling events following PI-3 kinase activation have only recently begun to be addressed. In B cells, the PH domain-containing kinases Btk and Akt are activated in response to BCR cross-linking in a PI-3 kinase-dependent manner (21,22). Using a specific pharmacological inhibitor of PI-3 kinase, LY294002, Bone and Williams (65) first demonstrated the vital role of PI-3 kinase in BCR-induced NF-{kappa}B DNA-binding activity. They also showed that PI3- kinase is critical in triggering NF-{kappa}B DNA-binding ability following LPS stimulation. Using the PI-3 kinase inhibitor LY294002, we also demonstrated that PI-3 kinase is required for anti-CD38- and anti-IgM-induced NF-{kappa}B activation (Fig. 3B). Due to a decrease in cell viability by LY294002, we could not examine its inhibitory effect on CD38-mediated germline {gamma}1 transcript expression. Our findings indicate a critical role of PI-3 kinase in the activation of NF-{kappa}B following CD38 ligation and BCR cross-linking. This is the first report which has highlighted a link between PI-3 kinase and NF-{kappa}B activation following CD38 ligation of B cells, and may add supporting evidence for the importance of PI-3 kinase activation not only for BCR-stimulated B cells, but also for CD38-activated B cells.

A major downstream effector of PI-3 kinase, Akt, has recently been shown to be involved in NF-{kappa}B activation (66). Together with two recent reports showing a direct interaction between Akt and IKK-{alpha} in cell lines (24,25), PI-3 kinase was proposed to regulate I{kappa}B degradation in splenic B cells via Akt. We observed that Akt phosphorylation was induced by anti-CD38 stimulation and blocked by PI-3 kinase inhibitor LY294002 (data not shown). However, addition of an Akt inhibitor did not affect anti-CD38-induced NF-{kappa}B activation (data not shown). Our study, while establishing the signaling pathway responsible for CD38-mediated NF-{kappa}B/Rel activation involves PI-3 kinase, indicates it is unlikely that PI-3 kinase-dependent CD38- and BCR-induced activation of NF-{kappa}B occurs via an Akt-dependent route.

Among several PKC family members capable of binding to the PH domain of the Btk, Btk phosphorylates PKC-ßI upon BCR stimulation. Phosphorylated PKC may, in turn, directly or indirectly down-regulate the kinase activity of Btk, as observed in the case of PKC-µ. PKC is necessary for BCR- but not anti-CD40-induced activation of NF-{kappa}B (67). Btk is in turn responsible for the activation of PKC and calcium mobilization, and the activation of NF-{kappa}B. We showed in Fig. 4 that specific PKC inhibitors Gö6983 and Gö6976 potently blocked CD38- and BCR-induced NF-{kappa}B activation. These results demonstrate a critical role of PKC in that process.

NF-{kappa}B activation is essential for the germline {gamma}1 expression
In primary splenic B cells, BCR cross-linking or CD40 ligation leads to nuclear translocation and DNA binding by NF-{kappa}B/Rel transcription factors. Phenotypic analysis of mice deficient in individual NF-{kappa}B/Rel family members has demonstrated the essential role of these transcription factors in the CD40, LPS and BCR pathways leading to B cell proliferation. NF-{kappa}B/Rel proteins are important for initiating CSR to IgG1 in response to T-dependent antigens. CD40 engagement contributes to this preferential isotype production by activating NF-{kappa}B/Rel to induce germline {gamma}1 transcripts, which are essential for CSR. While the CD40 signal transduction pathway leading to NF-{kappa}B activation has been well-characterized (8,68), there is no information about the molecular signaling events connecting the CD38 to NF-{kappa}B activation. It is quite important to clarify a link between NF-{kappa}B activation and germline {gamma}1 expression in anti-CD38-stimulated naive B cells.

As we demonstrated in Fig. 2, both anti-CD38 and anti-CD40 stimulation can activate p50 in wild-type B cells. However, RT-PCR analysis of germline {gamma}1 transcripts in p50–/– B cells revealed that anti-CD38-induced expression of germline {gamma}1 transcripts was diminished, while anti-CD40-induced expression of germline {gamma}1 transcripts was in the normal range (Fig. 6A). This may be due to kinetic differences or to the induction of RelB by anti-CD40, but not by anti-CD38. Interestingly, IgG1 production was profoundly impaired in response to anti-CD38 plus IL-5 and anti-CD40 plus IL-5 (Table 1). There are at least two possibilities to account for these observations. First, p50 may play a critical role in IL-5-induced CSR from Cµ to C{gamma}1 in both CD38- and CD40-activated B cells. Alternatively, p50 may play an important role in terminal maturation of sIgG1 B cells to IgG1-producing cells. We do not have concrete evidence at this moment to support either one of the possibilities. RelB, which can be activated in response to anti-CD40 (Fig. 2), may compensate germline {gamma}1 transcript expression in anti-CD40-stimulated p50–/– B cells. In c-Rel–/– B cells, both anti-CD38- and anti-CD40-induced expression of germline {gamma}1 transcripts was severely impaired (Fig. 6B). We infer from these results that NF-{kappa}B activation is indispensable for the expression of germline {gamma}1 transcripts in B cells stimulated with either anti-CD38 or anti-CD40. We recently found that BCR cross-linking and LPS were able to induce the expression of germline {gamma}1 transcripts.

CD38-mediated signaling is supposed to resemble BCR-mediated signaling (30). In our preliminary experiments, anti-CD38 seems not to activate BLNK, PLC-{gamma}2 and Vav, which are activated by anti-IgM under the same conditions (data not shown). These data suggest to us that a part of the CD38 signaling pathway may not be coupled with the BCR-mediated pathway. Therefore understanding the specific control of NF-{kappa}B activation could provide clues to the differential physiological outcomes of these B cell activation pathways.


    Acknowledgements
 
We are indebted to S. Takaki, S. Takasawa and T. Tamura for encouragement and valuable suggestions throughout this study; to F. W. Alt and for providing us Btk–/– mice; and to J. Inoue for his valuable advice about NF-{kappa}B EMSA. We also would like to thank Y. Kikuchi and M. Eguchi for technical support in the experiments using p50–/– and c-Rel–/– mice. This work was supported in part by a Research Grant from the Human Frontier Science Program (K. T.), and by a Grant-in-Aid for Scientific Research on Priority Areas (A) (K. T.) from the Ministry of Education, Science, Sports and Culture, in Japan. This was also supported, in part, by a Grant-in-Aid for Encouragement of Young Scientists (K. H.) from the Japan Society for the Promotion of Science.


    Abbreviations
 
Btk—Bruton’s tyrosine kinase

CS/2—agonistic anti-CD38 mAb

CSR—class switch recombination

EMSA—electrophoretic mobility shift assay

IL-5R{alpha}—IL-5 receptor {alpha} chain

LPS—lipopolysaccharide

PE—phycoerythrin

PH—pleckstrin homology

PI-3 kinase—phosphatidylinositol-3 kinase

PKC—protein kinase C

PLC—phospholipase C


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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