p38 MAPK Is Required for CD40-Induced Gene Expression and Proliferation in B Lymphocytes¹

CD40 is a 45- to 50-kDa transmembrane glycoprotein member of the rapidly expanding TNFR superfamily, which currently includes TNFR1 and -2, CD95/Fas, a low affinity nerve growth factor receptor (NGFR), CD27, CD30, death receptor (DR3³/TRAMP/WSL/APO-3), DR4 (TRAIL-R1), DR5 (TRAIL-R2), TRAIL receptor without an intracellular domain (TRID/TRAIL-R3), CAR1, OX40, ATAR, and the LTβ receptor (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). CD40 is expressed in late bone marrow B cells, mature B cells, follicular DCs, DCs, and some other cell types including activated monocytes and epithelial cells (11, 12, 13, 14, 15). The ligand for CD40 (CD40L) is expressed on activated CD4⁺ T cells and a number of other cell types including DCs (16, 17). A critical role for CD40-CD40L intercellular interactions in the humoral immune response has been demonstrated in patients with X-linked hyper IgM syndrome (HIM), who are defective in the CD40L gene and who both lack the ability to form germinal centers and undergo Ig isotype class switching from IgM to IgG, IgE, and IgA (18). A similar phenotype has been observed in either CD40 or CD40L “knock-out” mice (19, 20). Extensive studies indicate CD40 plays a critical role in the activation, proliferation, and differentiation of B lymphocytes and prevents both immature B cells from undergoing B cell Ag receptor (BCR)-induced apoptosis and germinal center B cells from spontaneous apoptosis (16, 21, 22). Hence, a detailed knowledge of the proximal signaling pathways triggered upon CD40 stimulation is critical to understanding the molecular basis of the diverse nature of CD40 responses.

Among the earliest detectable signaling events following CD40 engagement are activation of the Src protein tyrosine kinase (PTK) member Lyn, phosphoinositide 3-kinase (PI 3-kinase) stimulation and Ras activation (23, 24, 25). In addition, CD40 engagement activates two subfamilies of the stress-activated protein kinases, the c-jun amino-terminal kinase (JNK/SAPK; Refs. 26–29) and p38 MAPK (28, 30), while inducing little or no activation of the more distantly related extracellular signal-regulated mitogen-activated protein kinases (ERK; Refs. 26–28). However, the role(s) of either JNK or p38 MAPK in the biologic functions of CD40 remain uncharacterized.

Considerable attention has been focused toward investigating the role of p38 MAPK in the regulation of gene expression at both transcriptional and translational levels. Thus, CHOP (GADD 153), a stress-activated member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors (31), and MEF2C, a member of the myocyte enhancer factor 2 family of transcription factors (32), are phosphorylated by p38 MAPK. In addition, MAPKAPK-2, a downstream target for p38 MAPK, phosphorylates CREB in response to FGF and arsenite (33). At the transcriptional level, the p38 MAPK pathway is required for c-jun and c-fos mRNA expression in response to UV light and anisomycin (34) and IL-1β-induced cyclooxygenase-2 mRNA synthesis (35).

Thus, since engagement of the CD40 receptor leads to activation of a variety of transcription factors, including NF-κB, AP-1, NFAT, and STATs 3 and 6 (36, 37, 38, 39, 40, 41), we investigated the role of the p38 MAPK pathway in CD40-induced transcriptional activation using the specific, cell-permeable p38 MAPK inhibitor, SB203580 (42, 43). Furthermore, we tested the role of the p38 MAPK pathway in CD40-induced gene expression at the transcriptional level.

Our results demonstrate that CD40 activates the p38 MAPK pathway in both human tonsillar B cells and multiple B cell lines. We show that the p38 MAPK pathway is required, at least in part for CD40-induced NF-κB activation and that the induction of CD40-responsive genes occurs via both p38 MAPK-dependent and -independent pathways. Moreover, we describe a role for the p38 MAPK pathway in CD40-driven proliferation of mature tonsillar B cells.

The mAb used in these studies were: G28-5 (IgG1) to human CD40 (44), 1C10 to murine CD40 (45), G19-4 (IgG1) to CD3 (46), G28-7 (IgG1) to CD22 (47), G28-1 (IgG1) to CD37 (48), G28-8 (IgG1) to Bgp95, which is defined by our single agonistic Ab and does not yet have a CD designation (49), and LB-2 (IgG_2b) to CD54 (50). We elected to use the G28-5 mAb to CD40 in these studies since we have previously shown that G28-5 anti-CD40 induces equivalent activation of both NF-κB and SAPK when compared with CD40L (27, 37). F(ab′)₂ fragments of goat anti-mouse IgM were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit polyclonal anti-p38 MAPK antiserum prepared against the C-terminal peptide ISFVPPLDQEEMES was generated as described (51). Rabbit polyclonal anti-JNK1 (C-17) antiserum, rabbit polyclonal anti-p50, p65 and c-rel antiserum, and rabbit polyclonal anti-Stat1 (E-23) antiserum were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep polyclonal anti-MAPKAPK-2 antiserum was purchased from Upstate Biotechnology (Lake Placid, NY). Affinity-purified rabbit polyclonal anti-c-jun Ab was from New England Biolabs (Beverly, MA). In vitro transcription and RNase protection assay kits were obtained from PharMingen (San Diego, CA). GST-c-jun (5-89) and GST-ATF2 were expressed in Escherichia coli, and the fusion proteins were purified as described (27). SB203580 was purchased from Calbiochem (San Diego, CA). EMSA oligonucleotide probes synthesized by Life Technologies (Gaithersburg, MD), were annealed and end-labeled with [γ-³²P]ATP using T4 polynucleotide kinase (Promega, Madison, WI), followed by spin-column purification (Chromaspin TE-10, Clontech, Palo Alto, CA). Atlas human cDNA expression arrays were purchased from Clontech.

The murine B lymphoma cell line WEHI-231 and the human Burkitt’s lymphoma line Daudi were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 10 U/ml penicillin, 10 μg/ml streptomycin, 1 mM pyruvate, and nonessential amino acids. In addition, 50 μM 2-ME was added to all murine cell lines. Dense and buoyant human tonsillar B-enriched cells were isolated as described (52).

B cells (5–10 × 10⁶ per sample) were resuspended in complete RPMI 1640 medium to a density of 1 × 10⁶/ml and stimulated with either anti-CD40 (1 μg/ml) or goat anti-mouse IgM (10 μg/ml) for the indicated times. Incubations were terminated by rapid dilution with approximately 40 to 50 ml ice-cold PBS and centrifuged at 500 × g for 5 min at 4°C. The supernatants were aspirated, and cells were lysed by resuspension and brief vortexing (5 s) with 0.1 ml per 1 × 10⁶ cells of RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM Na₄P₂O₇, 25 mM sodium β-glycerophosphate, 1 mM EDTA, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM PMSF, 1 mM Na₃VO₄, 10 μM E-64, 1 μg/ml pepstatin, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Following incubation on ice for 15 min, the lysed cells were centrifuged at 16,000 × g for 10 min at 4°C. The lysates were added to 20 μl packed protein A-Sepharose beads, and 5 μl rabbit polyclonal anti-p38 MAPK antiserum was added. Following constant mixing by inversion for 3 h at 4°C, immune complexes were pelleted at 4°C by centrifugation at 16,000 × g for 5 min. The beads were washed twice each with i) 1 ml of RIPA, 0.1 mM Na₃VO₄ and ii) 1 ml of p38 MAPK assay buffer (25 mM HEPES, pH 7.4, 25 mM sodium β-glycerophosphate, 25 mM MgCl₂, 2 mM DTT, 0.1 mM Na₃VO₄). p38 MAPK activity was quantified by immune complex assay in a final volume of 90 μl p38 MAPK assay buffer at 25°C for 20 min using 3 μg GST-ATF2 and 20 μM ATP containing 10 μCi [γ-³²P]ATP. Incubations were terminated with an equal volume of 2× SDS sample buffer. After boiling for 5 min and brief centrifugation, samples were resolved by 10% SDS-PAGE. The gels were stained for 30 min with 50% methanol, 10% acetic acid, 0.005% Coomassie brilliant blue G-250, destained for 30 min with 40% methanol, 10% acetic acid, 3% glycerol, and dried before autoradiography at −70°C for 3 to 6 h.

JNK1 activity was quantified by immune complex assay using GST-c-jun (5–89) as a substrate, as described previously (27).

p38 MAPK immunoprecipitates prepared as described above were mixed with 15 μl 4× SDS-PAGE sample buffer and applied without boiling onto a 1.5-mm thick 10% SDS-PAGE minigel copolymerized with 0.2 mg/ml MBP. Following resolution, the gel was washed twice for 30 min each with 100 ml buffer A (50 mM HEPES, pH 7.6, 5 mM 2-ME) containing 20% (v/v) isopropanol to remove SDS. Isopropanol was subsequently removed by washing twice for 30 min each with 100 ml buffer A, and proteins were denatured by two consecutive washes for 15 min each with buffer A plus 6 M urea. The gel was washed sequentially for 15 min each at 4°C with i) buffer A plus 3 M urea, ii) buffer A plus 1.5 M urea, and iii) buffer A plus 0.75 M urea to renature proteins; urea was subsequently removed with three washes for 15 min each at 4°C and a final wash overnight at 4°C with buffer A containing 0.05% (w/v) Tween 20. Following two washes for 30 min each at 30°C with kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl₂, 2 mM DTT), the gel was incubated for 30 min at 30°C with 10 ml 20 μM ATP plus 100 μCi [γ-³²P]ATP. The gel was finally washed 6 to 10 times for 30 min each with 100 ml of 5% (w/v) TCA and 1% (w/v) Na₄P₂O₇ to remove unincorporated [γ-³²P]ATP; it was then stained with Coomassie brilliant blue, destained, dried, and exposed to film.

Cell lysates (100 μl per 1 × 10⁶ cells) were prepared from 5 to 10 × 10⁶ Daudi, WEHI-231, or human tonsillar B cells stimulated with either 1 μg/ml anti-CD40 or 10 μg/ml anti-IgM as described for JNK1 (27). Lysates were mixed with 2 to 4 μg anti-MAPKAPK-2 serum and incubated at 4°C with constant inversion for 3 h at 4°C with the addition of 10 μl packed protein G-Sepharose for the final 90 min. Immunoprecipitates were washed twice each with 1 ml JNK lysis buffer containing 1 mM DTT and 0.25 mM Na₃VO₄, 1 ml JNK assay buffer plus 1 mM DTT and 0.25 mM Na₃VO₄ and 1 ml MAPKAPK-2 assay buffer (20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 25 mM MgCl₂, 2 mM EGTA, 1 mM DTT, 0.25 mM Na₃VO₄). Following brief incubation of the immune complexes with 125 μM MAPKAPtide (KKLNRTLSVA) for 5 min at 30°C, in vitro kinase reactions (total volume 80 μl) were initiated by the addition of 0.1 mM ATP containing 10 μCi [γ-³²P]ATP. After 30 min at 30°C, an aliquot of the reaction mixture (60 μl) was spotted onto phosphocellulose paper (2.5 × 2.5 cm; Whatman P81) and washed 4 to 5 times for 10 min each with 75 mM phosphoric acid (approximately 20 ml/filter) to remove unincorporated [γ-³²P]ATP followed by a single wash with acetone. Filters were dried, and ³²P incorporation into MAPKAPtide was quantified by liquid scintillation counting.

B cells (5 × 10⁶ per sample) were resuspended in complete RPMI 1640 medium to a density of 2 × 10⁶/ml and pretreated with either solvent vehicle (DMSO) or SB203580 (0–20 μM) for 30 min at 37°C before stimulation with either anti-CD40 (1 μg/ml) or a combination of PMA (50 ng/ml) and ionomycin (250 ng/ml) for 15 min. Samples were immediately cooled on ice and centrifuged at 500 × g for 5 min at 4°C. Supernatants were aspirated, and 200 μl JNK lysis buffer containing 1× SDS sample buffer was added. Following transfer to microfuge tubes, samples were lysed by vortexing for 5 min at 4°C, heated at 95 to 100°C for 5 min, and briefly cooled on ice. Following centrifugation at 14,000 × g for 5 min, 50 μl aliquots were resolved by 10% SDS-PAGE. Resolved proteins were electrotransferred overnight to nitrocellulose membranes in 25 mM Tris, pH 8.5, 0.2 M glycine, 20% methanol at 25 V. Blots were blocked for at least 10 h with 1× TBST plus 5% nonfat dry milk, and c-jun phosphorylation was analyzed essentially according to the manufacturer’s instructions using a polyclonal anti-c-jun Ab, except that the primary Ab dilution was 1:750.

Nuclear extracts were prepared from Daudi cells as reported previously (37) and divided into 5- to 10-μl aliquots before storage at −70°C. Once thawed, individual aliquots were never refrozen and reused. For EMSA, binding reactions contained 10 mM HEPES, pH 8.0, 20 mM NaCl, 4 mM MgCl₂, 0.1 mM EDTA, 17.5% (v/v) glycerol, 0.5 μg poly(dI-dC), and 1 mM DTT. Upon brief thawing on ice, equal quantities of nuclear protein were immediately added to the binding buffer. After 10 min incubation at 25°C, 50,000 cpm ³²P-labeled oligonucleotide probe was added, and the binding reactions (final volume 10 μl) were continued for an additional 30 min at 25°C. Following the addition of 1 μl of 0.5× TBE, 10% (v/v) Ficoll, aliquots (0–2.5 μl) were loaded onto preelectrophoresed (90 min) 4% native polyacrylamide gels (1 mm thick) prepared in 0.5× TBE buffer and resolved for approximately 2 to 3 h with 120 V constant voltage at 4°C. Gels were fixed in 10% (v/v) acetic acid for 15 min, dried, and exposed to film at −70°C. Supershift analyses were performed by preincubating 1 μg of either anti-p50, anti-p65, anti-c-rel, or a nonspecific rabbit control Ab (anti-Stat1) with the nuclear extract for 30 min at 25°C before addition of the radiolabeled oligonucleotide probe.

Transient transfection of the Daudi B lymphoblastoid line was performed using DEAE-dextran as described previously (37), except that, following the addition of 9 vol RPMI/10% FCS, incubations were continued for 1 to 2 h at 37°C. After culture for 36 to 48 h, cells were pretreated with 0 to 20 μM SB203580 (final DMSO concentration 0.1% (v/v)) for 1 h before stimulation for 6 h at 37°C in a humidified incubator with either 0 to 1 μg/ml anti-CD40 or isotype-matched control Ab (anti-CD3, anti-CD22, or anti-CD37). Cell lysates were prepared according to the manufacturer’s instructions (Promega), and luciferase activity was quantified using a luminometer (Berthold Analytical Instruments, Nashua, NH). The data are expressed as the -fold increase in luciferase activity relative to enzyme activity from unstimulated cells using equivalent quantities of protein.

Daudi cells (50–150 × 10⁶; 1 × 10⁶ cells/ml) were stimulated for either 0 or 2 h with anti-CD40 (1 μg/ml) following a 30-min pretreatment with SB203580 (20 μM) or DMSO solvent vehicle (final concentration 0.1% (v/v)). Total RNA was isolated using a guanidine isothiocyanate-based method (Isoquick kit, Orca Biosciences, Bothell, WA) according to the manufacturer’s instructions except that residual genomic DNA was digested with 10 U RNase-free DNase and the mixture was subsequently re-extracted twice as described in the instruction manual. Poly(A)⁺ RNA was isolated from total RNA using a single round of oligo(dT) latex bead chromatography according to the manufacturer’s instructions (Oligotex Kit; Qiagen, Chatsworth, CA). [α-³² P]dATP-labeled cDNA was subsequently prepared from approximately 1 μg poly(A)⁺ RNA, purified to remove unincorporated [α-³² P]dATP using chromaspin 200 DEPC-H₂O columns, and equivalent quantities (5 × 10⁶ cpm) of [³²P]-labeled cDNA were hybridized for 16 to 20 h at 68°C with the human cDNA expression array in a hybridization oven according to the manufacturer’s instructions (Clontech). Following a series of washes at 68°C as recommended by the manufacturer, the expression arrays were exposed to BioMax MS film for 0 to 3 days at −70°C.

Total cellular RNA was prepared with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA (5 μg) was resolved by denaturing electrophoresis in 0.8% formaldehyde agarose gels, transferred to a nylon membrane by capillary blotting, and cross-linked by UV irradiation. Hybridization was performed at 42°C in 50 mM sodium phosphate, pH 6.5, 50% formamide, 1× Denhardt’s solution, 5× SSC, and 250 μg/ml denatured salmon sperm DNA. The blots were washed to a final stringency of 0.1 × SSC, 0.2% (w/v) SDS at 50°C, and exposed to autoradiographic film at −70°C. cDNA fragments of human CD54/ICAM-1 and CD40 were ³²P-labeled using random primers according to standard procedures.

RNA was isolated from Daudi cells (5–10 × 10 ⁶ cells) using a guanidine isothiocyanate-based method (Isoquick kit, Orca Biosciences), followed by a 30-min incubation at 37°C with 5 to 10 U RNase-free DNase (Promega) to digest residual genomic DNA. Total RNA was subsequently prepared using an RNA clean-up protocol supplied with the RNeasy kit (Qiagen). Total RNA was quantified by spectrophotometry at 260 nm, and 2 μg was dried by brief vacuum centrifugation. RNase protection assays were performed according to the manufacturer’s protocol, using either [³² P]-labeled hAPO-3 or hAPO-5 template sets, and protected fragments were resolved by denaturing PAGE in 0.5× TBE. After drying for 60 min at 80°C, gels were exposed to film at −70°C for varying times.

Since the engagement of TNF receptors such as TNFR2 and CD95/Fas stimulates JNK and p38 MAPK activities (53) and CD40 engagement itself strongly activates JNK (26, 27, 28, 29), we investigated whether CD40 ligation also stimulates p38 MAPK.

Daudi B cells were stimulated with anti-CD40 mAb for various times and proteins were immunoprecipitated with antiserum prepared against the carboxyl terminus of p38 MAPK (51). In vitro kinase assays were subsequently performed on the immune complexes using a GST-ATF2 fusion protein, a p38 MAPK substrate in vitro (53). Figure 1,A illustrates that CD40 cross-linking strongly activated p38 MAPK or other coimmunoprecipitated kinase(s), whereas other binding or nonbinding isotype-matched control Abs (anti-CD22, anti-CD37, and anti-CD3) either failed to stimulate or only weakly stimulated kinase activity. To evaluate the specificity of the anti-p38 MAPK antiserum, anti-p38 MAPK-immune complexes were resolved by SDS-PAGE, and an in gel kinase assay was performed in which MBP, an alternative p38 MAPK substrate in vitro (53), was copolymerized within the polyacrylamide gel. A single CD40-stimulated, 40-kDa immunoprecipitated MBP kinase was observed (Fig. 1,B), which is entirely consistent with p38 MAPK, which electrophoreses as a 40-kDa kinase on SDS-PAGE (42, 54); maximal activation was observed 15 min following CD40 engagement and persisted for at least 60 min. Both PMA and a combination of both PMA and ionomycin also stimulated the 40-kDa MBP kinase (Fig. 1,B), in accord with earlier studies (53). The 40-kDa MBP kinase was unequivocally resolved from both ERK1 (p44) and ERK2 (p42), two MAPKs of similar molecular masses, neither of which was stimulated by CD40 cross-linking and both of which were observed only in cell lysates and postimmunoprecipitate fractions (data not shown). A minor MBP kinase activity of 56 kDa, which was not activated upon CD40 ligation but was apparently stimulated by both PMA and PMA/ionomycin, was also coimmunoprecipitated with p38 MAPK immune complexes (Fig. 1 B); its identity remains to be elucidated. Sequential immunoprecipitation of other mammalian MAPKs (ERK1, ERK2, and JNK1), followed by in vitro kinase assays of anti-p38 MAPK immune complexes, resulted in no significant reduction of GST-ATF2 phosphorylation compared with assays performed with anti-p38 MAPK immunoprecipitates alone (data not shown), suggesting that the immunoprecipitated kinase was distinct from either ERK or JNK. In addition, anti-p38 MAPK immune complexes did not phosphorylate GST-c-jun, an excellent substrate for JNK (data not shown).

CD40 cross-linking also activated p38 MAPK strongly (approximately fivefold) in a variant of the murine B lymphoma M12 line transfected with human CD40 (M12/CD40; 55 whereas only a 1.5-fold stimulation was observed in the A20 cell line (data not shown). Freshly isolated dense human tonsillar B cells were also responsive to CD40 ligation, leading to a 2.5- to 3-fold stimulation of p38 MAPK activity (Fig. 1,C). IL-4, which is a potent regulator of human B cell growth and differentiation (56), did not significantly affect total anti-CD40-mediated p38 MAPK activation, although IL-4 treatment may induce earlier p38 MAPK stimulation in response to CD40 cross-linking (Fig. 1 C).

The murine immature B lymphoma cell line WEHI-231 represents a convenient model cell system for studying the functional interaction of BCR and CD40 signaling pathways, since cross-linking CD40 prevents BCR-induced growth arrest in the G₁ phase of the cell cycle and subsequent apoptosis via signaling pathway(s) involving Bcl-X_L induction (57, 58). Cross-linking CD40 with the 1C10 anti-CD40 mAb rapidly elevated p38 MAPK activity within 5 min, with a maximal fourfold activation observed after 15 min, followed by a steady decline in activity (Fig. 2). A similar transient activation of JNK1 upon CD40 ligation of WEHI-231 cells was also observed (data not shown; Refs. 28, 29). BCR engagement also transiently stimulated p38 MAPK, albeit less strongly than with anti-CD40, with a maximal twofold activation after 10 min (Fig. 2). Simultaneous engagement of the BCR and CD40 appeared to weakly potentiate CD40-induced p38 MAPK activation at all times following receptor cross-linking but failed to stimulate p38 MAPK in a sustained manner (Fig. 2). Our data suggest that rapid and transient p38 MAPK activation, at least in the WEHI-231 cell line, does not correlate with either the ability of BCR ligation to induce programmed cell death or the role of CD40 to rescue cells from BCR-induced apoptosis. These observations are consistent with the inability of the p38 MAPK inhibitor, SB203580, to block anti-IgM-induced apoptosis in WEHI-231 cells (30).

To independently confirm that both CD40 and BCR cross-linking stimulate the p38 MAPK pathway, we investigated whether anti-CD40 and anti-IgM activated MAPKAPK-2 activity in Daudi, WEHI-231, and human tonsillar B cells. Both MAPKAPK-2 and its recently identified homologue, MAPKAPK-3, are phosphorylated and activated by p38 MAPK in vivo (43, 59). CD40 cross-linking rapidly and transiently stimulated MAPKAPK-2 in Daudi cells, with a maximal three- to fourfold activation following 10 to 20 min stimulation (Fig. 3,A). Neither of the isotype-matched Abs to CD3 or CD22 significantly elevated MAPKAPK-2 activity, showing that the response to CD40 was stimulus specific (Fig. 3,B). Similarly, both CD40 and BCR engagement stimulated MAPKAPK-2 in WEHI-231 cells. Anti-CD40 induced a two- to threefold maximal activation whereas anti-IgM induced a smaller 1.5- to 2-fold increase in MAPKAPK-2 activity (Fig. 3,D), consistent with the ability of each stimulus to activate p38 MAPK (Fig. 2). The kinetics of both BCR- and CD40-induced MAPKAPK-2 activation were also similar to those for p38 MAPK, with peak stimulation after 10 to 20 min (Fig. 3, A and D).

To demonstrate that p38 MAPK was required for the activation of MAPKAPK-2 following both CD40 and BCR cross-linking in vivo, Daudi and WEHI-231 cells were pretreated with different concentrations of SB203580 before stimulation with either anti-CD40 or anti-IgM. SB203580 dose-dependently inhibited both anti-CD40- and anti-IgM-induced MAPKAPK-2 activation in either Daudi or WEHI-231 cells (Fig. 3, C and E); 1 μM SB203580 completely abolished MAPKAPK-2 stimulation in response to either BCR or CD40 ligation (Fig. 3, C and E). In contrast to both B cell lines examined, dense human tonsillar B cells expressed relatively higher levels of constitutive MAPKAPK-2 activity (Fig. 3,F). Nevertheless, anti-CD40 induced a 1.5-fold stimulation of MAPKAPK-2 activity in dense human tonsillar B lymphocytes (Fig. 3,F). Pretreatment with SB203580 dose-dependently inhibited MAPKAPK-2 activity to levels that were significantly less than those in unstimulated cells, showing that CD40-induced MAPKAPK-2 activity is strongly p38 MAPK dependent (Fig. 3 F).

Since CD40 cross-linking also strongly stimulates JNK (26, 27, 28, 29), a highly related MAPK family member, it was important to exclude the possibility that the p38 MAPK inhibitor SB203580 blocked CD40-induced JNK activation. Hence, since SB203580 is a reversible p38 MAPK inhibitor and is thus washed away from kinases during cell lysis and immunoprecipitation, we tested whether SB203580 abrogated anti-CD40-induced c-jun phosphorylation, a downstream target for JNK in vivo. Both anti-CD40 and PMA/ionomycin induced a similar transient JNK activation, with maximal stimulation after 15 min as detected by both in vitro immune complex kinase assays with GST-c-jun as an exogenous JNK substrate (data not shown) and c-jun Western blot analysis using anti-c-jun antiserum (Fig. 3,G). Moreover, SB203580 at concentrations (0–20 μM) that totally blocked anti-CD40-mediated MAPKAPK-2 activation, did not significantly attenuate either anti-CD40 or PMA/ionomycin-induced c-jun phosphorylation (Fig. 3 G).

In addition, although CD40 engagement does not stimulate either ERK1 or ERK2 in Daudi cells (Ref. 27; data not shown), we examined whether SB203580 inhibited the ERK pathway in B cells by testing the effect of SB203580 upon anti-IgM-induced rsk2 activation, which is a MEK-ERK-dependent pathway (data not shown), using an in vitro immune complex kinase assay with ribosomal S6 peptide as a specific substrate. While anti-IgM stimulated rsk2 activity three- to fourfold, enzyme activity was not inhibited by 0 to 20 μM SB203580 (data not shown). Thus, our results indicate that SB203580 does not inhibit either JNK or ERK signaling pathways and are consistent with earlier studies that show that SB203580 is a highly specific p38 MAPK inhibitor (42, 43).

Since the p38 MAPK pathway is required for both IL-2- and IL-7-driven T cell proliferation and a primary response of CD40-activated B cells is the stimulation of B cell growth (56, 60), we examined the effect of SB203580 on anti-CD40-induced proliferation of isolated buoyant and dense tonsillar B cells. Preincubation with SB203580, at concentrations that completely perturbed CD40-mediated MAPKAPK-2 activation (Fig. 3,F), dose-dependently inhibited anti-CD40-induced proliferation in both buoyant and dense tonsillar B cells with similar IC₅₀ values of approximately 1 μM (Fig. 4, A and B). In contrast, SB203580 strongly potentiated anti-IgM-mediated tonsillar B cell proliferation in either buoyant or dense B cell subfractions (Fig. 4, A and B). Equivalent doses of SB203580 either did not or moderately increased anti-Bgp95-driven proliferation in buoyant or dense tonsillar B cells under identical experimental conditions (Fig. 4, A and B), providing further support for the specificity and lack of cytotoxicity of the compound.

CD40 cross-linking in B lymphocytes induces rapid and persistent NF-κB activation (37). Since the p38 MAPK pathway is required for TNF-α-induced NF-κB activation (61) and CD40 is also a member of the same TNFR superfamily, we tested the effect of SB203580 on CD40-induced reporter gene expression of a construct containing four copies of a NF-κB binding site following transient transfection into Daudi B lymphocytes. CD40 cross-linking increased NF-κB-dependent reporter gene expression between 8- and 12-fold following stimulation for 6 h, similar to previous studies (Fig. 5,A; 37 . SB203580 pretreatment consistently reduced anti-CD40-induced NF-κB reporter gene expression by approximately 50% at all doses of anti-CD40 tested (Fig. 5,A), suggesting a partial requirement for the p38 MAPK pathway in CD40-mediated NF-κB transcriptional activation. In contrast, SB203580 had no effect on PMA-induced NF-κB-dependent reporter gene expression (Fig. 5 B), indicating that the requirement for the p38 MAPK pathway was stimulus specific. We also evaluated the potential contribution to NF-κB activation of another MAPK signaling pathway, the MEK-ERK pathway using the specific MEK1 inhibitor PD 98059, which prevents the activation of MEK1 by Raf (62). Under conditions where PD 98059 (100 μM) almost completely abrogated anti-IgM-induced ERK2 activation in Daudi B cells (data not shown), 0 to 100 μM PD 98059 either had no significant effect or slightly activated NF-κB reporter gene expression (data not shown).

The activation of NF-κB requires multiple biochemical events including inducible phosphorylation and subsequent degradation of IκB, nuclear translocation and binding of NF-κB to sequence-specific DNA elements, and inducible phosphorylation of NF-κB subunits such as p65/RelA (63). Thus, we tested the effect of SB203580 on CD40-induced NF-κB DNA binding in Daudi B cells by EMSA, using a NF-κB-specific [³²P]-labeled oligonucleotide. Anti-CD40 induced detectable increases in binding of at least three specific protein-DNA complexes within 15 min following stimulation (Fig. 5,C). EMSA supershift analysis with specific antiserum for different NF-κB/Rel family members suggested that the upper (I) and middle complexes (II) consisted of both p65 and c-rel whereas the lower complex (III) comprises a mixture of both p50 and c-rel (Fig. 5,C); the possibility that these complexes may contain other NF-κB/Rel members such as RelB or p52 cannot be excluded. Pretreatment of the Daudi cells with 0 to 10 μM SB203580 for 30 min failed to prevent CD40-induced NF-κB DNA binding, demonstrating that the p38 MAPK pathway does not appear to regulate either the release of IκB from NF-κB, the nuclear translocation of NF-κB, or its subsequent DNA binding (Fig. 5 D).

NF-κB plays an important role in the cytokine-inducible regulation of gene expression at the transcriptional level of various cell adhesion molecules, including CD54/ICAM-1 (64), CD62E/E-selectin (65, 66), and CD106/VCAM-1 (67). Thus, in preliminary studies the effect of SB203580 pretreatment upon CD40-induced expression of multiple genes including CD54/ICAM-1 was examined in Daudi B cells using an Atlas human cDNA expression array.

CD40 cross-linking strongly increased steady-state levels of both CD54/ICAM-1 and itself (CD40) at the transcriptional level within 2 h (Fig. 6,A). Moderate up-regulation of the C-C chemokines MIP-1α and MIP-1β and the chemokine and chemoattractant receptor CCR2 were also apparently evident following anti-CD40 treatment, while expression of the TNFR superfamily member, CD27, was moderately reduced (Fig. 6,A). A similar CD40-induced down-regulation of CD27 mRNA levels was previously reported in chronic lymphocytic leukemia (CLL) B cells (68). Moreover, SB203580 pretreatment of the Daudi cells significantly reduced anti-CD40-mediated CD54/ICAM-1 expression while exhibiting no effect upon CD40-induced up-regulation of its own mRNA (Fig. 6,B, circles 4 and 2, respectively). To quantitatively analyze the effect of SB203580 upon anti-CD40-induced CD54/ICAM-1 and CD40 RNA levels, Northern blot analysis was performed. Similar to results with the cDNA expression array, SB203580 again partially inhibited CD40-induced CD54/ICAM-1 expression by more than 50%, while anti-CD40-mediated up-regulation of its own transcript was unaffected (Fig. 6 C).

Initial experiments using the Atlas human cDNA expression array also suggested that CD40 engagement up-regulated expression of cIAP2 (data not shown), a member of the mammalian inhibitor of apoptosis (IAP) family of proteins that indirectly associates with TNFR2 via TRAF1 and TRAF2 (69). Thus, the effect of CD40 cross-linking upon expression of cIAP2 and other IAP and TRAF family members was investigated using multi-template RNase protection assays. CD40 cross-linking induced strong up-regulation of cIAP2 but not its family members XIAP or NAIP (data not shown), with little or no increase in cIAP1 expression (Fig. 7,A). In addition, anti-CD40 induced selective up-regulation of both TRAF1 and TRAF4/CART, whereas the steady-state mRNA levels of both TRAF2 and -3 remained unaffected (Fig. 7,A). Since the p38 MAPK pathway may be required for the differential up-regulation of IAP and TRAF family members, we tested the effect of SB203580 upon CD40-mediated elevations in cIAP2, TRAF1, and TRAF4/CART transcript levels. Pretreatment with SB203580, under conditions that blocked CD40-dependent MAPKAPK-2 activation, had no significant effect upon the up-regulation of either cIAP2, TRAF1, or TRAF4/CART expression in response to anti-CD40 stimulation (Fig. 7 A).

CD40 ligation also induces expression of at least one additional TNFR, CD95/Fas (70, 71). Hence, we examined the effect of SB203580 upon both CD40-induced CD95/Fas mRNA expression and other TNFR members (TNFR1, DR3) and TNFR-associated signaling intermediates (RIP, FADD, FAF, FAP, TRADD) using multiprobe RNase protection assays. CD40 engagement induced a strong and long-lived increase in CD95/Fas expression, which was maximal within 6 h and persisted for at least 24 h (Fig. 7,B). CD40 cross-linking also induced a weak, but reproducible, increase in steady-state levels of DR3, another TNFR with a death domain, while the mRNA levels of other TNFR members and associated proteins tested were not significantly affected (Fig. 7,B). However, pretreatment of the Daudi cells with SB203580 did not significantly reduce either anti-CD40-induced CD95/Fas or DR3 expression (Fig. 7 B). Collectively, these results indicate the the p38 MAPK pathway is required, at least in part, for CD40-mediated CD54/ICAM-1 expression but not CD40-induced cIAP2, TRAF1, TRAF4/CART, CD95/Fas, CD40 or DR3 expression.

The present study shows that CD40 engagement specifically activates the p38 MAPK pathway with rapid and transient kinetics in multiple B cell lines (Daudi, WEHI-231, M12/CD40, A20) using various complementary experimental approaches (in vitro p38 MAPK assays, in-gel MBP kinase assays, and in vitro MAPKAPK-2 assays). Importantly, anti-CD40 also stimulates both p38 MAPK and one of its substrates, MAPKAPK-2, in purified dense human tonsillar B lymphocytes (Figs. 1,C, 3F). Thus, our results both agree with and expand upon recent reports demonstrating that CD40 cross-linking transiently stimulates the p38 MAPK pathway in WEHI-231 lymphoma and mouse splenic B cells (28, 30). In the immature WEHI-231 cell line, CD40 and BCR engagement have opposing effects upon cell fate (57). Thus, the ability of both anti-CD40 and anti-IgM to activate p38 MAPK and one of its targets, MAPKAPK-2, indicates that activation of the p38 MAPK pathway does not correlate with either an apoptotic or survival signal (Figs. 2 and 3,D). This result is further supported by the inability of SB203580 to suppress BCR-mediated cell death in WEHI-231 cells (30). In apparent contrast, anti-IgM-induced apoptosis in human B104 lymphoma cells correlated with BCR-induced delayed and sustained activation of p38 MAPK (72). Moreover, anti-IgM-induced cell death was strongly inhibited by the p38 MAPK inhibitor, SB203580 (73). It seems plausible that the kinetics of BCR-induced p38 MAPK activation may correlate with the differential requirement for p38 MAPK in BCR-mediated apoptosis, or, alternatively, the requirement for p38 MAPK activation in anti-IgM-induced cell death is cell type specific. It is possible that a block in p38 MAPK-dependent cell death may contribute to the increase in BCR-induced proliferation observed in the presence of SB203580 with human tonsillar B cells (Fig. 4).

The recent identification of the pyridinyl imidazole, SB203580, as a highly specific and cell-permeable inhibitor of p38 MAPK has enabled us to study the role of the p38 MAPK pathway in CD40-responsive B cells. A role for the p38 MAPK pathway has previously been identified using SB203580 in diverse cellular processes such as LPS- and TNF-α-induced cytokine production (42, 61), UV- and anisomycin-induced c-jun and c-fos expression (34), IL-2- and IL-7-mediated T cell proliferation (60), glutamate- (74) and BCR-induced apoptosis (73), and FGF-, arsenite- and UVC-mediated CREB/ATF-1 phosphorylation (33, 75). SB203580 appears to be specific for p38 MAPK, since it selectively inhibits both p38 MAPKα and -β, but not the γ isoform, and exhibits no significant effect upon multiple other related kinases, including multiple members of the ERK and JNK families and their upstream activators (43, 76, 77, 78).

Our observation that SB203580 specifically blocked CD40-induced proliferation, under conditions where CD40-dependent MAPKAPK-2 activation was suppressed, while potentiating anti-IgM-driven proliferation of tonsillar B lymphocytes (Fig. 3 F, 4), suggests that the p38 MAPK pathway may either positively or negatively regulate proliferative responses in a stimulus-dependent manner. Moreover, the requirement for p38 MAPK activity in CD40-mediated proliferation supports the idea of a role for p38 MAPK in nonstress responses. A requirement for p38 MAPK activity in IL-2- and IL-7-induced T cell proliferation, another mitogenic response, has also been recently shown (60).

One important role of CD40 is its ability to regulate gene expression at the transcriptional level. CD40 engagement up-regulates steady-state levels of various transcripts including the anti-apoptotic Bcl-2 homologue Bcl-X_L (58), TNF and TNFR family members such as LTα and CD70 (68, 79), as well as adhesion molecules such as CD54/ICAM-1 (80). However, the signaling pathways and identities of key signaling intermediates which mediate CD40-induced gene expression are poorly characterized.

Under experimental conditions where SB203580 completely prevented anti-CD40-mediated activation of the immediate p38 MAPK target, MAPKAPK-2 (Fig. 3,C), the p38 MAPK inhibitor significantly reduced CD40-induced CD54/ICAM-1 expression, suggesting a role for the p38 MAPK pathway in CD40-regulated gene expression at the transcriptional level (Fig. 6). The possibility that SB203580 inhibits CD40-induced increases in steady-state CD54/ICAM-1 mRNA levels nonspecifically is unlikely since SB203580 failed to prevent the up-regulation of multiple other genes (CD40, CD95/Fas, DR3, cIAP2, TRAF1, TRAF4/CART) in response to CD40 engagement (Figs. 6 and 7). Furthermore, the p38 MAPK inhibitor did not inhibit either anti-CD40-induced NF-κB binding or anti-IgM-mediated CREB^Ser133 phosphorylation in the same cells (Fig. 5,D; data not shown). The possibility that SB203580 inhibits another CD40-responsive protein kinase is unlikely, since the inhibitor is without effect on either JNK or ERK or multiple other related protein kinases including the homologues, p38 MAPKγ (SAPK3) and SAPK4, which share 60% identity with p38 MAPKα and -β (Fig. 3 G; Refs. 43, 76, 81). However, the possibility of other targets cannot be completely excluded. In summary, these results suggest that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways in B lymphocytes.

Our results indicate that CD40-mediated NF-κB trans-activation is also a target for the p38 MAPK pathway (Fig. 5,A). However, CD40-induced NF-κB binding was not modulated by SB203580, suggesting that the p38 MAPK pathway selectively regulates the trans-activation potential of NF-κB (Fig. 5 D). A similar requirement for the p38 MAPK pathway in NF-κB activation via an unknown mechanism independent of its effect on DNA binding activity has also been reported in response to TNF-α (61, 82). While the p38 MAPK inhibitor did not prevent TNF-α-induced phosphorylation of either p65 and p50 subunits or its precursor p105 subunit (61), it remains possible that the p38 MAPK pathway may regulate CD40-dependent NF-κB activation via phosphorylation of the trans-activation domains of either p50, p65, or p105 subunits. Alternatively, the p38 MAPK pathway may regulate NF-κB activation in response to CD40 through phosphorylation of another transcription factor that is required for NF-κB trans-activation. Indeed, the p38 MAPK pathway is required for phosphorylation of other transcription factors such as ATF-1 and CREB (33, 75), CHOP (31), and myocyte enhancer factor MEF2C (32) in response to a variety of stimuli.

Our studies have also identified a number of novel CD40-responsive genes that may perform roles in the regulation of cell fate, including two TNF receptors, CD40 and DR3, the TNFR-associated factors TRAF1 and TRAF4/CART, and cIAP2, a member of the inhibitor of apoptosis protein family. CD40 is generally considered to play a positive role in B cell activation, proliferation and apoptotic rescue. Thus, the ability of CD40 to up-regulate DR3 expression appears paradoxical since DR3 both contains a cytoplasmic “death domain” and mediates cell death (3). However, these observations are analogous to the ability of CD40 ligation to up-regulate CD95/Fas expression and thereby enhance the sensitivity of B cells to CD95/Fas-mediated apoptosis (70, 71). Thus, CD40-induced DR3 expression may promote increased sensitivity to DR3 ligand-induced cell death, a possibility we are currently investigating.

CD40 engagement differentially increased steady-state mRNA levels of cIAP2, while not significantly modifying the levels of other family members (XIAP, NAIP, cIAP1; Fig. 7 A). Since TNF-α also selectively up-regulates cIAP2 expression in T lymphocytes (83), our findings suggest that NF-κB-inducing TNF receptors may share the capacity to up-regulate cIAP2 in response to ligand binding. Moreover, our results suggest that one putative mechanism whereby CD40 may mediate B cell rescue, in addition to the induction of Bcl-X_L expression (58), is via up-regulation of cIAP2. The possibility that IAPs also physically associate with the ligand-inducible CD40 receptor complex via interactions with TRAFs, similar to the TNFR complex, merits further study (69, 84). Thus, CD40 ligation may both make cells more susceptible to apoptosis in response to either CD95/Fas or DR3 receptor ligation, and also enhance their capacity to be rescued from apoptosis via increased expression of cIAP2.

Rather surprisingly, CD40 engagement specifically increased the steady-state mRNA levels of TRAF1 and TRAF4/CART, whereas the levels of TRAF2 and TRAF3, which associate with the CD40 cytoplasmic tail (85, 86, 87), were unaffected (Fig. 7 A). Although TRAF1 overexpression inhibits Ag-induced cell death of CD8⁺ T lymphocytes, the molecular details of its mechanism of action remain to be elucidated (88). Perhaps the up-regulation of TRAF1 in response to CD40 cross-linking also serves as an anti-apoptotic signal in B cells to either promote the recruitment of anti-apoptotic proteins, such as IAPs, or alternatively prevent the association of proapoptotic molecules with TRAF-TNFR complexes. The role of TRAF4/CART in CD40 function remains to be explored. In contrast to TRAF2 and TRAF3, which are ubiquitously expressed, TRAF4/CART transcripts are apparently highly restricted to some primary breast carcinomas and metastatic axillary lymph nodes (89).

In summary, we have shown that the stress-activated protein kinase, p38 MAPK, is rapidly and transiently stimulated following CD40 engagement in B lymphocytes. Furthermore, we have demonstrated a role for the p38 MAPK pathway in CD40-induced tonsillar B cell proliferation and NF-κB activation using the specific and cell-permeable p38 MAPK inhibitor, SB203580. In addition, we demonstrated that CD40 regulates gene expression via both p38 MAPK-dependent and -independent pathways.

We acknowledge the generous assistance of Marj Domenowske for the preparation of Figures and members of the Clark laboratory for helpful discussions.