CD40 Employs p38 MAP Kinase in IgE Isotype Switching

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Biochemical and Biophysical Research Communications 289, 276–281 (2001)

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D40 Employs p38 MAP Kinase in IgE Isotype Switching

evin Brady,*,1 Stephen Fitzgerald,*,1 Sigurdur Ingvarsson,† Carl A. K. Borrebaeck,†nd Paul N. Moynagh*,2

Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin,oster Avenue, Blackrock, Co. Dublin, Ireland; and †Department of Immunotechnology, Lund University, Sweden

eceived October 12, 2001

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IgE switching requires the prior induction of Ceermline transcripts which is mediated by the con-erted binding of STAT-6 and NFkB to the Ce pro-oter. These transcription factors are regulated by

L-4 and CD40, respectively. However the latter canffect other signaling pathways and the present studyxplores the role of p38 MAPK in induction of Ce germ-ine transcripts. CD40 and IL-4, both alone and in syn-rgy, were initially shown to activate the Ce promotern a B cell lymphoma cell line. Under the same condi-ions CD40 caused activation of p38 MAPK, whereasL-4 was ineffective. The p38 MAPK inhibitor,B203580, and a dominant negative form of p38 MAPKecreased the CD40 activation of the Ce promoter byeducing the ability of CD40 to increase the transacti-ation potential of NFkB. This study suggests that p38APK is crucially important in mediating CD40 acti-

ation of NFkB which acts to induce Ce germline tran-cripts, ultimately facilitating IgE switching. © 2001

cademic Press

Key Words: CD40; IgE; isotype switching; allergy; p38AP kinase; NFkB.

IgE is a central player in the generation of allergiciseases and thus the molecular mechanisms underly-ng IgE synthesis have become an area of intense in-erest (reviewed in 1). In order for a B lymphocyte toroduce IgE, it needs to undergo a process known assotype switching. This phenomenon involves the re-ombination of the VDJ gene region to a differenteavy chain C (CH) gene which will thus encode fornother heavy chain isotype with different effectorunctions but with the same antigen-binding specificity2). Such recombination takes place between the 59ntronic segment, known as the switch (S) region, of the

m gene and the corresponding S region of the appro-riate CH gene (3). In the case of switching to IgE,

1 These authors contributed equally to the work.2 To whom correspondence should be addressed. Fax: 1353-1-

692749. E-mail: [email protected].

276006-291X/01 $35.00opyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

ion, with deletion of the intervening DNA. However aecessary pre-requisite for this deletional recombina-ion is the prior transcription of the Ce gene whichtarts at the Ie exon, upstream of the Se region, andontinues through the S region and Ce gene (4). The Segion is removed by splicing, resulting in the joining ofhe Ie exon and Ce region which together form a germ-ine sterile transcript (5). The induction of these Ceermline transcripts is a determining step in isotypewitching to IgE since transcription through the Ceene segment makes that region accessible to switchecombination (6). The induction of Ce germline tran-cripts is regulated by a promoter region, upstream ofhe Ie exon, and it is activated by the concerted bindingf the transcription factors STAT-6 and NFkB (7–9).L-4 regulates the activation of STAT-6 and the impor-ance of this pathway in IgE synthesis is emphasisedy the lack of IgE switching in mice deficient forTAT-6 (10). The engagement of the B cell transmem-rane protein CD40 by its cognate ligand is sufficiento cause activation of NF-kB (8, 11) and this synergisesith STAT-6 to activate the Ce promoter (8, 9). The

rucial contribution of CD40 activation of NF-kB to IgEynthesis is reinforced by the observation that CD40igand-deficient patients (12), CD40 deficient mice (13)nd NF-kB deficient mice (14) are all defective in IgEsotype switching and lack serum IgE. However thectivation of NF-kB may not be the sole contributoryathway to mediating IgE switching in response toD40. The engagement of the latter has also beenhown to activate a plethora of intracellular signalingolecules including protein tyrosine kinases and PI-3

inase (15). In addition much recent interest has fo-ussed on the ability of CD40 to activate the mitogen-ctivated protein kinases (MAPKs), especially the-jun N-terminal kinase/stress-activated protein ki-ase (JNK/SAPK) (16, 17) and the p38 MAPK (18, 19).hile the functional consequences of CD40 activation

f these MAPKs remain to be fully resolved a recenteport has demonstrated a role for p38 in mediating

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Vol. 289, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

he CD40-induced proliferation of B cells and its induc-ion of genes such as ICAM-1 (20). Interestingly, theame report also described a lack of involvement of p38n mediating the CD40 induction of a wide range ofther proteins including CD40 itself, CD95/Fas, cIAP2,RAF1, TRAF4, and DR3 and thus concluded thatD40 induces gene expression via both p38 MAPK-ependent and independent pathways. This promptedhe present investigation into whether p38 is employedy CD40 in mediating one of its most important bio-ogical effects in B cells, namely its induction of Ceermline transcripts which acts as the key determiningtep in IgE isotype switching.The study exploited the specific cell-permeable p38APK inhibitor SB203580 (21) and a dominant nega-

ive form of p38 MAPK to show that p38 is cruciallynvolved in mediating the CD40 activation of the Ceromoter which regulates induction of Ce germlineranscripts. The results also suggest that p38 contrib-tes to the activation of the promoter by increasing theransactivation potential of NF-kB. The study thusoncludes that p38 MAPK is a crucial player in IgEsotype switching and may represent a valuable tar-et in controlling unwanted IgE synthesis in allergiceactions.

ATERIALS AND METHODS

Materials. RPMI 1640 and FCS were from Life Technologies,nc. (Paisley, UK). Glutamine, streptomycin, and HAT medium (100

FIG. 1. Activation of Ce promoter by IL-4 and engagement ofD40 in BJAB B cells. BJAB cells (1 3 107 cells) were electroporatedith e-luc (10 mg), a plasmid encoding a luciferase reporter gene

egulated by the Ce promoter. Electroporated cells were subse-uently treated for 24 h in the absence (Con) and presence of IL-4100 U/ml) and/or an anti-CD40 antibody (500 ng/ml) adsorbed ontoD32-transfected fibroblasts. Cell extracts were measured for lucif-rase activity and the results are expressed as fold induction ofuciferase relative to cells which were treated in the absence of IL-1nd anti-CD40 antibody. The data represent mean 1/2 SEM of fourndependent experiments.

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igma (Poole, UK). IL-4 was from R & D (Oxon, UK). Luciferaseubstrate and lysis buffer were from Promega Corp. (Madison, WI).he PhosphoPlus p38 MAP Kinase antibody kit was from New En-land Biolabs (Beverly, MA). SB203580 was from Calbiochem-ovabiochem Ltd. (Nottingham, UK). The EBV negative human B

ell lymphoma cell line was a gift from Dr. D. Walls, Dublin Cityniversity, Ireland. CD32-transfected fibroblasts (ATCC No. CRL-0680) and the anti-CD40 antibody are previously described (22).he e-luciferase reporter plasmid (e-luc) consists of the germline Ce

romoter (the region 2162/153 relative to the first initiation site forermline e-transcripts) ligated upstream of the luciferase reporterene and was a gift from Professor J. Stavnezer (University ofassachusetts Medical School, MA). The NFkB-luciferase reporter

lasmid (kB-luc) consists of five copies of the NFkB consensus siteloned into the luciferase reporter construct pGL3-Basic (Promegaorporation). The plasmid encoding dominant negative p38 MAPKas a gift from Professor R. J. Davis (University of Massachusettsedical Center, MA) and has been described previously (23).EGFP-N1 (in which EGFP stands for enhanced green fluorescentrotein) was from Clontech Laboratories (Palo Alto, CA).

Cell culture. Human BJAB cells were cultured in RPMI 1640edium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml

enicillin, and 100 mg/ml streptomycin. CD32-transfected fibroblastsATCC No. CRL-10680) were cultured in RPMI 1640 supplemented

FIG. 2. Engagement of CD40 activates p38 MAPK in BJAB cells.JAB cells (1 3 107 cells) were treated for 30 min in the absence andresence of IL-4 (100 U/ml) and/or an anti-CD40 antibody (500g/ml) adsorbed onto CD32-transfected fibroblasts. Cell lysates wererepared and analysed by Western immunoblotting for levels of (A)hosphorylated p38 MAPK and (B) total p38 MAPK using the Phos-hoPlus p38 MAP kinase antibody kit (New England Biolabs). Im-unoreactivity was visualised by enhanced chemiluminesence and

and intensity quantitated by densitometric scanning on a Molecularynamics 300S computing densitometer (Sunnyvale, CA) using Im-geQuant V3.0 software. The numbers under each lane represent theand intensity relative to the intensity of the band in cells which hadeen treated in the absence of IL-4 and anti-CD40.

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treptomycin, and HAT medium supplement. Both cell types wererown in a humidified atmosphere of 5% CO2 at 37°C.

Transfection of BJAB cells and assay of expression of luciferaseeporter genes. Aliquots (250 ml) of BJAB cells (4 3 107 cells/ml)ere electroporated (220 V, 960 mF) with e-luc (10 mg) or kB-luc (5g) in RPMI 1640 medium supplemented with 10% FCS, 2 mM-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin byeans of a Bio-Rad Gene Pulser equipped with a capacitance ex-

ender (Bio-Rad, Hercules, CA). In some experiments BJAB cellsere cotransfected with 15 mg of the expression vector encodingominant negative p38 MAPK, 5 mg of pEGFP-N1 and e-luc (10 mg)r kB-luc (5 mg). The electroporated cells were subsequently added toresh medium (5 ml) and allowed to recover for 24 h in a humidifiedtmosphere of 5% CO2 at 37°C. Aliquots (2.5 ml) of recovered cellsere incubated in the absence or presence of SB203580 (10 mM) forh prior to stimulation with or without anti-CD40 monoclonal an-

ibody (500 ng/ml) and recombinant human IL-4 (100 U/ml) for 5 mint room temperature. Cells were subsequently mixed with aliquots750 ml; 4 3 105 cells/ml) of CD32-transfected fibroblasts and aliquots250 ml) of the mixed BJAB/fibroblast suspension then seeded into6-well plates and incubated at 37°C for a further 24 h. Cells extractsere then generated and measured for luciferase activity using theuciferase Assay System with Reporter Lysis buffer from Promegaorporation. Transfection efficiency was measured by counting theumber of cells expressing the EGFP fluorescent protein using flowytometry as described previously (24).

Western blot analysis of p38 phosphorylation. Aliquots (2.5 ml) ofntransfected BJAB cells (4 3 106 cells/ml) were stimulated for 30in in the absence or presence of anti-CD40 monoclonal antibody

nd IL-4, as described above. Stimulation was terminated by centri-uging the cells at 1450g for 5 min and washing the cell pellet once inBS (1 ml). The cells were then lysed in SDS–PAGE sample buffer

100 ml) (50 mM Tris–HCl buffer, pH 6.8 containing 2% (w/v) SDS,0% glycerol, 1% (v/v) 2-mercaptoethanol and 0.1% bromophenollue). The lysates were sonicated for 10 s to reduce sample viscositynd then heated to 90°C for 5 min. Lysates were cleared of particu-ate material by centrifugation at 20,000g for 5 min. Aliqouts (25 ml)f the clear supernatants were subjected to SDS–PAGE on 12%olyacrylamide slab gels and the separated proteins electrophoreti-ally transferred from the gels to nitrocellulose. The nitrocelluloseas then probed for levels of phosphorylated and total p38 using thehosphoPlus p38 MAP Kinase (Thr180/Tyr182) antibody kit asecommended by the manufacturers (New England Biolabs). Immu-oreactivity was visualised by enhanced chemiluminesence. Densi-ometric scanning of enhanced chemiluminescence blots was per-ormed on a Molecular Dynamics 300S computing densitometerSunnyvale, CA) using ImageQuant V3.0 software.

Statistical analysis. Significance was evaluated by Student’s test for unpaired data.

ESULTS AND DISCUSSION

The overall objective of this study was to explore theotential role of p38 in IgE isotype switching in B

ogether with a pEGFP-N1 plasmid encoding constitutively ex-ressed EGFP (5 mg of DNA) and e-luc (10 mg). Electroporated cellsere subsequently incubated for 24 h as above in the absence andresence of IL-4 and anti-CD40. All cell extracts were measured foruciferase activity and the results are expressed as fold induction ofuciferase relative to control cells which were treated in the absencef IL-4 and anti-CD40 antibody. The data represent mean 1/2 SEMf four independent experiments. Significantly different (*P , 0.05,*P , 0.01, ***P , 0.001) when compared to control values.

FIG. 3. SB203580 and dominant negative p38 MAPK inhibitD40 activation of Ce promoter. (A) BJAB cells (1 3 107 cells) werelectroporated with e-luc (10 mg), a plasmid encoding a luciferaseeporter gene regulated by the Ce promoter. Electroporated cellsere treated with and without SB203580 (10 mM) for 1 h and sub-

equently stimulated for 24 h in the absence (Con) and presenceIL-41CD40) of IL-4 (100 U/ml) and anti-CD40 antibody (500 ng/ml).B) BJAB cells (1 3 107 cells) were cotransfected by electropora-ion with an empty expression vector (15 mg of DNA) or the samemount of expression vector encoding dominant negative p38 MAPK,

lymphocytes. Since the induction of Ce germline tran-spinfBssahaTcwstgwtCssdbwtcitpbTptlrtCsC

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cripts is the key step in initiating IgE switching thisrompted an investigation into the importance of p38n regulating this induction process. However, it wasecessary to initially establish a suitable model systemor investigation. The EBV negative B cell lymphomaJAB cell line was used as the model human B cellystem. The process of IgE switching requires the dualignals of IL-4 and activation of CD40 and this waschieved by treating the BJAB cells with exogenousuman recombinant IL-4 and an agonistic anti-CD40ntibody adsorbed onto CD32-trasfected fibroblasts.he latter has been shown to be an effective means ofross-linking and activating CD40 (22). BJAB cellsere initially examined for their responsiveness to

timulation by IL-4 and CD40 with respect to activa-ion of the promoter controlling expression of the Ceermline transcripts. The activity of the Ce promoteras determined by measuring the expression of a

ransfected luciferase gene ligated downstream of thee promoter (e-luc) (8). BJAB cells, in the absence oftimulation by IL-4 and CD40, displayed basal expres-ion of luciferase and any induction above this level isisplayed as fold induction. BJAB cells showed someasal activation of the Ce promoter but e-luc expressionas increased approximately twofold by stimulating

he cells alone with either IL-4 or CD40 (Fig. 1). Theombination of both stimuli caused a synergistic sixfoldnduction of luciferase. Such synergistic activation ofhe Ce promoter by IL-4 and CD40 is consistent with arevious report (8) and is explained by the concertedinding of STAT-6 and NF-kB to the Ce promoter (7–9).hese findings further validate a previous report whichromoted the BJAB cell line as a valuable model sys-em for exploring the regulation of expression of germ-ine Ce transcripts (25). Thus BJAB cells and the e-luceporter system were employed as a suitable paradigmo explore the role of p38 in mediating the induction ofe germline transcripts in B cells. However prior touch an exploration it was necessary to show thatD40 could activate p38 in BJAB cells.BJAB cells were stimulated in the presence and ab-

ence of IL-4 and agonistic anti-CD40 antibody andubsequently examined for activation of the p38 path-ay. This was judged by probing the cells for immuno-

eactivity with an antibody which detects doubly phos-horylated threonine 180 and tyrosine 182 of p38APK. Western immunoblotting demonstrated that

ells in the absence of stimulation by IL-4 and CD40,

L-4 and anti-CD40. All cell extracts were measured for luciferasectivity and the results are expressed as fold induction of luciferaseelative to control cells which were treated in the absence of IL-4 andnti-CD40 antibody. The data represent mean 1/2 SEM of fourndependent experiments. Significantly different (*P , 0.05) whenompared to control values.

FIG. 4. SB203580 and dominant negative p38 MAPK inhibitD40 induction of NFkB-regulated reporter gene. (A) BJAB cells

1 3 107 cells) were electroporated with kB-luc (5 mg), a plasmidncoding a luciferase reporter gene regulated by a promoter contain-ng five copies of the NFkB consensus site. Electroporated cells werereated with and without SB203580 (10 mM) for 1 h and subse-uently stimulated for 24 h in the absence (Con) and presence (IL-1CD40) of IL-4 (100 U/ml) and anti-CD40 antibody (500 ng/ml). (B)JAB cells (1 3 107 cells) were cotransfected by electroporation withn empty expression vector (15 mg of DNA) or the same amount ofxpression vector encoding dominant negative p38 MAPK, togetherith a pEGFP-N1 plasmid encoding constitutively expressed EGFP

5 mg of DNA) and kB-luc (5 mg). Electroporated cells were subse-uently incubated for 24 h as above in the absence and presence of

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hich reacted with the antibody indicative of someasal levels of phosphorylated p38 MAPK in BJABells (Fig. 2A). The engagement of CD40 alone wasufficient to at least double the intensity of the bandhus demonstrating that CD40 can activate the p38APK pathway in BJAB cells. The addition of IL-4

ailed to affect the degree of basal or CD40 inducedhosphorylation of p38 indicating that it does not con-ribute to the activation of the p38 MAPK pathway in

cells. As control for the assay, all samples showedquivalent levels of total p38 as judged by Westernmmunoblotting analysis using control antibodieshich recognise p38 irrespective of its phosphorylation

tatus (Fig. 2B). The ability of CD40 engagement toctivate the p38 MAPK pathway in B cells confirmsimilar previous findings (18–20). The lack of effect ofL-4 on p38 is not surprising since its primary role is toctivate the transcription factor STAT-6 via the unre-ated JAK-3 kinase (26).

The demonstration that CD40 activates p38 in BJABells allowed for the exploration of the contribution ofhis pathway to mediating CD40 induction of Ce germ-ine transcripts. This was achieved by initially evalu-ting the inhibitory effects of SB203580, the p38APK inhibitor, on the activity of the Ce promoter, as

udged by expression of the e-luc reporter gene (Fig.A). SB203580 reduced the basal expression of e-lucnd inhibited by over 50% the ability of CD40 to induce-luc. This indicates a key role for p38 in mediating theD40 induction of Ce germline transcripts, the pro-

ogue to IgE switching. This was further confirmed byrobing the effect of cotransfected dominant negative38 MAPK on the activity of the Ce promoter (Fig. 3B).ike SB203580, dominant negative p38 MAPK mark-dly reduced the basal and CD40-induced expression of-luc, confirming the crucial role of p38 MAPK in me-iating the activation of the Ce promoter by CD40.ominant negative p38 MAPK had no effect on trans-

ection efficiency since cells transfected with dominantegative p38 and control cells transfected with anmpty expression vector displayed comparable trans-ection efficiencies of 12%. The clear role of p38 inediating the CD40 activation of the Ce promoter

rompted an investigation into the underlying mecha-ism. The unequivocal importance of NFkB in theD40 signaling pathway leading to IgE swithing (8, 9,1, 14) coupled to recent reports describing a role for38 in regulating the transactivation potential ofFkB (20, 27) warranted an analysis of the involve-ent of p38 in modulating the activity of NFkB in the

resent experimental system.The activity of NFkB in the BJAB cells was deter-ined by measuring the induction of a transfectedFkB-regulated luciferase gene (kB-luc) (Fig. 4). The

timulation of CD40 in BJAB cells was sufficient tonduce an approximate twofold induction of luciferase.

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educed the ability of CD40 to induce kB-luc indicatingn important role for p38 in mediating the CD40 acti-ation of NFkB (Fig. 4A). This was further confirmedy the equally impressive inhibitory effects of cotrans-ected dominant negative p38 MAPK on the inductionf kB-luc by CD40 (Fig. 4B). Since SB203580 fails toffect the ability of CD40 to effect nuclear translocationnd DNA-binding of NFkB in BJAB cells (data nothown) these findings suggest that the CD40 activationf p38 facilitates increased NFkB activity by improvingts transactivation potential. This confirms a recenteport which proposes a similar mechanism by whichD40 activates NFkB and thus induces B cell prolifer-tion (20).In summary, the present study thus proposes thatD40 activates p38 MAPK in B cells which in turn

ncreases the ability of nuclear translocated NFkB toctivate the Ce promoter and induce Ce germline tran-cripts. This ultimately promotes IgE isotype switch-ng and thus p38 is a major player in facilitating IgEynthesis. Such findings promote p38 as a novel targeto exploit in the development of new therapeutic re-imes to counter the production of IgE in allergy-basediseases.

CKNOWLEDGMENTS

The work was supported by the European Commission 4th Frame-ork Biotechnology Programme (BIO4 CT96 0246), 5th Frameworkiotechnology Programme (QLK3-CT-2000-00270), and Enterprise

reland.

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CD40 Employs p38 MAP Kinase in IgE Isotype Switching

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