baff enhances chemotaxis of primary human b cells: a particular synergy between baff and cxcl13 on...

doi:10.1182/blood-2007-03-081232 Prepublished online Jan 2, 2008;2008 111: 2744-2754

Dessirier, Genevieve Lapree, Andreas Tsapis and Yolande Richard Gamal Badr, Gwenoline Borhis, Eric A. Lefevre, Nada Chaoul, Frederique Deshayes, Valérie

synergy between BAFF and CXCL13 on memory B cellsBAFF enhances chemotaxis of primary human B cells: a particular

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IMMUNOBIOLOGY

BAFF enhances chemotaxis of primary human B cells: a particular synergybetween BAFF and CXCL13 on memory B cellsGamal Badr,1 Gwenoline Borhis,1 Eric A. Lefevre,2 Nada Chaoul,1 Frederique Deshayes,3 Valerie Dessirier,4

Genevieve Lapree,4 Andreas Tsapis,4 and Yolande Richard1

1Service of Immuno-Virology, Commissariat a l’Energie Atomique, Departement des Sciences du Vivant, Institut des maladies emergentes et therapiesinnovantes, Unite Mixte de Recherche (UMR)-E1, Universite Paris-Sud, Orsay, France; 2Compton Laboratory, Institute for Animal Health, Compton, UnitedKingdom; 3UMR-Centre National de la Recherche Scientifique 7592, Paris, France; and 4Inserm U841 Team 2, Creteil, France

B-cell–activating factor of the TNF family,(BAFF), and a proliferation-inducing li-gand (APRIL) regulate B-lymphocyte sur-vival and activation. We report that BAFF,but not APRIL, increased the chemotacticresponse of primary human B cells toCCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxiswas totally abolished by blockade ofBAFF-R and was strongly dependent on

the activation of PI3K/AKT, NF-�B, andp38MAPK pathways. BAFF had similareffects on the chemotaxis of naive andmemory B cells in response to CCL21 butincreased more strongly that of memoryB cells to CXCL13 than that of naiveB cells. Our findings indicate a previouslyunreported role for the BAFF/BAFF-R pairin mature B-cell chemotaxis. The synergybetween CXCL13 and BAFF produced by

stromal cells and follicular dendritic cellsmay have important implications for B-cell homeostasis, the development of nor-mal B-cell areas, and for the formation ofgerminal center–like follicles that may beobserved in various autoimmune dis-eases. (Blood. 2008;111:2744-2754)

© 2008 by The American Society of Hematology

Introduction

B cell–activating factor belonging to the TNF family (BAFF) hasemerged as an important regulator of B-cell homeostasis andsurvival: it acts alone or in combination with B-cell receptor(BCR), IL-4, or CD40 ligands.1-4 BAFF binds 3 different TNFreceptors: BCMA (B-cell maturation),5,6 TACI (transmembraneactivator and CAML interactor),7 and BAFF-R/BR3 (BLys recep-tor 3).8 A highly similar homolog (called “a proliferation-inducingligand” or APRIL)1 also binds TACI and BCMA but not BAFF-R.9

BCMA, TACI, and BAFF-R are mostly found on B lympho-cytes,10-12 whereas BAFF-R is also present on a subset ofT cells.11,13 Accordingly, BAFF produced by antigen-presentingcells provides T-cell costimulation.13 The BAFF/BAFF-R pair isessential for survival of immature T2, B2, and marginal zone (MZ)B cells,14-16 but not for that of B1 cells,17,18 whereas TACI exerts anegative control over BAFF-mediated B2 cell survival.19,20 BCMAhas no obvious effect on mature B-cell survival, but is important forlong-term plasma cell biology10,12 and antigen presentation.21

BAFF- or BAFF-R–deficient mice form only rudimentarygerminal centers (GCs) and produce low levels of IgG in responseto T-dependent (TD) antigens.22,23 In contrast, TACI-deficient micedisplay an impaired response to type II T cell–independentantigens, suggesting that TACI is required for B1 cell survival.24

BAFF-R and TACI provide signals for isotype switching towardIgG and IgE, but the switch to IgA is mainly controlled byTACI.17,25 Many BAFF transgenic mice show signs of autoimmune-like diseases,2,26 whereas aged APRIL-transgenic mice display aprogressive expansion of B1 cells infiltrating the peritoneum andlymphoid organs.27 These various observations support a major

role for the TACI/APRIL and BAFF-R/BAFF pairs in B1 and B2cell physiology, respectively.

Like CD40L, BAFF mainly promotes NF-�B and MAPKactivation.28,29 Triggering BAFF-R results in activation of NF-�B2and NF-�B1 pathways, whereas triggering BCMA and TACI onlyactivate the NF-�B1 pathway.7,9,28,30,31 Different sets of MAPK andtranscription factors are activated downstream from BCMA, TACI,and BAFF-R.29,32,33 In particular, it has been shown that p38MAPKbut not ERK is stimulated early after BAFF-R triggering.34

Lymphocyte recirculation, which is essential for maintaining aneffective immune system, is tightly regulated by the expression ofadhesion molecules, chemoattractant receptors, and environmentalcytokines.35,36 Trafficking of human naive and memory B cells ismainly orchestrated by CXCR4/CXCL12, CXCR5/CXCL13, andCCR7/CCL21 (or CCL19) pairs.37,38 The efficiency of the humoralresponse depends on the chemotactic response of mature B cellsthat is modulated by BCR- and IL-4–receptor triggering andCD40/CD40L interactions.37,39-42 In particular, BCR triggeringenhances the chemotactic response of naive B cells to CCL21 butdecreases that to CXCL13. In contrast, CD40L enhances themigration of memory B cells to CXCL13 without modifying that ofCXCL12, CCL21, or CCL19.37,40 These modifications allow acoordinated relocalization of B cells during the various phases ofthe TD humoral response, characterized by GC foundation. Thereare substantial similarities between CD40 and BAFF-R signalingpathways and their biological effects on the B-cell response, so it isplausible that BAFF also modulates B-cell chemotaxis. We there-fore analyzed the effect of human BAFF on the expression of

Submitted March 22, 2007; accepted October 30, 2007. Prepublished online asBlood First Edition paper, January 2, 2008; DOI 10.1182/blood-2007-03-081232.

G. Badr and G. Borhis contributed equally to this work.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2008 by The American Society of Hematology

2744 BLOOD, 1 MARCH 2008 � VOLUME 111, NUMBER 5




CCR7, CXCR5, and CXCR4 by primary B cells and on theirmigratory capacity in response to CCL21, CXCL13, and CXCL12.We found that BAFF enhanced the migration of primary B cells tothese chemokines through BAFF-R triggering. BAFF did notmodify the basal expression or the ligand-induced internalizationof the chemokine receptors studied but modulated their chemokine-induced signaling. BAFF-induced BAFF-R stimulation led to thephosphorylation of I�B�, PI3K/AKT, and p38MAPK, and theprocessing of NF-�B2 (p100) to p52. The use of specific inhibitorsconfirmed that PI3K/PDK1, p38MAPK, and NF-�B pathwaysactivated downstream from BAFF-R were essential for the effect ofBAFF on primary B-cell chemotaxis. These findings establish anovel role for the BAFF/BAFF-R pair in the regulation of B-cellchemotaxis during the normal humoral response and potentiallyduring the recruitment of pathologic B cells into ectopic follicles.43

Methods

Flow cytometry

Cell-surface antigens were analyzed by single-parameter or multiparameterflow cytometric analysis using the following monoclonal antibodies(mAbs): CD19-phycoerythrin (PE), IgD-PE, and CD44-FITC (all from BDBiosciences, Le Pont de Claix, France). CCR7-PE, CXCR5-PE, andCXCR4-PE mAbs were purchased from R&D Systems (Abingdon, UnitedKingdom). Mouse isotype-matched FITC- and PE-conjugated control IgG1and IgG2a were purchased from BD Biosciences. The expression ofreceptors for APRIL and BAFF was detected by sequential addition ofFlag-APRIL and Flag-BAFF ligands (Alexis Biochemicals, Lausen, Swit-zerland, Coger, Paris, France; 100 ng/mL), mouse anti–Flag M2 antibody(Sigma-Aldrich Chimie, Isle d’Abeau Chesnes, France), and PE-conjugatedgoat F(ab�)2 anti–mouse IgG (Beckman Coulter, Marseille, France) aspreviously described.44 Cell-surface expression of BCMA and TACI wasdetected with rat anti–human BCMA (Vicky 1; Alexis) and rat anti–humanTACI (1A1; Alexis) Ab and FITC-conjugated mouse anti–rat IgG (BDBiosciences). Rat IgG1 (BCMA) and IgG2a (TACI) were purchased fromBeckman Coulter. BAFF-R expression was detected with goat anti–BAFF-R (R&D Systems) and PE-conjugated donkey F(ab�)2 anti–goat IgG(Beckman Coulter). A FACScan flow cytometer was used for dataacquisition and CellQuest software (BD Biosciences) was used for dataanalysis. After gating on viable cells, 10 000 cells/sample were analyzed.

B-cell preparation

B cell–enriched populations were obtained from palatine tonsils as previ-ously described.41 Briefly, after one cycle of rosette formation, residualT cells and monocytes were depleted with CD2 and CD14 magnetic beads(Dynabeads M-450; Dynal AS, Oslo, Norway). The total B-cell populationwas depleted of CD38� GC B cells by Percoll gradient separation aspreviously described.41 The resulting B-cell population, including onlynaive and memory B cells, is herein referred to as “B cells” and was98% (� 6%) CD19�, 93% (� 3%) CD44�, and 2% (� 3%) CD3� (n � 20).The viability of these cells was consistently higher than 98%.

B-cell cultures

For in vitro culture assays, B cells (2 � 106 cells/mL) were cultured inRPMI 1640–glutamax medium (Invitrogen SARL, Cergy-Pontoise, France)containing 10 mM HEPES, 100 U/mL penicillin, 100 �g/mL streptomycin,1 mM sodium pyruvate, and 10% heat-inactivated fetal calf serum (FCS)for various periods, with or without 25 to 100 ng/mL FLAG-APRIL,recombinant human BAFF (AbCys, Paris, France), or 100 ng/mLCD40MegaLigand (CD40MGL; Alexis). The concentration of endotoxin inthe culture medium and in the reagents used was consistently below1 ng/mL as indicated by the manufacturers.

In some experiments, neutralizing goat anti–BAFF-R Ab or goat IgG(R&D Systems), 100 nM wortmannin (WM; a PI3K/PI4K inhibitor),

10 �M SB203580 (SB; p38MAPK inhibitor), or a combination of thesewere added with BAFF for 16 hours before assaying B-cell chemotaxis.

In vitro chemotaxis assay

Chemotaxis assays were carried out as described.41 In brief, 5 � 105 B cellsin 100 �L of prewarmed RPMI 1640 containing 10 mM HEPES and 1%FCS were transmigrated through 5-�m pore size bare filters Transwellinserts (Costar, Cambridge, MA) for 3 hours at 37°C in response tochemokines (250 ng/mL CXCL12 or CCL21 or 500 ng/mL CXCL13). Afterexclusion of cell debris by forward and side scatter gating, the migratedcells were counted with a FACScan for 60 seconds. Results are shown as thepercentage of specific migration, from which background migration tocontrol medium was subtracted. To determine the phenotype of migratingcells, input cells and migrating cells were stained with CD44-FITC andIgD-PE mAbs and separately analyzed among gated sIgDhigh and sIgD

B cells by flow cytometry. The number of cells migrating to the lowerchamber is expressed as a percentage of the naive or memory B cells addedat the start of the assay (“input cells”).

To test the effect of inhibitors on the chemotaxis, B cells (2 �106 cells/mL) were cultured with medium (mock treatment) or 25 ng/mLBAFF for 16 hours, washed, and incubated for 1 hour with 1 �M WM,10 �M PD98059 (PD; MEK1/2 inhibitor), 10 �M SB, 250 nM U73122(PLC inhibitor) or its inactive control (U73343), 1 �M chelerythrinechloride (CC; inhibitor of all types of PKC), 1 �M SN50 (inhibitor ofNF-B1 [p50] nuclear translocation), 100 ng/mL Pertussis toxin (PTX;inhibitor of G�i proteins) and 20 �M Y27632 (ROCKI inhibitor; all fromCalbiochem, San Diego, CA), 10 �M SH5 (PDK1 inhibitor; Alexis, Coger)or DMSO as a control. The samples were then subjected to the chemotaxisassay. Alternatively, B cells were incubated with medium, 100 nM WM(dose-selectively inhibiting the class I PI3Ks41), 10 �M SB, or both in thepresence or absence of 25 ng/mL BAFF for 16 hours at 37°C before beingsubjected to the chemotaxis assay.

Ligand-induced chemokine receptor internalization

B cells (2 � 106 cells/mL) were cultured with 25 ng/mL BAFF or medium(mock treatment) for 16 hours at 37°C. BAFF- and mock-treated B cellswere subsequently incubated for 60 minutes with medium or 100 ng/mLchemokine at 37°C. After washing in ice-cold medium, cells were stainedwith CD44-FITC and PE-CXCR4, PE-CXCR5, PE-CCR7, or PE–controlIgG antibody for 30 minutes at 4°C. Fluorescence-activated cell sorter(FACS) analysis was performed on 10 000 viable cells after gating onCD44� cells.

Western blots

Freshly isolated B cells were resuspended at a density of 107cells/mL inprewarmed RPMI 1640 without FCS, starved for 2 hours, then stimulatedfor 2 minutes at 37°C with medium, 100 ng/mL CXCL13, 100 ng/mLCCL21, or 50 ng/mL BAFF. Alternatively, mock- and BAFF-treated B cellswere resuspended at a density of 107 cells/mL in prewarmed RPMI 1640without FCS, starved for 2 hours, then stimulated for 2 minutes at 37°C withmedium or 100 ng/mL chemokine. Lysates were prepared as previouslydescribed.41 Equal amounts of total cellular protein were loaded in eachlane of SDS–polyacrylamide gels and subjected to electrophoresis. Subse-quently, Western blotting was performed using antibodies recognizingphospho-AKT (S473), AKT, phospho-ERK1/2 (T202/Y204), phospho-I�B� (S32/S36), phospho-PLC�3 (S537), PLC�3, phospho-p38MAPK(T180/Y182), p38MAPK, and pan–phospho-PKC (all from New EnglandBiolabs, Ozyme, France), PKC�/�/�, I�B�, NF-�Bp52, ERK1/2, phospho-PKC , PKC , or actin (from Santa Cruz Biotechnology, Tebu, France),followed by incubation with horseradish peroxidase (HRP)–conjugatedsecondary antibodies. Protein bands were detected using enhanced chemilu-minescence (ECL) reagents (Supersignal Westpico chemilumiscent sub-strate; Pierce Biotechnologies, Illkirch, France), and the ECL signal wasrecorded on ECL Hyperfilm. To quantify band intensities, films werescanned, saved as TIFF files, and analyzed with National Institutes ofHealth (NIH) Image J free software.

BAFF ENHANCES HUMAN B-CELL CHEMOTAXIS 2745BLOOD, 1 MARCH 2008 � VOLUME 111, NUMBER 5




Immunoprecipitation

Lysates (corresponding to 800 �g total protein) were incubated overnight at4°C with 50 �L of packed agarose-conjugated antiphosphotyrosine beads(4G10; Millipore SAS, St Quentin-en-Yvelines, France). Immune com-plexes were washed 3 times before being analyzed by Western blot using aPLC�2 antibody (from Santa Cruz Biotechnology). A total of 40 �g proteinof each total lysate was also analyzed by Western blot to determine the totalPLC�2 content.

Statistical analysis

Data are expressed as means plus or minus SD unless otherwise indicated.Differences between groups were assessed using the Wilcoxon sum ranktest and P values less than .05 were considered significant.

Results

BAFF, but not APRIL, increases the chemotaxis of humanprimary B cells

We assessed the chemotactic response of B cells to CCL21,CXCL12, and CXCL13 following incubation for 16 hours withmedium (mock treatment), 25 ng/mL or 100 ng/mL BAFF, and25 ng/mL or 100 ng/mL APRIL. In the absence of chemokine,BAFF and APRIL had no significant effect on B-cell chemotaxis(data not shown). However, BAFF, but not APRIL, increased thechemotactic response to these 3 chemokines. BAFF at 25 ng/mLincreased the percentage of B cells specifically migrating to CCL21by 1.4-fold (16.7% � 5.1% increase; n � 6), to CXCL12 by1.3-fold (16% � 3% increase; n � 3), and to CXCL13 by 2.4-fold(19.7% � 2.5% increase; n � 3; Figure 1A).

Kinetic studies showed that the BAFF-mediated increase inB-cell chemotaxis reached a maximum at around 16 to 24 hoursand decreased thereafter, suggesting that the BAFF-mediatedincrease of B-cell chemotaxis was transitory. After 16 hours,specific migration to CXCL13 was 14% (� 3%) and 32% (� 3%,2.3-fold increase; n � 4) for mock- and BAFF-treated cells,respectively (Figure 1B). Similar kinetics were observed in re-sponse to CXCL12 and CCL21 (data not shown). Given thewell-known variability of chemokine responsiveness among indi-viduals, B cells from more donors were tested and responsessimilar to those shown on Figure 1B were obtained consistently.After 16 hours, BAFF increased the chemotactic response toCCL21 and CXCL12 by 1.6-fold (19.7% � 4.6% increase; n � 18,P � .005; and 18.8% � 5.9% increase; n � 9, P � .05, respec-tively) and to CXCL13 by 2.4-fold (24% � 5.9% increase; n � 20,P � .005; Figure 1C). CD40MGL and BAFF similarly increasedthe chemotaxis to CCL21 (1.5- and 1.4-fold increase, respectively)and CXCL13 (1.9- and 2-fold increase, respectively), whereasCD40MGL is less efficient than BAFF in increasing the chemotac-tic response to CXCL12 (1-fold vs 1.7-fold increase, respectively;Figure 1D). Viable cell recovery was similar in medium- andBAFF-treated B cells (73% � 5% and 77% � 5%, respectively)after 16 hours of culture (Figure 1E), so this increase was not due tothe antiapoptotic effect of BAFF.

We tested whether the BAFF-mediated increase of humanB-cell chemotaxis reflected increased chemokine receptor expres-sion. In the absence of their respective ligands, CCR7, CXCR4, andCXCR5 were similarly expressed on mock- and BAFF-treatedB cells (Figure 1F). CCL21-, CXCL12-, and CXCL13-inducedinternalization decreased the expression of CCR7, CXCR4, andCXCR5 by 65% (� 3%), 82% (� 1.9%), and 56% (� 3.1%) inmock-treated B cells and by 63% (� 4.9%), 83% (� 2.8%), and

58% (� 3.2%, n � 3) in BAFF-treated B cells, respectively. Thus,BAFF did not significantly change the basal expression or theligand-induced internalization of CCR7, CXCR4, or CCR5. Simi-larly, BAFF did not significantly modulate the expression ofvarious adhesion molecules, including �4, �7, or �1 integrins(Figure S1, available on the Blood website; see the SupplementalMaterials link at the top of the online article).

BAFF enhancement of B-cell chemotaxis is dependenton BAFF-R

Incubation with BAFF but not APRIL enhances B-cell chemo-taxis, so we compared the expression of TACI, BCMA, andBAFF-R (Figure 2A) and the binding of FLAG-BAFF andFLAG-APRIL (Figure 2B) on freshly isolated B cells and after16 hours of culture. Freshly isolated B cells strongly boundFLAG-BAFF but not FLAG-APRIL. Weak expression ofboth BCMA and TACI was detected on the membrane, andin contrast, BAFF-R was strongly expressed. After culture inmedium, the binding of FLAG-BAFF and the expression ofBAFF-R and BCMA were slightly higher, whereas both thebinding of FLAG-APRIL and the expression of TACI weresubstantially higher. The addition of a neutralizing BAFF-R Abtotally abolished the BAFF-mediated increase in B-cell chemo-taxis (Figure 2C), suggesting that the effect of BAFF on B-cellchemotaxis mainly resulted from BAFF-R stimulation.

BAFF increases the CXCL13-dependent chemotaxis of memoryB cells more strongly than that of naive B cells

Using the transmigration assay, we compared the ability of naive(CD44�IgD�) and memory (CD44�IgD) B cells from 6 donors tomigrate in response to chemokines following a 16-hour incubationwith medium or with 25 ng/mL BAFF (Figure 3). The percentagesof memory B cells in mock- and BAFF-treated input populationswere similar after a 16-hour incubation period (26% � 6.1% and25.6% � 7.1%, respectively) and comparable to that of untreatedB cells (28.5% � 6.7%; data not shown).

Results from one representative experiment are shown in Figure3A-F. A total of 15% of all B cells treated with medium (Figure 3E)and 43% of all B cells treated with BAFF (Figure 3F) specificallymigrated in response to CXCL13. This BAFF-induced increasewas due to an additional 19% of naive B cells (14% in mock-treated B cells vs 33% in BAFF-treated B cells) and 35% ofmemory B cells (19% in mock-treated B cells vs 54% inBAFF-treated B cells) specifically migrating to CXCL13 (Figure3E,F). The percentages of total B cells specifically migrating inresponse to CCL21 was 29% (medium) and 46% (BAFF), with anadditional 16% of naive and memory B cells in the migratingpopulation. Similarly, the percentage of total B cells migrating inresponse to CXCL12 was 35% in medium and 55% in those treatedwith BAFF, with 19% and 21% more naive and memory B cells,respectively, in the migrating population (Figure 3E,F).

Similar results were obtained in 6 independent experimentsshowing that BAFF similarly increased the chemotaxis of naiveand memory B cells to CCL21 and CXCL12. However, theBAFF-dependent increase of chemotaxis to CXCL13 was 1.8-foldhigher for memory B cells than naive B cells (19.7% � 5.3% naiveand 34% � 8.2% memory B cells migrated to CXCL13; P � .05;Figure 3G).

2746 BADR et al BLOOD, 1 MARCH 2008 � VOLUME 111, NUMBER 5




The BAFF-mediated increase in B-cell chemotaxis requiresp38MAPK, PI3K/PDK1, ROCK I, and NF-�B1

To determine which effectors are involved in the BAFF-inducedincrease in B-cell chemotaxis, we tested the effects of variousinhibitors added prior to the chemotaxis assay. Migration towardCXCL13 was similarly inhibited by the addition of 100 ng/mLPTX, 250 nM U73122, and 1 �M CC to B cells treated with andwithout BAFF from 5 different donors (Figure 4A). The addition of250 nM U73343, the inactive form of the PLC inhibitor U73122(data not shown), or of 10 �M PD98059 did not inhibit thechemotaxis of mock- or BAFF-treated B cells. In contrast, the

addition of 10 �M SB203580 did not impair the CXCL13-mediated migration of mock-treated B cells, but inhibited by38% (� 12%, P � .005) that of BAFF-treated B cells (Figure 4A).The addition of 1 �M WM (inhibiting all classes of PI3K),10 �M SH5, 20 �M Y27632, or 1 �M SN50 consistently inhibitedthe CXCL13-mediated specific migration of BAFF-treated cellsmore strongly than that of mock-treated cells. Similar results wereobtained for CCL21-mediated chemotaxis (Figure 4B). This sug-gested that BAFF-treated B cells are more dependent than mock-treated B cells on NF-�B and on the downstream effectors of PI3K,including PDK1 and ROCK I.

Figure 1. Dose- and time-dependent effect of BAFF on B-cell chemotaxis. (A) B cells were incubated for 16 hours with medium (�), 25 ng/mL (3) or 100 ng/mL (f) BAFF,and 25 ng/mL (z) or 100 ng/mL ( ) APRIL, and were then tested for migration to 250 ng/mL CCL21, 250 ng/mL CXCL12, or 500 ng/mL CXCL13. Basal migration of B cells was5% (� 1.4%, medium-treated), 4.7% (� 1.1%, 25 ng/mL BAFF–treated), 5.4% (� 0.5%, 100 ng/mL BAFF–treated), 4.3% (� 2%, 25 ng/mL APRIL–treated), and 5% (� 3%,100 ng/mL APRIL–treated) Results are expressed as the mean percentage plus or minus SD of cells specifically migrating in response to each chemokine. (B) B cells wereincubated for up to 48 hours with medium or 25 ng/mL BAFF. Mock-treated (�) and BAFF-treated (f) B cells were analyzed for migration to 500 ng/mL CXCL13. Theexperiment was performed using samples from 4 different donors, and basal migration ranged from 3% to 4% and 3.3% to 4.7% for mock- and BAFF-treated B cells,respectively. Results are expressed as the percentage plus or minus SD of specific chemotaxis. *P � .05; **P � .005. (C) B cells were treated for 16 hours with medium (�) or25 ng/mL BAFF (3) and analyzed for their migration to 250 ng/mL CCL21, 250 ng/mL CXCL12, and 500 ng/mL CXCL13. Basal migration was 4.4% (� 1.2%) and4.3% (� 1.1%) for mock- and BAFF-treated B cells, respectively. Results are expressed as the mean percentage plus or minus SD of specifically migrating cells obtained foreach donor. *P � .05; **P � .005. (D) B cells were treated for 16 hours with medium (data not shown), 25 ng/mL BAFF (3), or 100 ng/mL CD40MGL (f) and analyzed for theirmigration to 250 ng/mL CCL21, 250 ng/mL CXCL12, and 500 ng/mL CXCL13. The experiment was performed using samples from 3 different donors, and basal migration was3.3% (� 2.1%), 2.3% (� 1.5%), and 1.3% (� 0.6%) for mock-, BAFF-, and CD40MGL-treated B cells, respectively. Results are expressed as mean fold increase plus or minusSD. (E) The percentage of B-cell recovery after overnight culture with medium or 25 ng/mL BAFF was calculated for each donor. Results are expressed as median valuesobtained from 28 independent experiments. Error bars correspond to 95% confidence intervals about the median. (F) B cells from 3 different donors were incubated for16 hours at 37°C with medium (�) or 25 ng/mL BAFF (3) and then incubated for 60 minutes at 37°C with medium or 100 ng/mL chemokines. Cells were washed in ice-coldmedium and stained with PE-conjugated CCR7, CXCR4, or CXCR5 mAb for 30 minutes at 4°C. The mean channel fluorescence intensity (MFI) values for mock-treated cellswere between 57 and 123 for CCR7, 356 and 1067 for CXCR4, and 64 and 81 for CXCR5. Each of these individual values was considered as 100% expression. Data areexpressed as the mean plus or minus SD percentage of MFI values for remaining surface expression.





We next tested the effects on B-cell chemotaxis of PI3K andp38MAPK inhibitors added with BAFF. Neither 100 nM WM(selectively inhibiting the class I PI3Ks) nor SB203580 had anyeffect on mock-treated B-cell chemotaxis (data not shown). Thechemotactic response of BAFF-treated B cells to CXCL13 was

reduced by 57% by 100 nM WM, 62% by SB203580 and 86% byboth (Figure 4C); similarly, the BAFF-mediated increase ofB-cell chemotaxis to CCL21 was reduced by 65%, 57%, and100% by the same inhibitors (Figure 4D). These findingssuggested that class I PI3Ks and p38MAPK are activateddownstream from BAFF-R and are key effectors of the BAFF-mediated increase of B-cell chemotaxis.

BAFF and chemokines differently regulated thephosphorylation of p38MAPK, ERK1/2, AKT, and I�B� as wellas the processing of NF-�B2 (p100) to p52

Consistent with our previous findings for B cells,41 we observedthat CCL21 increased the phosphorylation of AKT by 9.2-fold, ofI�B� by 16-fold, and of p38MAPK by 2.1 fold (Figure 5).Similarly, CXCL13 increased the phosphorylation of AKT by6-fold, of I�B� by 7.6-fold, and of p38MAPK by 1.5-fold. Bothchemokines strongly induced the phosphorylation of ERK (1000-fold or greater increase) and of PKC (7.4- and 3.6-fold withCCL21 and CXCL13, respectively). Preliminary experimentsshowed that 50 ng/mL BAFF was optimal for assessing its effectson the phosphorylation of downstream effectors by biochemicalapproaches (data not shown). As expected from previous studies inmice and humans, BAFF induced both the phosphorylation ofI�B� (7.6-fold increase) and the processing of NF-�B2 (p100) top52 with a 4.7-fold increase of p52 content. This NF-�B2 (p100)processing was not significantly observed in response to chemo-kines. More surprisingly, BAFF also induced phosphorylation ofAKT (4-fold), PKC (3.6-fold), p38MAPK (1.8-fold), and PLC�2(4.2-fold), but not that of ERK or PLC�3.

Thus, in primary human B cells, BAFF triggered both thealternative and classic NF-�B pathways, whereas the chemokinesCXCL13 and CCL21 only stimulated the classic pathway. Inaddition, we report the first evidence in primary B cells that BAFFactivated the PI3K/PDK1 pathway: the induction of AKT andPKC phosphorylation.

Neutralizing BAFF-R Ab abolishes BAFF-inducedphosphorylation of AKT, p38MAPK, and I�B�,and p52 production

To confirm that BAFF-induced phosphorylation of the variouseffectors described occurred downstream from BAFF-R, we com-pared the BAFF-induced phosphorylation of these effectors follow-ing the incubation of B cells with or without a neutralizinganti–BAFF-R or with an isotype-control Ab. Incubation with theneutralizing anti–BAFF-R Ab but not goat IgG (data not shown)prior to BAFF stimulation totally prevented the phosphorylation ofAKT, p38MAPK, I�B�, and PKC , and strongly decreasedBAFF-induced p52 production (Figure 6).

BAFF increases the chemokine-induced phosphorylationof I�B�, p38MAPK, and AKT

We compared the chemokine-induced phosphorylation profiles ofvarious effectors in mock- and BAFF-treated B cells. The produc-tion of p52 was similarly increased in BAFF-treated and mock-treated cells, independent of their stimulation by medium (4.3-fold)or by chemokines (3.4- and 3.1-fold in the presence of CXCL13and CCL21, respectively; Figure 7). In contrast, CXCL13- andCCL21-induced phosphorylation of I�B� were differently in-creased by B-cell pretreatment with BAFF (by 3- and 1.3-fold,

Figure 2. BAFF increases B-cell chemotaxis through interactions with BAFF-R.(A) Surface expression of BCMA, TACI, and BAFF-R was analyzed by flow cytometryon freshly isolated B cells (0 hours) and on B cells incubated for 16 hours withmedium. MFI of specific staining (positive minus IgG control) was 17 (BCMA),17 (TACI), and 55 (BAFF-R) at 0 hours and 28 (BCMA), 32 (TACI), and 67 (BAFF-R)at 16 hours. Data are representative of 4 separate experiments. (B) Similarly, theability of BAFF-FLAG and APRIL-FLAG to bind B cells was analyzed by flowcytometry using both freshly isolated B cells (0 hours) and B cells incubated for16 hours with medium. MFI of specific staining was 7 (APRIL) and 39 (BAFF) at0 hours and 44 (APRIL) and 121 (BAFF) at 16 hours. Data are representative of4 separate experiments. (C) B cells were incubated for 16 hours with medium (�),10 �g/mL anti–BAFFR Ab (f), 25 ng/mL BAFF (3), or 10 �g/mL anti–BAFF-R mAband 25 ng/mL BAFF (z). B cells were analyzed for migration to 250 ng/mL CCL21,250 ng/mL CXCL12, and 500 ng/mL CXCL13. The experiment was performed withsamples from 3 different donors and results are expressed as the percentage plus orminus SD of specific chemotaxis. *P � .05.





respectively). BAFF also increased the CXCL13- and CCL21-induced phosphorylation of AKT by 2.8- and 1.9-fold and ofp38MAPK by 3.2- and 1.8-fold, respectively. BAFF- and CCL21-induced phosphorylation of p38MAPK and I�B� were mostlyadditive; there was a synergistic effect between BAFF- andCXCL13-induced phosphorylation. BAFF did not modulate thechemokine-induced phosphorylation of PLC�3.

Discussion

BAFF has a major role in B-cell maturation and survival, and we showhere that it also enhances the chemotactic response of mature B cells to

CXCL12, CXCL13, and CCL21. This effect is dose dependent,reaching a maximum at 25 ng/mL of BAFF. Like the CD40L-inducedmodulation of B-cell chemotaxis, the effect of BAFF is transitory andpeaks at around 16 to 24 hours.37 Although a short period of incubationin medium (mock treatment) progressively increases the chemotaxis offreshly isolated B cells,37,39,41 25 ng/mL of BAFF further increasesB-cell chemotaxis by 1.6-fold (CXCL12, CCL21) or 2.4-fold (CXCL13)after 16 hours of incubation. A comparable increase in CXCL13- andCCL21-mediated chemotaxis was observed in the presence ofCD40MGL. Consistent with BAFF-R being the major receptor forBAFF on mature B cells, the BAFF-mediated increase in B-cellchemotaxis is totally abolished by the addition of a neutralizinganti–BAFF-R Ab. In agreement with previous work,12 BAFF-R was

Figure 3. The BAFF-mediated increase of CXCL13-dependent chemotaxis is stronger with memory Bcells than with naive B cells. Mock- and BAFF- treatedB cells were analyzed for their migration to 250 ng/mLCCL21, 250 ng/mL CXCL12, and 500 ng/mL CXCL13.Input populations and migrated cell populations werestained with CD44-FITC and IgD-PE. Representativedot-plots of input cells (A,B) and transmigrated cells tomedium or CXCL13 (C,D) are shown. The numbers ofcells in the input and transmigrated populations fromeach B-cell subset are given in each quadrant (A-D). Thepercentages of naive (dotted bars) and memory B cells(hatched bars) that specifically migrated to the indicatedchemokines are shown in panels E and F for mock- andBAFF-treated cells, respectively. The percentages oftotal B cells migrating to each chemokine are indicatedbelow the histograms (E,F). Data from one representiveexperiment of 6 are shown (A-F). The experiment wasrepeated with samples from 6 different donors, andresults are expressed as the mean BAFF-induced per-centage increase plus or minus SD of total (f), naive(dotted bars), and memory B cells (hatched bars) thatspecifically migrated to the indicated chemokines. Basalmigration was 4.8% (� 0.8%), 4% (� 0.6%), and7.6% (� 1.8%) for total, naive, or memory B cells inmock-treated cultures, and 4.9% (� 0.7%), 3.8% (� 1%),and 7.9% (� 2.9%) in BAFF-treated cultures, respec-tively. (G). *P � .05.





similarly expressed on naive and memory B cells (data not shown), andits expression was slightly increased by incubation. Accordingly, BAFFbinding to freshly isolated B cells was substantial and only increasedslightly after B-cell incubation. BCMA was barely detectable bothbefore and after the incubation period, whereas the expression of TACIwas up-regulated during incubation. The binding of APRIL increasedsubstantially during incubation but remained 10-fold lower than that ofBAFF. Despite this increased binding, APRIL does not modulate B-cellchemotaxis, suggesting differences in the signaling pathways down-stream from BAFF-R and TACI. Although the percentage of naive andmemory B cells was not modified by the incubation, the BAFF-mediated increase of CXCL13-dependent chemotaxis was greater formemory B cells than for naive B cells. This phenomenon was observeddespite the fact that memory and naive B cells similarly expressCXCR5, TACI, and BAFF-R (data not shown). This difference insensitivity between memory and naive B cells was, however, specific toCXCL13: BAFF similarly increased the chemotactic response of naiveand memory B cells to CXCL12 and CCL21. These findings arereminiscent of data reported by Roy et al showing that a 16-hourincubation with CD40L specifically increases the CXCL13-mediatedchemotaxis of memory B cells.37,42 Similarly, we have previouslydescribed a hyper-responsiveness to CCL20 of memory B cells relativeto naive B cells,40 although they express similar levels of CCR6, and apreferential relative increase in the chemotaxis to CCL20, CXCL12, andCCL21 of type I IFN-treated memory B cells.41 The similarities betweenthe BAFF and CD40L signaling pathways are well known, so the sameeffectors may be responsible for the preferential enhancement ofCD40L- and CXCL13-induced chemotaxis in memory B cells.

CXCL13 is mainly produced by stromal cells and folliculardendritic cells (FDCs) in normal tissue and plays a unique role bothin B-cell entry into the lymphoid follicles36 and in the GCorganization during the TD response.45 CCL19 and CCL21 aremainly produced by dendritic cells within T-cell zones, and theratio between CCL19/21 and CXCL13 is determinant for therelocalization of naive B cells during the initiation phase of thehumoral response.45 As BAFF similarly increases the chemotacticresponse of naive B cells to CXCL13 and CCL21, it is unlikely thatBAFF impedes their relocalization, although it may speed up theirentry into lymphoid organs. In contrast, by increasing the chemotac-tic response of memory B cells to CXCL13 over that to CCL21,BAFF might favor their sequestration within follicles or theirre-entry into follicles during a secondary response. The synergybetween BAFF and CXCL13 might also favor the recruitment ofpathological B cells and their sequestration in follicle-like struc-tures during autoimmune diseases where CXCL13 and BAFF aresimultaneously overproduced.43 Interestingly, patients with Sjogrensyndrome have reduced numbers of memory B cells in the blood,whereas these cells accumulate in the salivary glands.46

The BAFF-mediated increase of B-cell chemotaxis does not corre-late with the increase in CXCR4, CXCR5, or CCR7 expression or withthe impairment of chemokine-induced receptor internalization. Thissuggests that BAFF/BAFF-R interactions modulate signaling down-stream from the chemokine receptors. Using various inhibitors ofdownstream effectors, we show that chemokines induce the activationof PLC�3, PI3K/AKT, RhoA/ROCKI, I�B�, p38MAPK, and ERK1/2in primary B cells.All but p38MAPK and ERK1/2 are crucial for B-cellchemotaxis.41 BAFF modifies the requirements for B-cell chemotaxis,such that it becomes dependent on p38MAPK and more dependent onPI3K/PDK1, RhoA/ROCKI, and NF-�B1 (Figure 4). In contrast, BAFFdoes not modify the involvement of G�i, PLC, or PKC in B-cellchemotaxis, and this is consistent with the observation that BAFF doesnot stimulate the heterotrimeric G proteins or the types of PLC involved

Figure 4. PI3K/AKT, p38MAPK, Rho-kinase, and NF-�B participate differently inthe chemotaxis of mock- and BAFF-treated B cells. Mock-treated (�) andBAFF-treated (3) B cells from 6 different donors were incubated for 1 hour at 37°Cwith various inhibitors or DMSO and were then subjected to the chemotaxis assay to500 ng/mL CXCL13 (A) or 250 ng/mL CCL21 (B). Results are expressed as the meanpercentage of inhibition plus or minus SD of the specific migration. *P � .05;**P � .005. B cells from 3 different donors were incubated with medium, 100 nM WM,10 �M SB203580, or both in the presence (3) or absence (�) of 25 ng/mL BAFF for16 hours at 37°C and were then subjected to the chemotaxis assay to 500 ng/mLCXCL13 (C) or 250 ng/mL CCL21 (D). Results are expressed as the meanpercentage plus or minus SD of specific migration.





in chemotaxis (ie, PLC�; Figure 5). However, we observed thatBAFF/BAFF-R interactions induce the phosphorylation ofPLC�2 in primary B cells. This agrees with data from Hikida etal showing that NF-�B is not activated downstream fromBAFF-R in PLC�2-deficient mice.47

The BAFF-mediated activation of p38MAPK was totally abolishedby the presence of a BAFF-R–neutralizing mAb, demonstrating therequirement for interaction between BAFF and BAFF-R. Consistentwith the involvement of p38MAPK in the BAFF-induced increase ofB-cell chemotaxis, preincubation with SB203580, an inhibitor of

Figure 5. BAFF and chemokines differently activateNF-�B1 and NF-�B2 pathways. Mock-treated B cellswere stimulated for 2 minutes with medium, 100 ng/mLCCL21, 100 ng/mL CXCL13, or 50 ng/mL BAFF. The p52contents of total cell lysates were corrected for the actincontents. Phosphorylation of AKT, I�B�, p38MAPK,ERK1/2, PKC , and PLC�3 were corrected for totalrelevant protein. Blots from one representative experi-ment are shown. Values reported for p52 contents orphosphorylation are mean values plus or minus SD from2 (PLC�3) or 3 independent experiments. Values re-ported for PLC�2 phosphorylation were calculated as theratio between PLC�2 contents in 4G10 immunoprecipi-tates and those in total lysates. Results are mean valuesplus or minus SD from 2 independent experiments. Blackdividing lines between lanes represent the cuts resultingfrom the grouping of images from different parts of thesame gel in order to optimize the order of the bandsand/or remove lanes corresponding to conditions notrelevant to the present work.





p38MAPK activity, inhibited the BAFF-mediated enhancement ofchemotaxis by 50% (Figure 4). The involvement of p38MAPK activityin the chemotaxis of BAFF-treated cells but not of mock-treated cells ispuzzling because both BAFF and chemokines induce its phosphoryla-tion. However, these 2 pathways may have subcellular distributions,leading to the phosphorylation of a different set of effectors, aspreviously reported for ERK.41,48 In addition, we show that BAFF,through the triggering of BAFF-R, induces the activation of thePI3K/PDK1 pathway (evidenced by AKT and PKC phosphorylation)and the phosphorylation of I�B�. In the light of recent studies ofTRAF6-dependent PI3K recruitment downstream from CD40,49 ourresults suggest that a similar mechanism occurs downstream fromBAFF-R. Patke et al also report the activation ofAKT downstream fromBAFF-R in mice.50 As a consequence, activated PI3K/PDK1 mayparticipate in the activation of RhoA/ROCK I and I�B�. Whereas100 nM WM and SB203580 added separately inhibited the effect of

BAFF on B-cell chemotaxis by 57% to 62%, their coaddition led tostronger inhibition (86% or greater), indicating that these 2 pathways areat least partially independent. In agreement with previous findings,BAFF-R triggering led to the activation of NF-�B1 and the processingof NF-�B2 (p100) to p52 in resting B lymphocytes.4 Indeed, addition ofa neutralizing anti–BAFF-R Ab decreased both the phosphorylation ofI�B� and the production of p52 protein. Data obtained in the presenceof SN50 and MG132 (a proteasome inhibitor; data not shown) alsoindicate that NF-�B participates in the BAFF-mediated enhancement ofB-cell chemotaxis. Therefore, PI3K/PDK1, p38MAPK, and NF-�Bpathways appear to act in concert to mediate the BAFF-induced effecton B-cell chemotaxis.

Our study demonstrates a previously unreported role forBAFF in stimulating B-cell chemotaxis and reveals a particularsynergy between CXCL13 and BAFF on memory B cells. Inphysiologic conditions, this BAFF-dependent effect might favor

Figure 6. BAFF-induced phosphorylation ofp38MAPK, AKT, I�B�, and PKC� depends on BAFF-Rtriggering. Mock-treated B cells were incubated for30 minutes at 4°C with medium or 10 �g/mL anti–BAFF-R Ab and then stimulated with medium or50 ng/mL BAFF. The p52 contents of total-cell lysateswere corrected for actin content. Phosphorylation of AKT,IB�, p38MAPK, classical PKC, and PKC were eachcorrected for total relevant protein. Blots from one repre-sentative experiment are shown. Values reported for p52contents and phosphorylation are mean values plus orminus SD from 2 independent experiments.





the sequestration or re-entry of memory B cells into folliclesduring primary or secondary immune responses, respectively.During autoimmune diseases (systemic lupus erythematosis,Sjogren syndrome, or rheumatoid arthritis), this synergy mightfavor the formation of ectopic follicles. This work also improvesour knowledge of signaling pathways downstream from BAFF-Rand, for the first time, evidences the BAFF-mediated activationof p38MAPK and PI3K/PDK1 in primary human B cells.

Acknowledgments

The authors thank Dr G. Gras for critical reading of the manuscript.This work was supported by grants from Inserm and the

Fondation de la Recherche Medicale (FRM). G. Borhis is sup-ported by a SIDACTION fellowship.

AuthorshipContribution: G. Badr and G. Borhis performed the research andwrote the paper. E.A.L., N.C., F.D., V.D., and G.L. participated inresearch. A.T. and Y.R. designed the research, analyzed the data,and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Yolande Richard, Service of Immuno-Virology,CEA, DSV/iMETI, 18 route du Panorama, 92260 Fontenay-aux-Roses,France; e-mail: [email protected].

Figure 7. BAFF increases CXCL13- and CCL21-induced phosphorylation of PI3K/AKT, p38MAPK,and I�B�. Mock- and BAFF-treated B cells were stimu-lated for 2 minutes with medium, 100 ng/mL CXCL13, or100 ng/mL CCL21. Phosphorylation of AKT, p38MAPK,PLC�3, and I�B� was corrected for total relevant protein.The p52 contents in total-cell lysates were corrected foractin contents. Blots from one representative experimentare shown. Values reported for p52 contents and phos-phorylation of AKT, p38MAPK, and I�B� are mean valuesplus or minus SD from 3 or 2 (PLC�3) independentexperiments.





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