Negative Selection Checkpoints Conferring ...

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IL-4 Regulates Bim Expression and Promotes B Cell Maturation in Synergy with BAFF Conferring Resistance to Cell Death at Negative Selection Checkpoints Alessandra Granato, Elize A. Hayashi, Barbara J. A. Baptista, Maria Bellio and Alberto Nobrega

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http://www.jimmunol.org/content/suppl/2014/05/16/jimmunol.130074 9.DCSupplemental This article cites 71 articles, 45 of which you can access for free at: http://www.jimmunol.org/content/192/12/5761.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2014; 192:5761-5775; Prepublished online 16 May 2014; doi: 10.4049/jimmunol.1300749 http://www.jimmunol.org/content/192/12/5761

The Journal of Immunology

IL-4 Regulates Bim Expression and Promotes B Cell Maturation in Synergy with BAFF Conferring Resistance to Cell Death at Negative Selection Checkpoints Alessandra Granato,1 Elize A. Hayashi,1 Barbara J. A. Baptista, Maria Bellio, and Alberto Nobrega

M

aturation of B cells in mice and humans is a hierarchic process in which newly formed immature B cells pass through a series of intermediary stages and selective checkpoints, both in bone marrow (BM) and spleen, before reaching a functionally competent mature state (1–3). During this process, BCR plays a dual role, delivering both positive and negative signals. A very significant number of B cells generated in the BM are autoreactive and negatively selected, undergoing receptor editing or deletion by apoptosis upon high-affinity/avidity Agspecific interaction in early stages before they emigrate to the periphery (2, 4, 5). B cells that do not encounter self-antigens in the BM or are only weakly self-reactive migrate into the spleen, where they are further exposed to peripheral self-antigens and tolerance mechanisms (6). Those cells that are allowed to continue their differentiation program need positive signals to push them forward to more mature stages. In this respect, tonic BCR sigDepartment of Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro 21941-590, Brazil 1

A.G. and E.A.H. contributed equally to this work.

Received for publication March 19, 2013. Accepted for publication April 18, 2014. This work was supported by grants from the Conselho Nacional de Pesquisa, the Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro, the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior, and the Financiadora de Estudos e Projetos. Address correspondence and reprint requests to Dr. Alberto Nobrega, Department of Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Avenida Carlos Chagas Filho, 373 CCS Bloco D, sala D-35, Rio de Janeiro 21941-902, Brazil. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BM, bone marrow; CsA, cyclosporin A; IL-4C, IL-4/anti–IL-4 mAb complex. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300749

naling or a low level of autoreactivity seems to be necessary, although not sufficient, for survival and differentiation of developing B lymphocytes as well as for maintenance of mature B cells (3, 7–9). The newly arrived immature B cells in the spleen are named T1 transitional B cells and must differentiate into T2 transitional stage before reaching a mature B cell compartment. The passage from the T1 to T2 stage, where upregulation of CD23 is a phenotypical hallmark (1, 10, 11), is dependent on BAFF, a cytokine belonging to the TNF family; indeed, BAFF- or BAFF-R–deficient mice have B cell development impaired at T1 stage in the spleen (12, 13). Owing to the profound effects in B cell homeostasis observed as a consequence of defective function of BCR and BAFF-R, it is thought that the signals from these two receptors, complementing or counteracting each other, form the central axis of the selective pressures responsible for shaping an equilibrated B cell repertoire sufficiently diverse to cover the wide variety of potential foreign Ags, but also restricted not to contain pathological self-reactive specificities (14). Overexpression of BAFF results in B cell–related autoimmune disorders similar to some human systemic autoimmune pathologies (15). Because of the multiple roles of BAFF in B cell maturation, survival, and activation, the molecular mechanisms and cellular stages involved in the disruption of B cell homeostasis are not clearly understood. However, even in BAFF-transgenic models, it has become evident that additional elements, such as competitiveness for restricted niches (16), B cell–intrinsic MyD88 recruitment (17), probably by TLR signaling (18, 19), and the action of other cytokines (20, 21), are necessary for complete breakdown of B cell tolerance checkpoints. It is thus relevant to identify those factors that complement the action of BAFF and contribute to the outbreak of autoimmunity. IL-4 is a cytokine with pleiotropic activity in the immune system (22), and it plays an essential role in the activation of mature

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IL-4 plays an essential role in the activation of mature B cells, but less is known about the role of IL-4 in B cell maturation and tolerance checkpoints. In this study, we analyzed the effect of IL-4 on in vitro B cell maturation, from immature to transitional stages, and its influence on BCR-mediated negative selection. Starting either from purified CD19+IgM2 B cell precursors, or sorted bone marrow immature (B220lowIgMlowCD232) and transitional (B220intIgMhighCD232) B cells from C57BL/6 mice, we compared the maturation effects of IL-4 and BAFF. We found that IL-4 stimulated the generation of CD23+ transitional B cells from CD232 B cells, and this effect was comparable to BAFF. IL-4 showed a unique protective effect against anti-IgM apoptotic signals on transitional B cell checkpoint, not observed with BAFF. IL-4 and BAFF strongly synergized to promote B cell maturation, and IL-4 also rendered it refractory to BCR-mediated cell death. IL-4 blocked upregulation of proapoptotic Bim protein levels induced by BCR crosslinking, suggesting that diminished levels of intracellular Bim promote protection to BCR-induced cell death. Evidence was obtained indicating that downmodulation of Bim by IL-4 occurred in a posttranscriptional manner. Consistent with data obtained in vitro, IL-4 in vivo was able to inhibit Bim upregulation and prevent cell death. These results contribute to the understanding of the role of IL-4 in B lymphocyte physiology, unveiling a previously undescribed activity of this cytokine on the maturation of B cells, which could have important implications on the breaking of B cell central tolerance in autoimmunity. The Journal of Immunology, 2014, 192: 5761–5775.

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IL-4 IN B CELL MATURATION Pharmingen, San Diego, CA); PE-Cy7 anti-CD23 (B3B4), PE-Cy7 antiB220 (RA3-6B2), PE-Cy7 anti-CD21 (7G6), allophycocyanin anti-CD93 (AA4.1) (eBioscience, San Diego, CA); Alexa Fluor 647 anti-mouse IgM (b-7-6), FITC anti-B220 (provided by Dr. John Cambier, University of Colorado Health Science Center, Aurora, CO); allophycocyanin anti-CD23 (B3B4) (Caltag Laboratories, San Francisco, CA); PE-Cy7 anti-CD93 (AA4.1), Alexa Fluor 647 anti–BAFF-R (eBioscience); biotin anti-IgD (11-26) (SouthernBiotech, Birmingham, AL); PE anti-IgD (11-26) (BD Pharmingen); FITC and DyLight 649 goat F(ab) fragments anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA); and biotin anti-CD93 (493 hybridoma provided by Dr. Antonius Rolink, Basel University, Basel, Switzerland; the mAb was purified and biotinconjugated according to standard protocols). Biotinilated Abs were revealed with Alexa Fluor 680-R-PE–conjugated streptavidin (Molecular Probes, Eugene, OR) or with allophycocyanin-conjugated streptavidin (Caltag Laboratories). Cells were incubated with Abs in FACS buffer (PBS, 5% FBS, 0.05% sodium azide) for 20 min at 4˚C and washed with FACS buffer. When biotinilated mAbs were used, another step of incubation with Alexa Fluor 680-PE-streptavidin or allophycocyaninstreptavidin were performed under the same conditions as described above. For intracellular staining, cells were fixed and permeabilized after surface staining using a Foxp3 transcription factor staining buffer set protocol and reagents from eBioscience. Abs with the following specificities were used: Alexa Fluor 488 anti-Bcl2 (BCL/10C4) (BioLegend, San Diego, CA), Alexa Fluor 488 anti–Bcl-xL (54H6) (Cell Signaling Technology, Beverly, MA), and purified anti-Bim (C34C5) (Cell Signaling Technology). To detect intracellular Bim, a step of secondary Ab incubation with DyLight 488 anti-rabbit IgG (Poly4064) (BioLegend) was performed according to the manufacturer’s instructions. Except for the fixed cell samples, propidium iodide was added at 0.5 mg/ml immediately before data acquisition for dead cell exclusion. Data were acquired by a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed using CellQuest software (BD Biosciences) or Summit (DakoCytomation, Glostrup, Denmark). A MoFlo flow cytometer (DakoCytomation) was used for sorting of immature (CD93+B220lowIgMlowCD232), CD232 transitional (CD93+B220dullIgMhighCD232), CD23+ transitional (CD93+B220dullIgMhigh CD23+) B cell populations from BM, as well as follicular (CD932CD19+ CD21lowCD23+), marginal zone (CD932CD19+CD21highCD232/low), T1 (CD93+CD19+CD232), and T2 (CD93+CD19+CD23+) B cell populations from spleen. After cell sorting, B cell populations showed purity .97%.

Materials and Methods

For cell proliferation analysis, cells were stained with CFSE immediately prior to the culture. Briefly, BM-sorted transitional B cells were incubated at 5 3 106 cells/ml with 0.5 mM carboxyfluorescein diacetate–succinimidyl ester (Molecular Probes) in prewarmed PBS for 5 min at 37˚C and washed twice with complete OptiMEM medium.

Mice and cells C57BL/6 and BALB/c mice, 8–10 wk of age, were obtained from animal facilities of the Federal University of Rio de Janeiro and the Federal Fluminense University. Experimental procedures were approved by the Comiteˆ de E´tica do Centro de Cieˆncias da Sau´de/Centro de Cieˆncia e Sau´de, Federal University of Rio de Janeiro. BM cells were flushed from femurs and tibias with ice-cooled complete medium (Opti-MEM supplemented with 10% FBS, 5 3 1025 M 2-ME, 100 mg/ml streptomycin, and 100 U/ml penicillin; Life Technologies, Grand Island, NY). Spleen cell suspensions were obtained by gently teasing spleens onto a cell strainer. Erythrocytes were depleted using ACK buffer (0.155 M NH4Cl, 10 mM KHCO3, 0.1 mM sodium EDTA). Cells were counted using a hemocytometer with exclusion of dead cells with trypan blue dye.

MACS A VarioMACS (Miltenyi Biotec, Bergisch Gladbach Germany) system was used for magnetic cell sorting of CD19+IgM2 B cell precursors. For depletion of IgM+ B lymphocytes, total BM cells were incubated with antimouse IgM MicroBeads (Miltenyi Biotec) according to manufacturer’s specifications. Cells were applied onto an LD depletion column (Miltenyi Biotec) and the effluent cells were collected as a BM IgM2 fraction. An IgM2 fraction was incubated with anti-CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s specifications and applied onto an MS sort column for positive selection. Viable cells were scored by trypan blue dye using a hemocytometer, and the purity of cell preparations was verified by flow cytometry. Usually, IgM+ cells corresponded to ,1% of B lineage cells after depletion, and CD19+ cells corresponded to .95% of recovered cells after positive selection.

CFSE staining

Cultures for B cell differentiation MACS-purified B220+IgM2 B cell precursor cells or FACS-sorted B cell populations were cultured in 96-well flat-bottom plates (Corning, Corning, NY) at 1.5–2 3 105 cells/well in 200 ml/well in OptiMEM supplemented with 10% FCS, 5 3 1025 M 2-ME, 100 mg/ml streptomycin, and 100 U/ml penicillin (Life Technologies) in humidified atmosphere of 5% CO2 at 37˚C. B cell precursors were cultured for 72 h and stimuli were added 16–18 h before the end of culture. Sorted B cell populations were cultured in the presence of indicated stimulus for 16–18 h. Stimulus included IL-4 either as 0.1% supernatant from cultures of XR63-4 cells or as 25 ng/ml (or indicated concentrations) of rIL-4 (eBioscience), 100 ng/ml (or indicated concentrations) of rBAFF (R&D Systems, Minneapolis, MN), 12.5 mg/ml ultrapure LPS from Escherichia coli 0111:B4 strain (InvivoGen, San Diego, CA), or anti-CD40 (clone 1C10; eBioscience) at 10 mg/ml. In some experiments, we added simultaneously to stimuli 200 ng/ml cyclosporin A (Sandimmune; Novartis Pharmaceuticals) and/or anti-IgM F(ab9)2 fragments (Jackson ImmunoResearch Laboratories) at concentrations ranging from 0.005 to 5 mg/ml. Recovered live cells were counted using a hemocytometer with exclusion of dead cells with trypan blue dye and processed for flow cytometric or quantitative RT-PCR analysis. Statistical analyses (unpaired Student t test) were done using Prism software (GraphPad Software, San Diego, CA); results were considered statistically significant when p , 0.05.

Quantitative RT-PCR analysis Cell staining, flow cytometry, and cell sorting Abs used for staining were PE anti-CD23 (B3B4), PE anti-B220 (RA36B2), FITC rat anti-mouse IgM (R6-60.2), FITC anti-CD21 (7G6) (BD

RNA was extracted using an RNeasy Micro kit (Qiagen, D€usseldorf, Germany) according to the manufacturer’s instructions. Total cDNA was prepared using random primers (Promega, Madison, WI) and an Improm-

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B cells as a cofactor for LPS, CD40L, and Ag stimulation to induce B cell differentiation, proliferation, and Ab secretion, mainly of IgG1 and IgE isotypes (22, 23). Additionally, IL-4 was shown to provide survival signals and inhibit spontaneous and BCR-mediated apoptosis of splenic B lymphocytes (10, 24, 25). Transgenic expression of IL-4 also results in B cell–related systemic autoimmune disorders (26). In contrast to the relatively well-resolved roles of IL-4 in mature B cell activation, less clear is its role in B cell maturation and whether it can interfere on the pivotal roles of BCR/BAFF-R with a potential cooperation with BAFF in the control of autoreactivity at negative selection checkpoints. We have previously described that TLR4 agonists can affect B cell maturation in vitro by stimulating generation of T2-like B cells from BM developing B cells and cooperating with BAFF to enhance this process (27, 28). These results suggested that TLR4 agonists can act as alternate/complementary factors for BAFF. In the present study, we used a similar system, based on short-term cultures of highly purified B cell populations, corresponding to defined maturation stages, to look for effects of IL-4 on B cell development. We found that IL-4 indeed affects early maturation stages of BM-derived B cells, namely BM CD232 transitional B cells, stimulating them to generate T2-like transitional B cells. IL-4 synergized with BAFF in B cell maturation, but, distinctly from BAFF, IL-4 rendered BM transitional B cells refractory to anti-IgM apoptotic signals. Resistance to BCRmediated cell death was a unique property of IL-4, which could not be conferred by CD40, LPS, or BAFF, although these stimuli have been previously associated with antiapoptotic activities (29– 31). We also found that IL-4 was able to decrease proapoptotic Bim protein levels induced by anti-IgM treatment in a posttranscriptional manner, suggesting that the negative regulation of Bim is the mechanism through which IL-4 prevents BCR-induced B cell death. The significance of these results to our current understanding of B cell tolerance and autoimmunity is discussed.

The Journal of Immunology II reverse transcription system (Promega) following the manufacturer’s instructions. Quantitative PCR reactions were performed in duplicate with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), and detection was done using an StepOne real-time PCR system (Applied Biosystems) and normalized to the amount of housekeeping gene 18S rRNA. For quantification of expression, the following primers were used: mcl1, forward 59-TGTAAGGACGAAACGGGACT-39, reverse, 59AAAGCCAGCAGCACATTTCT-39; bcl2, forward, 59-TGAGTACCTGAACCGGCATCT-39, reverse, 59-GCATCCCAGCCTCCGTTAT-39; bim, forward, 59-CAACACAAACCCCAAGTCCT-39, reverse, 59-GTTGAACTCGTCTCCGATCC-39; 18S, forward, 59- GGGCGTGGGGCGGAGATATGC-39, reverse, 59-CGCGGACACGAAGGCCCCAAA-39; bclxl, forward, 59-CTGGGACACTTTTGTGGATCTCT-39, reverse, 59-GAAGCGCTCCTGGCCTTT-39. Reactions were incubated at 95˚C for 10 min and then run through 45 cycles of 95˚C for 15 s and 60˚C for 1 min.

Preparation of IL-4/anti–IL-4 Ab complexes

Anti-IgD in vivo treatment BALB/c mice (four per group) were treated with PBS buffer containing 10% BALB/c serum (vehicle) or 50 mg azide-free anti-mouse IgD (clone 10.4.22, eBioscience) in the presence or absence of IL-4C i.v. into tail vein. Mice were sacrificed 1–3 d after anti-mouse IgD treatment. Spleen cells from individual mice were recovered analyzed by flow cytometry as previously described.

Results IL-4 stimulates in vitro B cell maturation To evaluate the activity of IL-4 on early B cell differentiation, we used an in vitro system in which CD19+IgM2 B cell precursors were purified from adult C57BL/6 mouse BM and cultured for 3 d with or without additional stimuli. As previously described (27, 28), even without addition of any supplemental growth factors, this system allows spontaneous differentiation of purified B cell precursors with generation of a heterogeneous population of IgM+ B cells (Fig. 1), constituted by a majority bearing a phenotype resembling BM CD232 fraction E, which we subdivided into early immature (IgMlowCD232) and CD232 transitional subsets (IgMhighCD232), and a minority (1–5%) bearing a more mature phenotype (IgMhighCD23+) resembling the recently resolved T2like BM CD23+ fraction E (11). Incubation of cells in the presence of BAFF for the last 18 h of culture in this system induces a 2- to 4-fold increase in the percentage and number of the CD23+ B cells compared with unstimulated control (Fig. 1A, 1C), in accordance with the well-known role of BAFF in B cell development at a transitional stage (13–15, 28). Using BAFF as our comparative standard, we tested the effect of the incubation of B cells with IL-4 for the last 18 h of culture on developing B lymphocytes, analyzing changes in the expression of surface maturation markers by flow cytometry. We found that IL-4, similarly to BAFF, induced a 2- to 4-fold increase in percentage and numbers of T2-like IgM+CD23+ B cells relative to the unstimulated control (Fig. 1A, 1C). To verify whether the CD23+ B cell appearance induced by IL-4 corresponded to a maturation event, we further analyzed expression of other developmental markers in the three major B lymphocyte subpopulations generated in culture, that is, IgMlowCD232, IgMhighCD232, and IgMhighCD23+ (Fig. 1B). Upon IL-4 stimulation, IgMhighCD23+ B cells exhibited lower expression of CD93 and higher expression of CD21, B220, and IgD when compared with the CD232 population. This phenotype was similar to the phenotype expressed by the equivalent subsets in BAFF-stimulated cultures (Fig. 1B). More importantly, modulation of markers in the subsets

generated in vitro follows the pattern of maturational sequence observed in vivo (1, 2, 11). These results indicate that IL-4 is able to stimulate a complex set of phenotypical changes in B cells that is compatible with progression of maturation process. BCR signaling in developing B cells is differentially modulated by IL-4 and BAFF We have previously found that the generation of IgMhighCD23+ B cells in vitro, induced by BAFF or LPS, was blocked by the addition of soluble anti-IgM F(ab9)2 fragments, showing that BCR crosslinking resulted in the inhibition of in vitro B cell maturation (28). These results indicate that those factors cannot break central tolerance mechanisms in vitro, mimicked by anti-IgM F(ab9)2. We then addressed the impact of BCR crosslinking on IL-4–induced B cell maturation. Fig. 2A shows the results of experiments where anti-IgM F(ab9)2 fragments, mimicking high-affinity autoantigen interaction, were added into the cultures for B cell differentiation from BM-purified precursors, in the presence or absence of IL-4 or BAFF. Whereas in nonstimulated as well as in BAFF-stimulated cultures there was a strong inhibition of the generation of CD23+ B cells, even upon treatment with low doses (0.63 mg/ml) of antiIgM (∼8- to 10-fold inhibition compared with the respective untreated controls), in IL-4–stimulated cultures saturating doses of anti-IgM were poorly able to inhibit the generation of CD23+ B cells (Fig. 2A). Comparative dose–response curve analyses for BAFF and IL-4, in the absence and presence of a saturating dose of anti-IgM (5 mg/ml), were performed to verify whether the distinct outcomes for BAFF and IL-4–stimulated cultures were due to the use of insufficient doses of BAFF (Fig. 2B). Even saturating concentrations of BAFF were not able to overcome the inhibitory effect of anti-IgM on the B cell maturation. Alternatively, even low doses of IL-4, ranging from 0.025 to 25 ng/ml, were able to strongly inhibit the deleterious effect of anti-IgM (Fig. 2B). Those results clearly show that IL-4 and BAFF have distinct impact on BCR signaling in maturing B cells. Activation of calcium-dependent signaling in immature B cell stages has important roles in negative selection events (34–36), and Ca2+-dependent calcineurin activity is greatly responsible for the BCR-mediated inhibition of LPS- or BAFF-induced in vitro maturation (28). We then evaluated the effect of blocking the calcineurin pathway on B cell maturation induced by IL-4. To do so, we added cyclosporin A (CsA), a calcineurin inhibitor, to B cell differentiation cultures of purified precursors in the presence of BAFF or IL-4, with or without anti-IgM (Fig. 2C). CsA was able to significantly revert the inhibition of B cell maturation by anti-IgM in cultures with BAFF or medium alone, as expected, and had no effects on the action of IL-4 in anti-IgM–stimulated cultures (Fig. 2C). Interestingly, the presence of CsA led to a slight decrease of the activity of IL-4 alone (Fig. 2C), maybe suggesting that BCR tonic signaling may participate in IL-4– driven B cell maturation. T cell help, either through IL-4 secretion or engagement of membrane CD40L, has previously been described as a potent costimulus that promotes rescue of BCR-induced apoptosis in splenic B lymphocytes (10, 37). We tested the effect of CD40 engagement by anti-CD40 agonist mAb in the B cell differentiation system and found that anti-CD40 was also able to stimulate generation of CD23+ B cells, similarly to BAFF, LPS, and IL-4 (Fig. 2D). Nonetheless, anti-CD40 was not able to block inhibition of B cell maturation by anti-IgM (Fig. 2E). IL-4 responsiveness is developmentally regulated B cell precursors in culture continuously generate immature B cells, producing a nonsynchronous population of cells with

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rIL-4 (eBioscience) (1 mg/mouse) was mixed at a 1:1 volume ratio with neutralizing anti–IL-4 mAb (BVD4-1D11.2; eBioscience) (10 mg/mouse) to freshly prepare IL-4/anti–IL-4 mAb complexes (IL-4C), which greatly increase the in vivo t1/2 and activity of IL-4 as previously described (32, 33). After 5 min at room temperature, complexes were diluted with 1% BALB/c serum for injection i.v. into mice tail vein.

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IL-4 IN B CELL MATURATION

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FIGURE 1. Comparative analysis of in vitro B lymphocyte differentiation in the presence of BAFF and IL-4. B cell precursors (CD19+IgM2) purified from mouse BM were cultured for 72 h. Stimuli (BAFF at 100 ng/ml and IL-4 at 0.1% of supernatant from XR63-4 cell line cultures) were added 18 h before the end of culture (54 h), and the cells were recovered for viable cell counting and analysis by flow cytometry. (A) CD23 versus IgM profile of viable cells at 0 h and recovered from cultures just before the stimuli (54 h) and unstimulated control and stimulated cultures after 72 h. Numbers indicate IgMlow CD232, IgMhighCD232, and IgMhighCD23+ B cell percentages relative to total viable population. (B) Histograms show expression levels of CD93, CD21, B220, and IgD of the B cell populations, defined as shown in (A), recovered from the indicated cultures. Dead cells were excluded from analyses by propidium iodide labeling. Results are representative of three independent experiments. (C) Absolute numbers of total viable (left), IgM+ (center), and CD23+ B (right) cells per well were defined based on counting of trypan2 cells and the relative percentages of the respective subsets. Bars represent means 6 SEM of three independent experiments. *p , 0.05.

The Journal of Immunology

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different developmental stages at the moment of stimulation with IL-4. In this experimental system we cannot precisely identify at which developmental stage immature B cells become prone to respond to IL-4. In an attempt to better define the effect of IL-4 on different subpopulations of developing B cells, early immature (B220lowIgMlowCD232) and CD232 transitional (B220intIgMhigh CD232) B cells were sorted by FACS from mouse BM (Fig. 3A) and cultured for 16–18 h with or without BAFF or IL-4. We found that IL-4, as well as BAFF, was able to induce an increase in the percentage and numbers of CD23+ B cells, both in sorted BM immature and CD232 transitional B cells, relative to control cultures (Fig. 3B, 3C). However, sorted CD232 transitional B cells, more advanced in maturation, were more prone to produce

CD23+ B cells than sorted immature B cells upon stimulation with IL-4. Total live cell numbers recovered from IL-4–stimulated immature and transitional B cell cultures were equivalent to the control conditions (Fig. 3C), showing that IL-4 does not provide any protection against spontaneous apoptosis for those populations during this short period of incubation. Those results together indicate that the activity of IL-4 is under developmental control, and IL-4 stimulates the generation of CD23+ B cells by induction of differentiation of CD232 transitional B cells, their immediate earlier counterparts. BM transitional B cells seem to be particularly susceptible to deletion by apoptosis upon BCR crosslinking (2). To verify whether the protective activity of IL-4 could affect BCR-mediated

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FIGURE 2. Developing B cell response to in vitro anti-IgM treatment in the presence of IL-4. (A and B) B cell precursors (CD19+IgM2) purified from mouse BM were cultured for 72 h. Stimuli and anti-IgM were added 18 h before the end of culture, and the cells were recovered for viable cell counting and analysis by flow cytometry. (A) Dose–response curve of anti-IgM treatment on the percentage of CD23+ cells in the presence or absence of BAFF (100 ng/ml) or IL-4 (25 ng/ml). Relative percentages were normalized considering 100% the percentage of CD23+ cells among IgM+ cells obtained with each stimulus in the absence of anti-IgM. (B) Dose–response curve of BAFF (left) and IL-4 (right) on the percentage of CD23+ cells among IgM+ cells generated in untreated or anti-IgM– treated cultures (5 mg/ml). (C) The indicated stimuli (100 ng/ml BAFF and 25 ng/ml IL-4) were added 18 h before the end of culture with or without anti-IgM (5 mg/ml) and/or CsA (200 ng/ml). Bars indicate mean percentage of CD23+ B cells among IgM+ cells of duplicate samples 6 SD. (D) The stimuli (100 ng/ml BAFF, 12.5 mg/ml LPS, 25 ng/ml IL-4, and 10 mg/ml anti-CD40) were added 18 h before the end of culture without or with 5 mg/ml anti-IgM. Relative percentages shown in (D) were normalized considering 100% of the average percentage of CD23+ cells obtained with each stimulus in the absence of anti-IgM. Bars represent means 6 SEM of at least two independent experiments. *p , 0.05 (E) Dose–response curve of anti-IgM treatment on the percentage of CD23+ cells among IgM+ cells in the presence or absence of BAFF (100 ng/ml), IL-4 (25 ng/ml), and anti-CD40 (10 mg/ml). Relative percentages were normalized considering 100% of the percentage of CD23+ cells obtained with each stimulus in the absence of anti-IgM. Data are representative of al least three independent experiments.

5766 cell death of that subpopulation in our system, sorted CD232 transitional B cells were submitted to anti-IgM treatment, in the presence or absence of IL-4 or BAFF, for 16–18 h. Rates of apoptosis in the absence of anti-IgM were relatively constant among the different treatments, reinforcing the notion that IL-4 stimulus does not provide survival advantages against spontaneous cell death mechanisms in our short-term culture system. Although BAFF was poorly able to avoid BCR-mediated apoptosis, IL-4 protected B cells from cell death (Fig. 3D) and allowed full generation of CD23+ cells in the presence of BCR signaling (Fig. 3D). These results indicate that BM CD232 transitional B cells become resistant to BCR-mediated negative selection upon IL-4 protective stimulus. IL-4 synergizes with BAFF, stimulating B cell maturation

IL-4 prevents the upregulation of Bim protein by anti-IgM Pro- and antiapoptotic members of the Bcl2 family are known to be key players in balancing death and survival in B cell development (38–41). To assess the role of these molecules in the IL-4– mediated protection against BCR-induced cell death, we analyzed the mRNA expression of antiapoptotic bcl2, bcl_xL, and mcl1 genes in sorted BM CD232 transitional B cells submitted to antiIgM treatment in the presence or absence of IL-4 (Fig. 5). IL-4 was unable to modulate bcl2 and bcl_xL gene expression (Fig. 5A) or their intracellular protein levels (Fig. 5B). Whereas mcl1 gene expression was slightly increased in the presence of IL-4 alone, mcl1 expression was suppressed by anti-IgM, both in the presence and the absence of IL-4 (Fig. 5A). Bim is a critical player in BCR-mediated apoptosis both in vitro and in vivo (42). In the present study, we found that bim mRNA and intracellular Bim protein levels were augmented in anti-IgM–

treated cultures of CD232 B cells (Fig. 6A, 6B). Unexpectedly, we observed an even higher increase of bim gene expression after anti-IgM treatment in the presence of IL-4. However, IL-4 was able to prevent intracellular Bim protein upregulation after antiIgM treatment (Fig. 6B), suggesting a posttranscriptional regulation of this proapoptotic molecule levels by IL-4. The Bim mRNA level after anti-IgM treatment was increased in BM CD23+ transitional B cells, where it was even more pronounced (Fig. 6C) and also accompanied by high amounts of intracellular Bim protein (Fig. 6D). Again, as observed in CD232 transitional B cell cultures, Bim protein levels were inhibited by IL-4 treatment on the CD23+ B cell population (Fig. 6D). Percentages of live cells in BM CD23+ transitional B cell cultures are shown in Fig. 6E. Splenic transitional B cells gave similar results (Supplemental Fig. 1). IL-4 prevents Bim protein level upregulation by anti-IgM in mature follicular B cells The observation of downmodulation of Bim protein expression by IL-4 in BM transitional B cells (Fig. 6) led us to investigate the involvement of bim in the prevention of apoptosis of mature splenic B cells as well. It is known that IL-4 protects transitional splenic B cells from BCR-induced apoptosis, but the mechanisms have not been elucidated (37). In mature B cells, IL-4 renders activated B cells insensitive to Fas ligation (43–45), and some studies suggest a role for STAT6-dependent Bcl_xL upregulation (46), but the molecular events induced by IL-4 in mature B cell survival are not understood. In this study, we observed that IL-4 was also able to protect follicular B cells from BCR-induced mortality (Fig. 7A). The data obtained showed that IL-4 does not modulate antiapoptotic bcl2 and bcl_xL gene expression (Fig. 7B) or intracellular protein levels (Fig. 7C) after BCR crosslinking on sorted splenic follicular B cells. Alternatively, differently from sorted BM transitional B cells, mcl1 gene expression was increased under anti-IgM treatment; IL-4 also induced mcl1 upregulation, which was equivalent in the absence or presence of BCR crosslinking (Fig. 7B). As for BM transitional B cells, augmented expression of proapoptotic bim gene mRNA was observed after anti-IgM treatment of mature follicular B cells both in the presence and absence of IL-4 (Fig. 7D). However, as in transitional B cell populations, IL-4 prevented intracellular Bim protein accumulation upon anti-IgM treatment in follicular B cells (Fig. 7E), again indicating a posttranscriptional regulation of this key proapoptotic molecule. These results suggest that IL-4 is able to prevent BCR-induced apoptosis by decreasing Bim intracellular levels, both in BM transitional B cells and mature splenic follicular B cells. Interestingly, BAFF treatment partially inhibited BCR-induced cell death of mature follicular B cells (Fig. 7F), which was associated with a partial decrease of intracellular Bim protein (Fig. 7G); this result contrast from what was observed with BM transitional B cells, where BAFF was unable to counteract BCR-induced apoptosis, indicating a dependence on the maturation stage for the response to BAFF. IL-4 prevents in vivo Bim protein level upregulation after anti-IgD treatment in mature B cells In this study, anti-IgM was used in vitro as a surrogate for Ag stimulus and evaluation of tolerance checkpoints. In vivo, anti-IgM cannot be used in the same manner in adult mice because of the high levels of serum IgM. To circumvent this, we chose a system in which treatment of BALB/c mice with an anti-IgD causes deletion of mature B cells (33, 47, 48) to determine whether IL-4–mediated protection could be observed in vivo in a normal B cell repertoire model. Previous studies observed that IL-4 in vivo treatment

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We next evaluated how developing B cells react to the combination of IL-4 and BAFF, and whether the concurrent stimulation by those factors interferes in the inhibition of B cell maturation following BCR engagement. Purified CD232 transitional B cells were cultured for 16–18 h with BAFF or IL-4 separately or in combination, in the presence or absence of anti-IgM (Fig. 4). As shown in Fig. 4A, the concomitant presence of IL-4 and BAFF led to an additive effect on the generation of CD23+ B cells, but also to the rescue from BCR-induced inhibition of CD23+ generation (Fig. 4A) and apoptosis (Fig. 4B), characterizing a synergic activity of IL-4 with BAFF, pushing forward B cell differentiation despite BCR engagement. In the combination of BAFF and IL-4, most recovered cells (∼60%) were CD23+ (Fig. 4A) without detectable change of cell survival rates (Fig. 4B). IL-4 did not induce transitional B cell proliferation even in combination with other factors, as verified in CFSE staining assay (Fig. 4C); additional proliferation control with mature (fraction F) B cells are also shown (Fig. 4D). Thus, generation of CD23+ B cells observed upon the simultaneous stimulation with BAFF and IL-4 is mainly due to a massive induction of B cell differentiation toward a more mature stage and does not involve proliferation. Given the potent synergy observed in the present study between IL-4 and BAFF, we investigated whether this effect was due to upregulation of BAFF-R on developing B cells. We then analyzed BAFF-R expression of developing B cell populations recovered from BM B cell precursor cultures, stimulated or not with IL-4. As shown in the Fig. 4C, none of the developing B cell populations upregulated BAFF-R expression upon IL-4 stimulation; on the contrary, a slight downmodulation of BAFF-R on transitional B cells was observed in IL-4 cultures. Therefore, the role of IL-4 in this synergic interaction, providing protection from BCR-mediated cell death, seems to rely directly on IL-4R signaling, possibly controlling intracellular mediators acting on cell survival and death.

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FIGURE 3. Generation of CD23+ B cell from FACS-sorted BM immature and transitional B lymphocytes in the presence of IL-4. Immature and CD232 transitional B lymphocytes were sorted by FACS from mouse BM and cultured for 16–18 h in the presence or absence of BAFF (100 ng/ml) or IL-4 (25 ng/ml). After culture, cells were restained for analysis of CD23, IgM, and B220 expression by flow cytometry. (A and B) Plots show CD23 versus B220 profiles of viable cells. Numbers indicate percentage of CD23+ B cells before (A) and after (B) stimulation. (C) Numbers of CD23+ (left) and total viable (right) B cells per well in 0 h and after 16 h of culture of sorted immature and transitional B cells, based on counting of trypan2 cells and the relative CD23+ B cell percentages. Bars represent means 6 SEM of three independent experiments. (D) Sorted BM CD232 transitional B lymphocytes were cultured for 16 h in the presence or absence of BAFF (100 ng/ml) or IL-4 (25 ng/ml), treated or not with anti-IgM (5 mg/ml). After culture, cells were restained for analysis of CD23, IgM, and B220 expression by flow cytometry. Graphs show live cell percentages assessed by propidium iodide2 cells and the percentage of CD23+ B cells in the viable population. Bars represent means 6 SEM of three independent experiments.

prevents deletion of autoreactive transgenic B cells or wild-type mature B cells treated with anti-IgD (33), suggesting that IL-4

might contribute to autoimmunity by inhibiting Ag-induced deletion of autoreactive B cells. As shown in Fig. 8A, treatment with

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anti-IgD greatly decreased splenic B cell numbers after 48 h, as previously described (47). Consistent with the in vitro results, administration of IL-4C was able to protect B cell death at this time point (Fig. 8A). In this model, we could observe Bim upregulation on follicular and marginal zone B cells after 24 h of anti-IgD treatment (Fig. 8B, 8C), anticipating B cell death at later time points (47). Interestingly, we could not detect any differences of Bim expression at transitional BM (data not shown) and splenic B cell populations (Fig. 8B, 8C) after anti-IgD treatment, suggesting rapid dynamics of these populations in vivo. Together with B cell death protection, IL-4 prevents Bim upregulation at mature B cell stages after 24 h of anti-IgD treatment (Fig. 8B, 8C). Importantly, note that the loss of surface IgD is faster than splenic B cell death, indicating IgD internalization and signaling (47). Despite that anti-IgD treatment was able to significantly decrease the expression of surface IgD, IL-4 cannot rescue IgD expression on the B cell membrane (Fig. 8C). These results indicate that IL-4 does not prevent BCR signaling but interferes at specific steps as accumulation of Bim intracellular levels.

Discussion In the present study we described the action of IL-4 upon B cell development in vitro and characterized several key points: 1) IL-4 stimulates maturation of BM-derived developing B cells in vitro, very similarly to BAFF, acting preferentially on CD232 CD93+ transitional B cells; 2) IL-4 has unique properties of ren-

dering BM transitional B cells refractory to anti-IgM apoptotic signals; 3) IL-4 synergizes with BAFF to stimulate B cell maturation and resistance to apoptosis upon BCR crosslinking; and 4) IL-4 prevents the increase of Bim intracellular levels induced by BCR crosslinking in immature and mature B cells, acting in a posttranscriptional manner. In vitro culture systems of B cell development have been instrumental in characterizing the distinct differentiation stages and elucidating their relationship during the process of maturation of B cell precursors. Early studies using transformed cell lines, thought to represent specific stages of differentiation, were performed to investigate the control of variable Ig gene rearrangement in B cell development (49). Although important results could be obtained with these models, it became clear that transformed cells could not reveal the physiological condition of the network of transcription factors that guide B cell development and maturation. Later, the identification of a series of B cell surface markers allowed the resolution and isolation by multiparameter flow cytometry of distinct stages of maturing B cells in normal B cell development (50). Importantly, the sorting of these populations, followed by short-term in vitro cultures, reflected the behavior of the cells more faithful to their in vivo counterparts. Although transformed cells are still used to study signal transduction (51), it has been increasingly appreciated that signaling is also critically influenced by the maturation stage of the B cell, with BCR signaling being the canonical example (35); therefore, the results

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FIGURE 4. Effect of different combinations of IL-4 and BAFF on in vitro differentiation of developing B cells. (A and B) Sorted BM CD232 transitional B lymphocytes were cultured in the presence or absence of BAFF (100 ng/ml), IL-4 (25 ng/ml), or both, treated or not with anti-IgM (5 mg/ml). After 16 h, cells were restained for analysis of CD23, IgM, and B220 expression by flow cytometry. (A) Percentage of CD23+ B cells and (B) percentage of live cells (propidium iodide2 cells) of the population recovered from the cultures of BM-sorted transitional B cells. Bars represent means 6 SEM of at least two independent experiments. *p , 0.05, **p , 0.01. (C) Sorted BM CD232 transitional B lymphocytes were stained with CFSE and cultured in the presence or absence of BAFF (100 ng/ml), IL-4 (25 ng/ml), or BAFF plus IL-4 plus LPS (12.5 mg/ml); histograms show CFSE staining of CD232 and CD23+ subsets recovered from the cultures at indicated time points. (D) Sorted BM mature, fraction F B cells were stained with CFSE and cultured with LPS; histograms show CFSE staining of cells recovered from the cultures at indicated time points. (E) B cell precursors purified from mouse BM were cultured for 72 h. IL-4 was added 18 h before the end of culture, and the recovered cells were analyzed by flow cytometry of the BAFF-R expression. Histograms show the BAFFR expression levels for the immature (upper), CD232 transitional (middle), and CD23+ transitional B cell subsets in unstimulated control (gray line) and IL4 (black line) cultures. Results are representative of two independent experiments.

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obtained with these models have to be taken carefully and confirmed with normal B lymphocytes. Taking into account these considerations, we thought that the ideal system to study B cell maturation in vitro would be short-term cultures of defined and highly purified populations obtained by FACS and/or magnetic cell sorting (28, 50, 52). Following these guidelines, we preferred to avoid the enrichment of B cell precursors through cultures with IL-7, typically for 5–7 d (53), to keep the cells closer to their physiological state, as these precursors will certainly accumulate a series of alterations along the multiple cycles of proliferation. In the present study, in our culture system B cell precursors were directly purified from BM and cultured immediately for 48–72 h; little proliferation was observed in control cultures (medium alone). However, addition of IL-4, but not BAFF, induced the proliferation of IgM2 B cell precursors in 48–72 h (data not shown); therefore, we decided to perform most of the maturation experiments with FACS-sorted immature B cells, or transitional B cells, which do not proliferate with IL-4 in our shortterm culture assay (Fig. 4C). We found that IL-4 induced an increase both in the percentage and numbers of CD23+ B cells in vitro. IL-4 is known to be able to upregulate CD23 in mature B cells (54). Thus, it was not completely unexpected that IL-4 was able to induce the appearance of CD23+ B cells during differentiation cultures. However, in a careful comparative evaluation of the expression of other maturation markers (B220, CD21, CD93, and IgM) in cell populations from each culture condition, we found that the phenotype of CD23+ B cells from IL-4 cultures fairly corresponded to CD23+ populations in control and BAFF cultures, as well as to ex vivo BM CD23+ fraction E or splenic transitional T2 B cells, bearing a more mature overall phenotype relative to their CD232 counterparts (1, 11). The equivalence in the expression of maturation markers in B cells from IL-4 cultures, with the corresponding

populations from BAFF or unstimulated cultures, argues against the hypothesis that IL-4 is inducing a simple burst of expression of CD23. Furthermore, the finding that sorted BM immature B cells generate much less CD23+ B cells than do the sorted BM CD232 transitional B cells upon IL-4 stimulation indicates that the expression of this maturation marker is stage-restricted and respects the sequence of events in the B cell developmental program. Collectively, these data strongly support the notion that IL-4 can promote B cell maturation of transitional BM B cells; similar results with splenic transitional T1 B cells were also obtained (data not shown). IL-4 and BAFF are known to provide survival signals to B lymphocytes at different stages (22, 24, 37), and thus it is possible that survival signals by these cytokines might also contribute to the enhanced recovery of CD23+ B cells, which could be dying fast in control cultures with medium alone (54). However, given that cultures of CD232 transitional B cells in the absence of anti-IgM had very similar low levels of spontaneous cell death in the presence or absence of IL-4 or BAFF, despite largely varying in CD23+ B cell percentages (Figs. 3, 4), we conclude that there must be an active generation of CD23+ B cells in vitro by IL-4, as well as by BAFF. Additionally, at this time point of culture, proliferation was not observed in any culture conditions (Fig. 4C), ruling out the possibility that higher percentages of CD23+ B cells could be due to specific proliferative response of that subpopulation. Taken together, our results indicate that IL-4 affects developing B cells in vitro through induction and/or acceleration of a coordinated differentiation process compatible with maturation, similar to BAFF. In contrast to BAFF (30), IL-4 conferred to sorted BM transitional B cells full resistance to both BCR-mediated apoptosis and blockade of B cell differentiation. Resistance to both effects could not be conferred either by LPS or CD40L. Distinctly from

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FIGURE 5. Regulation of antiapoptotic molecules by anti-IgM treatment in the presence of IL-4 in BM CD232 transitional B cells. Sorted BM CD232 B lymphocytes were cultured for 16–18 h in the presence or absence of IL-4 (25 ng/ml) treated or not with anti-IgM (5 mg/ml). (A) Quantitative PCR analysis of bcl2, bcl_xL, and mcl1 mRNA in BM CD232 transitional B cells cultured under indicated conditions. One experiment representative of three is shown. Error bars represent the means 6 SEM of duplicates. Samples were normalized for the expression of 18S. (B) Intracellular Bcl2 and Bcl_xL protein levels in BM CD232 transitional B cells assessed by FACS staining after culture under anti-IgM treatment in the absence (open solid histograms) or presence of IL-4 (dotted histograms). Control cultures are represented by gray-filled histograms. Isotype control staining is represented by gray open histograms. One experiment representative of at least two is shown.

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CD40 engagement, or LPS treatment, as we have previously reported (28), B cell maturation induced by IL-4 was not inhibited by antiIgM (Fig. 2). Although previous works have shown that T cell help by CD40L could inhibit BCR-induced apoptosis in splenic transitional B cells (10, 37), we found that CD40 engagement was able to increase the numbers of CD23+ B cells, but was unable to protect from BCR-induced cell death and blockade of B cell differentiation. Only IL-4 showed protective activity against apoptotic signals triggered by BCR and allowed maturation to proceed in the presence of anti-IgM F(ab9)2. Interestingly, note that BAFF was able to partially revert BCR-induced cell death in follicular B cells but not in BM transitional B cells, demonstrating a stage-specific difference in the response to that cytokine, pos-

sibly related to alterations in intracellular signaling. Alternatively, IL-4 sensitivity to protection against BCR-induced cell death seems to arise at an earlier developmental stage (BM CD232 transitional), being sustained all along B cell maturation. Consistent data support the notion that BM can sustain maturation up to late steps of transitional B cells, in parallel to the spleen (11, 55), and that activated CD4+ T lymphocytes present in the BM of healthy mice, including IL-4–producing cells, can participate in homeostasis of BM B lineage cells, as demonstrated in the T cell–deficient mice (56). Nonetheless, the great majority of the studies on IL-4 activity have been focused on splenic B cell subsets (10, 37, 43), and evaluation of antiapoptotic effects of IL-4 in BM-derived maturing B cells have not been reported.

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FIGURE 6. Regulation of Bim expression by anti-IgM treatment in the presence of IL-4 in BM transitional B cells. Sorted BM CD232 and CD23+ B lymphocytes were cultured for 16–18 h in the presence or absence of IL-4 (25 ng/ml) treated or not with anti-IgM (5 mg/ml). (A and C) Quantitative PCR analysis of bim mRNA in BM CD232 (A) and CD23+ (C) transitional B cells cultured under indicated conditions. One experiment representative of three is shown. Error bars represent the means 6 SEM of duplicates. Samples were normalized for the expression of 18S. (B and D) Intracellular Bim protein levels in BM CD232 (B) and CD23+ (D) transitional B cells assessed by FACS staining after culture under anti-IgM treatment in the absence (open solid histograms) or presence of IL-4 (dotted histograms). Control cultures are represented by filled histograms. One experiment representative of at least two is shown. Graphs in (E) show live cells percentage on BM CD23+ transitional B cell cultures assessed by propidium iodide2 cells.

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FIGURE 7. Regulation of anti- and proapoptotic molecules by anti-IgM treatment in the presence of IL-4 in mature follicular B cells. Mature follicular B lymphocytes were sorted by FACS from mouse spleen and cultured for 16–18 h in the presence or absence of IL-4 (25 ng/ml) treated or not with anti-IgM (5 mg/ml). (A) Live cells percentage on splenic mature follicular B cell cultures assessed by propidium iodide2 cells. Bars represent means 6 SEM of two independent experiments. (B) Quantitative PCR analysis of bcl2, bcl_xL, and mcl1 mRNA in mature follicular B cells cultured under indicated conditions. One experiment representative of three is shown. Error bars represent the means 6 SEM of duplicates. Samples were normalized for the expression of 18S. (C) Intracellular Bcl2 and Bcl_xL protein levels of mature follicular B cells assessed by FACS staining after culture under anti-IgM treatment in the absence (open solid histograms) or presence of IL-4 (dotted histograms). Control cultures are represented by gray-filled histograms. (Figure legend continues)

5772

and ubiquitination. Expression of a microRNA-17–92 cluster was found to be essential during B cell development, peaking at pre– B cell stage, where it inhibited cell death through the downregulation of Bim (60). Phosphorylation of Bim, mediated by MAPK kinases, promotes changes in its proapoptotic activity, and ERKinduced phosphorylation of Bim leads to its ubiquitination and subsequent degradation via the proteasome (61–63). All these mechanisms remain as possible candidates to mediate the action of IL-4 described in the present study. It is interesting to speculate whether this action could be related to alternate BCR signaling in the presence of IL-4 (64). Finally, our findings on the activity of IL-4, counteracting BCRmediated inhibition of B cell maturation, allows some speculation on the role of this cytokine in the process of tolerance breaking in autoimmune diseases. Establishment of B cell autoimmunity depends on failing of final regulatory mechanisms of B cell activation that lead autoreactive B cells to be recruited into the effector compartment of plasmacytes (65); nevertheless, deficient regulation at early checkpoints of central tolerance mechanisms has also been associated with autoimmune conditions in mice and humans as well (66, 67), and it probably contributes cumulatively to an increased predisposition to pathogenesis. BAFF transgenic mice present a systemic autoimmune syndrome, but little is known about the mechanisms that mediate the break of tolerance to self in these animals. Interestingly, BAFF overexpression, which leads to rescue of autoreactive B cells from peripheral tolerance, is not able to rescue autoreactive cells submitted to central tolerance mechanisms (16), suggesting that BAFF plays a minor role in the negative selection process taking place in the BM. It is clear that others factors besides BAFF are necessary to tolerance breakdown; for example, MyD88 signaling seems to be required (17). It is tempting to speculate about a role for IL-4 in BAFF transgenic mice, as it has been reported that overexpression of IL-4 is also present in systemic autoimmunity (26). In the physiological context, the role of IL-4 in BM B cell development has been poorly addressed. No gross alterations on B cell development and maturation in IL-4 knockout mice have been reported (68). However, studying the progeny of BAFF transgenic animals with IL-4 knockout may reveal a critical role for IL-4 in the disruption of tolerance. Another interesting set of data has been reported from chronic the graft-versus-host model of systemic lupus erythematosus, where B cells participating in the pathological response require continuous “nurturing” of CD4+ T cells since very early stages of post-irradiated BM autoreconstitution (69, 70). In this model, IL-4 was able to replace this CD4+ T cell–dependent effect (69), implying that this cytokine plays key roles along distinct steps of the ontogeny of these autoreactive B cells. Interestingly, T cell–deficient animals have a severe reduction in B cell lymphopoiesis, which can be reverted by T cell transfers; whether IL4 is involved in this recovery remains to be determined (56, 71). Our data bring new insights to this scenario, providing evidence that IL-4 can stimulate positive selection of BM-derived developing B lymphocytes into the CD23+ B cell compartment, making

Isotype control staining is represented by gray open histograms. One experiment representative of at least two is shown. (D) Quantitative PCR analysis of bim mRNA in mature follicular B cells cultured under indicated conditions. One experiment representative of three is shown. Error bars represent the means 6 SEM of duplicates. Samples were normalized for the expression of 18S. (E) Intracellular Bim protein levels in mature follicular B cells assessed by FACS staining after culture under anti-IgM treatment in the absence (open solid histograms) or presence of IL-4 (dotted histograms). Control cultures are represented by filled histograms. One experiment representative of at least two is shown. (F) Live cells percentage on sorted splenic mature follicular B cells cultured under BAFF (100 ng/ml) and/or anti-IgM (5 mg/ml) treatment and assessed by propidium iodide2 cells. Bars represent means 6 SEM of two independent experiments. (G) Intracellular Bim protein levels in mature follicular B cells assessed by FACS staining after culture under anti-IgM treatment in the absence (open solid histograms) or presence of BAFF (dotted histograms). Control cultures are represented by filled histograms. One experiment representative of at least two is shown.

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Moreover, it remains unclear whether IL-4 could have other physiological effects in developing B cells and how it could interfere in the activity of BAFF, the key maturation and survival factor for B cells, on different developmental stages. The results we obtained in the present study showed that the combination of IL-4 with BAFF led to enhanced generation of transitional CD23+ B cells, and, more importantly, rendered this process refractory to anti-IgM inhibition. To our knowledge, this is the first description of this activity impacting on maturing B cells. As discussed bellow, these results may have important consequences for our understanding of B cell–mediated autoimmunity. The intracellular mechanisms involved in IL-4–mediated protection of BCR-induced cell death are not well understood. It was reported that Stat6-deficient B cells undergo apoptosis in the presence of IL-4, which has been linked to impaired Bcl_xL induction (46). In our short-term culture model, we could not observe any important change induced by IL-4 in antiapoptotic Bcl2, Bcl_xL, and Mcl1 expression levels either in BM transitional or splenic follicular B cell populations (Figs. 6, 7). However, IL-4 was able to prevent the increase of Bim intracellular levels induced by BCR crosslinking, despite bim mRNA upregulation (Figs. 6, 7). Moreover, downmodulation of Bim protein expression by IL-4 was observed not only in BM transitional B cells, but also in follicular mature B cells as well. Interestingly, in mature follicular B cell cultures, BAFF provided significant protection against BCR-induced cell death and, accordingly, Bim protein expression was partially downmodulated (Fig. 7G). These results strongly argue for a role of Bim modulation in the protection of BCR-mediated cell death by IL-4. We also obtained evidence in vivo, using anti-IgD–treated mice, for the role of IL-4 on the inhibition of the expression of Bim and B cell death (Fig. 8). These in vivo findings are consistent with our results obtained in vitro. In agreement with our findings, BCR-induced apoptosis is strongly reduced in immature and mature B cells from Bim knockout mice, and the deletion of autoreactive B cells is also inhibited (42). Furthermore, autoantigen-stimulated B cells from Ig/hen egg lysozyme double transgenic mice express elevated levels of bim mRNA and protein (57). Intriguingly, in T cells, IL-4 provides survival signals but simultaneously promotes the augmented expression of Bim protein, indicating a different mode of action on B cells (58). Bim expression can be regulated at both transcriptional and posttranscriptional levels. In response to growth factor withdrawal and concomitant blockade of the PI3K/Akt pathway, bim mRNA levels are upregulated by activation of the forkhead-like transcription factor Foxo3A/FKHRL-1 (59). Alternatively, the molecular mechanisms linking BCR signaling to the upregulation of bim mRNA have not been elucidated (42). In the present study, we obtained clear evidence for a posttranscriptional control of Bim protein expression by IL-4; however, the mechanisms have not been investigated. The posttranscriptional control of Bim has been previously reported and three different mechanisms were described: microRNA-mediated RNA interference, phosphorylation,

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FIGURE 8. In vivo modulation of Bim expression after anti-IgD treatment in the presence of IL-4. BALB/c mice were injected i.v. into the tail vein with PBS buffer containing 10% BALB/c serum (vehicle) or 40 mg azide-free anti-mouse IgDa (10.4.22, eBioscience) in the presence or absence of the IL-4C complex. Mice were sacrificed 1 or 2 d after anti-mouse IgD treatment and spleen cells from individual mice were recovered. (A) Percentage of splenic CD19+ B cell after 48 h of anti-IgD treatment. (B and C) Intracellular Bim expression and (D) surface IgD expression on T1, T2, follicular (FO), and marginal zone (MZ) B cell populations after 24 h of anti-IgD treatment assessed by FACS staining. (B) Intracellular Bim protein levels on indicated B cell population after anti-IgD treatment in the absence (open solid histograms) or presence of IL-4C (dotted histograms). Control mice injected with vehicle are represented by gray-filled histograms. One representative mouse of at least three is shown. (C and D) Bars represent mean fluorescence intensity 6 SEM of at least three individual mice/group. One representative experiment of two is shown. *p , 0.001.

this process extremely resistant to BCR-mediated apoptosis. Given that not only the specificity but also the frequency of positively selected autoreactive clones in the emerging repertoire are

probably relevant to determine susceptibility to autoimmunity, the synergy of BAFF and IL-4 enhancing the rate of B cells resistant to BCR-mediated negative selection could be an important

5774 tolerance-breaking mechanism working at the transitional B cell stage both in the BM and in the spleen.

Acknowledgments We thank Dr. John Cambier for providing FITC anti-B220 and Alexa Fluor 647 anti-mouse IgM Abs and Dr. Antonius Rolink for providing anti-CD93– producing 493 hybridoma.

Disclosures The authors have no financial conflicts of interest.

References

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