BAFF and APRIL Expression in B Cells in Vitro and in Vivo

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In Vitro and In Vivo Activation Induces BAFF and APRIL Expression in B Cells Van Trung Chu, Philipp Enghard, Gabriela Riemekasten and Claudia Berek This information is current as of September 27, 2021. J Immunol 2007; 179:5947-5957; ; doi: 10.4049/jimmunol.179.9.5947 http://www.jimmunol.org/content/179/9/5947 Downloaded from

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

In Vitro and In Vivo Activation Induces BAFF and APRIL Expression in B Cells1

Van Trung Chu,* Philipp Enghard,† Gabriela Riemekasten,† and Claudia Berek2*

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88- dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up- regulated membrane expression of BAFF. The ﬁnding that in vitro activation of B cells is inhibited by the antagonist transmem-

brane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture Downloaded from supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo ؋ activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB NZW)F1 mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of ؋ BAFF. In (NZB NZW)F1 mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease. The Journal of Immunology, 2007, 179: 5947–5957. http://www.jimmunol.org/ wo closely related cytokines of the TNF superfamily, B BAFF is produced by a number of different cell types. Expres- cell-activating factor (BAFF,3 also termed BLyS, sion of BAFF by follicular dendritic cells (FDC) may be essential T TALL-1, zTNF-4, THANK, and TNF13B) and a prolif- for B cell homeostasis (12–14). However, the main sources of eration-inducing ligand (APRIL), are central players in B cell de- BAFF are monocytes, macrophages, dendritic cells, and neutro- velopment and homeostasis. BAFF-deficient mice have an almost phils, although subpopulations of B and T cells have also been complete loss of follicular (FO) and MZ B cells, although there is shown to express it (15–21). BAFF is found to be membrane as- normal development of early B cells in the bone marrow (1–4). sociated and its expression is enhanced by cytokines, such as Normal numbers of newly formed immature B cells leave the bone IFN-␣, IFN-␥, and IL-10 or growth factors (17, 19, 20, 22). marrow and develop into the transitional T1 stage. In the absence Much less is known about APRIL which has no essential func- by guest on September 27, 2021 of BAFF. B cells do not progress past the transitional T2 stage, tion in normal B cell development and plays only a minor role in resulting in impaired humoral immune responses (5). However, B cell homeostasis (23). In immune responses APRIL acts as a when BAFF is overexpressed, for example in BAFF- transgenic costimulator for B and T cell proliferation and supports class mice, self-reactive B cells may be rescued from peripheral deletion switch (2, 4, 22). (6, 7). As a consequence, BAFF-transgenic mice develop a lupus- BAFF binds to three separate receptors, the BAFF receptor like autoimmune disease (8–11). From these data, it is apparent (BAFF-R, BR3), the transmembrane activator and calcium mod- that the level of BAFF has to be tightly regulated to ensure B cell ulator ligand interactor (TACI), and the B cell maturation Ag (24– survival on the one hand and to prevent autoimmunity on the other. 27). APRIL binds only to TACI and B cell maturation Ag. All three receptors are expressed on B cells, although their expression level changes with B cell maturation. The analysis of human B cell tumor lines, such as B cell chronic *Deutsches Rheuma ForschungsZentrum; and †Department of Rheumatology and Clinical Immunology, Charite University Hospital, Berlin, Germany lymphatic leukemia (B-CLL), multiple myeloma, and Hodgkin’s lymphoma cells, suggested that BAFF and APRIL support tumor Received for publication April 6, 2007. Accepted for publication August 21, 2007. survival by an autocrine pathway (21, 28–30). There is also evi- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance dence that normal nonmalignant human B cells up-regulate BAFF with 18 U.S.C. Section 1734 solely to indicate this fact. and APRIL upon activation (21, 28). However, it is thought that 1 This work was supported by a grant from the Government of Vietnam and the murine B cells do not express BAFF or APRIL (3), although an DAAD (to V.T.C.), the Bundesministerium fu¨r Bildung und Forschung Grant NGFN2 analysis of early murine B cell development suggested that B cell and the SFB 650. The DRFZ is supported by the Berlin Senate of Research and Education. survival in the bone marrow may be supported by an autocrine 2 Address correspondence and reprint requests to Dr. Claudia Berek, Deutsches pathway (31). Rheuma ForschungsZentrum, Chariteplatz 1, Berlin, Germany. E-mail address: In this study, we show a detailed analysis of BAFF and APRIL [email protected] expression in different B cell subsets isolated from the bone mar- 3 Abbreviations used in this paper: BAFF, B cell-activating factor; APRIL, a prolif- row, spleen, and peritoneal cavity. In vitro cultures demonstrate eration-inducing ligand; MZ, marginal zone; FO, follicular; GC, germinal center; FDC, follicular dendritic cells; CMK, chloromethylketone; phOx, 2-phenyl-ox- that upon stimulation with LPS or CpG-oligodeoxynucleotides azolone; PNA, peanut agglutinin; MFI, mean fluorescence intensity; TACI, trans- (CpG-ODN), splenic B2 cells as well as peritoneal B1 cells up- membrane activator and calcium modulator ligand interactor; ODN, oligodeoxynucle- regulate BAFF and APRIL expression. MyD88-deficient mice otide; BAFF-R, BAFF receptor. demonstrated that BAFF expression in B cells is regulated by the Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 TLR signaling pathway. Immunization with the T-dependent Ag www.jimmunol.org 5948 ACTIVATED B CELLS EXPRESS BAFF AND APRIL

FIGURE 1. Expression of BAFF and APRIL in B cells. A, To determine the level of BAFF and APRIL expression, B cell subsets were sorted by FACS. Gating for the isolation of pro-, pre-, immature (Imm) bone marrow; T1, T2, MZ, and FO (spleen); B1 and B2 (peritoneal cavity) B cell subsets is indicated. Representative dot blots show the purity of pro-, pre-, and immature B cells (Post-sort). B, B cell subsets isolated from the bone marrow and the peritoneal cavity (PC) express APRIL cDNA. Amplification was done for 40 cycles. C, The relative level of BAFF mRNA was determined by real- time PCR. Three mice per group were analyzed. Results of three independent experiments are shown as mean values plus SD. D, Expression of BAFF, APRIL, and CD11c mRNA in defined numbers of MZϪ,FOϪ, ϩ peritoneal B1 B, and CD11c cells. Number Downloaded from of sorted cells is indicated. For each cell population, one-fifth of the cDNA was used for PCR amplification of BAFF, APRIL, CD11c, and ␤-actin. E, Splenocytes and peritoneal cells from BALB/c mice were triple stained for CD5, B220, and BAFF

(Buffy-2). Dot blots show the gating for http://www.jimmunol.org/ splenic newly formed B220low (NF-B), mature (M-B), and B1 B cells and for peritoneal (PC) B1a, B1b and B2 subsets. Histograms in the upper row show staining of splenic B cells with anti-BAFF-R (left panel) and with BAFF-speciﬁc Abs (middle and right panels). Surface expression of BAFF before (normal line) and after incubation of B cells with soluble BAFF (heavy line) is shown. Isotype by guest on September 27, 2021 control (shaded area) is included. The different B cell subsets (spleen and peritoneal cavity) were stained with the mAb Buffy-2 only. Data are representative of three (spleen) to ﬁve (peritoneum) experiments.

2-phenyl-oxazolone (phOx) induced BAFF expression in germinal with polyclonal rabbit anti-BAFF (Sigma-Aldrich) or anti-APRIL (Stress- ϫ gen) Ab, respectively. As secondary Ab Alexa Fluor 546-conjugated goat center (GC) B cells. In (NZB NZW)F1 mice, expression of BAFF and APRIL mRNA in MZ and B1 cells was strongly up- anti-rabbit IgG (Molecular Probes) was used. To control for the specificity of the BAFF-specific mAb Buffy-2, purified B cells were incubated for 2 h regulated with increasing age and onset of disease. In addition, with 200 ng of soluble BAFF and then stained with the mAb Buffy-2 or strong expression of BAFF and APRIL protein was found in with polyclonal rabbit anti-BAFF Abs. The expression of the BAFF-R was plasma cells. Thus, expression of these cytokines in activated and controlled by staining with the biotinylated rat mAb 204406 (R&D differentiated B cells may support the development of autoimmune Systems). The antagonist TACI Ig was prepared by fusing the extracellular domain disease. of human TACI (aa 1–154) to the Fc region of the human IgG1 H chain (32). The protein was expressed in the HEK 293T cell line and isolated Materials and Methods from supernatants by protein G chromatography. Mice FITC-, PE-, Cy5-, PE-Cy7-, or biotin-conjugated anti-B220 (RA3-6B2), biotinylated anti-CD11b (MI/70.15.11), anti-CD11c (N418) and anti-CD90 Experiments were performed with BALB/c, C57BL/6, MyD88Ϫ/Ϫ (T24), FITC and Cy5 anti-CD19 (ID3) and anti-CD21 (7G6), Cy5 anti-IgM ϫ ␬ (C57BL/6 background), and (NZB NZW)F1 mice. One to 2-mo-old (M14), and biotinylated anti- L chain (187.1) Abs were provided by the BALB/c mice were immunized i.p. with a single injection of 100 ␮gof Deutsches Rheuma ForschungsZentrum (DRFZ). FITC or biotinylated anti- alum-precipitated phOx coupled to the carrier protein chicken serum albu- CD43 (1B11) was obtained from Biolegend, PE or biotinylated anti-CD5 ϫ min. (NZB NZW)F1 mice were purchased from The Jackson Laboratory from eBioscience, FITC or PE anti-CD23 (B3B4) and PE anti-CD138 and bred in the Bundesinstitut fu¨r gesundheitlichen Verbraucherschutz und (281–2) from BD, Pharmingen, and biotinylated and FITC-peanut agglu- Veterina¨rmedizin, Berlin-Marienfelde. Animal experiments were approved tinin (PNA) from Vector laboratories. To visualize biotinylated Ab Alexa by the institutional animal care and use committee. Fluor 488-, PE-, or allophycocyanin-conjugated streptavidin was used (Molecular Probes and BD Biosciences). Abs and reagents Isolation of lymphocytes Surface expression of BAFF on B cells was determined using a FITC- labeled anti-BAFF mAb (Buffy-2; Alexis). For isotype control, FITC-la- Suspensions of bone marrow cells were flushed from tibias and femurs of beled rat IgM (Southern Biotechnology Associates) was used. To detect 6–10-wk-old BALB/c mice and stained with B220-PE-, CD43-FITC-, and BAFF and APRIL expression, cytospins and frozen sections were stained IgM-Cy5-specific Abs. To isolate pro-B cells, lymphocytes were gated on The Journal of Immunology 5949

IgMϪ cells and B220ϩCD43ϩ cells were sorted as shown in Fig. 1A.To isolate pre- and immature B cells, lymphocytes were gated on B220ϩ CD43Ϫ and the IgM-negative (pre-B) and -positive (immature B cell) frac- tions sorted. After cell sorting, cells were controlled for purity (Fig. 1A). Spleen cells were stained with biotinylated Ab specific for CD43, CD11b, CD11c, and CD90 to remove myeloid, stromal, and T cells. After incubation with antibiotin microbeads (Miltenyi Biotec), B cells were enriched by MACS (Miltenyi Biotec) to Ͼ96% purity. To isolate total splenic B cells for setting up in vitro tissue cultures, the enriched fraction was stained with anti-B220-Cy5 and B cells sorted to Ͼ99% purity (Fig. 2A). For the isolation of T1/T2, FO, and MZ B cells, the MACS-enriched B cell fraction was stained with FITC-conjugated anti-B220, Cy5, anti- CD21/35, and PE anti-CD23 (Fig. 1A). The different B cell subsets, MZ (CD21highCD23Ϫ), FO (CD21intCD23ϩ), transitional T1 (CD21ϪCD23Ϫ), and T2 (CD21highCD23ϩ) B cells were sorted by FACS (BD Biosciences). B1 cells were isolated from the peritoneal cavity and sorted as CD5ϩB220lowIgMϩ cells (Fig. 1A). Purity of sorted cells was in the range of 98–99%. Cell culture and stimulation Spleen cells of three animals were pooled and duplicate cultures (106 sorted splenic B cells/ml) set up in RPMI 1640 supplemented with 10% Downloaded from FCS, 50 ␮M 2-ME, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. B cells were stimulated with 25 ␮g/ml LPS (Sigma-Aldrich) or 10 ␮g/ml CpG-ODN 1826 (InvivoGen) for 2–4 days. Expression of mRNA for FIGURE 2. In vitro- activated splenic B cells up-regulate BAFF and BAFF and APRIL was measured by semiquantitative RT-PCR or real-time APRIL. A, In vitro-activated B cells were analyzed by FACS. Represen- PCR. Surface expression of BAFF on B cells was determined by FACS tative stainings are shown. B, The presence of BAFF, APRIL, and CD11c using the monoclonal anti-BAFF (Buffy-2) Ab. mRNA was determined by RT-PCR. cDNA was amplified for 40, 45, or 35 To test whether B cells express biologically active BAFF and/or APRIL, cycles, respectively. As positive control, splenocytes (Spl) were used, as sorted B cells were activated for 3 days with different concentrations of negative control (control) amplification without DNA. C, B cells from http://www.jimmunol.org/ ␮ LPS (1 and 5 g/ml). Cultures were set up in the presence or in the absence BALB/c (n ϭ 4), C57BL/6 (n ϭ 3), and MyD88 (n ϭ 4)-deficient mice of the TACI Ig (20 ␮g/culture) fusion protein. As a control protein, human were stimulated with LPS or CPG-ODN for 2 days and BAFF surface IgG was added. To monitor proliferation, cells were labeled with the CSFE expression was determined. Histograms show overlays of BAFF expres- using a standard protocol. The percentage of cells in S-G2-M phase was determined by FACS analysis. Briefly, after 3 days of activation, B cells sion in unstimulated (dotted line), stimulated B cells (solid line), and iso- were harvested, washed with PBS, and fixed overnight in 70% ethanol at type control (shaded histogram). 4°C. Fixed cells were incubated for 30 min in 50 ␮g/ml propidium iodide at 37°C before analysis. Furthermore, cells were cultured for 3 days with LPS or CpG-ODN in the absence or presence of 50 ␮M furin-like convertase inhibitor decanoyl- in triplicate. The unit number showing relative mRNA level in each sample Arg-Val-Lys-Arg-chloromethylketone (CMK; Alexis). For these experi- was determined as a value of mRNA normalized against ␤-actin. by guest on September 27, 2021 ments, cell cultures were set up using sorted splenic B2 or peritoneal B1 cells isolated from individual BALB/c mice. FACS analysis Detection of BAFF and APRIL mRNA Cells were stained in FACS buffer with directly labeled or biotinylated Ab. Unspecific staining was inhibited by blocking for 10 min at 4°C with rat Expression of mRNA for BAFF and APRIL was measured by semiquan- IgG (Sigma-Aldrich) and the mAb 2.4G2/75 specific for the Fc␥R (CD16/ titative RT-PCR or real-time PCR. Total RNA was prepared from 2 ϫ 106 32). For intracellular staining, surface-labeled cells were washed twice and sorted cells using a RNeasy Mini Kit (Qiagen). RNA was treated with fixed in 2% (w/v) paraformaldehyde (Merck) for 20 min at room temper- RNase-free DNase I (Qiagen) according to the manufacturer’s instruction. ature. After washing, cells were permeabilized in PBS supplemented with Concentration of total RNA was measured by NaNoDrop (Biotech Inter- 0.5% saponin (Sigma-Aldrich) and 0.5% BSA for 10 min at room temper- national) and reverse transcribed into cDNA using an Ominiscript RT kit ature and stained with BAFF or ␬-specific Abs for 30 min in the dark. (Qiagen). cDNA was amplified with BAFF-, APRIL-, or CD11c-specific Before analyses, cells were washed with 0.5% saponin buffer. Stained cells primers using 1.25 U/reaction AmpliTaq Gold polymerase (Applied Bio- were analyzed using FACSCalibur or LSRII (BD Biosciences) and the systems). The number of cycles is indicated for the different experiments. CellQuest (BD Biosciences) or FlowJo software (Tree Star). For the ␤-actin control, cDNA was amplified for 30 cycles. Each cycle consisted of 1 min at 94°C, 45 s at 63°C, and 20 s at 72°C. Amplified Cytospin, fluorescence staining, and confocal microscopy cDNA was visualized on agarose gels and specificity was checked by B cells activated in vitro for 3 days with different stimuli were harvested. sequencing. To remove receptor-bound BAFF and APRIL, cells were incubated with To control the purity of sorted cells, defined numbers of MZ, FO, and 0.1% sodium citrate/0.1% Triton X-100 (pH 5.2) overnight at 4°C. After peritoneal B1 cells were prepared as described and sorted into PCR tubes ␮ three washes with PBS/0.5% BSA, cells were centrifuged onto glass slides containing 20 l of reverse transcriptase buffer (Qiagen). In addition, a (Menzel). Spleens were frozen in Tissue-Tec OCT compound and stored at second spleen was gently homogenized and the suspension was digested in Ϫ ␮ ␮ 70°C. Frozen tissue sections of 7 m were prepared, fixed in cold ace- RPMI 1640/10% FCS containing 1 g/ml collagenase for 45 min at 37°C. tone for 10 min, and air dried. After three washes, cells were incubated with Cy5-conjugated anti-CD11c ϩ To detect BAFF and APRIL expression, cytospins and sections were mAb (N418) and defined numbers of CD11c cells were sorted again into double stained with polyclonal rabbit anti-BAFF or anti-APRIL Ab and PCR tubes. For each of the cell populations, cDNA was directly transcribed anti-␬ Ab. Nuclei were counterstained with 4Ј,6-diamidino-2-phenylin- using 10 ␮M oligo(dT) as primer and one-fifth of the reaction mixture was ␤ dole. To analyze BAFF expression in GC, tissue sections were double used to amplify BAFF, APRIL, CD11c, or -actin cDNA in a seminested stained with polyclonal rabbit anti-BAFF and biotinylated FDC-specific PCR (Qiagen). The first amplification consisted of 25 cycles of 1 min at mAb M2 (Serotec) or biotinylated PNA (Vector Laboratories). Fluorescent 94°C, 1 min at 63°C, and 30 s at 72°C and the second round of 40 cycles microscopy images were captured with a SPOT RT camera (Diagnostic for APRIL, 35 cycles for BAFF, 30 cycles for CD11c, and 25 cycles for Instruments) or by confocal microscopy using Leica software. ␤-actin. Quantitative PCR to determine the relative amount of BAFF mRNA was ELISA for soluble BAFF performed with a LightCycler System (Roche Diagnostics) using the Ligh- Cycler FastStart DNA Master SYBR Green I (Roche Diagnostics). The Soluble BAFF in supernatants was detected using a mouse anti-BAFF real-time PCR products were analyzed on 4% agarose gels to check purify ELISA kit (APO-54N-019-KI01; Apotech) according to the manufactur- and specificity. Each sample from three independent experiments was run er’s instruction The sensitivity of the kit is 0.2 ng/ml. ODs at 450 nm were 5950 ACTIVATED B CELLS EXPRESS BAFF AND APRIL

FIGURE 3. In vitro and in vivo activation induces BAFF and APRIL expression. Sorted splenic B cells were in vitro activated with medium alone, LPS, or CpG-ODN for 3 days. Cytospins were prepared and costained with anti-␬ (green) and polyclonal rabbit anti-BAFF (red; A) or anti-APRIL Ab (red; B). Nuclei were counterstained with 4Ј,6-dia-

midino-2-phenylindole (DAPI; blue). Downloaded from Single staining and overlays are shown. C, Splenic tissue sections from immunized (C1–3), naive BALB/c (C4), and 3- to 4-mo-old ϫ (NZB NZW)F1 mice (D) are shown. Consecutive sections (C1,2) were double stained with anti-BAFF http://www.jimmunol.org/ and either anti-FDC (M2) or PNA. Light zone (LZ) and dark zone (DZ) are indicated. E, Splenic tissue sections from 6- to 7-mo-old (NZB ϫ ␬ NZW)F1 mice were stained for , BAFF, and APRIL. Single staining and overlays of plasma cells are shown. Original magniﬁcation, ϫ40.

Representative results from three in- by guest on September 27, 2021 dependent experiments are shown.

measured with a microplate spectrophotometer (Spectrarax 190). The mu- leave the bone marrow and differentiate into transitional T1/T2 rine A20 cell line was used as a positive control for BAFF release. After B cells, APRIL mRNA is down-regulated and is no longer de- Ϯ 3 days in culture, without activation, a concentration of 1.6 0.02 ng/ml tectable in FO (CD21intCD23ϩ) and MZ (CD21highCD23Ϫ)B soluble BAFF was measured (see Fig. 5). cells. Although no APRIL mRNA was detected in mature B2 Statistical analysis cells, B1 cells isolated from the peritoneal cavity expressed it at All statistical analyses were performed using the Student t test; statis- high levels (Fig. 1B). tical significance of difference was determined as p Ͻ 0.05 or p Ͻ 0.01 Surprisingly, all murine B cell subsets analyzed expressed or p Ͻ 0.001. BAFF mRNA (Fig. 1C). A comparative analysis showed that pro-, pre-, and immature B cells isolated from the bone marrow express Results 4- to10-fold higher levels of BAFF mRNA than T1/T2, mature FO B cells express APRIL and BAFF mRNA and MZ B cells (Fig. 1C). With the differentiation of the immature Both B cell lymphoma lines and normal human B cells have been B cell into the transitional T1/T2 stage, BAFF mRNA is down- reported to express BAFF and APRIL (8, 21, 29–31). This regulated. However, this is different for peritoneal B1 cells that prompted us to analyze whether normal murine B cells also ex- expressed high levels of BAFF mRNA. The level was comparable press these cytokines. We found that during early B cell develop- to that seen in the immature B cells isolated from the bone marrow ment in the bone marrow, pro-B cells (B220ϩCD43ϩIgMϪ), pre-B (Fig. 1C). cells (B220ϩCD43ϪIgMϪ), and immature B cells (B220ϩCD43Ϫ It is unlikely that BAFF and APRIL mRNA are derived from IgMϩ) express APRIL mRNA (Fig. 1B). When immature B cells a contamination by monocytes/macrophages or dendritic cells The Journal of Immunology 5951 since the purity of the sorted B cells was between 98 and 99%. To exclude the possibility of a few contaminating myeloid cells, defined numbers of splenic FO, MZ B cells, peritoneal B1 cells, and CD11cϩ cells were sorted by FACS and tested for BAFF, APRIL and CD11c mRNA expression using seminested PCR. Fig. 1D shows that as few as 10 MZ B cells expressed sufficient BAFF mRNA to give a positive signal. The intensity of the band was further increased when 100 MZ B cells were sorted. Ten FO B cells showed only a very weak band. However, the intensity increased when 100 FO B cells were sorted. As described, FO and MZ B cells were negative for APRIL mRNA expression. In contrast, high levels of BAFF and APRIL mRNA were seen in peritoneal B1 cells. Independent of whether 10 or 100 FO, MZ, or peritoneal B1 B cells were sorted, no PCR signal for CD11c was detectable (Fig. 1D). Since already 10 CD11ϩ cells express high levels of CD11c (Fig. 1D), it is unlikely that BAFF mRNA expression in FO, MZ, and peritoneal B1 B cells is due to a contamination with CD11c myeloid cells. Downloaded from High levels of BAFF mRNA expression is associated with FIGURE 4. CMK enhances membrane-bound BAFF expression in ac- protein expression on the surface of B cells tivated B cells. Splenic B (A) or peritoneal B1 (B) cells from BALB/c mice ϭ ϭ To determine whether murine B cells express BAFF, bone marrow (n 4 and n 6, respectively) were sorted and activated with medium Ϫ cells were triple stained with B220, anti-IgM, and the BAFF-spe- alone, LPS, or CpG-ODN either in the absence ( CMK) or in the presence (ϩCMK) of inhibitor. After 3 days in culture, cells were harvested and cific mAb Buffy-2. The subset of IgMϪ B220low pre- and pro-B stained for CD19 and BAFF (Buffy-2). Expression levels of membrane- cells showed little BAFF expression. The signal was only slightly bound BAFF were measured by FACS. Representative dot plots are shown; http://www.jimmunol.org/ higher than in the isotype control. Comparably weak expression numbers on each panel indicate the percentage of BAFFhigh B cells. levels were seen for the subset of IgMϩB220low immature B cells (data not shown). To control for the specificity of the mAb Buffy-2, sorted splenic in vitro with different stimuli. RNA was isolated and cytokine B cells were incubated for 2 h with soluble BAFF (Fig. 1E, upper expression was analyzed. After 3 days in culture, the purity of B row of histograms). Before and after incubation, B cells were cells was rechecked by staining with CD19, CD3, CD11c, and stained with BAFF-R (Fig. 1E, left panel) or BAFF-specific Abs CD11b. Because FACS analysis showed a purity of Ͼ99% B cells (Fig. 1E, middle and right panels). Receptor bound BAFF was (Fig. 2A) and because RT-PCR gave no signal for CD11c (Fig. only detectable with BAFF specific polyclonal rabbit Abs (Fig. 1E, 2B), it is unlikely that cultured B cells are contaminated by my- by guest on September 27, 2021 right panel). In addition, pre-incubation with BAFF slightly re- eloid cells. duced the signal obtained with anti-BAFF-R Abs (Fig. 1E, left RT-PCR showed an up-regulation of BAFF and APRIL mRNA panel). These data show that the mAb Buffy-2 does not recognize when B cells were activated with CpG-ODN or with LPS (Fig. BAFF when it is bound by its receptors. In contrast to the poly- 2B). The elevated levels of BAFF mRNA expression in LPS- or clonal rabbit anti-BAFF Abs, the mAb Buffy-2 recognizes only CpG-ODN-activated B cells correlated with the expression of membrane-expressed BAFF. The B cell lymphoma lines WEHI- BAFF on the surface of the cells. Splenic B cells expressed mem- 231 and A20 were used as positive controls and clearly showed brane-bound BAFF already 2 days after in vitro activation (Fig. membrane-associated BAFF expression (Fig. 1E, second row of 2C). Surface expression of BAFF was confirmed by using biotin- histograms). ylated TACI Ig (data not shown). Splenic B cells were enriched by MACS to a purity Ͼ96% and A control experiment using MyD88-deficient mice demon- stained with the BAFF-specific mAb Buffy-2. FACS analysis strated that the enhancement of BAFF expression is dependent on showed that newly formed (B220low) and the majority of mature the TLR signaling pathway (Fig. 2C). The negative result seen in splenic B cells were negative for BAFF surface expression (Fig. the MyD88-deficient mice is not due to the C57BL/6 genetic back- 1E, third row of histograms). Only a small fraction (1.38 Ϯ 0.3%) ground, since splenic B cells isolated from both BALB/c and of splenic B cells expressed membrane-bound BAFF (data not C57BL/6 mice up-regulated BAFF expression when activated with shown). The low frequency suggested that BAFF-positive cells may CpG-ODN or LPS. be splenic B1 cells. To further analyze whether splenic B1 cells ex- Activation of splenic B cells with LPS or CpG-ODN induces press membrane-bound BAFF, a triple staining with Ab specific for differentiation into plasma cells and expression of intracellular BAFF, CD5, and B220 was performed (Fig. 1E). Gating on BAFF and APRIL. After 3 days in culture, B cells were harvested CD5ϩB220low cells confirmed that B1 cells express low levels of by cytospin and stained with BAFF- or APRIL-specific Ab. BAFF in their membrane, whereas no expression was seen on splenic Costaining with ␬-specific Ab demonstrated high levels of APRIL mature and newly formed B2 cells (Fig. 1E, third row). When peri- and BAFF expression in the cytoplasm of the newly generated toneal B1 cells were analyzed for BAFF surface expression, the signal plasma cells (Fig. 3, A and B). Comparable results were found was again marginally higher than in the isotype control (Fig. 1E, last when peritoneal B1 cells were activated with LPS or with CpG- row), supporting that B1 cells express membrane-bound BAFF. ODN. B1 cells expressed low levels of BAFF and APRIL already before in vitro activation and after 3 days in culture with LPS or In vitro activation of B cells induces APRIL and BAFF CpG-ODN a strong up-regulation of both cytokines was found. expression Again, double staining with ␬-specific Ab showed BAFF and To analyze whether activation of B cells induces APRIL and APRIL expression in the cytoplasm of plasma cells (data not BAFF protein expression, splenic B cells were sorted and cultured shown). 5952 ACTIVATED B CELLS EXPRESS BAFF AND APRIL

FIGURE 5. In vitro-activated B cells secrete biologically active BAFF and/or APRIL. A, The presence of soluble BAFF in supernatants collected from 3-day tissue cultures using a BAFF-speciﬁc ELISA kit. B, Three days after LPS activation in the Downloaded from presence or absence of TACI Ig, B cells were harvested and the number of living cells was determined. Dotted line shows cell numbers at the start of culture. Inhibition of B cell activation was controlled by adding human IgG http://www.jimmunol.org/ into the cultures. Mean values and SDs of four experiments are shown. C, The percentage of proliferating B cells (upper graph), B cells in

S-G2-M phase (middle graph), and CD138high plasma cells was determined. To analyze cell proliferation, sorted B cells were labeled with CFSE. To determine the frequency of cycling cells, harvested B cells were by guest on September 27, 2021 stained with propidium iodide. Mean values of three independent experiments are shown. Values of p are indicated.

CMK enhances the expression of membrane-bound BAFF on the absence of CMK increased the MFI from 4.25 Ϯ 0.24 (medium activated B cells alone) to 12.86 Ϯ 0.39 or 10.39 Ϯ 1.36. In the presence of CMK, Ϯ Ϯ Sorted splenic B cells and also peritoneal B1 cells were activated the MFI increased to 20.29 1.92 or 17.8 1.08, respectively. A with LPS or with CpG-ODN in the presence or absence of CMK, similar up-regulation of BAFF expression was observed when an inhibitor of furin-like ecto-peptidases which inhibits cleavage peritoneal B1 cells were activated. When incubated with LPS, the of BAFF from the cell surface and thus release of soluble BAFF MFI was 19.23 Ϯ 1.48 in the absence of CMK and 26.64 Ϯ 2.61 (20). After 3 days in culture, B cells were harvested and stained in its presence. Activation of B1 cells with CpG-ODN increased with the BAFF-specific mAb Buffy-2. FACS analysis showed that the MFI of BAFF expression to 24.94 Ϯ 3.29 in the absence and activated B cells up-regulate membrane-bound BAFF expression 38.54 Ϯ 3.59 in the presence of CMK. (Fig. 4). In cultures with inhibitor, the mean fluorescence intensity Furthermore, when B2 cells were activated in the presence of (MFI) for BAFF expression was higher than in cultures without CMK, a significant increase in the fraction of BAFFhigh B cells (Fig. 4). Activation of splenic B cells with LPS or CpG-ODN in was seen (ϳ6% of total B cells; Fig. 4A). A similar result was The Journal of Immunology 5953

FIGURE 6. GC B cells up-regulate BAFF expression. Splenocytes from immunized BALB/c and ϫ (NZB NZW)F1 mice of different ages were stained with PNA-, BAFF (Buffy-2)-, and B220- speciﬁc Abs. The MFI of BAFF expression on naive (B220ϩPNAlow) and GC B cells (B220ϩPNAhigh) was compared (left panel). Bar graphs show the increase in the MFI (fold change) in compari- Downloaded from son to the isotype control. Intracellu- lar staining for BAFF is only shown for the group of 6- to 7- mo-old ϫ (NZB NZW)F1 mice. For each group, ﬁve animals were analyzed. Values of p are indicated. Represen- tative histograms showing BAFF http://www.jimmunol.org/ staining of naive and GC B cells are included (right panel). by guest on September 27, 2021

found for B1 cells, in particular when they were activated with cells were counted as at the start of cultures (Fig. 5B). In the CpG-ODN (Fig. 4B). The finding of elevated levels of membrane- presence of TACI Ig, the frequency of cycling B cells was signif- bound BAFF expression in the presence of CMK gives further icantly reduced (Fig. 5C, middle graph). Furthermore, the differ- evidence that activated B cells express BAFF. These findings in- entiation of activated B cells into CD138high plasma cells was dicate that upon activation B cells release soluble BAFF into the inhibited (Fig. 5C, lower graph). The finding that in the presence culture supernatant. of the antagonist TACI Ig, B cell survival, proliferation, and differentiation are all inhibited indicates that in vitro activation of B In vitro activation of B cells induces secretion of BAFF and/or cells induces secretion of biologically active BAFF and/or APRIL. APRIL To look for the release of soluble BAFF, tissue culture superna- In vivo activation of B cells and up-regulation of BAFF tants of splenic B cells activated for 3 days with LPS or CpG-ODN expression were tested for the presence of soluble BAFF using an ELISA kit. In primary follicles of BALB/c mice, FDC do not express BAFF The increase in the level of soluble BAFF was low, but detectable (Fig. 3C, C3). Staining with APRIL-specific Ab was also negative ( p Ͻ 0.01). Activation with LPS or CpG-ODN yielded a concen- (data not shown). To induce a T-dependent immune response, tration of 1.2 Ϯ 0.1 or 0.9 Ϯ 0.1 ng/ml soluble BAFF, respectively BALB/c mice were immunized with phOx and 10 days after in- (Fig. 5A). jection of Ag, GC formation was observed (Fig. 3C). The question arises whether the BAFF and APRIL expressed by Double staining with the FDC-specific M2 Ab and anti-BAFF B cells is of biological significance. To address this question, in- showed that BAFF expression is up-regulated in FDC of the GC hibition assays using the fusion protein TACI Ig were performed. light zone (Fig. 3C, C2). An analysis of BAFF expression at high Sorted B cells were incubated with different amounts of LPS in the magnification suggests that BAFF is also expressed in GC B cells, presence or absence of TACI Ig. Fig. 5, B and C, shows that in the as B cells in the dark zone of the GC costained for PNA and BAFF presence of TACI Ig there is a significant reduction in B cell ac- (Fig. 3C, C3). From staining of tissue sections, it is difficult to say tivation. Although a low dose of 1 ␮g/ml LPS is sufficient to in- whether BAFF is bound by its receptors or indeed expressed on the duce B cell proliferation (Fig. 5C, upper graph), the number of B surface of B cells. However, FACS analysis of GC B cells showed cells does not increase in the presence of TACI Ig. After 3 days of up-regulation of membrane-bound BAFF and intracellular staining activation with 1 ␮g/ml LPS, approximately the same number of B for BAFF confirmed endogenous production of BAFF by GC B 5954 ACTIVATED B CELLS EXPRESS BAFF AND APRIL Downloaded from http://www.jimmunol.org/

ϩ high

ϫ by guest on September 27, 2021 FIGURE 7. Chronic activation of B cells in (NZB NZW)F1 mice. A, Contour plots show the frequency of GC (B220 PNA )(upper row) and low ϩ ϫ plasma cells (CD19 CD138 )(lower row) in the spleen of (NZB NZW)F1 mice. B, With age, a signiﬁcant increase in the frequency and the absolute numbers of GC B cells and plasma cells was found. At each time point, ﬁve mice were analyzed.

cells (Fig. 6). In accordance with previous results, staining with fore the onset of autoimmune disease (Fig. 8A). The level of APRIL-specific Ab showed no significant expression of APRIL in APRIL mRNA increased further with the development of dis- the FDC network and in GC B cells (14). ease (Fig. 8A). Enhanced levels of BAFF mRNA expression were mainly seen Enhanced levels of BAFF and APRIL mRNA expression in B in MZ B cells. A significant increase was found in female animals ϫ cells of (NZB NZW)F1 mice at the age of 2–4 mo and in male animals at the age of 6- mo (Fig. ϫ (NZB NZW)F1 mice, which at the age of 3–4 mo spontaneously 8B). For FO B cells, no significant up-regulation in BAFF mRNA develop a lupus-like syndrome, were used as a disease model for was found. In B1 B cells, an increase in the level of BAFF mRNA chronic autoimmunity. An increase in MZ and CD5ϩ B cell pop- expression with age was found. However, in 6- to 7-mo-old ϫ ulations was found with increasing age and onset of disease (33). (NZB NZW)F1 mice, the level of BAFF expression was not Furthermore, the frequency and the absolute numbers of both significantly different from that in BALB/c mice (Fig. 8B). plasma cells and GC B cells increased with age (Fig. 7, A and B). The finding of both an expansion of the MZ and the spontaneous ϫ development of GC suggests that B cells are continuously acti- BAFF expression is up-regulated in GC of (NZB NZW)F1 ϫ mice vated in (NZB NZW)F1 mice. ϫ To test whether the activation of the immune system affects In 3- to 4-mo-old (NZB NZW)F1 female mice, staining of APRIL and BAFF expression in B cells, spleens were prepared splenic tissue sections showed large GC with strong BAFF ex- ϫ from 4- to 6-wk-old (NZB NZW)F1 mice before the onset of pression in their dark and light zones (Fig. 3D). A comparison of autoimmune disease, from 3- to 4-mo- old animals at the time fully developed GC from immunized BALB/c mice with those ϫ point when the disease becomes apparent and from 6-mo-old mice from (NZB NZW)F1 mice suggested enhanced BAFF expres- with established autoimmune disease. In a first step, MZ and FO sion in GC (Fig. 3, C and D). However, when BAFF expression in cells were isolated and the level of BAFF and APRIL mRNA was GC B cells was analyzed by FACS, no significant difference was ϫ ϫ compared in young and old (NZB NZW)F1 mice (Fig. 8). Both, found (Fig. 6). GC B cells from BALB/c, old and young (NZB female and male mice were analyzed. In contrast to BALB/c mice, NZW)F1 mice showed a 3- to 4-fold increase in their MFI when where no APRIL expression was seen in FO and MZ B cells, an compared with naive B cells (Fig. 6). Intracellular staining con- ϫ up-regulation was evident in (NZB NZW)F1 mice even be- firmed that GC B cells express high levels of BAFF (Fig. 6). The Journal of Immunology 5955

FIGURE 8. Up-regulation of BAFF and APRIL mRNA in B cells from ϫ (NZB NZW)F1 mice. FO, MZ, and peritoneal B1 cells were sorted as described in Materials and Methods. A, The presence of APRIL (40 cycles) and ␤-actin mRNA was determined by RT-PCR. B, The level of BAFF mRNA was determined by real-time PCR. Relative units of BAFF mRNA were calculated by normalizing values against ␤-actin. Downloaded from http://www.jimmunol.org/ Staining of splenic tissue sections of 3- to 4-mo-old (NZB ϫ and B). We therefore analyzed whether in vivo-generated

NZW)F1 mice showed APRIL expression in the light zone of GC. plasma cells also express BAFF and APRIL. Splenic tissue sec- ϫ Using confocal microscopy, colocalization of the FDC-M2 signal tions of 6- to 7-mo-old (NZB NZW)F1 mice were prepared with APRIL expression was found (data not shown). Whether and stained for BAFF or APRIL expression. Costaining with ␬- APRIL expression is also enhanced in GC B cells could not be specific Ab demonstrated that the majority of plasma cells in the ϫ determined. spleen of (NZB NZW)F1 mice express both BAFF and APRIL (Fig. 3E). FACS analysis confirmed that in (NZB ϫ ϫ In the spleen of (NZB NZW)F1 mice plasma cells express NZW)F1 mice with established autoimmune disease practically BAFF and APRIL all splenic plasma cells (CD19lowCD138ϩ) express high levels by guest on September 27, 2021 In vitro activation of B cells with LPS or CpG-ODN induced of both membrane-bound and also intracellular BAFF (Fig. 9). BAFF and APRIL expression. The newly developing plasma cells showed high cytoplasmic expression of these cytokines (Fig. 3, A Discussion BAFF is a fundamental survival factor for B cells and plays an essential role in the homeostatic regulation of the naive peripheral B cell pools (2–4). In the absence of BAFF or BAFF-R, only a few transitional B cells will differentiate into FO and MZ B cells (5, 24–26). APRIL has no essential function in B cell development. However, there is evidence that it supports B cell proliferation and acts as cofactor in class switch (2). In this study, we show for the first time that BAFF and/or APRIL produced by murine B cells themselves support B cell development and survival. Our analysis shows that B cells at all stages of differentiation express BAFF mRNA, while APRIL mRNA was restricted to early B cell development in the bone marrow and to peritoneal B1 B cells. As described for human peripheral B cells, no membrane-bound expression of BAFF was detectable on resting mature B cells (21, 28, 29), but both in vitro and in vivo activation resulted in up-regulation of BAFF and APRIL. Using MyD88- deficient mice, we show that the up-regulation of BAFF and APRIL expression following treatment with LPS or CpG-ODN depends on MyD88-TLR4 and TLR9 complex signaling (Fig. 2). One might argue that BAFF may be released by contaminating FIGURE 9. Plasma cells in the spleen of old (NZB ϫ NZW)F mice 1 myeloid cells and that the increased level of BAFF surface expres- express BAFF. Spleen cell suspensions were stained for CD19 and CD138 (left panel). Histograms (right panels) indicate expression levels of mem- sion which we see is due to up-regulation of BAFF-R. Were that brane-bound (M-BAFF) and intracellular (I-BAFF) BAFF in to be the case, no increase in the level of BAFF should be observed CD19ϩCD138Ϫ B cells (R1) and CD19lowCD138ϩ plasma cells (R2). In when B cells are activated in the presence of the protease inhibitor addition, intracellular staining for ␬ (I-␬) is shown. The isotype control is CMK (Fig. 4). However the opposite was found. When B cells indicated. A representative result is shown. were activated with LPS or CpG-ODN in the presence of CMK, 5956 ACTIVATED B CELLS EXPRESS BAFF AND APRIL the MFI of BAFF expression increased significantly ( p Ͻ 0.01). Disclosures These results suggest that B cells release soluble BAFF which may The authors have no financial conflict of interest. then support an autocrine survival pathway; however, the mecha- nisms are unclear. References In general, cytokines of the TNF superfamily are functional both 1. Schneider, P., F. MacKay, V. Steiner, K. Hofmann, J. L. Bodmer, N. Holler, in their membrane-bound and in soluble form when shed by en- C. Ambrose, P. Lawton, S. Bixler, H. Acha-Orbea, et al. 1999. 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