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BAFF and APRIL Expression in B Cells
In Vitro and In Vivo Activation Induces
Van Trung Chu, Philipp Enghard, Gabriela Riemekasten and
2007; 179:5947-5957; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 1, 2013
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
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)F1mice with
established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of
BAFF. In (NZB ? NZW)F1mice, 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.
eration-inducing ligand (APRIL), are central players in B cell de-
velopment and homeostasis. BAFF-deficient mice have an almost
complete loss of follicular (FO) and MZ B cells, although there is
normal development of early B cells in the bone marrow (1–4).
Normal numbers of newly formed immature B cells leave the bone
marrow and develop into the transitional T1 stage. In the absence
of BAFF. B cells do not progress past the transitional T2 stage,
resulting in impaired humoral immune responses (5). However,
when BAFF is overexpressed, for example in BAFF- transgenic
mice, self-reactive B cells may be rescued from peripheral deletion
(6, 7). As a consequence, BAFF-transgenic mice develop a lupus-
like autoimmune disease (8–11). From these data, it is apparent
that the level of BAFF has to be tightly regulated to ensure B cell
survival on the one hand and to prevent autoimmunity on the other.
wo closely related cytokines of the TNF superfamily, B
TALL-1, zTNF-4, THANK, and TNF13B) and a prolif-
BAFF is produced by a number of different cell types. Expres-
sion of BAFF by follicular dendritic cells (FDC) may be essential
for B cell homeostasis (12–14). However, the main sources of
BAFF are monocytes, macrophages, dendritic cells, and neutro-
phils, although subpopulations of B and T cells have also been
shown to express it (15–21). BAFF is found to be membrane as-
sociated and its expression is enhanced by cytokines, such as
IFN-?, IFN-?, and IL-10 or growth factors (17, 19, 20, 22).
Much less is known about APRIL which has no essential func-
tion in normal B cell development and plays only a minor role in
B cell homeostasis (23). In immune responses APRIL acts as a
costimulator for B and T cell proliferation and supports class
switch (2, 4, 22).
BAFF binds to three separate receptors, the BAFF receptor
(BAFF-R, BR3), the transmembrane activator and calcium mod-
ulator ligand interactor (TACI), and the B cell maturation Ag (24–
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
lymphatic leukemia (B-CLL), multiple myeloma, and Hodgkin’s
lymphoma cells, suggested that BAFF and APRIL support tumor
survival by an autocrine pathway (21, 28–30). There is also evi-
dence that normal nonmalignant human B cells up-regulate BAFF
and APRIL upon activation (21, 28). However, it is thought that
murine B cells do not express BAFF or APRIL (3), although an
analysis of early murine B cell development suggested that B cell
survival in the bone marrow may be supported by an autocrine
In this study, we show a detailed analysis of BAFF and APRIL
expression in different B cell subsets isolated from the bone mar-
row, spleen, and peritoneal cavity. In vitro cultures demonstrate
that upon stimulation with LPS or CpG-oligodeoxynucleotides
(CpG-ODN), splenic B2 cells as well as peritoneal B1 cells up-
regulate BAFF and APRIL expression. MyD88-deficient mice
demonstrated that BAFF expression in B cells is regulated by the
TLR signaling pathway. Immunization with the T-dependent Ag
*Deutsches Rheuma ForschungsZentrum; and†Department of Rheumatology and
Clinical Immunology, Charite University Hospital, Berlin, Germany
Received for publication April 6, 2007. Accepted for publication August 21, 2007.
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
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by a grant from the Government of Vietnam and the
DAAD (to V.T.C.), the Bundesministerium fu ¨r Bildung und Forschung Grant NGFN2
and the SFB 650. The DRFZ is supported by the Berlin Senate of Research and
2Address correspondence and reprint requests to Dr. Claudia Berek, Deutsches
Rheuma ForschungsZentrum, Chariteplatz 1, Berlin, Germany. E-mail address:
3Abbreviations used in this paper: BAFF, B cell-activating factor; APRIL, a prolif-
eration-inducing ligand; MZ, marginal zone; FO, follicular; GC, germinal center;
FDC, follicular dendritic cells; CMK, chloromethylketone; phOx, 2-phenyl-ox-
azolone; PNA, peanut agglutinin; MFI, mean fluorescence intensity; TACI, trans-
membrane activator and calcium modulator ligand interactor; ODN, oligodeoxynucle-
otide; BAFF-R, BAFF receptor.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 1, 2013
2-phenyl-oxazolone (phOx) induced BAFF expression in germinal
center (GC) B cells. In (NZB ? NZW)F1mice, expression of
BAFF and APRIL mRNA in MZ and B1 cells was strongly up-
regulated with increasing age and onset of disease. In addition,
strong expression of BAFF and APRIL protein was found in
plasma cells. Thus, expression of these cytokines in activated and
differentiated B cells may support the development of autoimmune
Materials and Methods
Experiments were performed with BALB/c, C57BL/6, MyD88?/?
(C57BL/6 background), and (NZB ? NZW)F1mice. One to 2-mo-old
BALB/c mice were immunized i.p. with a single injection of 100 ?g of
alum-precipitated phOx coupled to the carrier protein chicken serum albu-
min. (NZB ? NZW)F1mice were purchased from The Jackson Laboratory
and bred in the Bundesinstitut fu ¨r gesundheitlichen Verbraucherschutz und
Veterina ¨rmedizin, Berlin-Marienfelde. Animal experiments were approved
by the institutional animal care and use committee.
Abs and reagents
Surface expression of BAFF on B cells was determined using a FITC-
labeled anti-BAFF mAb (Buffy-2; Alexis). For isotype control, FITC-la-
beled rat IgM (Southern Biotechnology Associates) was used. To detect
BAFF and APRIL expression, cytospins and frozen sections were stained
with polyclonal rabbit anti-BAFF (Sigma-Aldrich) or anti-APRIL (Stress-
gen) Ab, respectively. As secondary Ab Alexa Fluor 546-conjugated goat
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
with 200 ng of soluble BAFF and then stained with the mAb Buffy-2 or
with polyclonal rabbit anti-BAFF Abs. The expression of the BAFF-R was
controlled by staining with the biotinylated rat mAb 204406 (R&D
The antagonist TACI Ig was prepared by fusing the extracellular domain
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
from supernatants by protein G chromatography.
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
(T24), FITC and Cy5 anti-CD19 (ID3) and anti-CD21 (7G6), Cy5 anti-IgM
(M14), and biotinylated anti-? L chain (187.1) Abs were provided by the
Deutsches Rheuma ForschungsZentrum (DRFZ). FITC or biotinylated anti-
CD43 (1B11) was obtained from Biolegend, PE or biotinylated anti-CD5
from eBioscience, FITC or PE anti-CD23 (B3B4) and PE anti-CD138
(281–2) from BD, Pharmingen, and biotinylated and FITC-peanut agglu-
tinin (PNA) from Vector laboratories. To visualize biotinylated Ab Alexa
Fluor 488-, PE-, or allophycocyanin-conjugated streptavidin was used
(Molecular Probes and BD Biosciences).
Isolation of lymphocytes
Suspensions of bone marrow cells were flushed from tibias and femurs of
6–10-wk-old BALB/c mice and stained with B220-PE-, CD43-FITC-, and
IgM-Cy5-specific Abs. To isolate pro-B cells, lymphocytes were gated on
APRIL in B cells. A, To determine the level
of BAFF and APRIL expression, B cell sub-
sets 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 ana-
lyzed. Results of three independent experi-
ments are shown as mean values plus SD. D,
Expression of BAFF, APRIL, and CD11c
mRNA in defined numbers of MZ?, FO?,
peritoneal B1 B, and CD11c?cells. Number
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 tri-
ple stained for CD5, B220, and BAFF
(Buffy-2). Dot blots show the gating for
splenic newly formed B220low(NF-B), ma-
ture (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 pan-
els). Surface expression of BAFF before (nor-
mal line) and after incubation of B cells with
soluble BAFF (heavy line) is shown. Isotype
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
Expression of BAFF and
5948ACTIVATED B CELLS EXPRESS BAFF AND APRIL
by guest on June 1, 2013
IgM?cells and B220?CD43?cells were sorted as shown in Fig. 1A. To
isolate pre- and immature B cells, lymphocytes were gated on B220?
CD43?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 en-
riched by MACS (Miltenyi Biotec) to ?96% 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 ?99% 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
(CD21highCD23?), FO (CD21intCD23?), transitional T1 (CD21?CD23?),
and T2 (CD21highCD23?) B cells were sorted by FACS (BD Biosciences).
B1 cells were isolated from the peritoneal cavity and sorted as
CD5?B220lowIgM?cells (Fig. 1A). Purity of sorted cells was in the range
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%
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
BAFF and APRIL was measured by semiquantitative RT-PCR or real-time
PCR. Surface expression of BAFF on B cells was determined by FACS
using the monoclonal anti-BAFF (Buffy-2) Ab.
To test whether B cells express biologically active BAFF and/or APRIL,
sorted B cells were activated for 3 days with different concentrations of
LPS (1 and 5 ?g/ml). Cultures were set up in the presence or in the absence
of the TACI Ig (20 ?g/culture) fusion protein. As a control protein, human
IgG was added. To monitor proliferation, cells were labeled with the CSFE
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
were harvested, washed with PBS, and fixed overnight in 70% ethanol at
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-
Arg-Val-Lys-Arg-chloromethylketone (CMK; Alexis). For these experi-
ments, cell cultures were set up using sorted splenic B2 or peritoneal B1
cells isolated from individual BALB/c mice.
Detection of BAFF and APRIL mRNA
Expression of mRNA for BAFF and APRIL was measured by semiquan-
titative RT-PCR or real-time PCR. Total RNA was prepared from 2 ? 106
sorted cells using a RNeasy Mini Kit (Qiagen). RNA was treated with
RNase-free DNase I (Qiagen) according to the manufacturer’s instruction.
Concentration of total RNA was measured by NaNoDrop (Biotech Inter-
national) and reverse transcribed into cDNA using an Ominiscript RT kit
(Qiagen). cDNA was amplified with BAFF-, APRIL-, or CD11c-specific
primers using 1.25 U/reaction AmpliTaq Gold polymerase (Applied Bio-
systems). The number of cycles is indicated for the different experiments.
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
cDNA was visualized on agarose gels and specificity was checked by
To control the purity of sorted cells, defined numbers of MZ, FO, and
peritoneal B1 cells were prepared as described and sorted into PCR tubes
containing 20 ?l of reverse transcriptase buffer (Qiagen). In addition, a
second spleen was gently homogenized and the suspension was digested in
RPMI 1640/10% FCS containing 1 ?g/ml collagenase for 45 min at 37°C.
After three washes, cells were incubated with Cy5-conjugated anti-CD11c
mAb (N418) and defined numbers of CD11c?cells were sorted again into
PCR tubes. For each of the cell populations, cDNA was directly transcribed
using 10 ?M oligo(dT) as primer and one-fifth of the reaction mixture was
used to amplify BAFF, APRIL, CD11c, or ?-actin cDNA in a seminested
PCR (Qiagen). The first amplification consisted of 25 cycles of 1 min at
94°C, 1 min at 63°C, and 30 s at 72°C and the second round of 40 cycles
for APRIL, 35 cycles for BAFF, 30 cycles for CD11c, and 25 cycles for
Quantitative PCR to determine the relative amount of BAFF mRNA was
performed with a LightCycler System (Roche Diagnostics) using the Ligh-
Cycler FastStart DNA Master SYBR Green I (Roche Diagnostics). The
real-time PCR products were analyzed on 4% agarose gels to check purify
and specificity. Each sample from three independent experiments was run
in triplicate. The unit number showing relative mRNA level in each sample
was determined as a value of mRNA normalized against ?-actin.
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
IgG (Sigma-Aldrich) and the mAb 2.4G2/75 specific for the Fc?R (CD16/
32). For intracellular staining, surface-labeled cells were washed twice and
fixed in 2% (w/v) paraformaldehyde (Merck) for 20 min at room temper-
ature. After washing, cells were permeabilized in PBS supplemented with
0.5% saponin (Sigma-Aldrich) and 0.5% BSA for 10 min at room temper-
ature and stained with BAFF or ?-specific Abs for 30 min in the dark.
Before analyses, cells were washed with 0.5% saponin buffer. Stained cells
were analyzed using FACSCalibur or LSRII (BD Biosciences) and the
CellQuest (BD Biosciences) or FlowJo software (Tree Star).
Cytospin, fluorescence staining, and confocal microscopy
B cells activated in vitro for 3 days with different stimuli were harvested.
To remove receptor-bound BAFF and APRIL, cells were incubated with
0.1% sodium citrate/0.1% Triton X-100 (pH 5.2) overnight at 4°C. After
three washes with PBS/0.5% BSA, cells were centrifuged onto glass slides
(Menzel). Spleens were frozen in Tissue-Tec OCT compound and stored at
?70°C. Frozen tissue sections of 7 ?m were prepared, fixed in cold ace-
tone for 10 min, and air dried.
To detect BAFF and APRIL expression, cytospins and sections were
double stained with polyclonal rabbit anti-BAFF or anti-APRIL Ab and
anti-? Ab. Nuclei were counterstained with 4?,6-diamidino-2-phenylin-
dole. To analyze BAFF expression in GC, tissue sections were double
stained with polyclonal rabbit anti-BAFF and biotinylated FDC-specific
mAb M2 (Serotec) or biotinylated PNA (Vector Laboratories). Fluorescent
microscopy images were captured with a SPOT RT camera (Diagnostic
Instruments) or by confocal microscopy using Leica software.
ELISA for soluble BAFF
Soluble BAFF in supernatants was detected using a mouse anti-BAFF
ELISA kit (APO-54N-019-KI01; Apotech) according to the manufactur-
er’s instruction The sensitivity of the kit is 0.2 ng/ml. ODs at 450 nm were
APRIL. A, In vitro-activated B cells were analyzed by FACS. Represen-
tative stainings are shown. B, The presence of BAFF, APRIL, and CD11c
mRNA was determined by RT-PCR. cDNA was amplified for 40, 45, or 35
cycles, respectively. As positive control, splenocytes (Spl) were used, as
negative control (control) amplification without DNA. C, B cells from
BALB/c (n ? 4), C57BL/6 (n ? 3), and MyD88 (n ? 4)-deficient mice
were stimulated with LPS or CPG-ODN for 2 days and BAFF surface
expression was determined. Histograms show overlays of BAFF expres-
sion in unstimulated (dotted line), stimulated B cells (solid line), and iso-
type control (shaded histogram).
In vitro- activated splenic B cells up-regulate BAFF and
5949 The Journal of Immunology
by guest on June 1, 2013
measured with a microplate spectrophotometer (Spectrarax 190). The mu-
rine A20 cell line was used as a positive control for BAFF release. After
3 days in culture, without activation, a concentration of 1.6 ? 0.02 ng/ml
soluble BAFF was measured (see Fig. 5).
All statistical analyses were performed using the Student t test; statis-
tical significance of difference was determined as p ? 0.05 or p ? 0.01
or p ? 0.001.
B cells express APRIL and BAFF mRNA
Both B cell lymphoma lines and normal human B cells have been
reported to express BAFF and APRIL (8, 21, 29–31). This
prompted us to analyze whether normal murine B cells also ex-
press these cytokines. We found that during early B cell develop-
ment in the bone marrow, pro-B cells (B220?CD43?IgM?), pre-B
cells (B220?CD43?IgM?), and immature B cells (B220?CD43?
IgM?) express APRIL mRNA (Fig. 1B). When immature B cells
leave the bone marrow and differentiate into transitional T1/T2
B cells, APRIL mRNA is down-regulated and is no longer de-
tectable in FO (CD21intCD23?) and MZ (CD21highCD23?) B
cells. Although no APRIL mRNA was detected in mature B2
cells, B1 cells isolated from the peritoneal cavity expressed it at
high levels (Fig. 1B).
Surprisingly, all murine B cell subsets analyzed expressed
BAFF mRNA (Fig. 1C). A comparative analysis showed that pro-,
pre-, and immature B cells isolated from the bone marrow express
4- to10-fold higher levels of BAFF mRNA than T1/T2, mature FO
and MZ B cells (Fig. 1C). With the differentiation of the immature
B cell into the transitional T1/T2 stage, BAFF mRNA is down-
regulated. However, this is different for peritoneal B1 cells that
expressed high levels of BAFF mRNA. The level was comparable
to that seen in the immature B cells isolated from the bone marrow
It is unlikely that BAFF and APRIL mRNA are derived from
a contamination by monocytes/macrophages or dendritic cells
activation induces BAFF and APRIL
expression. Sorted splenic B cells
were in vitro activated with medium
alone, LPS, or CpG-ODN for 3 days.
costained with anti-? (green) and
polyclonal rabbit anti-BAFF (red; A)
or anti-APRIL Ab (red; B). Nuclei
were counterstained with 4?,6-dia-
midino-2-phenylindole (DAPI; blue).
Single staining and overlays are
shown. C, Splenic tissue sections
BALB/c (C4), and 3- to 4-mo-old
(NZB ? NZW)F1 mice (D) are
shown. Consecutive sections (C1,2)
were double stained with anti-BAFF
and either anti-FDC (M2) or PNA.
Light zone (LZ) and dark zone (DZ)
are indicated. E, Splenic tissue sec-
tions from 6- to 7-mo-old (NZB ?
NZW)F1mice were stained for ?,
BAFF, and APRIL. Single staining
and overlays of plasma cells are
shown. Original magnification, ?40.
Representative results from three in-
dependent experiments are shown.
In vitro and in vivo
5950 ACTIVATED B CELLS EXPRESS BAFF AND APRIL
by guest on June 1, 2013
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 CD11c?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 de-
scribed, 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
CD11?cells express high levels of CD11c (Fig. 1D), it is un-
likely that BAFF mRNA expression in FO, MZ, and peritoneal
B1 B cells is due to a contamination with CD11c myeloid cells.
High levels of BAFF mRNA expression is associated with
protein expression on the surface of B cells
To determine whether murine B cells express BAFF, bone marrow
cells were triple stained with B220, anti-IgM, and the BAFF-spe-
cific mAb Buffy-2. The subset of IgM?B220lowpre- and pro-B
cells showed little BAFF expression. The signal was only slightly
higher than in the isotype control. Comparably weak expression
levels were seen for the subset of IgM?B220lowimmature B cells
(data not shown).
To control for the specificity of the mAb Buffy-2, sorted splenic
B cells were incubated for 2 h with soluble BAFF (Fig. 1E, upper
row of histograms). Before and after incubation, B cells were
stained with BAFF-R (Fig. 1E, left panel) or BAFF-specific Abs
(Fig. 1E, middle and right panels). Receptor bound BAFF was
only detectable with BAFF specific polyclonal rabbit Abs (Fig. 1E,
right panel). In addition, pre-incubation with BAFF slightly re-
duced the signal obtained with anti-BAFF-R Abs (Fig. 1E, left
panel). These data show that the mAb Buffy-2 does not recognize
BAFF when it is bound by its receptors. In contrast to the poly-
clonal rabbit anti-BAFF Abs, the mAb Buffy-2 recognizes only
membrane-expressed BAFF. The B cell lymphoma lines WEHI-
231 and A20 were used as positive controls and clearly showed
membrane-associated BAFF expression (Fig. 1E, second row of
Splenic B cells were enriched by MACS to a purity ?96% and
stained with the BAFF-specific mAb Buffy-2. FACS analysis
showed that newly formed (B220low) and the majority of mature
splenic B cells were negative for BAFF surface expression (Fig.
1E, third row of histograms). Only a small fraction (1.38 ? 0.3%)
of splenic B cells expressed membrane-bound BAFF (data not
shown). The low frequency suggested that BAFF-positive cells may
be splenic B1 cells. To further analyze whether splenic B1 cells ex-
press membrane-bound BAFF, a triple staining with Ab specific for
BAFF, CD5, and B220 was performed (Fig. 1E). Gating on
CD5?B220lowcells confirmed that B1 cells express low levels of
BAFF in their membrane, whereas no expression was seen on splenic
mature and newly formed B2 cells (Fig. 1E, third row). When peri-
was again marginally higher than in the isotype control (Fig. 1E, last
row), supporting that B1 cells express membrane-bound BAFF.
In vitro activation of B cells induces APRIL and BAFF
To analyze whether activation of B cells induces APRIL and
BAFF protein expression, splenic B cells were sorted and cultured
in vitro with different stimuli. RNA was isolated and cytokine
expression was analyzed. After 3 days in culture, the purity of B
cells was rechecked by staining with CD19, CD3, CD11c, and
CD11b. Because FACS analysis showed a purity of ?99% B cells
(Fig. 2A) and because RT-PCR gave no signal for CD11c (Fig.
2B), it is unlikely that cultured B cells are contaminated by my-
RT-PCR showed an up-regulation of BAFF and APRIL mRNA
when B cells were activated with CpG-ODN or with LPS (Fig.
2B). The elevated levels of BAFF mRNA expression in LPS- or
CpG-ODN-activated B cells correlated with the expression of
BAFF on the surface of the cells. Splenic B cells expressed mem-
brane-bound BAFF already 2 days after in vitro activation (Fig.
2C). Surface expression of BAFF was confirmed by using biotin-
ylated TACI Ig (data not shown).
A control experiment using MyD88-deficient mice demon-
strated that the enhancement of BAFF expression is dependent on
the TLR signaling pathway (Fig. 2C). The negative result seen in
the MyD88-deficient mice is not due to the C57BL/6 genetic back-
ground, since splenic B cells isolated from both BALB/c and
C57BL/6 mice up-regulated BAFF expression when activated with
CpG-ODN or LPS.
Activation of splenic B cells with LPS or CpG-ODN induces
differentiation into plasma cells and expression of intracellular
BAFF and APRIL. After 3 days in culture, B cells were harvested
by cytospin and stained with BAFF- or APRIL-specific Ab.
Costaining with ?-specific Ab demonstrated high levels of APRIL
and BAFF expression in the cytoplasm of the newly generated
plasma cells (Fig. 3, A and B). Comparable results were found
when peritoneal B1 cells were activated with LPS or with CpG-
ODN. B1 cells expressed low levels of BAFF and APRIL already
before in vitro activation and after 3 days in culture with LPS or
CpG-ODN a strong up-regulation of both cytokines was found.
Again, double staining with ?-specific Ab showed BAFF and
APRIL expression in the cytoplasm of plasma cells (data not
tivated B cells. Splenic B (A) or peritoneal B1 (B) cells from BALB/c mice
(n ? 4 and n ? 6, respectively) were sorted and activated with medium
alone, LPS, or CpG-ODN either in the absence (?CMK) or in the presence
(?CMK) of inhibitor. After 3 days in culture, cells were harvested and
stained for CD19 and BAFF (Buffy-2). Expression levels of membrane-
bound BAFF were measured by FACS. Representative dot plots are shown;
numbers on each panel indicate the percentage of BAFFhighB cells.
CMK enhances membrane-bound BAFF expression in ac-
5951The Journal of Immunology
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CMK enhances the expression of membrane-bound BAFF on
activated B cells
Sorted splenic B cells and also peritoneal B1 cells were activated
with LPS or with CpG-ODN in the presence or absence of CMK,
an inhibitor of furin-like ecto-peptidases which inhibits cleavage
of BAFF from the cell surface and thus release of soluble BAFF
(20). After 3 days in culture, B cells were harvested and stained
with the BAFF-specific mAb Buffy-2. FACS analysis showed that
activated B cells up-regulate membrane-bound BAFF expression
(Fig. 4). In cultures with inhibitor, the mean fluorescence intensity
(MFI) for BAFF expression was higher than in cultures without
(Fig. 4). Activation of splenic B cells with LPS or CpG-ODN in
the absence of CMK increased the MFI from 4.25 ? 0.24 (medium
alone) to 12.86 ? 0.39 or 10.39 ? 1.36. In the presence of CMK,
the MFI increased to 20.29 ? 1.92 or 17.8 ? 1.08, respectively. A
similar up-regulation of BAFF expression was observed when
peritoneal B1 cells were activated. When incubated with LPS, the
MFI was 19.23 ? 1.48 in the absence of CMK and 26.64 ? 2.61
in its presence. Activation of B1 cells with CpG-ODN increased
the MFI of BAFF expression to 24.94 ? 3.29 in the absence and
38.54 ? 3.59 in the presence of CMK.
Furthermore, when B2 cells were activated in the presence of
CMK, a significant increase in the fraction of BAFFhighB cells
was seen (?6% of total B cells; Fig. 4A). A similar result was
BAFF and/or APRIL. A, The pres-
ence 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
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
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
CD138highplasma cells was deter-
mined. To analyze cell proliferation,
sorted B cells were labeled with
CFSE. To determine the frequency of
cycling cells, harvested B cells were
stained with propidium iodide. Mean
values of three independent experi-
ments are shown. Values of p are
In vitro-activated B
5952ACTIVATED B CELLS EXPRESS BAFF AND APRIL
by guest on June 1, 2013
found for B1 cells, in particular when they were activated with
CpG-ODN (Fig. 4B). The finding of elevated levels of membrane-
bound BAFF expression in the presence of CMK gives further
evidence that activated B cells express BAFF. These findings in-
dicate that upon activation B cells release soluble BAFF into the
In vitro activation of B cells induces secretion of BAFF and/or
To look for the release of soluble BAFF, tissue culture superna-
tants of splenic B cells activated for 3 days with LPS or CpG-ODN
were tested for the presence of soluble BAFF using an ELISA kit.
The increase in the level of soluble BAFF was low, but detectable
(p ? 0.01). Activation with LPS or CpG-ODN yielded a concen-
tration of 1.2 ? 0.1 or 0.9 ? 0.1 ng/ml soluble BAFF, respectively
The question arises whether the BAFF and APRIL expressed by
B cells is of biological significance. To address this question, in-
hibition assays using the fusion protein TACI Ig were performed.
Sorted B cells were incubated with different amounts of LPS in the
presence or absence of TACI Ig. Fig. 5, B and C, shows that in the
presence of TACI Ig there is a significant reduction in B cell ac-
tivation. Although a low dose of 1 ?g/ml LPS is sufficient to in-
duce B cell proliferation (Fig. 5C, upper graph), the number of B
cells does not increase in the presence of TACI Ig. After 3 days of
activation with 1 ?g/ml LPS, approximately the same number of B
cells were counted as at the start of cultures (Fig. 5B). In the
presence of TACI Ig, the frequency of cycling B cells was signif-
icantly reduced (Fig. 5C, middle graph). Furthermore, the differ-
entiation of activated B cells into CD138highplasma cells was
inhibited (Fig. 5C, lower graph). The finding that in the presence
of the antagonist TACI Ig, B cell survival, proliferation, and dif-
ferentiation are all inhibited indicates that in vitro activation of B
cells induces secretion of biologically active BAFF and/or APRIL.
In vivo activation of B cells and up-regulation of BAFF
In primary follicles of BALB/c mice, FDC do not express BAFF
(Fig. 3C, C3). Staining with APRIL-specific Ab was also negative
(data not shown). To induce a T-dependent immune response,
BALB/c mice were immunized with phOx and 10 days after in-
jection of Ag, GC formation was observed (Fig. 3C).
Double staining with the FDC-specific M2 Ab and anti-BAFF
showed that BAFF expression is up-regulated in FDC of the GC
light zone (Fig. 3C, C2). An analysis of BAFF expression at high
magnification suggests that BAFF is also expressed in GC B cells,
as B cells in the dark zone of the GC costained for PNA and BAFF
(Fig. 3C, C3). From staining of tissue sections, it is difficult to say
whether BAFF is bound by its receptors or indeed expressed on the
surface of B cells. However, FACS analysis of GC B cells showed
up-regulation of membrane-bound BAFF and intracellular staining
for BAFF confirmed endogenous production of BAFF by GC B
late BAFF expression. Splenocytes
(NZB ? NZW)F1mice of different
ages were stained with PNA-, BAFF
(Buffy-2)-, and B220- specific Abs.
The MFI of BAFF expression on na-
ive (B220?PNAlow) and GC B cells
(B220?PNAhigh) was compared (left
panel). Bar graphs show the increase
in the MFI (fold change) in compari-
son to the isotype control. Intracellu-
lar staining for BAFF is only shown
for the group of 6- to 7- mo-old
(NZB ? NZW)F1mice. For each
group, five animals were analyzed.
Values of p are indicated. Represen-
tative histograms showing BAFF
staining of naive and GC B cells are
included (right panel).
GC B cells up-regu-
5953The Journal of Immunology
by guest on June 1, 2013
cells (Fig. 6). In accordance with previous results, staining with
APRIL-specific Ab showed no significant expression of APRIL in
the FDC network and in GC B cells (14).
Enhanced levels of BAFF and APRIL mRNA expression in B
cells of (NZB ? NZW)F1mice
(NZB ? NZW)F1mice, which at the age of 3–4 mo spontaneously
develop a lupus-like syndrome, were used as a disease model for
chronic autoimmunity. An increase in MZ and CD5?B cell pop-
ulations was found with increasing age and onset of disease (33).
Furthermore, the frequency and the absolute numbers of both
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
development of GC suggests that B cells are continuously acti-
vated in (NZB ? NZW)F1mice.
To test whether the activation of the immune system affects
APRIL and BAFF expression in B cells, spleens were prepared
from 4- to 6-wk-old (NZB ? NZW)F1mice before the onset of
autoimmune disease, from 3- to 4-mo- old animals at the time
point when the disease becomes apparent and from 6-mo-old mice
with established autoimmune disease. In a first step, MZ and FO
cells were isolated and the level of BAFF and APRIL mRNA was
compared in young and old (NZB ? NZW)F1mice (Fig. 8). Both,
female and male mice were analyzed. In contrast to BALB/c mice,
where no APRIL expression was seen in FO and MZ B cells, an
up-regulation was evident in (NZB ? NZW)F1mice even be-
fore the onset of autoimmune disease (Fig. 8A). The level of
APRIL mRNA increased further with the development of dis-
ease (Fig. 8A).
Enhanced levels of BAFF mRNA expression were mainly seen
in MZ B cells. A significant increase was found in female animals
at the age of 2–4 mo and in male animals at the age of 6- mo (Fig.
8B). For FO B cells, no significant up-regulation in BAFF mRNA
was found. In B1 B cells, an increase in the level of BAFF mRNA
expression with age was found. However, in 6- to 7-mo-old
(NZB ? NZW)F1mice, the level of BAFF expression was not
significantly different from that in BALB/c mice (Fig. 8B).
BAFF expression is up-regulated in GC of (NZB ? NZW)F1
In 3- to 4-mo-old (NZB ? NZW)F1female mice, staining of
splenic tissue sections showed large GC with strong BAFF ex-
pression in their dark and light zones (Fig. 3D). A comparison of
fully developed GC from immunized BALB/c mice with those
from (NZB ? NZW)F1mice suggested enhanced BAFF expres-
sion in GC (Fig. 3, C and D). However, when BAFF expression in
GC B cells was analyzed by FACS, no significant difference was
found (Fig. 6). GC B cells from BALB/c, old and young (NZB ?
NZW)F1mice showed a 3- to 4-fold increase in their MFI when
compared with naive B cells (Fig. 6). Intracellular staining con-
firmed that GC B cells express high levels of BAFF (Fig. 6).
plasma cells (CD19lowCD138?) (lower row) in the spleen of (NZB ? NZW)F1mice. 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.
Chronic activation of B cells in (NZB ? NZW)F1mice. A, Contour plots show the frequency of GC (B220?PNAhigh) (upper row) and
5954 ACTIVATED B CELLS EXPRESS BAFF AND APRIL
by guest on June 1, 2013
Staining of splenic tissue sections of 3- to 4-mo-old (NZB ?
NZW)F1mice showed APRIL expression in the light zone of GC.
Using confocal microscopy, colocalization of the FDC-M2 signal
with APRIL expression was found (data not shown). Whether
APRIL expression is also enhanced in GC B cells could not be
In the spleen of (NZB ? NZW)F1mice plasma cells express
BAFF and APRIL
In vitro activation of B cells with LPS or CpG-ODN induced
BAFF and APRIL expression. The newly developing plasma cells
showed high cytoplasmic expression of these cytokines (Fig. 3, A
and B). We therefore analyzed whether in vivo-generated
plasma cells also express BAFF and APRIL. Splenic tissue sec-
tions of 6- to 7-mo-old (NZB ? NZW)F1mice were prepared
and stained for BAFF or APRIL expression. Costaining with ?-
specific Ab demonstrated that the majority of plasma cells in the
spleen of (NZB ? NZW)F1mice express both BAFF and
APRIL (Fig. 3E). FACS analysis confirmed that in (NZB ?
NZW)F1mice with established autoimmune disease practically
all splenic plasma cells (CD19lowCD138?) express high levels
of both membrane-bound and also intracellular BAFF (Fig. 9).
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 mem-
brane-bound expression of BAFF was detectable on resting ma-
ture 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
One might argue that BAFF may be released by contaminating
myeloid cells and that the increased level of BAFF surface expres-
sion which we see is due to up-regulation of BAFF-R. Were that
to be the case, no increase in the level of BAFF should be observed
when B cells are activated in the presence of the protease inhibitor
CMK (Fig. 4). However the opposite was found. When B cells
were activated with LPS or CpG-ODN in the presence of CMK,
and APRIL mRNA in B cells from
(NZB ? NZW)F1mice. FO, MZ, and
peritoneal B1 cells were sorted as de-
scribed 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 val-
ues against ?-actin.
Up-regulation of BAFF
express BAFF. Spleen cell suspensions were stained for CD19 and CD138
(left panel). Histograms (right panels) indicate expression levels of mem-
CD19?CD138?B cells (R1) and CD19lowCD138?plasma cells (R2). In
addition, intracellular staining for ? (I-?) is shown. The isotype control is
indicated. A representative result is shown.
Plasma cells in the spleen of old (NZB ? NZW)F1mice
5955 The Journal of Immunology
by guest on June 1, 2013
the MFI of BAFF expression increased significantly (p ? 0.01).
These results suggest that B cells release soluble BAFF which may
then support an autocrine survival pathway; however, the mecha-
nisms are unclear.
In general, cytokines of the TNF superfamily are functional both
in their membrane-bound and in soluble form when shed by en-
zymatic cleavage from the surface through a furin-like convertase
(34). An alternative direct secretion pathway for BAFF has been
reported, but it is unique to neutrophils (19). Also, for APRIL it
was found that it is secreted following intracellular processing in
the Golgi apparatus (35). Our data demonstrate membrane-bound
and intracellular cytoplasmic BAFF and APRIL expression in B
cells. It is therefore possible that the survival of B cells and, in
particular, of plasma cells may be supported by autocrine cyto-
kines signaling both through receptors in the outer membrane and
in the cytosolic compartments (8). Our finding that in the presence
of the antagonist TACI Ig B cell activation is inhibited (Fig. 5)
indicates that B cells are supported by an autocrine pathway. Upon
stimulation, B cells themselves release biologically active BAFF
and/or APRIL into the culture supernatants.
The analysis of the GC reaction suggested that BAFF and
APRIL are required for both the development of the mature FDC
network in the GC and for the maintenance of the GC (26, 36, 37).
Our data show that immunization of BALB/c mice with a T-de-
pendent Ag up-regulates BAFF expression both in FDC and in GC
B cells (Fig. 3). High-affinity interaction between the Ag and the
BCR may up-regulate BAFF expression and help the Ag-selected
B cell to survive and to differentiate into an effector cell. The
differentiation of GC B cells seems to be independent of APRIL
because we see no significant expression of it in GC. This is in
accordance with a previous report (14).
In T-independent responses, the simultaneous up-regulation of
both BAFF and APRIL in TLR-activated B cells may help to com-
pete with Ag-inexperienced B cells for short-term survival niches.
The autocrine pathway may thus provide a mechanism that pro-
motes survival and differentiation of Ag-activated B cells in de-
fined microenvironments within the peripheral lymphoid organs.
Our analysis of (NZB ? NZW)F1mice shows that the chronic
activation of the immune system in these animals enhances BAFF
and APRIL expression in B cells. In particular, plasma cells ex-
pressed high levels of BAFF and APRIL and this may enhance
their life span in the spleen. Furthermore, cytokine secretion by
plasma cells may also contribute to the increased levels of BAFF
in sera of the (NZB ? NZW)F1mouse.
Similarly, in patients with autoimmune diseases like lupus ery-
thematosus, Sjo ¨gren’s syndrome, and rheumatoid arthritis, ele-
vated levels of BAFF may be generated by activated B cells (38–
40). Autocrine BAFF will then become part of a vicious circle in
which enhanced serum levels of BAFF lower the threshold of af-
finity-based selection and result in an increase in the frequency of
autoreactive B cells.
We find that the majority of plasma cells in autoimmune
(NZB ? NZW)F1mice express high levels of BAFF and APRIL.
This observation is potentially of clinical importance, particularly
with respect to ongoing clinical trials of BAFF and APRIL antag-
onists. These trials aim to deplete pathogenic autoreactive B and
plasma cells. However, if these cells themselves produce their own
survival factors, they may turn out to be resistant to this type of
We are thankful for technical support by members of the DRFZ, in particular
S. Schu ¨rer and G. Steinhauser for their help in preparing TACI Ig. We also
thank S. Fillatreau, R. S. Jack, and A. Radbruch for critical discussion.
The authors have no financial conflict of interest.
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