In vitro and in vivo activation induces BAFF and APRIL expression in B cells. J Immunol

Charité Universitätsmedizin Berlin, Berlín, Berlin, Germany
The Journal of Immunology (Impact Factor: 4.92). 12/2007; 179(9):5947-57. DOI: 10.4049/jimmunol.179.9.5947
Source: PubMed
ABSTRACT
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 transmembrane 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 x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.

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BAFF and APRIL Expression in B Cells
In Vitro and In Vivo Activation Induces
Claudia Berek
Van Trung Chu, Philipp Enghard, Gabriela Riemekasten and
http://www.jimmunol.org/content/179/9/5947
2007; 179:5947-5957; ;J Immunol
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
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In Vitro and In Vivo Activation Induces BAFF and APRIL
Expression in B Cells
1
Van Trung Chu,* Philipp Enghard,
Gabriela Riemekasten,
and Claudia Berek
2
*
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)F
1
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)F
1
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.
T
wo closely related cytokines of the TNF superfamily, B
cell-activating factor (BAFF,
3
also termed BLyS,
TALL-1, zTNF-4, THANK, and TNF13B) and a prolif-
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.
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
pathway (31).
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.
1
This 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
Education.
2
Address correspondence and reprint requests to Dr. Claudia Berek, Deutsches
Rheuma ForschungsZentrum, Chariteplatz 1, Berlin, Germany. E-mail address:
berek@drfz.de
3
Abbreviations 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
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2-phenyl-oxazolone (phOx) induced BAFF expression in germinal
center (GC) B cells. In (NZB NZW)F
1
mice, 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
disease.
Materials and Methods
Mice
Experiments were performed with BALB/c, C57BL/6, MyD88
/
(C57BL/6 background), and (NZB NZW)F
1
mice. One to 2-mo-old
BALB/c mice were immunized i.p. with a single injection of 100
gof
alum-precipitated phOx coupled to the carrier protein chicken serum albu-
min. (NZB NZW)F
1
mice 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
Systems).
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
FIGURE 1. Expression of BAFF and
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 B220
low
(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
(peritoneum) experiments.
5948 ACTIVATED B CELLS EXPRESS BAFF AND APRIL
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IgM
cells and B220
CD43
cells were sorted as shown in Fig. 1
A.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
(CD21
high
CD23
), FO (CD21
int
CD23
), transitional T1 (CD21
CD23
),
and T2 (CD21
high
CD23
) B cells were sorted by FACS (BD Biosciences).
B1 cells were isolated from the peritoneal cavity and sorted as
CD5
B220
low
IgM
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 (10
6
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