Development and Function of Murine B Cells
Lacking RANK
Thomas Perlot and Josef M. Penninger
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Supplementary
Material
J Immunol 2012; 188:1201-1205; Prepublished online 4
January 2012;
doi: 10.4049/jimmunol.1102063
http://www.jimmunol.org/content/188/3/1201
The Journal of Immunology
Development and Function of Murine B Cells Lacking RANK
Thomas Perlot and Josef M. Penninger
RANKL–RANK signaling regulates numerous physiologic processes such as bone remodeling, lymph node organogenesis, central
thermoregulation, and formation of a lactating mammary gland in pregnancy. Recently, a receptor activator of NF-kB ligand
(RANKL)-blocking Ab has been approved for human use in potentially millions of osteoporosis and cancer patients. However,
germline deficiencies in RANKL or receptor activator of NF-kB (RANK) also lead to strong B cell defects in mice and human
patients, suggesting that RANKL–RANK inhibition could interfere with B cell physiology and thereby trigger immunologic sideeffects. To address this key question—that is, whether RANKL–RANK signaling affects B cell physiology directly or the observed
defects are secondary because of the severe osteopetrosis—we generated B cell-specific RANK knockout mice. We show that
B cells deficient for RANK undergo normal development and do not show any obvious defects in Ab secretion, class switch
recombination, or somatic hypermutation. Our data indicate that ablation of the RANKL–RANK pathway has no direct adverse
effect on B cell physiology. The Journal of Immunology, 2012, 188: 1201–1205.
Institute of Molecular Biotechnology of the Austrian Academy of Sciences, 1030
Vienna, Austria
Received for publication July 15, 2011. Accepted for publication November 28,
2011.
J.M.P. is supported by grants from the Institute of Molecular Biotechnology of
the Austrian Academy of Sciences, the Austrian Ministry of Sciences, the Austrian
Academy of Sciences, Genome Research in Austria (AustroMouse), and a European
Union European Research Council Advanced Grant. T.P. is supported by a Marie
Curie International Incoming Fellowship. The research leading to these results has
received funding from the European Community’s Seventh Framework Programme
(FP7/2007-2013) under Grant Agreement 252210.
Address correspondence and reprint requests to Prof. Josef M. Penninger, Institute
of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohrgasse 3,
1030 Vienna, Austria. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: CSR, class switch recombination; OPG, osteoprotegerin; PNA, peanut agglutinin; RANK, receptor activator of NF-kB; RANKL,
receptor activator of NF-kB ligand; SHM, somatic hypermutation; SRBC, sheep RBC.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102063
gammaglobulinemia, or impaired Ab responses to Ags (11).
Denosumab is a fully human mAb against RANKL that has recently been approved as a treatment against osteoporosis of
postmenopausal women, bone loss in men undergoing hormone
ablation therapy against prostate cancer, and the effects of bone
metastases from solid tumors (12–19). It has therefore been proposed that blocking RANKL might directly interfere with B cell
function. However, both mice and human patients lacking functional RANK or RANKL develop osteopetrosis because of the
absence of osteoclasts (2, 3, 11, 20). Osteopetrotic bones are
denser than normal bones and exhibit a lack of bone marrow
cavities, which are the regular environment for B cell development
(1). Therefore, it is also conceivable that B cell defects in RANK
deficient mice and human patients are secondary to the absence of
the natural site of B cell development—the bone marrow cavities.
To address this question, we generated mice that lack RANK
specifically in B cells. In this study, we report that mice that lack
RANK in B cells generate regular primary and secondary lymphoid
organs, and RANK-deleted B cells undergo development normally.
Furthermore, we demonstrate that basic B cell functions involved
in humoral immunity such as Ab secretion, Ig class switch recombination (CSR), or somatic hypermutation (SHM) are undisturbed
in the absence of RANK signaling. These data indicate that
RANKL/RANK have no direct involvement in B cell physiology.
Materials and Methods
Mice
Mice carrying the conditional RANKFlox (RANKF) allele and the RANKD
allele have been described previously (21). The mutant RANK alleles were
backcrossed to C57BL/6 more than ten times. MB1-cre mice (22) were
obtained on a C57BL/6 background. We crossed RANKF/F females with
mb1-cre+ RANKD/+ males to obtain mb1-cre+ RANKD/F experimental mice
and RANKF/+ control animals. All mice were maintained at the animal
colony of Institute of Molecular Biotechnology according to institutional
guidelines.
Fluorescence activated cell sorting
The following biotin-, FITC-, PE-, CyChrome-, PE Cy5-, or allophycocyanin-coupled Abs were used for flow cytometry: anti-B220 (RA3-6B2),
CD3ε (145-2C11), CD5 (53-7.3), CD8a (53-6.7), CD11b/Mac-1 (M1/70),
CD11c (HL3), CD21 (7G6), CD23 (B3B4), CD25 (PC61), CD28 (37.51),
CD95/Fas (Jo2), CD117/c-Kit (2B8), CD138 (281-2), IgD (11-26c.2a),
IgG1 (356), IgG3 (R40-82), Igk (187.1), Igl (R26-46) (all Abs from BD),
anti-CD4 (RM4-5), CD93 (AA4.1), IgM (II/41; Abs from eBioscience),
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
R
eceptor activator of NF-kB ligand (RANKL, also known
as ODF, TRANCE, OPGL, and TNFSF11) is the activating ligand of the receptor RANK (receptor activator
of NF-kB, also known as TRANCE-R and TNFRSF11A), whereas
osteoprotegerin (OPG; also known as OCIF, TNFRSF11B, TR1,
FDRC1) acts as a natural decoy receptor for RANKL and,
therefore, as a negative regulator of RANKL–RANK signaling
(1). The RANKL/RANK/OPG system was shown to regulate
a number of physiologic processes. RANKL–RANK signaling is
essential for bone remodeling by regulating osteoclast development and function (1). Moreover, RANKL and RANK are indispensable for lymph node organogenesis (2, 3), for the
development of a lactating mammary gland (4), the formation of
AIRE+ medullary thymic epithelial cells (5), and were shown to
regulate the fever response in the CNS (6). In addition, RANKL–
RANK can promote progestin driven breast cancer (7), can act as
a soil factor for cancer cells metastasizing to bone (8), or drive
local invasion of experimental breast and prostate tumors (9).
Interestingly, it was also reported that B lymphocytes express
RANK (10). The analysis of mice carrying a germline deletion
of either RANK or RANKL revealed dramatic defects in B cell
development, resulting in reduced numbers of peripheral B cells
(2, 3). Importantly, human patients with a mutation in RANK also
exhibit B cell defects such as reduced serum Ig levels, hypo-
1202
RANK IN B CELLS
anti-CD19 (6D5; from BioLegend), and fluorescein-coupled peanut agglutinin (PNA) from Vector Laboratories. Single-cell suspensions were
preincubated with CD16/CD32 Fc block (BD) and stained with the respective Abs. Plasma cells were identified as CD138hiCD28+Lin2 (CD42
CD8a2CD11b2CD192) where lineage depletion was performed by
MACS sorting (Miltenyi Biotec). FACS analysis was performed on a
FACSCalibur (BD Biosciences), a FACSCanto (BD Biosciences), and on
an LSRFortessa (BD Biosciences) apparatus. Cell sorts were performed
using a FACSAria (BD Biosciences) apparatus.
Absolute splenocyte numbers were determined by counting total splenocytes after RBC lysis with a CASY1 counter and subsequent calculation
of T cell and B cell numbers based on ratios from FACS experiments.
Histology
Cryosections from spleens were fixed with acetone and blocked in TBS,
0.1% BSA. Samples were incubated with biotin-coupled TCR-b (H57-597)
or FITC-coupled anti-B220 (RA3-6B2) or IgD (11-26c.2a) Abs (BD) or
with biotinylated PNA, followed by streptavidin-HRP (BD) and anti
Fluorescein alkaline phosphatase Fab Fragments (Roche). Fast Diaminobenzidine tablets (Sigma) and Fast Blue RR salt (Sigma) were used for
detection according to the manufacturers’ instructions.
Immunizations
Fresh sheep RBCs (Innovative Research) were washed in PBS three times;
1 3 108 cells per mouse were injected i.p. Analysis was done after 12 d.
Somatic hypermutation assay
Efficiency of SHM was assesses as described previously (23). B220+PNAhi
CD95+ splenic germinal center B cells were sorted from sheep RBC
(SRBC)-immunized mice. Upon DNA extraction, the VHJ558–JH4 intragenic region was PCR amplified with Pfu DNA polymerase (Stratagene),
cloned into Zero Blunt-TOPO vector (Invitrogen), sequenced, and analyzed in comparison with nonimmunized splenocytes.
Class switch recombination
CD432 B cells were isolated from spleens by MACS (Miltenyi Biotec) and
stimulated for 5 d with LPS (20 mg/ml) or IL-4 (50 ng/ml) plus aCD40
(1 mg/ml) to induce switching to IgG3 or IgG1, respectively. Percentages
of switched B lymphocytes were assessed by flow cytometry (24).
PCR analyses
RANK wt (256 bp), floxed (390 bp), and D (566 bp) alleles were identified
using PCR. The following primers and amplification conditions were used:
59-GGCAGAACTCGGATGCACAGATTGG-39, 59-AGTGTGCCTGGCATGTGCAGACCTT-39, 59-CTGGTGGTTGTTCTCCTGGTGTCAT-39 with
35 cycles of 95˚C for 30 min, 60˚C for 30 min, and 72˚C for 30 min. For
detection of RANK mRNA expression, RNA was isolated with Trizol
(Invitrogen), followed by reverse transcription with SuperscriptII (Invitrogen) and random hexamers (Roche). Real time PCR was performed using
the oligonucleotides 59-CCAGGAGAGGCATTATGAG-39 and 59-CATTCCAGGTGTCCAAGTA-39 (40 cycles of 95˚C for 10 min, 60˚C for 60 min).
Determination of Ig levels
Results
Serum levels of IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA were assessed
using the Milliplex Map Kit (Millipore) with subsequent analysis on a
Luminex test instrumentation. IgE serum ELISA (BioLegend) was performed according to the manufacturer’s instructions.
Deletion of RANK in B cells
Table I.
To analyze a direct effect of RANK deficiency on B cells, we used
the conditional RANK allele in which exons 2 and 3 are flanked by
Spleen cellularity
Cell Number (3106)
RANKDB
Control
Total Splenocytes
CD3ε+ Splenic T Cells
CD19+ Splenic B Cells
IgM+ Splenic B Cells
40.6 6 10.4
36.9 6 9.5
12.6 6 4.1
9.7 6 2.5
20.8 6 6.3
19.8 6 4.5
20.5 6 6.1
19.4 6 4.3
Total splenocyte numbers, as well as CD3ε+ splenic T cell, CD19+, or IgM+ splenic B cell numbers are shown. Each value
represents the average 6 SD from four RANKDB and seven control mice at 5–8 wk of age. Numbers from RANKDB and control
animals do not show statistical significant differences (two-tailed t test).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. B cell development. A,
FACS analysis of pro-B cells (IgM2
CD19+c-kit+), pre-B cells (IgM2CD19+
CD25+), and immature B cells (IgM+
B220int) in bone marrow. B, FACS
analysis of immature (B220+CD93+)
and mature B cells (B220+CD932; left
panels); T1 (IgM+CD232), T2 (IgM+
CD23+), and T3 (IgMloCD23+) transitional B cells (middle panels), and follicular B cells (B220+CD21+CD23+)
and marginal zone B cells (CD21hi
CD23lo; right panels) in spleen. C,
FACS analysis of B-1B cells (B220+
CD11b+IgM+CD5+) isolated from the
peritoneal cavity. D, FACS analysis of
plasma cells (Lin2CD28+CD138+) in
bone marrow (left panels) and spleen
(right panels). FACS blots are representative of at least three independent
experiments and at least three mice per
group, 6–12 wk of age.
The Journal of Immunology
loxP sites. Cre-mediated deletion of exons 2 and 3 results in a frame
shift and a RANK null allele (Supplemental Fig. 1A) (21). For a
specific deletion in B cells, we crossed the conditional RANK
allele to mb1-cre mice, which efficiently delete in the complete
B cell lineage from the earliest pro B cell stage onwards (22).
Efficiency of Cre-mediated deletion of RANK exons 2 and 3 and
consequent loss of RANK expression in B cells was confirmed by
PCR for the deleted and floxed alleles (Supplemental Fig. 1B) and
the absence of RANK mRNA expression (Supplemental Fig. 1C).
In all the experiments described below, we analyzed mb1-cre+
RANKD/F mice (hereafter termed RANKDB) that lack RANK
in B cells, and, as controls, RANKF/+ animals. Both experimental
mice and controls were obtained as littermates by crossing
RANKF/F females to mb1-cre+ RANKD/+ males.
Normal B cell development
It has been reported that absence of RANKL–RANK signaling
in mice can lead to a block in B cell development (2, 3). We
therefore analyzed B lymphocytes in bone marrow, spleen, and
FIGURE 3. Ig levels in serum. Serum Ig levels
of all Ig isotypes from nonimmunized (A) or
SRBC-immunized (B) mice were assessed. Values
obtained from RANKDB animals are shown as
triangles, values obtained from control animals
are shown as squares and are shown for each Ig
isotype for both nonimmunized (A) and SRBCimmunized (B) mice. Triangles and squares represent values from individual animals. Data are
shown as mean 6 SEM. Data are shown from
three independent experiments. Three to 11 mice,
6–14 wk of age, were used per group.
lymph nodes by FACS analysis. B cells develop in the bone
marrow from hematopoietic stem cells to pro-B cells (IgM2
CD19+c-kit+), pre-B cells (IgM2CD19+CD25+), and immature
B cells (IgM+B220int). Mice lacking Rank in B cells display
similar frequencies of pro-B cells, pre-B cells, and immature
B cells in bone marrow when compared with control RANKF/+
animals (Fig. 1A). In spleens, we analyzed population frequencies
of immature (B220+CD93+) and mature B cells (B220+CD932).
B220+CD93+ immature B cells can be subdivided into T1 (IgM+
CD232), T2 (IgM+CD23+), and T3 (IgMloCD23+) transitional
B cell subsets. Furthermore, we analyzed the mature B cell subsets
of follicular B cells (B220+CD21+CD23+) and marginal zone
B cells (CD21hiCD23lo). All of those B cell populations were
present at comparable ratios in RANKDB and control mice (Fig.
1B). Moreover, ratios of Igk and Igl expressing B cells in bone
marrow and spleen were similar in RANKDB and control mice
(Supplemental Fig. 2). We finally analyzed peritoneal B-1 B cells
(B220+CD11b+IgM+CD5+; Fig. 1C) and plasma cells (Lin2
CD28+CD138+) in bone marrow and spleen (Fig. 1D). Again, all
those B cell populations showed similar frequencies in the presence and absence of RANK. Finally, assessment of total splenocyte numbers, as well as CD3ε+ splenic T cell, CD19+, or IgM+
splenic B cell numbers resulted in similar values (two-tailed t test)
in RANKDB and control animals (Table I). Therefore, we conclude
that RANK deletion in B cells does not affect B cell development
in bone marrow, spleen, and the peritoneal cavity.
Formation of secondary lymphoid organs
In RANKL-deficient mice, it was observed that B cell zones in
spleens were dramatically reduced in size (3) (Fig. 2A). However, it
was not clear whether this phenotype could be directly attributed
to the lack of RANKL–RANK signaling in B cells or whether
it was secondary for example to the osteopetrosis phenotype and
a resulting defect in lymphocyte development. To answer this
question directly, we first analyzed spleen sections of RANKDB
mice. Histologic stainings of B and T lymphocytes revealed normal B and T cell zones in splenic white pulp in RANKDB mice
(Fig. 2B). Moreover, we observed normal development of splenic
germinal centers upon immunization with SRBCs (Fig. 2C). This
result was corroborated by quantification of B220+PNAhi germinal center B cells by FACS analysis (Supplemental Fig. 3).
Therefore, splenic architecture and organization of B cell and
T cell zones do not depend on RANK signaling in B lymphocytes.
It was reported that mice with a germline deletion of RANK or
RANKL do not develop any lymph nodes, but do develop Peyer’s
patches at reduced numbers and size (2, 3). Moreover, human
patients with germline mutations in RANK do not show palpable
lymph nodes. Therefore, we examined axillary, inguinal, mesenteric, and popliteal lymph nodes as well as Peyer’s patches but did
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FIGURE 2. Histologic analysis of RANKL2/2 and RANKDB spleen
sections. A, White pulp in spleens from RANKL2/2 and wild type mice
stained for IgD (brown). Original magnification 350. B, White pulp in
spleens from RANKDB and control mice stained with anti-B220 (blue) and
anti–TCR-b (brown) Abs to identify B cell and T cell zones. Original
magnification 320. C, White pulp in spleens from SRBC immunized
RANKDB and control mice stained with an anti-IgD (blue) Ab and PNA
(brown) to identify B cell follicles and germinal centers, respectively.
Original magnification 320. Representative results are shown from at least
three independent experiments and at least four mice per group, 8–12 wk
of age, with similar results.
1203
1204
not find an obvious defect in morphology or in numbers of these
organs in RANKDB mice (not shown). Ratios of B cells and T cells
in Peyer’s patches and lymph nodes were comparable in RANKDB
and control animals (Supplemental Fig. 4). Therefore, we conclude that RANKL–RANK signaling in B cells is dispensable for
the generation of lymph nodes.
Intact B cell functions in RANKDB mice
Discussion
We show that B cell intrinsic expression of RANK is not required
for normal development or homeostasis of B lymphocytes in bone
marrow and in the peripheral lymphoid organs. RANKDB mice
exhibit normal populations of pro-B cells, pre-B cells, immature
or transitional B cells, mature B cells such as marginal zone
B cells and follicular B cells, and plasma cells in bone marrow and
spleen as well as normal numbers of B-1 B cells in the peritoneum. Our data differ from previously published work on mice
carrying a germline mutation for either RANK or RANKL (2, 3).
FIGURE 4. Class switch recombination. FACS analysis of splenic
B cells from RANKDB and control mice stimulated with IL-4/anti-CD40
(A) or with LPS (B) inducing class switch recombination to IgG1 (A)
or IgG3 (B), respectively. FACS blots are representative of four independent
experiments. Four mice per group, 7–12 wk of age, were used with similar
results.
Table II. Somatic hypermutation
Mouse
DB
RANK 1
RANKDB 2
Control 1
Control 2
Independent
Sequences
Bp Analyzed
Mutations
73
82
38
45
40,953
44,880
21,318
25,245
490
463
132
214
Mutation Rate
1.2
1.0
0.6
0.8
3
3
3
3
1022
1022
1022
1022
B220+PNAhiCD95+ activated germinal center B cells from spleens of SRBCimmunized RANKDB and control mice were sorted by FACS. After DNA isolation
and amplification, the mutation rate (mutations per bp) was assessed by DNA sequencing. Only independent sequences were included in the analysis.
In these mice, a dramatic reduction of peripheral B cells was
observed, combined with a partial developmental block from the
pro- to the pre-B cell stage in RANKL-deficient mice (3). Because
such whole body RANKL and RANK mutant mice exhibit severe
osteopetrosis, it has been difficult to ascertain whether RANKL–
RANK act directly in B cells. Osteopetrotic bones of RANKL and
RANK knockout mice are denser than normal bones and, importantly, exhibit an almost complete lack of bone marrow cavities. The bone marrow is the natural environment for B cell
development as well as the residence of hematopoietic stem cells
from which all immune cell types are derived (25). Therefore, one
feasible theory has always been that the observed B cell defects
are secondary to the absence of bone marrow cavities in RANKLand RANK-deficient mice rather than being a direct effect of
absent RANKL–RANK signaling on B lymphocytes. Our data
strongly support an argument against a direct effect of RANKL–
RANK signaling on B cell development.
In RANKDB mice, we found Peyer’s patches and lymph nodes
of normal morphology with normal B cell and T cell numbers. In
contrast, germline mutations of RANKL or RANK result in the
complete absence of lymph nodes in mice (2, 3). Similarly, human
patients carrying inactivating RANKL and RANK mutations
lack palpable lymph nodes (11, 20). Our data demonstrate that
RANKL–RANK signaling in B cells is not a requirement for the
development of lymph nodes. The exact cellular and molecular
mechanisms by which RANKL and RANK control lymph node
organogenesis during embryogenesis remain largely elusive
(1, 26).
It was reported that human patients carrying RANK mutations
can exhibit disturbed B cell functions resulting in conditions
such as hypogammaglobulinemia, a lack of Ab response to Ag,
or in the presence of reduced serum Ig levels (11). Because we
observed high RANK expression levels in IL-4/anti-CD40 activated B cells (Supplemental Fig. 1C), we investigated various
functions of activated B cells, including CSR, SHM, and Ab secretion to serum with or without immunization. Again, RANKDB
mice did not display any obvious defects, arguing against an essential role for RANKL–RANK signaling in B lymphocyte
functions.
Our work examines various aspects of B cell biology in the
absence of RANKL–RANK signaling, without identifying an essential function of RANK in B lymphocytes. That leaves us with
the question of the actual biologic function of the observed expression of RANK in B cells. One possible explanation is a
functional redundancy of RANK signaling in B cells. Alternatively, although we performed comprehensive analyses of B cell
development and basic B cell functions, the potential role of
RANK signaling in B cells could be hidden in more complex
functions of B cells that were not addressed in this study. Overall,
RANK signaling does not seem to have a major role in B cell
physiology.
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It was reported that RANK mutations in human patients can lead
to reduced Ig levels in serum and to impaired Ab responses to Ags.
To assess whether RANK deficiency in B cells can cause such
phenotypes, we measured serum Ig levels in nonimmunized and
in SRBC-immunized mice. We determined levels of serum IgM,
IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE in RANKDB and control
mice, but did not detect a significant difference in any of the
Ig isotypes, neither in nonimmunized nor in SRBC-immunized
mice (Fig. 3). Another way to investigate efficiency of Ig CSR is
to stimulate splenic B cells in vitro. Stimulation with LPS leads
to switching to IgG2b and IgG3, whereas stimulation with IL-4
and aCD40 results in switching to IgG1 and IgE. Efficiency of
CSR was similar in B cells from RANKDB and from control
mice (Fig. 4). Finally, we determined SHM in RANKDB mice. To
this end, we immunized mice with SRBCs and FACS sorted
B220+PNAhiCD95+ activated B cells that undergo SHM (23).
We did not observe an SHM defect in B cells lacking RANK,
as the mutation rates were comparable in RANKDB and control
animals (Table II). These data indicate that RANK signaling in
B cells is dispensable for Ab production, efficient Ig CSR, and
SHM.
RANK IN B CELLS
The Journal of Immunology
Acknowledgments
We thank Dr. Shane Cronin, Dr. Ulrich Elling, Dr. Reiko Hanada,
Dr. Toshikatsu Hanada, Dr. Gregory Neeley, Dr. Daniel Schramek, and
Dr. Gerald Wirnsberger for helpful comments and discussions, Qiong Sun
for help with histologic stainings, and the Institute of Molecular Biotechnology service departments, especially Gerald Schmauss and Harald Scheuch,
for technical support.
Disclosures
J.M.P. owns stock of Amgen. The other author has no financial conflicts
of interest.
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