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Direct B cell stimulation by dendritic cells in a mouse model of lupus.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 6, June 2008, pp 1741–1750
DOI 10.1002/art.23515
© 2008, American College of Rheumatology
Direct B Cell Stimulation by Dendritic Cells in
a Mouse Model of Lupus
Suigui Wan, Zhenhai Zhou, Biyan Duan, and Laurence Morel
Objective. Dendritic cells (DCs) play a major role
in regulating lymphocytes, including B cells, and defective DC functions have been implicated in lupus. The
purpose of this study was to assess the contribution of
DCs to B cell hyperactivity in the B6.Sle1.Sle2.Sle3
(B6.TC) murine lupus model.
Methods. We compared the effects of B6 and
B6.TC bone marrow–derived DCs on naive B cells
cocultured in the presence of lipopolysaccharide (LPS),
anti-CD40, or anti-IgM. We measured the proliferation,
antibody production, and expression of activation markers and chemokine receptors for the B cells, as well as
DC cytokine production. B cell proliferation was also
assessed in Transwell experiments and in response to
activated DC supernatants or exosomes. The role of
DC-produced cytokines was evaluated with blocking
antibodies and transgenic mice.
Results. LPS-stimulated or anti-CD40–stimulated
DCs from B6.TC mice increased B cell proliferation,
antibody production, and chemokine receptor expression as compared with DCs from B6 mice. Cell-to-cell
contact was not necessary for the augmented effect of
the lupus-prone DCs. Anti-CD40 treatment induced a
higher production of interleukin-6 (IL-6), soluble IL-6
receptor (sIL-6R), IL-10, and tumor necrosis factor ␣ in
B6.TC DCs. Blocking these individual cytokines, however, did not abrogate the effects of B6.TC DCs. Addi-
tional experiments also ruled out involvement of BAFF,
IL-12, and interferon-␣.
Conclusion. Activated DCs from B6.TC mice directly increase B cell effector functions. This effect
depends on soluble factors released by activated DCs,
but none of the single major DC-produced cytokines
known to affect B cells are necessary. Increased sIL-6R
production suggests that increased sensitivity to IL-6
may be involved.
Lupus has been described as a disease of B cell
hyperactivity (1), based on data accumulated both in
patients and in mouse models of the disease, and this
body of work has provided the rationale for the various
B cell–targeting therapies currently under consideration
(2). Intrinsic defects in B cell tolerance have been
demonstrated in lupus-prone mice (3). Multiple studies,
however, have shown that T cell help was necessary for
B cell hyperactivity and the production of pathogenic
autoantibodies (4). The role of other cellular components of the immune system in inducing the lupus B cell
phenotypes has been given less attention. A growing
number of studies concur in showing that dendritic cells
(DCs) play a critical role in B cell development, mostly
through the production of survival factors, such as
BAFF, or cytokines, such as interleukin-12 (IL-12), IL-6,
and interferon-␣ (IFN␣) (5).
More recently, intravital microscopy has demonstrated direct cognate antigen transfer from DCs to B
cells (6). DCs have been implicated in the pathogenesis
of lupus by a number of studies, including one conducted
in the B6.Sle1.Sle2.Sle3 (B6.TC) congenic mouse strain
derived from the NZM2410 model (7). Increased numbers of DCs accumulate in the lymphoid organs of
B6.TC and other lupus-prone mice (8–12). Abnormal
costimulatory profiles have also been reported in DCs
from lupus patients (13–15) and in (NZB ⫻ NZW)F1
(BWF1), NZM2410, and B6.TC mice (12,16). Injections
of BWF1 syngeneic DCs primed with apoptotic cells
Dr. Morel’s work was supported by NIH grants R01-AI045050 and R01-AI-058150.
Suigui Wan, MD (current address: Xuanwu Hospital, Capital
University of Medical Science, Beijing, China), Zhenhai Zhou, MD,
Biyan Duan, PhD (current address: University of Texas Southwestern
Medical Center, Dallas), Laurence Morel, PhD: University of Florida,
Gainesville.
Drs. Wan and Zhou contributed equally to this work.
Address correspondence and reprint requests to Laurence
Morel, PhD, Department of Pathology, Immunology and Laboratory
Medicine, University of Florida, PO Box 100275, Gainesville, FL
32610-0275. E-mail: morel@ufl.edu.
Submitted for publication July 10, 2007; accepted in revised
form February 25, 2008.
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accelerated disease (17). Furthermore, injections of
Sle3-expressing DCs into nonautoimmune C57BL/6 (B6)
mice induced the production of anti-DNA antibodies
(18). However, most of these studies have been interpreted as lupus-associated DCs producing altered signals and amplifying autoreactive specificities in T cells,
which, in turn, provide help to autoreactive B cells.
DCs can directly regulate B cells through the
production of IFN␣, whose elevated production has
been proposed to be at the core of lupus pathogenesis
(19). IFN␣ lowers the B cell receptor threshold of
activation (20) and accelerates differentiation into
plasma cells (21). In contrast to humans with lupus,
there is little evidence for a type I IFN signature in
murine models of spontaneous disease (22). In fact, the
Sle2 locus, which contains the Ifa gene cluster, corresponds to a lower IFN␣ production (23). We have,
however, shown that DCs from B6.TC mice produce
large amounts of IL-6 in response to lipopolysaccharide
(LPS) (12) or anti-CD40 stimulation (the present study),
and this cytokine prevents CD4⫹,CD25⫹ Treg cells
from exerting their inhibitory function (12). This IL-6–
dependent blockade of Treg function maps at least
partly to Sle1a (24). IL-6 is a proinflammatory cytokine
that induces B cell proliferation and differentiation into
plasma cells (25,26). We therefore assessed the effect of
B6.TC-derived DCs on B cell proliferation and differentiation.
Our congenic experimental system allows the
direct comparison of B6, B6.TC, and B6.Sle1a DCs on
the same B cells, either B6-derived or B6.TC-derived.
B6 and B6.TC mice share ⬎95% of their genome,
including their major histocompatibility complex
(MHC), B cell, and T cell receptor genes, and therefore represent a well-controlled experimental system.
We show here that LPS- or anti-CD40–stimulated
DCs from B6.TC mice supported a stronger B cell
proliferation and antibody production than DCs from
B6 mice. Furthermore, DCs from the lupus-prone
mice induced an increased expression of the chemokine receptors that direct the B cells toward the
follicles, T cell zones, and germinal centers. Cell-tocell contact between DCs and B cells increased overall
B cell proliferation, but was not necessary for the
differential effect of the lupus-prone DCs, and supernatant from activated B6.TC DCs was sufficient to
induce a more robust B cell proliferation.
Anti-CD40 treatment induced a higher production of IL-6, IL-10, and tumor necrosis factor ␣
(TNF␣) in DCs from B6.TC mice than in those from
B6 mice. Blocking these individual cytokines, how-
WAN ET AL
ever, did not abrogate the increased effect of the
B6.TC DCs. Additional experiments also ruled out
involvement of BAFF, IL-12, and IFN␣. Activated
B6.TC DCs also produced higher levels of soluble
IL-6 receptor (sIL-6R), suggesting that IL-6 transsignaling may be involved in the lupus DC–B cell
interactions.
Our findings show that B6.TC DCs act directly on
B cells to increase their effector function and that the
Sle1a locus plays a major role in this phenotype. This
effect depends on soluble factors produced by activated
DCs, but none of the major DC-produced cytokines that
are known to affect B cell growth and differentiation are
necessary.
MATERIALS AND METHODS
Mice. The production of the triple congenic
B6.NZM2410-Sle1.Sle2.Sle3 (B6.TC), B6.NZM2410-Sle1a
(B6.Sle1a), and B6.NZM2410-Sle1.Sle2.Sle3.STAT-4 –/–
(B6.TC.STAT-4–/–) mouse strains has been described previously (7,27,28). C57BL/6J (B6) mice were bred at the University of Florida at Gainesville. B6.129S6-Il6tm1Kopf (B6.IL-6–/–)
and 129S1/SvImJ (referred to as 129) mice were obtained from
The Jackson Laboratory (Bar Harbor, ME). Mice of the
129/Sv.IFNAR–/–strain (29) were a kind gift from Dr. Westley
Reeves (University of Florida, Gainesville, FL). All mice used
in our experiments were 2–3-month-old females. All experiments were conducted according to protocols approved by the
Institutional Animal Care and Use Committee of the University of Florida.
DC and B cell isolation. Single-cell suspensions of
bone marrow (BM) cells were cultured in complete RPMI
1640 containing 10% fetal calf serum (FCS), 10 ng/ml of
granulocyte–macrophage colony-stimulating factor, and 10
ng/ml of IL-4 (R&D Systems, Minneapolis, MN). Every 2 days,
50% of the culture medium was replaced with fresh medium.
Cells were harvested after 6 days of culture, ascertained by
flow cytometry, and then CD11c⫹ cells were positively selected with magnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Purity of the
CD11c⫹ fraction was ⬎90%. Splenic B cells were purified by
CD43 negative selection (B cell isolation kit; Miltenyi Biotec),
and the purity of the B220high fraction was typically ⬎95%.
CXCL13 (B lymphocyte chemoattractant [BLC]) production
by DCs was assessed by quantitative reverse transcription–
polymerase chain reaction (RT-PCR), as described by Ishikawa et al (9).
Flow cytometry. Single-cell suspensions were treated
with Fc receptor block (2.4G2) and then stained with
monoclonal antibodies to mouse B220 (RA3-6B2), CD11c
(HL3), CD80 (16-10A1), CD86 (GL1), CXCR4 (2B11),
CXCR5 (2G8), or I-Ab (AF6-120.1) (all from BD PharMingen, San Diego, CA), or BAFF receptor (BAFF-R; clone
204406) or CCR7 (4B12) (both from R&D Systems), or
isotype controls. Intracellular cytokine production was assayed by adding GolgiPlug (BD PharMingen) for the last 5
B CELL STIMULATION OF DCs IN MURINE LUPUS
hours of stimulation. Cells were then stained with the
appropriate antibodies to gate on the population of interest, and with anti–IL-6 (MP5-20F3; BD PharMingen) or
BAFF (clone 121808; R&D Systems) after cell permeabilization.
At least 50,000 cells were acquired per sample using
a FACSCalibur cytometer (BD Biosciences, Mountain
View, CA), and dead cells were excluded by forward and
side scatter characteristics. ModFit software (Verity Software House, Topsham, ME) was used to analyze the 5,6carboxyfluorescein succinimidyl ester (CFSE; Invitrogen,
Carlsbad, CA) dilution.
DC and B cell cocultures. BM-derived DCs (2 ⫻ 104)
from B6 or B6.TC mice were cocultured with 105 B cells from
B6, B6.TC, B6.IL-6–/–, B6.TC.STAT-4–/–, or 129 mice in 96
round-well Costar plates (Sigma-Aldrich, St. Louis, MO) for 5
days in complete RPMI 1640 in the presence of either anti-IgM
Fab (R&D Systems) or anti-CD40 (3/23; BD PharMingen) at
10 ␮g/ml each or in the presence of 1 ␮g/ml of LPS (SigmaAldrich). In some experiments, 2 ⫻ 104 DCs were stimulated
first with anti-CD40 or LPS for 24 hours, and then the
supernatant was added to 105 B cells, which were then cultured
for 5 days.
For Transwell experiments, 3 ⫻ 104 B cells were
placed in the bottom wells and 1.5 ⫻ 104 DCs were placed
in the top wells in the presence of anti-CD40 in both wells.
After 5 days of culture, B cell proliferation was measured
according to the CFSE dilution or with the CellTiter 96
AQueous One Solution Cell Proliferation Assay (Promega,
Madison, WI); 1 ␮Ci of 3H-thymidine was added to each
well for the last 16 hours of culture. In some assays,
anti–IL-6 (MP5-20F3; BD PharMingen), anti–IL-10 (A5-4;
BD PharMingen), or anti-TNF␣ (MP6-XT22; R&D Systems) blocking antibodies or isotype controls were added at
5 ␮g/ml. After 72 hours of culture, IL-6, IL-10, IL-12, TNF␣,
and sIL-6R (CD126) were measured in supernatants by
sandwich enzyme-linked immunosorbent assay (ELISA)
(R&D Systems).
Exosomes were isolated as previously described (30).
Briefly, DC supernatants were harvested and centrifuged at
300g for 10 minutes, 1,200g for 20 minutes, and 10,000g for 30
minutes to remove cell debris. Exosomes were then pelleted at
100,000g for 1 hour at 4°C and resuspended in cold phosphate
buffered saline. The presence of exosomes was confirmed by
measuring the protein concentration in the pelleted samples.
Exosomes collected from the supernatant of 2 ⫻ 104 DCs were
added to 105 B cells, which were then cultured as described
above. IgM and IgG production after 4 days of coculture was
measured by sandwich ELISA, as previously described (7), in
supernatants diluted 1:10. All experiments were performed at
least twice, and representative results are shown.
Statistical analysis. Statistical analysis was performed with the GraphPad Prism 4 package (GraphPad
Software, San Diego, CA). Data were compared using t-tests
after verification of normal distribution. Dunnett’s tests
were used when multiple comparisons were performed in
the same experiment. P values less than or equal to 0.05
were considered significant.
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RESULTS
Proliferation of, and production of antibodies by,
naive B cells in the presence of activated DCs from
lupus-prone mice. CD43– naive B cells from B6 mice
proliferated significantly more when cocultured with
anti-CD40–stimulated BM-derived DCs from lupusprone B6.TC than those from B6 controls (Figure 1A).
We have previously shown that Sle1a results in increased
DC activation (24). We therefore tested the effects of
B6.Sle1a DCs on B cells, and the results were the same
as with DCs from the B6.TC mice (Figure 1B), indicating that this locus plays a significant role in this phenotype. DCs from B6 (P ⫽ 0.006) and B6.TC (P ⫽ 0.026)
mice increased B cell proliferation induced by B cell
receptor cross-linking with anti-IgM. However, no difference was consistently observed between these 2 types
of DCs, although in a few assays (see Figure 6E below),
proliferation was increased with B6.TC DCs in the
presence of anti-IgM.
This result suggested that the differential proliferation was mediated by DCs that had been activated
through CD40 signaling. This was confirmed by the fact
that DCs from B6 and B6.TC mice did not show
increased B cell proliferation over that of medium alone
in the absence of anti-CD40 (Figure 1E) and that DCs
from neither strain proliferated in response to antiCD40 (Figure 1D). As expected, B cells proliferated to
anti-CD40 treatment alone (Figure 1E), but the presence of B6.TC DCs significantly enhanced the process.
Direct evidence of increased B cell proliferation
was obtained from CFSE dilution studies of B220⫹
gated cells after coculture in the presence of anti-CD40
and DCs from B6, B6.TC, or B6.Sle1a mice (Figure 1C).
Similar results were obtained with B cells derived from
B6.TC mice (Figure 1D). In the absence of DCs, B cells
from B6.TC mice proliferated more to CD40 stimulation
than did B cells from B6 mice (compare Figures 1C and
D), but the addition of DCs resulted in a similar
response in B cells from both strains. This result ruled
out the possibility that the differential response to
B6.TC DCs was due to alloreactivity between the 2
strains. No significant proliferation was observed in DCs
from either B6 or B6.TC mice in response to anti-CD40
(data not shown). The increased ability of B6.TC DCs to
induce B cell proliferation was not specific to stimulation through CD40 since LPS stimulation produced the
same effect (Figure 1F). These data indicate that activation induces in B6.TC DCs an enhanced ability to
promote B cell proliferation.
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WAN ET AL
Figure 1. Induction of stronger B cell proliferation by activated dendritic cells (DCs) from
B6.TC and B6.Sle1a mice. A and B, Induction of thymidine incorporation. B6.TC (TC) or
B6.Sle1a (Sle1a) bone marrow–derived DCs induced greater thymidine incorporation in B6
B cells in the presence of anti-CD40 (␣CD40), but not anti-IgM (␣IgM), than did B6 DCs.
C and D, Proliferation of B cells from B6 (C) or B6.TC (D) mice, as measured by the
5,6-carboxyfluorescein succinimidyl ester (CFSE) dilution in the last 4 divisions of B220⫹
gated cells in the presence of B6 or B6.TC DCs and anti-CD40. Representative flow
cytometry plots are shown at the right. Shaded areas show the cell division peaks predicted
by the ModFit software. Horizontal bars show the gate for CFSElow. E, Proliferation of B
cells from B6 mice. DCs did not promote B6 B cell proliferation in the absence of anti-CD40
stimulation. Anti-IgM– and anti-CD40–stimulated B cells in the absence of DCs were used
as controls. F, Proliferation of B cells from B6 mice with and without lipopolysaccharide
(LPS; 1 ␮g/ml) stimulation. LPS stimulation of DCs from B6.TC mice induced B6 B cells to
proliferate significantly more than did those from B6 mice. Values are the mean and SEM
of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 for the comparisons
indicated or versus B6 DCs, by t-test.
B CELL STIMULATION OF DCs IN MURINE LUPUS
Figure 2. Induction of stronger antibody production by dendritic cells
(DCs) from B6.TC mice in the presence of anti-CD40. Production of
A, total IgM and B, total IgG by B cells from B6 mice after 4 days in
culture with B6 or B6.TC (TC) DCs in the presence of anti-IgM
(␣IgM) or anti-CD40 (␣CD40). OD ⫽ optical density (at 405 nm). C,
Plasma cell differentiation B cells from B6 mice cocultured with B6 or
B6.TC DCs, as measured by CD138 expression. D, B6.TC DC induction of a lower surface IgM expression on CD138⫹ B6 B cells in the
presence of anti-CD40. Values are the mean and SEM of 4 mice per
group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by t-test.
Naive B cells from B6 mice produced significantly
more IgM and IgG antibodies in the presence of antiCD40–activated DCs than with B cell receptor crosslinking and DCs (Figures 2A and B). Anti-CD40–
stimulated DCs from B6.TC mice showed significantly
higher production of both IgM and IgG than those from
B6 mice. The induction of plasma cell differentiation, as
measured by CD138 induction, was not appreciably
different in DCs from either strain (Figure 2C), but
surface IgM expression on CD138⫹ cells was significantly reduced in the presence of B6.TC DCs (Figure
2D), indicating faster progression to terminal plasma
cell differentiation.
Chemokine receptors play a critical role in directing B cells toward the various microenvironments of the
lymphoid tissues. CXCR5 ligation induces B cells to
move into the follicles (31), and relative levels of
CXCR5 and CXCR4 regulate movements within the
germinal centers (32). CCR7 expression is required for
B cells to enter the T cell zone (31). We found that
anti-CD40–activated DCs from B6.TC mice induced
1745
significantly higher levels of CXCR5, CXCR4, and
CCR7 expression on B6 B cells than on B6 DCs (Figures
3A, B, and C, respectively). Interestingly, B cells from
B6.TC mice expressed higher levels of CXCR5 and
CXCR4 than did B cells from B6 mice in the presence of
either B6 or B6.TC DCs.
Although DCs from old BWF1 mice secrete
CXCL13 (9,33), we were not able to detect CXCL13
expression by quantitative RT-PCR in BM-derived DCs
from young B6 or young B6.TC mice (data not shown).
In addition, we have previously shown that SDF-1 levels
were equivalent between B6 and B6.TC mice (34).
Therefore, these 2 chemokines cannot account for the
activation of B cells by B6.TC DCs. In contrast to
chemokine receptors, coculture with DCs did not increase B cell activation, as shown for class II MHC in the
present study (Figure 3D). Activation marker expression
was higher on B cells from B6.TC mice than on those
from B6 mice (as previously shown [7]), but it was
similar with either B6.TC or B6 mouse DCs. Similar
results were obtained with CD80 and CD86 stimulation
(data not shown).
DC stimulation of B cells through both contactdependent and contact-independent mechanisms. To
assess whether the increased B cell proliferation induced
Figure 3. Induction of increased expression of chemokine receptors,
but not activation markers, on B6 B cells by dendritic cells (DCs) from
B6.TC mice. The expression of A, CXCR5, B, CXCR4, C, CCR7, and
D, class II major histocompatibility complex I-Ab, as determined by
flow cytometry on B220⫹ gated B cells from B6 or B6.TC (TC) mice
after 5 days of coculture with either B6 or B6.TC DCs, is shown.
Values are the mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ
⫽ P ⬍ 0.01, by t-test.
1746
WAN ET AL
a significantly higher proliferation than supernatants
from B6 DCs (Figure 4B). The same results were
obtained with LPS-stimulated B6.TC DCs (data not
shown).
This effect was not due to DC-released exosomes
present in these supernatants (Figure 4C and data not
shown for exosomes extracted from LPS-activated DCs).
In fact, exosomes, which have been shown to carry
surface markers from the DC membrane (30), did not
induce B cell proliferation above background, implying
that soluble factors produced by activated B6.TC DCs
are responsible for the enhanced effector function on B
cells. These soluble factors do not involve the BAFF
pathway, since intracellular production of BAFF did not
differ between DCs from B6 and B6.TC mice after CD40
stimulation (mean ⫾ SEM 38.28 ⫾ 2.32%
CD11c⫹,BAFF⫹ cells for B6 DCs versus 41.15 ⫾ 2.89%
CD11c⫹,BAFF⫹ cells for B6.TC DCs) (Figure 5A).
Similarly, BAFF-R expression on the B cell surface did
not differ after culture with either B6 or B6.TC DCs
(Figure 5B).
Figure 4. Contact-independent enhancement of B cell proliferation
by dendritic cells (DCs) from B6.TC mice. A, Proliferation of B cells
from B6 mice after 5 days in simultaneous coculture with either B6 or
B6.TC DCs mixed together (left) or separated in Transwells (right). B,
Induction of B cell proliferation by anti-CD40 in supernatants and by
direct cell contact. Supernatants from anti-CD40–stimulated B6.TC
DCs induced B cell proliferation to a similar level as DCs cocultured
with B cells in the presence of anti-CD40. Absorbance was read at 450
nm. C, Induction of B cell proliferation by anti-CD40 in exosomes and
supernatants. Exosomes collected from anti-CD40–stimulated DCs
failed to induce B cell proliferation, as compared with supernatants
collected from the same cells. Values are the mean and SEM of 4 mice
per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by t-test.
by DCs from B6.TC mice was contact dependent, we
performed simultaneous Transwell and standard coculture experiments (Figure 4A). As expected, IgM stimulation in the presence of DCs was minimally reduced
when contact between B cells and DCs was prevented.
However, proliferation in the presence of anti-CD40
stimulation greatly decreased when contact was prevented between the B cells and DCs. This indicated that
despite the fact that the known effect of anti-CD40–
activated DCs on B cells is through the production of
cytokines, cell-to-cell contact is important. However, the
reduction was proportionally similar with DCs from both
strains (mean ⫾ SEM proliferation in Transwells as the
percentage of proliferation in conventional wells
45.07 ⫾ 4.17% for B6 DCs versus 39.48 ⫾ 3.99% B6.TC
DCs; P ⫽ 0.18). Moreover, supernatants from B6.TC
DCs activated for 24 hours with anti-CD40 still induced
Figure 5. Lack of involvement of BAFF in the activation of B6.TC
mouse dendritic cells (DCs) on mouse B cells. A, Representative
intracellular BAFF staining relative to CD11c expression in B6 B cell
cocultures with DCs from B6 or B6.TC (TC) mice. APC ⫽ antigenpresenting cells; PE ⫽ phycoerythrin. B, Surface expression of BAFF
receptor (BAFF-R) on B220⫹ cells after 5 days of coculture in the
presence of anti-CD40 (␣CD40). The histogram (right) shows representative BAFF-R expression on B220⫹ gated B6 (solid) or B6.TC
(open) DCs. Horizontal bar shows the gate for BAFF-R⫹. FITC ⫽
fluorescein isothiocyanate. Values are the mean and SEM of 4 mice
per group.
B CELL STIMULATION OF DCs IN MURINE LUPUS
In contrast, B6.TC DCs produced significantly
more IL-6 than did B6 DCs after anti-CD40 stimulation
of cocultures (Figure 6A). We further examined the
origin of IL-6 by intracellular staining and showed that
the B cells in the cocultures produced minimal amounts
of IL-6, which was similar in the presence of DCs from
either B6 or B6.TC mice (data not shown). Furthermore,
the same response to anti-CD40–stimulated B6.TC DCs
was similar in B cells from IL-6–deficient mice and B6
controls (data not shown), indicating that IL-6 did not
act in an autocrine manner in our system.
When IL-6 was neutralized with blocking antibodies at a concentration that was sufficient to abrogate
the effect of B6.TC DCs on Treg cells (12), B6.TC DCs
still induced greater proliferation than did B6 DCs
(Figure 6E). Soluble IL-6R can enhance IL-6 signaling
through a process known as trans-signaling (35), and
increased IL-6 production has been shown to increase
the production of sIL-6R (36). Interestingly, B6.TC DCs
produced significantly more sIL-6R than did B6 DCs
after anti-CD40 stimulation (Figure 6D), which could
lower the B cell threshold response to IL-6.
DCs from B6.TC mice also produced significantly
more IL-10 and TNF␣ than did DCs from B6 mice after
CD40 stimulation of cocultures (Figures 6B and C).
Blocking IL-10 or TNF␣ significantly decreased B cell
proliferation, but it did not abrogate the difference
between B6.TC and B6 DCs (Figure 6F). Under our
experimental conditions, IL-12, another DC-produced
cytokine that regulates B cell differentiation, was produced in lower but similar amounts by DCs from B6 or
B6.TC mice (data not shown).
We tested the involvement of IL-12 and IFN␣
indirectly by using B cells from STAT-4–deficient (Figure 6G) and IFN␣/␤/␻ receptor 1 (IFNAR-1)–deficient
(Figure 6H) mice, respectively. B cells from
B6.TC.STAT-4–/– mice proliferated more than did B
cells from B6.TC mice in the presence of anti-CD40–
stimulated DCs from either B6 or B6.TC mice (Figure
6G). It is not clear at this point why coculture with
B6.Sle1a DCs did not affect the proliferation of
B6.STAT-4–/– B cells. To test the involvement of type I
IFN, we used IFNAR-1–deficient mice, which have the
129/Sv genetic background. With both B6.TC and
B6.Sle1a DCs, 129.Sv.IFNAR-1–/– B cells proliferated
significantly more than did 129.Sv B cells as compared
with B6 DCs. A similar result, although not statistically
significant, was obtained with 129/Sv DCs. This latter
control, as well as the fact that these experiments were
conducted in Transwells, rules out alloreactivity as a
mechanism for the increased proliferation. Overall,
1747
these results rule out IL-12 and IFN␣ as being responsible for the differential effect of B6.TC and B6 DCs on
B cells.
DISCUSSION
The B6.TC congenic mouse strain is a model of
lupus that contains the 3 NZM2410-derived major susceptibility loci on a nonautoimmune B6 background
(B6.Sle1.Sle2.Sle3) (7). B6.TC mice accumulate DCs in
their lymphoid organs with age, and these DCs result in
higher proliferation of CD4⫹ T cells and inhibit the
function of normal regulatory T cells (12). We have
recently shown that DCs expressing Sle1a, one of the
lupus susceptibility loci expressed in B6.TC mice, presented the same CD4⫹ T cell–activating phenotype as
B6.TC DCs (24). In the present study, we found that
B6.TC DCs directly contribute to B cell hyperactivity by
increasing their proliferation, antibody production, and
chemokine receptor expression. This phenotype was also
present in Sle1a-expressing DCs. This indicates that
Sle1a, which we initially characterized as a locus that
contributes to lupus susceptibility through the production of autoreactive T cells (37), also contributes to lupus
through the abnormal activation of T and B cells by DCs.
The up-regulation of B cell activity by DCs from
B6.TC or B6.Sle1a mice occurred in the presence of
CD40 stimulation, which is critical for the differentiation
and development of effector functions in B cells (38) and
DCs (39). CD40L is overexpressed in B6.TC and
B6.Sle1a CD4⫹ T cells (37). This suggests that reciprocal interactions occur between CD4⫹ T cells, DCs, and
B cells in the B6.TC model, all of which contribute to
enhanced effector phenotypes in both T and B lymphocytes. Our results also showed that the CD40 activation
pathway is not unique and that B6.TC DCs activated by
other stimuli, such as LPS, also stimulated B cells to a
higher degree. We have previously shown that Toll-like
receptor ligands induced a higher level of activation and
cytokine production in DCs from B6.TC mice than in
those from B6 mice (12). This suggests a mechanism by
which subthreshold levels of activation may induce
B6.TC DCs to activate B cells in vivo.
Sle1a-expressing B cells express a higher level of
activation markers (24,27), but we have not yet determined whether and to what extent it is B cell intrinsic.
We have shown here that B6.TC or B6.Sle1a BMderived DCs, i.e., differentiated in the absence of T cells,
up-regulated B cell functions, suggesting that these DCs
have an abnormal intrinsic function. The results presented herein show that DC activation contributes to
1748
WAN ET AL
Figure 6. Production of cytokines by mouse dendritic cells (DCs). DCs from B6.TC (TC) mice produced significantly more A, interleukin-6 (IL-6),
B, IL-10, and C, tumor necrosis factor ␣ (TNF␣) than did DCs from B6 mice in the presence of B6 B cells and anti-CD40 (␣CD40), and produced
more D, soluble IL-6 receptor (sIL-6R) in the presence of anti-CD40. This differential cytokine production was not responsible for the increased
B cell proliferation, as shown in Transwell experiments in the presence of E, anti–IL-6 (␣IL-6) or F, anti–IL-10 or anti-TNF␣ (␣TNF␣) blocking
antibodies. Ab ⫽ antibody. The involvement of IL-12 and interferon-␣ (IFN␣) was tested by comparing the responses induced by B6 and B6.TC DCs
in G, B6.TC and B6.TC.STAT-4–/– B cells or H, 129/Sv and 129/SV.IFNAR-1–/– B cells in Transwell experiments. Results in G are expressed as the
percentage change in B6.TC.STAT-4–/– relative to B6.TC B cell responses to B6, B6.TC, or B6.Sle1a (Sle1a) DCs; those in H are expressed as the
percentage change in 129/Sv.IFNAR-1–/– relative to 129/Sv B cell response to 129/Sv, B6, B6.TC, or B6.Sle1a DCs. WT ⫽ wild-type. Values are the
mean and SEM of 4 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by t-test.
B CELL STIMULATION OF DCs IN MURINE LUPUS
these effector phenotypes in B6.TC and Sle1a B cells.
Using Transwell experiments, we have shown that cellto-cell contact enhanced the effect of both B6 and
B6.TC DCs on B cells, but the enhanced effect of B6.TC
DCs was contact independent. DCs release membranebound exosomes, which have been shown to perform a
number of effector functions in vivo and in vitro (40,41).
Exosomes from either anti-CD40– or LPS-activated
DCs were not able to induce B cell proliferation in our
system, but the supernatants from the same cells induced
proliferation to a level similar to that in the cocultures of
B cells and DCs. These results demonstrate that soluble
factors released by activated B6.TC DCs enhance B cell
effector phenotypes.
Analysis of the cytokine production showed that
B6.TC DCs responded differently from B6 DCs to the
same anti-CD40 stimulation by producing more IL-6,
TNF␣, and IL-10. Decreased production of IL-12 was
observed, however, which corresponds to the defective
IL-12 response of NZM2410 macrophages to LPS (42),
but differs from Sle3-only expressing DCs (18). Blocking
of any of these individual cytokines did not alter these
effects. These results indicate either that several of these
4 cytokines are involved, but they can substitute for each
other, or that an as-yet-undetermined soluble factor is
responsible. The higher expression of sIL-6R by antiCD40–stimulated B6.TC DCs could be due to an autocrine effect of high IL-6 production by these cells (36).
IL-6 trans-signaling has recently been shown to inhibit
the induction of forkhead box P3 in naive CD4⫹ T cells
(43), and evidence for its role in chronic inflammation
and autoimmunity is accumulating (35). We hypothesize
that in our specific model, sIL-6R increases B cell
responses to IL-6, and we are currently testing this
hypothesis.
Myeloid cells have previously been shown to play
a major role in disease pathogenesis in the NZM2410
lupus model through the expression of Sle3 (18). Sle3expressing DCs are associated with defective CD4⫹ T
cell activation–induced cell death and the production of
anti–double-stranded DNA antibodies. As we have
shown here for B6.TC DCs, Sle3-expressing DCs produce high levels of proinflammatory cytokines (but not
IL-6). In spite of these cytokines, the precise mechanisms by which either Sle3 or B6.TC DCs induce B cells
to produce autoantibodies (for Sle3) or to display enhanced effector functions (for B6.TC) have not been yet
determined. For Sle3, a direct interaction between DCs
and B cells has not been examined. For B6.TC, we have
now shown that DCs directly regulate CD4⫹ T cell and
B cell hyperactivity and that Sle1a plays a major role in
the process. Consequently, DCs should be considered an
1749
integral part of lupus pathogenesis, at least in the
NZM2410 model. Moreover, Sle1a is a locus that affects
both the intrinsic phenotype of CD4⫹ T cells (31,37)
and DCs (present study), and these DCs regulate the
functions of both CD4⫹ T cells and B cells. The Sle1a
congenic interval represents a 2.49–2.96 Mb NZM2410derived segment that contains a maximum of 19 genes
(http://www.ensembl.org/Mus_musculus/). The same
gene in this locus could be responsible for all these
phenotypes in CD4⫹ T cells and DCs. Alternatively,
different genes may be involved, and we are currently
investigating this using recombinant congenics within
the Sle1a interval.
ACKNOWLEDGMENTS
We thank Leilani Zeumer and Xuekong Su for excellent animal care, Dr. Westley Reeves for the 129/Sv.IFNAR–/–
mice, Dr. Eric Sobel and Carla Cuda for critical review of the
manuscript, and members of Dr. Morel’s laboratory for helpful
discussions.
AUTHOR CONTRIBUTIONS
Dr. Morel had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Wan, Zhou, Duan, Morel.
Acquisition of data. Wan, Zhou, Duan.
Analysis and interpretation of data. Wan, Zhou, Duan, Morel.
Manuscript preparation. Morel.
Statistical analysis. Wan, Zhou, Duan.
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