Interleukin-10-producing CD5+ B cells inhibit mast cells during immunoglobulin E-mediated allergic responses
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RESEARCH ARTICLE
IMMUNOLOGY
Interleukin-10–producing CD5+ B cells inhibit
mast cells during immunoglobulin E–mediated
allergic responses
Hyuk Soon Kim,1* A-Ram Kim,1* Do Kyun Kim,1 Hyun Woo Kim,1 Young Hwan Park,1
Geun Hyo Jang,1 Bokyung Kim,1 Yeong Min Park,1 Jueng Soo You,1 Hyung Sik Kim,2
Michael A. Beaven,3 Young Mi Kim,4 Wahn Soo Choi1†
Subsets of B cells inhibit various immune responses through their production of the cytokine interleukin-
10 (IL-10). We found that IL-10–producing CD5+ B cells suppressed the immunoglobulin E (IgE)– and
antigen-mediated activation of mast cells in vitro as well as allergic responses in mice in an IL-10–
dependent manner. Furthermore, the suppressive effect of these B cells on mast cells in vitro and in vivo
depended on direct cell-to-cell contact through the costimulatory receptor CD40 on CD5+ B cells and the
CD40 ligand on mast cells. This contact enhanced the production of IL-10 by the CD5+ B cells. Through
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activation of the Janus-activated kinase–signal transducer and activator of transcription 3 pathway, IL-10
decreased the abundance of the kinases Fyn and Fgr and inhibited the activation of the downstream ki-
nase Syk in mast cells. Together, these findings suggest that an important function of IL-10–producing
CD5+ B cells is inhibiting mast cells and IgE-mediated allergic responses.
INTRODUCTION
tivalent binding of antigen to FceRI-bound IgE, with the subsequent re-
Allergic disorders are widespread, particularly in developed countries (1). lease of various allergic mediators, including histamine, eicosanoids, and
Allergic responses are associated with increases in the number of T helper inflammatory cytokines. Release of these mediators leads to immediate,
2 (TH2) cells and immunoglobulin E (IgE) antibody production. Presen- and sometimes delayed, symptoms of allergic diseases, such as allergic rhini-
tation of allergen to antigen-presenting cells (APCs) causes TH2 cells to tis, anaphylaxis, and atopic dermatitis (17, 18). We found that CD5+ B cells
produce TH2-type cytokines. In particular, the cytokine interleukin-4 (IL-4) inhibited IgE-mediated mast cell activation and anaphylaxis in mice in an IL-
is essential for the isotype switching of B cells to produce allergen-specific 10–dependent manner. Furthermore, we found that IL-10–producing CD5+ B
IgE antibodies (2), which then bind specifically to the multimeric high- cells inhibited the activation of the tyrosine kinase Syk (spleen tyrosine ki-
affinity IgE receptor FceRI (3) on mast cells and basophils. nase) in mast cells. Together, our findings suggest that IL-10–producing CD5+
B cells are generally known for their capacity to regulate effector T cell B cells inhibit IgE-mediated allergic responses in physiological settings.
responses and to produce antibodies (4); however, studies in murine dis-
ease models revealed some distinct B cell subsets that exhibit immunosup-
pressive functions (5–8) and thus are named regulatory B (Breg) cells (9). RESULTS
Other subsets of Breg cells have also been identified to suppress various
immune responses in an IL-10–dependent manner (10), whereas helminth CD5+ B cells suppress IgE-mediated anaphylaxis
infection–induced IL-10–producing B cells inhibit allergic reactions in in vivo and mast cell activation in vitro
animal models, specifically ovalbumin-mediated anaphylaxis (11) and al- IgE-dependent mast cell activation is regarded as one of the cardinal me-
lergic asthma (12). IL-10 also suppresses mast cell activation in vitro and chanisms in the development of anaphylaxis. Here, we administrated IgE
in vivo and may thus counteract their excessive activation and the devel- antibody and antigen intravenously to mice to induce passive systemic
opment of chronic inflammation (13, 14). Despite these reports, and anaphylaxis (PSA), and these responses are essentially associated with
given the role of mast cells in these and other allergic diseases (15, 16), extensive mast cell activation in vivo. We found that the numbers of IL-
surprisingly little is known about the mechanism of interaction between 10–producing CD5+ B cells were increased in the spleen, peritoneal cavity,
Breg cells and mast cells or about the consequences of these interactions lymph node, and blood of IgE- and antigen-induced PSA mice (fig. S1A),
for IgE-mediated allergic responses. which suggests that these cells are associated with the progression of symp-
Mast cells are the key effector cells in IgE-mediated allergic reactions. toms. We next examined CD19-decifient mice in which IL-10–producing
These cells are widely distributed in vascularized tissues, especially near B cells are substantially depleted (fig. S1B) (7). We found that IgE-mediated
surfaces exposed to the environment, such as the skin, airways, and the anaphylaxis responses (Fig. 1A) and increases in the concentration of his-
gastrointestinal tract (15). Mast cells are commonly activated by the mul- tamine in the blood (Fig. 1B) were substantially enhanced in the CD19-
deficient mice compared to wild-type mice.
1
School of Medicine, Konkuk University, Chungju 380-701, Korea. 2College of To assess the effects of CD5+ B cells in IgE-mediated allergic re-
Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea. 3Laboratory of sponses in vivo, we first induced PSA in CD19-deficient mice. Three days
Molecular Immunology, National Heart, Lung, and Blood Institute, National after they received purified CD5+ or CD5− B cells from wild-type mice
Institutes of Health, Bethesda, MD 20892, USA. 4College of Pharmacy, Duksung
Women’s University, Seoul 132-714, Korea.
by adoptive transfer (fig. S1C), IgE-sensitized CD19-deficient mice were
*These authors contributed equally to this work. intravenously challenged with antigen. The presence of CD5+ B cells,
†Corresponding author. E-mail: wahnchoi@kku.ac.kr but not CD5− B cells, markedly alleviated the decline in temperature in
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Fig. 1. CD5+ B cells suppress IgE-mediated activation of mouse mast cells the absence or presence of CD5+ or CD5− B cells at a BMMC/B cell ratio of
in vivo and in culture. (A and B) PSA was induced by injecting wild-type 1:5 for the indicated times (E) or CD5+ or CD5− B cells at the indicated
(WT) mice or CD19−/− mice with dinitrophenyl (DNP)–IgE (3 mg) 24 hours BMMC/B cell ratios for 24 hours (F). Cells were then treated with or with-
before injecting them with DNP–bovine serum albumin (DNP-BSA; 250 mg; out IgE and antigen (Ag) as indicated, and the extent of release of b-
Ag) as indicated. Mice were then subjected to analysis of rectal tempera- hexosaminidase from the BMMCs was determined. (G to I) BMMCs were
tures at the indicated times (A) and serum concentrations of histamine 30 min cultured alone or together with CD5+ or CD5− B cells and then were treated
after stimulation (B) (n ≥ 5 mice per experiment). (C and D) PSA was induced with the indicated combinations of IgE and antigen. The amounts of hista-
in CD19−/− mice 3 days after they received CD5+ B cells or CD5− B cells by mine (G), tumor necrosis factor–a (TNF-a) (H), and IL-4 (I) released into the
adoptive transfer. Mice were then subjected to analysis of rectal tempera- culture medium were determined by enzyme-linked immunosorbent assay
tures (C) and serum concentrations of histamine (D) (n ≥ 5 mice per experiment). ELISA). Data are means ± SEM of three independent experiments. *P < 0.05;
(E and F) Bone marrow–derived mast cells (BMMCs) were preincubated in **P < 0.01; n.s., not significant.
response to antigen (Fig. 1C). Serum histamine concentrations were in- from the spleen of BALB/c mice also inhibited the antigen-stimulated de-
creased in antigen-challenged mice compared to those in unchallenged granulation of BMMCs (fig. S1, D and E).
mice, but this increase was substantially reduced after the adoptive transfer
of CD5+ cells (Fig. 1D). IL-10 from CD5+ B cells is critical for mast cell inhibition
Next, we found that culturing BMMCs with CD5+ B cells, but not The immunoregulatory role of Breg cells, also called B10 cells, is gen-
CD5− B cells, substantially inhibited their degranulation in response to erally dependent on IL-10 (10). We found that the passive cutaneous an-
antigen in a time- and cell concentration–dependent manner (Fig. 1, E aphylaxis (PCA) reaction was increased in IL-10−/− mice compared to that
and F), as well as inhibited their release of other allergic mediators, such in wild-type mice (fig. S2, A and B, top). The numbers of degranulated
as histamine, TNF-a, and IL-4 (Fig. 1, G to I). This inhibitory action of mast cells in the ear tissues of IL-10−/− mice were increased, albeit not
CD5+ B cells was not dependent on mouse strain or tissue source because statistically significantly, compared to those in the ear tissues of wild-
CD5+ B cells isolated from the peritoneal cavity of C57BL/6 mice and type mice (fig. S2B, bottom). These results led us to investigate whether
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IL-10 generated by CD5+ B cells inhibited mast cell activation and IgE- CD5+ B cells from either wild-type or IL-10−/− mice showed no statisti-
mediated anaphylaxis. First, we found that monoclonal antibodies cally significant differences in the amounts of their cell surface markers,
against IL-10 or the IL-10 receptor (IL-10R) blocked the suppressive including IgD, IgM, CD19, CD21, CD1d, CD11b, CD40, and B220 (fig.
effect of CD5+ B cells on the antigen-induced degranulation of BMMCs S2C). Third, CD5+ B cells also inhibited the antigen-stimulated degra-
(Fig. 2A). Second, CD5+ B cells from IL-10−/− mice failed to inhibit the nulation of IL-10−/− BMMCs (fig. S2D). Notably, flow cytometric anal-
IgE- and antigen-stimulated degranulation of BMMCs in vitro (Fig. 2B). ysis revealed that IL-10–producing B cells were found mostly within a
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Fig. 2. IL-10 production by CD5+ B cells is critical for the suppression of mast PSA was induced in IL-10−/− mice 3 days after they received CD5+ or CD5−
cell activation. (A and B) BMMCs were preincubated for 24 hours with CD5+ B cells from WT or IL-10−/− mice by adoptive transfer, as indicated. Mice
B cells in the presence or absence of the indicated combinations of anti- were then analyzed to determine rectal temperatures at the indicated times
IL-10 (a-IL-10), anti-IL-10R (a-IL-10R), or isotype control monoclonal anti- (E) and serum histamine concentrations at 30 min (F) (n ≥ 5 mice per ex-
body (mAb) (A) or with the indicated combinations of CD5+ or CD5− B cells periment). (G to I) Equal numbers of CD5+ or CD5− B cells were incubated
from WT or IL-10−/− mice (B). Cells were then treated with the indicated alone or in the presence of unstimulated or IgE- and antigen-stimulated
combinations of IgE and antigen, and the amount of b-hexosaminidase re- BMMCs from WT (G) or IL-10−/− mice (H and I), as indicated. Cells were
leased into the culture medium was determined. Data are means ± SEM of then analyzed by flow cytometry to determine the percentages of IL-10+ B
three independent experiments. (C and D) PSA was induced in IL-19−/− cells (CD19+) (G), the percentages of CD5+ or CD5− B cells that contained
mice 3 days after they had received CD5+ or CD5− B cells from WT or IL-10 (were IL-10+) (H), and the amount of IL-10 in the culture medium (I).
IL-10−/− mice, as indicated, by adoptive transfer. Mice were then analyzed Data in (G) are representative of three independent experiments. Data in
to determine rectal temperatures at the indicated times (C) and serum his- (H) and (I) are means ± SEM of three independent experiments. *P < 0.05;
tamine concentrations at 30 min (D) (n ≥ 5 mice per experiment). (E and F) **P < 0.01.
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Fig. 3. Suppression of mast cell activation by IL-10–producing CD5+ B
cells requires cell-to-cell contact. (A) BMMCs were cultured for 24 hours
alone or together with CD5+ or CD5− B cells either in direct contact (filled
bars) or separated in transwell plates (empty bars). Cells were then treated
with the indicated combinations of IgE and antigen before b-hexosaminidase
release was determined. (B and C) CD5+ B cells were incubated in vitro
alone or with BMMCs that were untreated, treated with IgE, or treated with
IgE and antigen, as indicated. (B) After 45 min, the cocultured cells were
analyzed by flow cytometry. (C) The percentages of BMMC-CD5+ B cell
conjugates that formed were calculated. (D) Spleens of PSA-induced WT
mice were analyzed by immunohistochemistry to detect CD5 [black, nickel-
diaminobenzidine (N-DAB)], CD19 (red, Novared), and mast cells (pur-
ple, toluidine blue). CD5+ B cells are indicated by green arrows; mast
cells are indicated by red arrows. Images are shown at ×400 magnifica-
tion; however, the area in the yellow box is shown in the bottom right panel
at a magnification of ×1000. Scale bar, 100 mm. (E and F) CD5+ or CD5− B
cells were cultured for 24 hours alone or with unstimulated or IgE- and
antigen-stimulated BMMCs under conditions of cell-to-cell contact or in
transwells. (E) B cells were analyzed by flow cytometry to detect intracel-
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lular IL-10. (F) Top: The percentages of IL-10+ B cells were determined by
flow cytometry. Bottom: The amounts of IL-10 in the culture medium of the
indicated cells were determined by ELISA. Data in (B), (D), and (E) are rep-
resentative of three independent experiments. Data in (A), (C), and (F) are
means ± SEM of three independent experiments. *P < 0.05; **P < 0.01.
CD5+CD19high B cell subset (fig. S2E). We also found that the amounts of
IgM, CD1d, and CD21 were greater on the surface of IL-10+ CD19+ B
cells compared with the amounts of those markers on IL-10− CD19+ B
cells (fig. S2F). Finally, the adoptive transfer of wild-type CD5+ B cells,
but not IL-10−/− CD5+ B cells, suppressed PSA reactions and blocked in-
creases in serum histamine concentrations in CD19-deficient mice (Fig. 2,
C and D) and IL-10−/− mice (Fig. 2, E and F), which supports the idea
that IL-10 produced by CD5+ B cells inhibits mast cell activation and
IgE-mediated anaphylaxis.
BMMCs stimulate the production of IL-10 from
CD5+ B cells
We next found that the percentage of IL-10–producing B cells among the
whole population of B cells was increased by the coculture with BMMCs
and was even further increased if the cocultured BMMCs were stimulated with
IgE and antigen (fig. S3A). However, the production of IL-10 by the BMMCs
themselves was minimal when they were cocultured with B cells (fig. S3, B
and C), indicating that most of the IL-10 in cocultures was produced by the B
cells. We further found that BMMCs enhanced the production of IL-10 from
CD5+ B cells, but not from CD5− B cells (Fig. 2, G and H), and that this ~25%) by the addition of antigen (Fig. 3, B and C). We also observed
increased production was further enhanced by stimulating wild-type or conjugation between CD5− B cells and mast cells (fig. S3F), which was
IL-10−/− BMMCs with antigen (Fig. 2, H and I). The stimulatory effect of similar to that between CD5+ B cells and mast cells. Furthermore, immu-
BMMCs on IL-10 production was also apparent with CD5+ B cells from nohistochemical analysis of mouse spleens after PSA was induced revealed
the spleen, inguinal lymph node, and blood, but not from the peritoneal that some CD5+CD19+ B cells were in close proximity to mast cells, which
cavity (fig. S3, D and E). These results suggest that BMMCs stimulate IL-10 raises the possibility of crosstalk between these two cell types in vivo (Fig.
production by CD5+ B cells from various lymphoid organs. 3D). Similarly, the increase in IL-10 production by CD5+ B cells was ob-
served only in cocultures and not when both cell types were separated in
Direct cell-cell contact is essential to inhibit mast cell transwell flasks (Fig. 3, E and F).
activation and enhance IL-10 production by CD5+ B cells
Although degranulation was inhibited when mast cells were cultured with CD40 on CD5+ B cells and CD40 ligand (CD154) on
CD5+ B cells, this was not the case when the cell types were physically mast cells are required for the production of IL-10 by CD5+
separated in transwell culture flasks (Fig. 3A). Note that conjugation of B cells and the suppression of mast cell activation
CD5+ B cells and mast cells with or without IgE (~20% of cells conjugated) CD40-generated signals in IL-10–producing B cells participate in the reg-
was observed in cocultures, and the extent of conjugation was increased (to ulation of various inflammatory diseases and possibly in the production
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of IL-10 (10). Because the abundance of CD40 ligand (CD40L) on the or both were substituted for the corresponding wild-type cells (Fig. 5,
surface of mast cells is increased by stimulation with antigen (19), we A and B), suggesting that CD40-CD40L contact was necessary for
determined whether the enhanced IL-10 production by B cells was such conjugation to occur. A similar pattern of effects on the produc-
dependent on the CD40-CD40L interaction. The abundances of CD40 tion of IL-10 by CD5+ B cells was observed when cells deficient in
on B cells and IL-10 in B cells, as well as the amount of IL-10 secreted CD40 or CD40L were substituted for the corresponding wild-type cells
from B cells, were substantially increased by recombinant CD40L (Fig. 4, (Fig. 5, C and D), which was consistent with the data obtained from exper-
A to D). Whereas CD40 abundance and IL-10 production in whole B cells iments with recombinant CD40L and the anti-CD40L antibody (Fig. 4), as
were increased when they were cocultured with BMMCs (fig. S4, A and well as with the requirement of CD40-CD40L interaction for IL-10 pro-
B), IL-10 production by B cells was largely blocked in the presence of an duction by CD5+ B cells. Moreover, this interaction appeared to be nec-
anti-CD40L monoclonal antibody (fig. S4, B and C). In addition, in co- essary irrespective of whether the BMMCs were stimulated with antigen
cultures of CD5+ B cells and BMMCs, the increased abundances of cell (Fig. 5, C and D). Loss of either CD40 or CD40L did not alter the pat-
surface CD40 (Fig. 4E) and IL-10 (Fig. 4, F and G) in CD5+ B cells were terns of cell surface markers of CD5+ B cells or BMMCs, respectively
markedly inhibited by the anti-CD40L antibody, and the suppressive (fig. S4D).
action of CD5+ B cells on BMMC degranulation was also blocked by this With respect to mast cell function, the IgE- and antigen-induced de-
antibody (Fig. 4H). granulation of BMMCs was no longer suppressed when CD40−/− CD5+ B
cells or CD40L−/− BMMCs were substituted for the corresponding wild-
The interaction between CD5+ B cells and mast cells type cells (Fig. 5E). Furthermore, the adoptive transfer of wild-type CD5+
through CD40-CD40L is critical for the suppression of B cells, but not CD40−/− CD5+ B cells, into CD19-deficient mice suppressed
IgE-mediated mast cell activation and anaphylaxis the decline in rectal temperatures when PSA was induced (Fig. 5F). To
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We next observed whether CD5+ B cells and mast cells interacted di- further determine whether the CD40-CD40L interaction between CD5+
rectly under cocultured conditions. The extent of conjugation of CD5+ B cells and mast cells occurs in vivo, we transferred wild-type or
B cells and BMMCs in coculture that we noted earlier (Fig. 3B) was CD40L-deficient BMMCs into KitW-sh/W-sh mice, which do not contain mast
substantially reduced when CD40−/− CD5+ B cells, CD40L−/− BMMCs, cells and, thus, do not exhibit PSA symptoms in response to stimulation
Fig. 4. IL-10 production by mouse CD5+ B cells is dependent on CD40- (E) and the percentages of IL-10+ B cells (F and G, top) were determined
CD40L interactions. (A to D) Total B cells from WT mice were left unstim- by flow cytometric analysis. (G, bottom) The amounts of IL-10 in the cul-
ulated or were stimulated with recombinant CD40L (rCD40L;1 mg/ml) ture medium were determined by ELISA. (H) BMMCs preincubated in
for the indicated times. The mean fluorescence intensity (MFI) of CD40 the absence or presence of the indicated combinations of CD5+ B cells,
staining on B cells (A) and the percentages of IL-10+ B cells (B and C) were anti-CD40L antibody, and isotype control antibody were stimulated
determined by flow cytometric analysis. (D) The amounts of IL-10 in the with IgE and antigen, as indicated. Twenty-four hours later, the extent of
culture medium were determined by ELISA. (E to G) CD5+ B cells were b-hexosaminidase release into the culture medium was determined. Plots
cultured alone or with either unstimulated or IgE- and antigen-stimulated in (B) and (F) are representative of three independent experiments. Data
BMMCs in the presence or absence of anti-CD40L antibody or an iso- in (A), (C) to (E), (G), and (H) are means ± SEM of three independent
type control antibody for 24 hours. The MFI of CD40 staining on B cells experiments. *P < 0.05; **P < 0.01; n.s., not significant.
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Fig. 5. Suppression of mast cell activation and PSA by CD5+ B cells depends from WT or CD40L−/− mice, as indicated. The extent of b-hexosaminidase
on CD40-CD40L interactions. (A and B) CD5+ B cells from WT or CD40−/− release into the culture medium was determined. (F) CD19−/− mice were
mice were incubated in a 1:1 ratio with BMMCs from WT or CD40L−/− mice, left untreated or received CD5+ or CD5− B cells from WT or CD40−/− mice
as indicated. (A) After 1 hour, BMMC–B cell conjugates were detected by by adoptive transfer. Three days later, the mice were treated with IgE and
flow cytometric analysis. (B) The percentages of BMMC–B cell conjugates antigen to induce PSA, and rectal temperatures in the indicated mice were
that formed under the four indicated conditions (a to d) were calculated. measured over time (n = 5 mice per experiment). (G) Mast cell–deficient
(C and D) CD5+ B cells from WT or CD40−/− mice were incubated alone or mice (KitW-sh/W-sh mice) were left untreated or received BMMCs from WT or
in a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs from CD40L−/− mice by adoptive transfer. Three days later, the mice were
WT or CD40L−/− mice, as indicated. (C) Cells were subjected to flow cyto- treated with the indicated combinations of IgE and antigen, and the rec-
metric analysis to identify IL-10+CD5+ B cells. (D) The percentages of tal temperatures of the mice were measured over time. Plots in (A) and
IL-10+CD5+ B cells under the indicated conditions were calculated. (E) (C) are representative of three independent experiments. Data in (B) and
CD5+ B cells from WT or CD40−/− mice were incubated for 24 hours alone (D) to (G) are means ± SEM of three independent experiments. *P <
or in a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs 0.05; **P < 0.01.
with IgE and antigen (20). Although the antigen-stimulated degranulation inhibited the antigen-induced tyrosine phosphorylation of Syk in BMMCs
of CD40L-deficient BMMCs was comparable to that of wild-type (Fig. 6, F and G), which suggests that wild-type CD5+ B cells can inhibit
BMMCs (Fig. 5E), IgE-mediated anaphylaxis responses became more se- the activation of mast cells by reducing the abundances of Fyn and Fgr.
vere when the KitW-sh/W-sh mice received CD40L−/− BMMCs instead of These observations were further confirmed by incubating BMMCs with re-
wild-type BMMCs (Fig. 5G). Together, these results suggest that the combinant IL-10 for 24 or 72 hours (fig S5, A to D). The essential require-
CD40-CD40L interaction between CD5+ B cells and mast cells is critical ment for an interaction between CD40 (on CD5+ B cells) and CD40L (on
for the suppression of mast cell activation and IgE-mediated PSA re- mast cells) to suppress the phosphorylation of the tyrosine kinases after
sponses in vivo. IgE- and antigen-mediated stimulation was verified in experiments with
an anti-CD40L antibody (fig. S5, E and F).
IL-10–producing CD5+ B cells inhibit FceRI-mediated The inhibition of mast cells by IL-10 is dependent on signal transducer
signaling in mast cells and activator of transcription 3 (STAT3) signaling, which leads to the re-
We did not observe any decrease in the cell surface abundances of the a, duced activation of tyrosine kinases involved in early signaling events
b, or g subunits of FceRI on BMMCs that were cocultured with CD5+ B (14). We observed that IL-10 stimulated the phosphorylation of STAT3,
cells for 24 hours (Fig. 6, A and B). However, we observed alterations in which was inhibited by the Janus-activated kinase (JAK) inhibitor AG490
the abundances of some tyrosine kinases that transduce initial signals after (Fig. 6H). In addition, AG490 restored the abundance of Fyn and Fgr (Fig.
antigen-dependent cross-linking of IgE bound to FceRI. Coculture with 6I) as well as the extent of tyrosine phosphorylation of Syk in IL-10–treated
wild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, substantially reduced BMMCs (Fig. 6J). Further experiments showed that STAT3-specific small
the abundances of the kinases Fyn and Fgr in BMMCs, whereas Lyn and interfering RNA (siRNA) restored the amounts of Fyn and Fgr, as well as
Syk were unaffected (Fig. 6, C and D). The lack of change in the abundance the phosphorylation of Syk, in IL-10–treated mast cells (Fig. 6, K and L),
of Syk was further confirmed by flow cytometric analysis (Fig. 6E). Fur- which suggests that the suppression of mast cells by CD5+ B cell–derived
thermore, wild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, markedly IL-10 is mediated through the JAK-STAT3 pathway.
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Fig. 6. CD5+ B cell–derived IL-10 reduces Fyn and Fgr abundance and nant IL-10 (rIL-10; 100 ng/ml) in the absence or presence of 25 mM AG490
inhibits downstream activation of Syk in BMMCs. (A and B) BMMCs from for 24 hours and were then were left unstimulated or were stimulated with
WT mice were cultured alone or with CD5+ or CD5− B cells, as indicated. antigen for 15 min. Cells were then analyzed by flow cytometry to determine
(A) Twenty-four hours later, the cells were analyzed by flow cytometry to the MFIs of total STAT3 (top) and pSTAT3 (Tyr705; bottom). (I and J) BMMCs
detect the cell surface expression of the indicated FceRI subunits. (B) were cultured for 24 hours in medium containing IL-3 in the absence or
The relative MFIs of the indicated FceRI subunits on the BMMCs were presence of rIL-10 (100 ng/ml) or 25 mM AG490. The cells were then stimu-
calculated. (C and D) BMMCs were cultured alone or with CD5+ B cells lated with the indicated combinations of IgE and antigen for 7 min before
from WT or IL-10−/− mice, as indicated. (C) BMMCs were then analyzed being analyzed by Western blotting to detect total Lyn, Fyn, and Fgr pro-
by Western blotting with antibodies against the indicated proteins. (D) teins (I) and total Syk and phosphorylated Syk proteins (J). Band densities
Quantification of the relative abundances of the indicated proteins was per- are shown as the mean values from three independent experiments in each
formed by densitometric analysis of Western blots. (E) BMMCs were lower panel. (K and L) BMMCs were transfected with STAT3-specific siRNAs
cultured alone or with CD5+ B cells from WT or IL-10−/− mice at a 1:5 ra- (siSTAT3) or control siRNAs (siCtrl) 48 hours before the experiment. The
tio. Twenty-four hours later, the cells were treated with the indicated com- BMMCs were then incubated with the indicated combinations of rIL-10
binations of IgE and antigen before being analyzed by flow cytometry to (100 ng/ml) and IgE (500 ng/ml) for 24 hours before being left untreated
determine the MFI of Syk. (F and G) BMMCs cultured alone or with WT or stimulated with antigen for 7 min. (K) BMMCs were analyzed by Western
or IL-10−/− CD5+ B cells at a 1:5 ratio for 24 hours were then treated for blotting with antibodies specific for the indicated proteins. Western blots are
7 min with the indicated combinations of IgE and antigen. (F) The BMMCs representative of three independent experiments. (L) Densitometric analysis
were analyzed by flow cytometry to detect tyrosine-phosphorylated Syk of the relative abundances of total Fyn and Fgr proteins and of pSyk. Data in
(Tyr352). (G) The MFIs of pSyk in BMMCs under the indicated conditions (B), (D), (E), (G), (I and J, lower panels), and (L) are means ± SEM of three
were determined. (H) BMMCs were incubated with or without recombi- independent experiments. *P < 0.05; **P < 0.01.
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DISCUSSION which in turn suppress mast cell activation and anaphylaxis (Fig. 5). No-
tably, the anaphylaxis responses stimulated by IgE and antigen were re-
B cells have the capacity to produce antibodies, function as APCs, and duced in mice that received wild-type CD5+ B cells, but not CD40−/−
regulate the activation of CD4+ T cells (21–23). In addition, there is ev- CD5+ B cells, by adoptive transfer (Fig. 5F). To further determine whether
idence that B cells have a regulatory role in various immune responses CD40L on mast cells was critical for a direct interaction with CD40 on
through their production of cytokines. Katz et al. (24) were the first to CD5+ B cells, we performed experiments with mast cell–deficient mice
demonstrate that the delayed-type hypersensitivity reaction was exacer- (KitW-sh/W-sh mice), which do not show any PSA in response to IgE and
bated by the depletion of B cells. Mizoguchi and Bhan (9) introduced antigen (20). Although IgE-mediated anaphylaxis was not observed in
the term “regulatory B cells” to designate negative regulatory subpop- KitW-sh/W-sh mice (Fig. 5G) (20), the responses became much more severe
ulations of B cells. Breg cells are now recognized as one of the key regu- in mice that received CD40L−/− BMMCs by adoptive transfer compared to
latory cell types that suppress inflammatory disorders (10) and various those that received wild-type BMMCs (Fig. 5G). Together, these results
immune cells, including dendritic cells (DCs), macrophages, and TH cells suggest that CD40L on mast cells is critical to the induction of IL-10–
(25, 26). producing CD5+ B cells in physiological settings.
IL-10 was originally identified as the TH2 cell–derived cytokine syn- The precise details by which IL-10–producing Breg cells suppress al-
thesis inhibitory factor (27), and it has broad anti-inflammatory actions. lergic and inflammatory responses are unclear. Our results provide some
IL-10 suppresses the effector function of T cells and macrophages (28, 29). insight with regard to the interaction of the B cells with mast cells. Early
With respect to Breg cells (B10 cells), IL-10 enables the suppressive func- signaling events in antigen-stimulated mast cells include the recruitment of
tions of these cells in various immune disease models (10). Moreover, sub- Lyn and other Src family kinases, such as Fyn (41) and Fgr (42), to FceRI,
sets of IL-10–producing Breg cells suppress TH2 cell–mediated allergic which results in the phosphorylation and activation of Syk and the activa-
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responses, including contact hypersensitivity and allergic airway disease tion of mast cells (43). In our experimental system, coculturing CD5+ B
in mouse models (7, 12). Although mast cells can produce IL-10 through cells with BMMCs for 24 hours reduced the abundance of Fyn and Fgr in
FceRIII signaling after the receptor is increased in abundance by IL-4 (30), mast cells, and consequently decreased the phosphorylation of Syk (Fig.
IL-10 is not normally produced by mast cells through IgE- and antigen- 6, C to G), but had no effect on the abundances of individual FceRI
mediated stimulation (fig. S3, B and C). subunits (Fig. 6B). Similar results were obtained from experiments in
Mast cells are the critical effector cells in food allergies, allergic asthma, which BMMCs were treated with recombinant IL-10 under similar
and allergic rhinitis (16, 18). Although present in multiple tissues, mast cells conditions. However, more prolonged incubation (72 hours) of mast cells
are distributed mainly at the major immunologic interfaces, such as the skin, with IL-10 resulted in a reduction in the amounts of the FceRI subunits, in
gut, and lungs. Secondary lymphoid organs including spleens, tonsils, and addition to a reduction in the abundances of Syk, Fyn, and Fgr in mast
lymph nodes harbor modest numbers of mast cells in physiological settings cells (fig. S5, B and D), consistent with previous findings (14). We further
(19, 31) where they could also regulate inflammation. We previously re- demonstrated that the effects of CD5+ B cells on the abundances of Fyn
ported that the prevalence of IL-10–producing CD5+ peripheral blood and Fgr and on the phosphorylation of Syk were blocked by inhibiting the
B cells increased in healthy donors but decreased in patients with milk JAK-STAT3 pathway with a typical JAK inhibitor, AG490 (Fig. 6, H to J),
allergy after challenge with milk antigen (32). Our present finding that and by siRNAs specific for STAT3 in mast cells (Fig. 6, K and L). These
CD5+ B cells inhibited IgE-mediated mast cell activation and anaphylaxis observations led us to suggest that CD5+ B cells inhibit mast cell activa-
in mice in an IL-10–dependent manner suggests that the interaction be- tion through IL-10 by reducing the abundances of Fyn and Fgr through
tween IL-10–producing CD5+ B cells and mast cells provides a mecha- the JAK-STAT3 signaling pathway. It would be also of interest to determine
nism for counteracting allergic phenomena. IL-10, in particular, may
provide another link between IL-10–producing CD5+ B cells and various
immune cells, such as regulatory T cells, DCs, and eosinophils, in that it
inhibits mast cell activation as well as allergic reactions (14, 33, 34). As
we reported here, production of IL-10 by CD5+ B cells is enhanced upon
coculture with mast cells (Fig. 2, G to I). The possible physical association
between mast cells and CD5+ B cells was apparent from immunohisto-
chemical analysis of the spleens of mice with PSA (Fig. 3D) and was verified
by the observation that these cells form conjugates in coculture (Fig. 3,
B and C). Therefore, the potential exists for crosstalk between these
two cell types in physiological settings. Furthermore, direct cell-to-cell
contact was essential for the production of IL-10 by CD5+ B cells, as well
as for the inhibition of mast cell activation by CD5+ B cells.
The interaction between CD40L and CD40 on T cells and B cells,
respectively, is critical for CD4+ T cell activation and the effector func-
tions of B cells (35). Such an interaction may also stimulate the proliferation
of IL-10–producing Breg cells in mice (36) and in patients with systemic
lupus erythematosus (SLE) (37, 38) and thereby suppress the humoral re- Fig. 7. Proposed scheme for the suppression of mast cell activation by IL-10–
sponse. Mast cells were reported to communicate with B cells and astro- producing CD5+ B cells. Suppression is dependent on direct cell-to-cell con-
cytes through the CD40-CD40L interaction (39, 40). Here, we propose tact through the interaction of CD40L on mast cells and CD40 on CD5+
that mast cells may similarly regulate CD5+ B cell function on the basis B cells. This interaction results in the production of IL-10 by the CD5+ B cells.
that mast cells express CD40L (fig. S5D) and increase the cell surface IL-10 signaling reduces the abundances of Fyn and Fgr in the mast cells,
abundance of CD40 on CD5+ B cells, and that the CD40L-CD40 interac- which thus reduces the extent of activation of Syk, resulting in the inhibition
tion leads to an increase in the number of IL-10–producing CD5+ B cells, of mast cell degranulation. Tyk, tyrosine kinase.
www.SCIENCESIGNALING.org 17 March 2015 Vol 8 Issue 368 ra28 8RESEARCH ARTICLE
whether the inhibitory effects of IL-10–producing B cells extend to other conjugated and dead cells were excluded from the analysis on the basis
mast cell stimulants such as IgG1, which may be involved in analphylactic of forward and side light scatter parameters and staining with Fixable Vi-
reactions. ability Dye (eBioscience). Cells were fixed and permeabilized with a
In summary, our results demonstrate a mechanism by which IL-10– Cytofix/Cytoperm kit (BD Biosciences) and then were incubated with anti–
producing CD5+ B cells inhibit mast cell function as follows: (i) Both IL-10 monoclonal antibody (JES5-16E3, eBioscience) at 4°C for 30 min.
cell types form cell-cell conjugates through an interaction between The antibodies against cell surface proteins were as follows: anti-CD1d
CD40 (on CD5+ B cells) and CD40L (on mast cells). (ii) This interac- (1B1), anti-CD4 (RM4-5), anti-CD5 (53–7.3), anti-CD11b (M1/70), anti-
tion stimulates IL-10 production by CD5+ B cells. (iii) IL-10 produced CD19 (eBio1D3), anti-CD21/CD35 (eBioBD9), anti-CD23 (B3B4), anti-
by CD5+ B cells inhibits the abundance of Fyn and Fgr in mast cells CD25 (PC61.5), anti-CD40 (HM40-3), anti-CD86 (GL1), anti-B220
through activation of the JAK-STAT3 pathway. (iv) As a result, mast (RA3-6B2), anti-IgD (11–26), anti-IgM (eB121-15 F9), anti–c-Kit
cell activation through FceRI is suppressed by the diminished quanti- (2B8), and anti-CD40L (MR1), which were purchased from eBioscience,
ties of Fyn and Fgr in mast cells (Fig. 7). These findings suggest that and anti-FceRI (anti-IgE, R35-72), which was purchased from BD Bio-
IL-10–producing CD5+ B cells may provide an additional therapeutic sciences. To detect FceRI subunits and intracellular Syk, fixed BMMCs
target to treat IgE-mediated allergic diseases. were stained with antibodies against FceRIa (G-14), FceRIb (N-18),
FceRIg (H-5), and Syk (N-19), which were obtained from Santa Cruz
Biotechnology, and with anti-STAT3 antibody (M59-50), which was ob-
MATERIALS AND METHODS tained from BD Biosciences. To evaluate the extent of phosphorylation of
Syk or STAT3, BMMCs were primed with DNP-specific IgE (500 ng/ml)
Mice and cultured with CD5+ B cells for 24 hours before being stimulated with
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Wild-type (~6- to 8-week-old male C57BL/6 mice), CD19−/− [Cd19tm1(cre)Cgn], DNP-BSA (100 ng/ml) for 7 min (for pSyk) or 15 min (for pSTAT3).
IL-10−/− (Il10tm1Cgn), CD40−/− (Cd40tm1Kik), CD40L−/− (Cd40lgtm1Imx), The BMMCs were immediately fixed and permeabilized and then were
and KitW-sh/W-sh mice were purchased from The Jackson Laboratory, housed stained with anti-CD19 (eBio1D3), anti–c-Kit (2B8, eBioscience), and ei-
in a specific pathogen–free animal facility at Konkuk University (Seoul, Korea) ther anti-ZAP70(Tyr319)/Syk(Tyr352) (17A/P-ZAP70) or anti-STAT3
and fed with a sterilized diet and autoclaved water before being used for (Tyr705) (4/P-STAT3) antibodies (BD Biosciences). Briefly, after CD19+
experiments. All animal experiments were approved by the Institutional B cells were excluded from gated mast cells because of staining with an anti-
Animal Care and Use Committee (IACUC) at Konkuk University. CD19 monoclonal antibody, c-Kit+ cells that stained with anti-ZAP70(Tyr319)/
Syk(Tyr352)+ or anti-STAT3 (Tyr705)+ were analyzed. Cells were analyzed
Preparation and adoptive transfer of B cell subsets with a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo version
Splenic B cells were presorted with CD19 microbeads (Miltenyi Biotec). 10 software (TreeStar).
Then CD5+ or CD5− B cells were isolated with a FACSAria flow cytom-
eter (BD Biosciences). For in vivo adoptive transfer of B cell subsets, BMMC differentiation and transfection with
the isolated B cells were transferred intravenously [2 × 106 cells/0.2 ml of STAT3-specific siRNA
phosphate-buffered saline (PBS)] into recipient mice. Three days after the BMMCs derived from C57BL/6 or BALB/c mice were cultured in RPMI
adoptive transfer of cells, PSA was induced in the recipient mice. 1640 medium containing 2 mM L-glutamine, 0.1 mM nonessential amino
acids, antibiotics, 10% fetal bovine serum (FBS), and IL-3 (10 ng/ml;
Induction of PSA or PCA PeproTech Inc.). After 4 weeks, >98% of the cells were verified as
Mice were primed with 3 mg of DNP-specific IgE (SPE-7; Sigma) by in- BMMCs, as previously described (44). BMMCs (5 × 106 cells) were trans-
travenous injection. On the next day, the mice were injected intravenously fected with 100 nM STAT3-specific siRNA or scrambled siRNA with an
with 250 mg of DNP-BSA (Sigma) in 200 ml of PBS or as indicated in Amaxa Nucleofector (Lonza Cologne AG) with program T-5 in Dulbecco’s
the figure legends. Rectal temperatures of the mice were measured every modified Eagle’s medium with 20% FBS and 50 mM Hepes (pH 7.5).
10 min for 1 hour and 30 min after they were injected with antigen. For Cells were used within 48 hours of transfection.
the histamine assay, the mice were euthanized with CO2 30 min after
they were injected with antigen, and serum was obtained by cardiac punc- Measurement of degranulation and release of cytokines
ture. The concentration of histamine in the serum was measured by BMMCs were primed for 4 hours with DNP-specific IgE (500 ng/ml; Sigma).
ELISA according to the manufacturer’s instructions (Beckman Coulter). The IgE-primed BMMCs were then stimulated with antigen [DNP-BSA
PCA was induced as described previously (43). All experiments with (100 ng/ml); Sigma] in Tyrode-BSA buffer [20 mM Hepes (pH 7.4), 135
mice were performed three times, with five mice for each condition used mM NaCl, 5 mM potassium chloride, 1.8 mM calcium chloride, 1 mM
per experiment. magnesium chloride, 5.6 mM glucose, and 0.05% BSA] for 15 min in the
presence or absence of the B cell subsets indicated in the figure legends.
Flow cytometric analysis Degranulation was determined by measuring the release of the granule
Single-cell suspensions were isolated from the spleen, inguinal lymph node, marker b-hexosaminidase as previously described (45). In some coculture
peritoneal cavity, and blood. To detect intracellular IL-10 in B cells from experiments, BMMCs and B cell subsets were separated by 3.0-mm trans-
each site, isolated B cells were cultured with medium alone or with medium well membrane plates (Corning Life Sciences). Cells were stimulated with
containing BMMCs, IgE-treated BMMCs, IgE- and antigen-treated BMMCs, antigen for 24 hours (or the times indicated in the figure legends) in
or CD40L (1 mg/ml; R&D Systems) for 24 hours or the times indicated in complete medium to measure the secretion of TNF-a, IL-4, and IL-10 with
the figure legends, and phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ELISA kits from Invitrogen (BioSource) or R&D Systems Inc.
ionomycin (500 ng/ml; Sigma), and brefeldin A (3 mg/ml; eBioscience)
were added during the last 5 hours of incubation. Before cell surface Immunohistochemistry
markers were stained, Fcg receptors were blocked with anti-CD16 and Paraffin-embedded spleen sections were subjected to immunohistochem-
anti-CD32 monoclonal antibodies (2.4G2, BD Biosciences), and ical analysis with specific antibodies and isotype controls according to a
www.SCIENCESIGNALING.org 17 March 2015 Vol 8 Issue 368 ra28 9RESEARCH ARTICLE
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S. H. Bernstein, C. M. Magro, A. D. Williams, R. P. Hall, E. W. St Clair, T. F. Tedder, Char- Blood Institute, NIH. Author contributions: W.S.C. and Y.M.K. designed the experiments,
acterization of a rare IL-10–competent B-cell subset in humans that parallels mouse analyzed the data, and wrote the paper; Hyuk S.K. and A.-R.K. performed most of the ex-
regulatory B10 cells. Blood 117, 530–541 (2011). periments; D.K.K. and G.H.J. collected and analyzed flow cytometry data; H.W.K. and Y.H.P.
39. D. Y. Kim, D. Jeoung, J. Y. Ro, Signaling pathways in the activation of mast cells cocul- performed in vivo experiments; J.S.Y. and Hyung S.K. performed all experiments for cell
tured with astrocytes and colocalization of both cells in experimental allergic encephalo- signaling analysis; B.K. and Y.M.P. contributed reagents and knockout animals and designed
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