Research ArticleImmunology

Interleukin-10–producing CD5+ B cells inhibit mast cells during immunoglobulin E–mediated allergic responses

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Sci. Signal.  17 Mar 2015:
Vol. 8, Issue 368, pp. ra28
DOI: 10.1126/scisignal.2005861

Limiting allergic responses with B cells

B cells promote immune responses by producing antibodies; however, subsets of B cells secrete the anti-inflammatory cytokine interleukin-10 (IL-10) and have immunosuppressive properties. Kim et al. found that these B cells inhibited the activation of mast cells, immune cells that are critical regulators of allergic reactions. Indeed, mice lacking these special B cells had more severe symptoms of anaphylaxis. Mast cell inhibition required physical contact with the B cells, which stimulated the B cells to produce more IL-10. B cell–mediated inhibition of mast cells depended on IL-10, which inhibited tyrosine kinase signaling in mast cells. Thus, IL-10–producing B cells might provide a therapeutic target to treat allergic diseases.

Abstract

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 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 kinase 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

Allergic disorders are widespread, particularly in developed countries (1). Allergic responses are associated with increases in the number of T helper 2 (TH2) cells and immunoglobulin E (IgE) antibody production. Presentation of allergen to antigen-presenting cells (APCs) causes TH2 cells to produce TH2-type cytokines. In particular, the cytokine interleukin-4 (IL-4) is essential for the isotype switching of B cells to produce allergen-specific IgE antibodies (2), which then bind specifically to the multimeric high-affinity IgE receptor FcεRI (3) on mast cells and basophils.

B cells are generally known for their capacity to regulate effector T cell responses and to produce antibodies (4); however, studies in murine disease models revealed some distinct B cell subsets that exhibit immunosuppressive functions (58) and thus are named regulatory B (Breg) cells (9). Other subsets of Breg cells have also been identified to suppress various immune responses in an IL-10–dependent manner (10), whereas helminth infection–induced IL-10–producing B cells inhibit allergic reactions in animal models, specifically ovalbumin-mediated anaphylaxis (11) and allergic asthma (12). IL-10 also suppresses mast cell activation in vitro and in vivo and may thus counteract their excessive activation and the development of chronic inflammation (13, 14). Despite these reports, and given the role of mast cells in these and other allergic diseases (15, 16), surprisingly little is known about the mechanism of interaction between Breg cells and mast cells or about the consequences of these interactions for IgE-mediated allergic responses.

Mast cells are the key effector cells in IgE-mediated allergic reactions. These cells are widely distributed in vascularized tissues, especially near surfaces exposed to the environment, such as the skin, airways, and the gastrointestinal tract (15). Mast cells are commonly activated by the multivalent binding of antigen to FcεRI-bound IgE, with the subsequent release of various allergic mediators, including histamine, eicosanoids, and inflammatory cytokines. Release of these mediators leads to immediate, and sometimes delayed, symptoms of allergic diseases, such as allergic rhinitis, anaphylaxis, and atopic dermatitis (17, 18). We found that CD5+ B cells inhibited IgE-mediated mast cell activation and anaphylaxis in mice in an IL-10–dependent manner. Furthermore, we found that IL-10–producing CD5+ B cells inhibited the activation of the tyrosine kinase Syk (spleen tyrosine kinase) in mast cells. Together, our findings suggest that IL-10–producing CD5+ B cells inhibit IgE-mediated allergic responses in physiological settings.

RESULTS

CD5+ B cells suppress IgE-mediated anaphylaxis in vivo and mast cell activation in vitro

IgE-dependent mast cell activation is regarded as one of the cardinal mechanisms in the development of anaphylaxis. Here, we administrated IgE antibody and antigen intravenously to mice to induce passive systemic anaphylaxis (PSA), and these responses are essentially associated with extensive mast cell activation in vivo. We found that the numbers of IL-10–producing CD5+ B cells were increased in the spleen, peritoneal cavity, lymph node, and blood of IgE- and antigen-induced PSA mice (fig. S1A), which suggests that these cells are associated with the progression of symptoms. We next examined CD19-decifient mice in which IL-10–producing B cells are substantially depleted (fig. S1B) (7). We found that IgE-mediated anaphylaxis responses (Fig. 1A) and increases in the concentration of histamine in the blood (Fig. 1B) were substantially enhanced in the CD19-deficient mice compared to wild-type mice.

Fig. 1 CD5+ B cells suppress IgE-mediated activation of mouse mast cells in vivo and in culture.

(A and B) PSA was induced by injecting wild-type (WT) mice or CD19−/− mice with dinitrophenyl (DNP)–IgE (3 μg) 24 hours before injecting them with DNP–bovine serum albumin (DNP-BSA; 250 μg; Ag) as indicated. Mice were then subjected to analysis of rectal temperatures at the indicated times (A) and serum concentrations of histamine 30 min after stimulation (B) (n ≥ 5 mice per experiment). (C and D) PSA was induced in CD19−/− mice 3 days after they received CD5+ B cells or CD5 B cells by adoptive transfer. Mice were then subjected to analysis of rectal temperatures (C) and serum concentrations of histamine (D) (n ≥ 5 mice per experiment). (E and F) Bone marrow–derived mast cells (BMMCs) were preincubated in the absence or presence of CD5+ or CD5 B cells at a BMMC/B cell ratio of 1:5 for the indicated times (E) or CD5+ or CD5 B cells at the indicated BMMC/B cell ratios for 24 hours (F). Cells were then treated with or without IgE and antigen (Ag) as indicated, and the extent of release of β-hexosaminidase from the BMMCs was determined. (G to I) BMMCs were cultured alone or together with CD5+ or CD5 B cells and then were treated with the indicated combinations of IgE and antigen. The amounts of histamine (G), tumor necrosis factor–α (TNF-α) (H), and IL-4 (I) released into the culture medium were determined by enzyme-linked immunosorbent assay ELISA). Data are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; n.s., not significant.

To assess the effects of CD5+ B cells in IgE-mediated allergic responses in vivo, we first induced PSA in CD19-deficient mice. Three days after they received purified CD5+ or CD5 B cells from wild-type mice by adoptive transfer (fig. S1C), IgE-sensitized CD19-deficient mice were intravenously challenged with antigen. The presence of CD5+ B cells, but not CD5 B cells, markedly alleviated the decline in temperature in response to antigen (Fig. 1C). Serum histamine concentrations were increased in antigen-challenged mice compared to those in unchallenged mice, but this increase was substantially reduced after the adoptive transfer of CD5+ cells (Fig. 1D).

Next, we found that culturing BMMCs with CD5+ B cells, but not CD5 B cells, substantially inhibited their degranulation in response to antigen in a time- and cell concentration–dependent manner (Fig. 1, E and F), as well as inhibited their release of other allergic mediators, such as histamine, TNF-α, and IL-4 (Fig. 1, G to I). This inhibitory action of CD5+ B cells was not dependent on mouse strain or tissue source because CD5+ B cells isolated from the peritoneal cavity of C57BL/6 mice and from the spleen of BALB/c mice also inhibited the antigen-stimulated degranulation of BMMCs (fig. S1, D and E).

IL-10 from CD5+ B cells is critical for mast cell inhibition

The immunoregulatory role of Breg cells, also called B10 cells, is generally dependent on IL-10 (10). We found that the passive cutaneous anaphylaxis (PCA) reaction was increased in IL-10−/− mice compared to that in wild-type mice (fig. S2, A and B, top). The numbers of degranulated mast cells in the ear tissues of IL-10−/− mice were increased, albeit not statistically significantly, compared to those in the ear tissues of wild-type mice (fig. S2B, bottom). These results led us to investigate whether IL-10 generated by CD5+ B cells inhibited mast cell activation and IgE-mediated anaphylaxis. First, we found that monoclonal antibodies against IL-10 or the IL-10 receptor (IL-10R) blocked the suppressive effect of CD5+ B cells on the antigen-induced degranulation of BMMCs (Fig. 2A). Second, CD5+ B cells from IL-10−/− mice failed to inhibit the IgE- and antigen-stimulated degranulation of BMMCs in vitro (Fig. 2B). CD5+ B cells from either wild-type or IL-10−/− mice showed no statistically significant differences in the amounts of their cell surface markers, including IgD, IgM, CD19, CD21, CD1d, CD11b, CD40, and B220 (fig. S2C). Third, CD5+ B cells also inhibited the antigen-stimulated degranulation of IL-10−/− BMMCs (fig. S2D). Notably, flow cytometric analysis revealed that IL-10–producing B cells were found mostly within a 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 increases 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.

Fig. 2 IL-10 production by CD5+ B cells is critical for the suppression of mast cell activation.

(A and B) BMMCs were preincubated for 24 hours with CD5+ B cells in the presence or absence of the indicated combinations of anti-IL-10 (α-IL-10), anti-IL-10R (α-IL-10R), or isotype control monoclonal antibody (mAb) (A) or with the indicated combinations of CD5+ or CD5 B cells from WT or IL-10−/− mice (B). Cells were then treated with the indicated combinations of IgE and antigen, and the amount of β-hexosaminidase released into the culture medium was determined. Data are means ± SEM of three independent experiments. (C and D) PSA was induced in IL-19−/− mice 3 days after they had received CD5+ or CD5 B cells from WT or IL-10−/− mice, as indicated, by adoptive transfer. Mice were then analyzed to determine rectal temperatures at the indicated times (C) and serum histamine concentrations at 30 min (D) (n ≥ 5 mice per experiment). (E and F) PSA was induced in IL-10−/− mice 3 days after they received CD5+ or CD5 B cells from WT or IL-10−/− mice by adoptive transfer, as indicated. Mice were then analyzed to determine rectal temperatures at the indicated times (E) and serum histamine concentrations at 30 min (F) (n ≥ 5 mice per experiment). (G to I) Equal numbers of CD5+ or CD5 B cells were incubated alone or in the presence of unstimulated or IgE- and antigen-stimulated BMMCs from WT (G) or IL-10−/− mice (H and I), as indicated. Cells were then analyzed by flow cytometry to determine the percentages of IL-10+ B cells (CD19+) (G), the percentages of CD5+ or CD5 B cells that contained IL-10 (were IL-10+) (H), and the amount of IL-10 in the culture medium (I). Data in (G) are representative of three independent experiments. Data in (H) and (I) are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

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 increased production was further enhanced by stimulating wild-type or IL-10−/− BMMCs with antigen (Fig. 2, H and I). The stimulatory effect of BMMCs on IL-10 production was also apparent with CD5+ B cells from the spleen, inguinal lymph node, and blood, but not from the peritoneal cavity (fig. S3, D and E). These results suggest that BMMCs stimulate IL-10 production by CD5+ B cells from various lymphoid organs.

Direct cell-cell contact is essential to inhibit mast cell activation and enhance IL-10 production by CD5+ B cells

Although degranulation was inhibited when mast cells were cultured with CD5+ B cells, this was not the case when the cell types were physically separated in transwell culture flasks (Fig. 3A). Note that conjugation of CD5+ B cells and mast cells with or without IgE (~20% of cells conjugated) was observed in cocultures, and the extent of conjugation was increased (to ~25%) by the addition of antigen (Fig. 3, B and C). We also observed conjugation between CD5 B cells and mast cells (fig. S3F), which was similar to that between CD5+ B cells and mast cells. Furthermore, immunohistochemical analysis of mouse spleens after PSA was induced revealed that some CD5+CD19+ B cells were in close proximity to mast cells, which raises the possibility of crosstalk between these two cell types in vivo (Fig. 3D). Similarly, the increase in IL-10 production by CD5+ B cells was observed only in cocultures and not when both cell types were separated in transwell flasks (Fig. 3, E and F).

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 β-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 (purple, toluidine blue). CD5+ B cells are indicated by green arrows; mast cells are indicated by red arrows. Images are shown at ×400 magnification; however, the area in the yellow box is shown in the bottom right panel at a magnification of ×1000. Scale bar, 100 μm. (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 intracellular 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 representative of three independent experiments. Data in (A), (C), and (F) are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

CD40 on CD5+ B cells and CD40 ligand (CD154) on mast cells are required for the production of IL-10 by CD5+ B cells and the suppression of mast cell activation

CD40-generated signals in IL-10–producing B cells participate in the regulation of various inflammatory diseases and possibly in the production of IL-10 (10). Because the abundance of CD40 ligand (CD40L) on the surface of mast cells is increased by stimulation with antigen (19), we determined whether the enhanced IL-10 production by B cells was dependent on the CD40-CD40L interaction. The abundances of CD40 on B cells and IL-10 in B cells, as well as the amount of IL-10 secreted from B cells, were substantially increased by recombinant CD40L (Fig. 4, A to D). Whereas CD40 abundance and IL-10 production in whole B cells were increased when they were cocultured with BMMCs (fig. S4, A and B), IL-10 production by B cells was largely blocked in the presence of an anti-CD40L monoclonal antibody (fig. S4, B and C). In addition, in cocultures of CD5+ B cells and BMMCs, the increased abundances of cell surface CD40 (Fig. 4E) and IL-10 (Fig. 4, F and G) in CD5+ B cells were markedly inhibited by the anti-CD40L antibody, and the suppressive action of CD5+ B cells on BMMC degranulation was also blocked by this antibody (Fig. 4H).

Fig. 4 IL-10 production by mouse CD5+ B cells is dependent on CD40-CD40L interactions.

(A to D) Total B cells from WT mice were left unstimulated or were stimulated with recombinant CD40L (rCD40L;1 μg/ml) for the indicated times. The mean fluorescence intensity (MFI) of CD40 staining on B cells (A) and the percentages of IL-10+ B cells (B and C) were determined by flow cytometric analysis. (D) The amounts of IL-10 in the culture medium were determined by ELISA. (E to G) CD5+ B cells were cultured alone or with either unstimulated or IgE- and antigen-stimulated BMMCs in the presence or absence of anti-CD40L antibody or an isotype control antibody for 24 hours. The MFI of CD40 staining on B cells (E) and the percentages of IL-10+ B cells (F and G, top) were determined by flow cytometric analysis. (G, bottom) The amounts of IL-10 in the culture medium were determined by ELISA. (H) BMMCs preincubated in the absence or presence of the indicated combinations of CD5+ B cells, anti-CD40L antibody, and isotype control antibody were stimulated with IgE and antigen, as indicated. Twenty-four hours later, the extent of β-hexosaminidase release into the culture medium was determined. Plots in (B) and (F) are representative of three independent experiments. Data in (A), (C) to (E), (G), and (H) are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; n.s., not significant.

The interaction between CD5+ B cells and mast cells through CD40-CD40L is critical for the suppression of IgE-mediated mast cell activation and anaphylaxis

We next observed whether CD5+ B cells and mast cells interacted directly under cocultured conditions. The extent of conjugation of CD5+ B cells and BMMCs in coculture that we noted earlier (Fig. 3B) was substantially reduced when CD40−/− CD5+ B cells, CD40L−/− BMMCs, or both were substituted for the corresponding wild-type cells (Fig. 5, A and B), suggesting that CD40-CD40L contact was necessary for such conjugation to occur. A similar pattern of effects on the production of IL-10 by CD5+ B cells was observed when cells deficient in CD40 or CD40L were substituted for the corresponding wild-type cells (Fig. 5, C and D), which was consistent with the data obtained from experiments with recombinant CD40L and the anti-CD40L antibody (Fig. 4), as well as with the requirement of CD40-CD40L interaction for IL-10 production by CD5+ B cells. Moreover, this interaction appeared to be necessary irrespective of whether the BMMCs were stimulated with antigen (Fig. 5, C and D). Loss of either CD40 or CD40L did not alter the patterns of cell surface markers of CD5+ B cells or BMMCs, respectively (fig. S4D).

Fig. 5 Suppression of mast cell activation and PSA by CD5+ B cells depends on CD40-CD40L interactions.

(A and B) CD5+ B cells from WT or CD40−/− mice were incubated in a 1:1 ratio with BMMCs from WT or CD40L−/− mice, as indicated. (A) After 1 hour, BMMC–B cell conjugates were detected by flow cytometric analysis. (B) The percentages of BMMC–B cell conjugates that formed under the four indicated conditions (a to d) were calculated. (C and D) CD5+ B cells from WT or CD40−/− mice were incubated alone or in a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs from WT or CD40L−/− mice, as indicated. (C) Cells were subjected to flow cytometric analysis to identify IL-10+CD5+ B cells. (D) The percentages of IL-10+CD5+ B cells under the indicated conditions were calculated. (E) CD5+ B cells from WT or CD40−/− mice were incubated for 24 hours alone or in a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs from WT or CD40L−/− mice, as indicated. The extent of β-hexosaminidase release into the culture medium was determined. (F) CD19−/− mice were left untreated or received CD5+ or CD5 B cells from WT or CD40−/− mice by adoptive transfer. Three days later, the mice were treated with IgE and antigen to induce PSA, and rectal temperatures in the indicated mice were measured over time (n = 5 mice per experiment). (G) Mast cell–deficient mice (KitW-sh/W-sh mice) were left untreated or received BMMCs from WT or CD40L−/− mice by adoptive transfer. Three days later, the mice were treated with the indicated combinations of IgE and antigen, and the rectal temperatures of the mice were measured over time. Plots in (A) and (C) are representative of three independent experiments. Data in (B) and (D) to (G) are means ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

With respect to mast cell function, the IgE- and antigen-induced degranulation of BMMCs was no longer suppressed when CD40−/− CD5+ B cells or CD40L−/− BMMCs were substituted for the corresponding wild-type cells (Fig. 5E). Furthermore, the adoptive transfer of wild-type CD5+ B cells, but not CD40−/− CD5+ B cells, into CD19-deficient mice suppressed the decline in rectal temperatures when PSA was induced (Fig. 5F). To further determine whether the CD40-CD40L interaction between CD5+ B cells and mast cells occurs in vivo, we transferred wild-type or CD40L-deficient BMMCs into KitW-sh/W-sh mice, which do not contain mast cells and, thus, do not exhibit PSA symptoms in response to stimulation with IgE and antigen (20). Although the antigen-stimulated degranulation of CD40L-deficient BMMCs was comparable to that of wild-type BMMCs (Fig. 5E), IgE-mediated anaphylaxis responses became more severe when the KitW-sh/W-sh mice received CD40L−/− BMMCs instead of wild-type BMMCs (Fig. 5G). Together, these results suggest that the CD40-CD40L interaction between CD5+ B cells and mast cells is critical for the suppression of mast cell activation and IgE-mediated PSA responses in vivo.

IL-10–producing CD5+ B cells inhibit FcεRI-mediated signaling in mast cells

We did not observe any decrease in the cell surface abundances of the α, β, or γ subunits of FcεRI on BMMCs that were cocultured with CD5+ B cells for 24 hours (Fig. 6, A and B). However, we observed alterations in the abundances of some tyrosine kinases that transduce initial signals after antigen-dependent cross-linking of IgE bound to FcεRI. Coculture with wild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, substantially reduced the abundances of the kinases Fyn and Fgr in BMMCs, whereas Lyn and Syk were unaffected (Fig. 6, C and D). The lack of change in the abundance of Syk was further confirmed by flow cytometric analysis (Fig. 6E). Furthermore, wild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, markedly inhibited the antigen-induced tyrosine phosphorylation of Syk in BMMCs (Fig. 6, F and G), which suggests that wild-type CD5+ B cells can inhibit the activation of mast cells by reducing the abundances of Fyn and Fgr. These observations were further confirmed by incubating BMMCs with recombinant IL-10 for 24 or 72 hours (fig S5, A to D). The essential requirement for an interaction between CD40 (on CD5+ B cells) and CD40L (on mast cells) to suppress the phosphorylation of the tyrosine kinases after IgE- and antigen-mediated stimulation was verified in experiments with an anti-CD40L antibody (fig. S5, E and F).

Fig. 6 CD5+ B cell–derived IL-10 reduces Fyn and Fgr abundance and inhibits downstream activation of Syk in BMMCs.

(A and B) BMMCs from WT mice were cultured alone or with CD5+ or CD5 B cells, as indicated. (A) Twenty-four hours later, the cells were analyzed by flow cytometry to detect the cell surface expression of the indicated FcεRI subunits. (B) The relative MFIs of the indicated FcεRI subunits on the BMMCs were calculated. (C and D) BMMCs were cultured alone or with CD5+ B cells from WT or IL-10−/− mice, as indicated. (C) BMMCs were then analyzed by Western blotting with antibodies against the indicated proteins. (D) Quantification of the relative abundances of the indicated proteins was performed by densitometric analysis of Western blots. (E) BMMCs were cultured alone or with CD5+ B cells from WT or IL-10−/− mice at a 1:5 ratio. Twenty-four hours later, the cells were treated with the indicated combinations of IgE and antigen before being analyzed by flow cytometry to determine the MFI of Syk. (F and G) BMMCs cultured alone or with WT or IL-10−/− CD5+ B cells at a 1:5 ratio for 24 hours were then treated for 7 min with the indicated combinations of IgE and antigen. (F) The BMMCs were analyzed by flow cytometry to detect tyrosine-phosphorylated Syk (Tyr352). (G) The MFIs of pSyk in BMMCs under the indicated conditions were determined. (H) BMMCs were incubated with or without recombinant IL-10 (rIL-10; 100 ng/ml) in the absence or presence of 25 μM AG490 for 24 hours and were then were left unstimulated or were stimulated with antigen for 15 min. Cells were then analyzed by flow cytometry to determine the MFIs of total STAT3 (top) and pSTAT3 (Tyr705; bottom). (I and J) BMMCs were cultured for 24 hours in medium containing IL-3 in the absence or presence of rIL-10 (100 ng/ml) or 25 μM AG490. The cells were then stimulated with the indicated combinations of IgE and antigen for 7 min before being analyzed by Western blotting to detect total Lyn, Fyn, and Fgr proteins (I) and total Syk and phosphorylated Syk proteins (J). Band densities are shown as the mean values from three independent experiments in each lower panel. (K and L) BMMCs were transfected with STAT3-specific siRNAs (siSTAT3) or control siRNAs (siCtrl) 48 hours before the experiment. The BMMCs were then incubated with the indicated combinations of rIL-10 (100 ng/ml) and IgE (500 ng/ml) for 24 hours before being left untreated or stimulated with antigen for 7 min. (K) BMMCs were analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are representative of three independent experiments. (L) Densitometric analysis of the relative abundances of total Fyn and Fgr proteins and of pSyk. Data in (B), (D), (E), (G), (I and J, lower panels), and (L) are means ± SEM of three independent experiments. *P < < 0.05; **P < 0.01.

The inhibition of mast cells by IL-10 is dependent on signal transducer and activator of transcription 3 (STAT3) signaling, which leads to the reduced activation of tyrosine kinases involved in early signaling events (14). We observed that IL-10 stimulated the phosphorylation of STAT3, which was inhibited by the Janus-activated kinase (JAK) inhibitor AG490 (Fig. 6H). In addition, AG490 restored the abundance of Fyn and Fgr (Fig. 6I) as well as the extent of tyrosine phosphorylation of Syk in IL-10–treated BMMCs (Fig. 6J). Further experiments showed that STAT3-specific small interfering RNA (siRNA) restored the amounts of Fyn and Fgr, as well as the phosphorylation of Syk, in IL-10–treated mast cells (Fig. 6, K and L), which suggests that the suppression of mast cells by CD5+ B cell–derived IL-10 is mediated through the JAK-STAT3 pathway.

DISCUSSION

B cells have the capacity to produce antibodies, function as APCs, and regulate the activation of CD4+ T cells (2123). In addition, there is evidence that B cells have a regulatory role in various immune responses through their production of cytokines. Katz et al. (24) were the first to demonstrate that the delayed-type hypersensitivity reaction was exacerbated by the depletion of B cells. Mizoguchi and Bhan (9) introduced the term “regulatory B cells” to designate negative regulatory subpopulations of B cells. Breg cells are now recognized as one of the key regulatory cell types that suppress inflammatory disorders (10) and various immune cells, including dendritic cells (DCs), macrophages, and TH cells (25, 26).

IL-10 was originally identified as the TH2 cell–derived cytokine synthesis inhibitory factor (27), and it has broad anti-inflammatory actions. IL-10 suppresses the effector function of T cells and macrophages (28, 29). With respect to Breg cells (B10 cells), IL-10 enables the suppressive functions of these cells in various immune disease models (10). Moreover, subsets of IL-10–producing Breg cells suppress TH2 cell–mediated allergic responses, including contact hypersensitivity and allergic airway disease in mouse models (7, 12). Although mast cells can produce IL-10 through FcεRIII signaling after the receptor is increased in abundance by IL-4 (30), IL-10 is not normally produced by mast cells through IgE- and antigen-mediated stimulation (fig. S3, B and C).

Mast cells are the critical effector cells in food allergies, allergic asthma, and allergic rhinitis (16, 18). Although present in multiple tissues, mast cells are distributed mainly at the major immunologic interfaces, such as the skin, gut, and lungs. Secondary lymphoid organs including spleens, tonsils, and lymph nodes harbor modest numbers of mast cells in physiological settings (19, 31) where they could also regulate inflammation. We previously reported that the prevalence of IL-10–producing CD5+ peripheral blood B cells increased in healthy donors but decreased in patients with milk allergy after challenge with milk antigen (32). Our present finding that CD5+ B cells inhibited IgE-mediated mast cell activation and anaphylaxis in mice in an IL-10–dependent manner suggests that the interaction between IL-10–producing CD5+ B cells and mast cells provides a mechanism 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 immunohistochemical 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 functions 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 response. Mast cells were reported to communicate with B cells and astrocytes through the CD40-CD40L interaction (39, 40). Here, we propose that mast cells may similarly regulate CD5+ B cell function on the basis that mast cells express CD40L (fig. S5D) and increase the cell surface abundance of CD40 on CD5+ B cells, and that the CD40L-CD40 interaction leads to an increase in the number of IL-10–producing CD5+ B cells, which in turn suppress mast cell activation and anaphylaxis (Fig. 5). Notably, the anaphylaxis responses stimulated by IgE and antigen were reduced in mice that received wild-type CD5+ B cells, but not CD40−/− CD5+ B cells, by adoptive transfer (Fig. 5F). To further determine whether CD40L on mast cells was critical for a direct interaction with CD40 on CD5+ B cells, we performed experiments with mast cell–deficient mice (KitW-sh/W-sh mice), which do not show any PSA in response to IgE and antigen (20). Although IgE-mediated anaphylaxis was not observed in KitW-sh/W-sh mice (Fig. 5G) (20), the responses became much more severe in mice that received CD40L−/− BMMCs by adoptive transfer compared to those that received wild-type BMMCs (Fig. 5G). Together, these results suggest that CD40L on mast cells is critical to the induction of IL-10–producing CD5+ B cells in physiological settings.

The precise details by which IL-10–producing Breg cells suppress allergic and inflammatory responses are unclear. Our results provide some insight with regard to the interaction of the B cells with mast cells. Early signaling events in antigen-stimulated mast cells include the recruitment of Lyn and other Src family kinases, such as Fyn (41) and Fgr (42), to FcεRI, which results in the phosphorylation and activation of Syk and the activation of mast cells (43). In our experimental system, coculturing CD5+ B cells with BMMCs for 24 hours reduced the abundance of Fyn and Fgr in mast cells, and consequently decreased the phosphorylation of Syk (Fig. 6, C to G), but had no effect on the abundances of individual FcεRI subunits (Fig. 6B). Similar results were obtained from experiments in which BMMCs were treated with recombinant IL-10 under similar conditions. However, more prolonged incubation (72 hours) of mast cells with IL-10 resulted in a reduction in the amounts of the FcεRI subunits, in addition to a reduction in the abundances of Syk, Fyn, and Fgr in mast cells (fig. S5, B and D), consistent with previous findings (14). We further demonstrated that the effects of CD5+ B cells on the abundances of Fyn and Fgr and on the phosphorylation of Syk were blocked by inhibiting the JAK-STAT3 pathway with a typical JAK inhibitor, AG490 (Fig. 6, H to J), and by siRNAs specific for STAT3 in mast cells (Fig. 6, K and L). These observations led us to suggest that CD5+ B cells inhibit mast cell activation through IL-10 by reducing the abundances of Fyn and Fgr through the JAK-STAT3 signaling pathway. It would be also of interest to determine whether the inhibitory effects of IL-10–producing B cells extend to other mast cell stimulants such as IgG1, which may be involved in analphylactic reactions.

In summary, our results demonstrate a mechanism by which IL-10–producing CD5+ B cells inhibit mast cell function as follows: (i) Both cell types form cell-cell conjugates through an interaction between CD40 (on CD5+ B cells) and CD40L (on mast cells). (ii) This interaction stimulates IL-10 production by CD5+ B cells. (iii) IL-10 produced by CD5+ B cells inhibits the abundance of Fyn and Fgr in mast cells through activation of the JAK-STAT3 pathway. (iv) As a result, mast cell activation through FcεRI is suppressed by the diminished quantities of Fyn and Fgr in mast cells (Fig. 7). These findings suggest that IL-10–producing CD5+ B cells may provide an additional therapeutic target to treat IgE-mediated allergic diseases.

Fig. 7 Proposed scheme for the suppression of mast cell activation by IL-10–producing CD5+ B cells.

Suppression is dependent on direct cell-to-cell contact through the interaction of CD40L on mast cells and CD40 on CD5+ B cells. This interaction results in the production of IL-10 by the CD5+ B cells. IL-10 signaling reduces the abundances of Fyn and Fgr in the mast cells, which thus reduces the extent of activation of Syk, resulting in the inhibition of mast cell degranulation. Tyk, tyrosine kinase.

MATERIALS AND METHODS

Mice

Wild-type (~6- to 8-week-old male C57BL/6 mice), CD19−/− [Cd19tm1(cre)Cgn], IL-10−/− (Il10tm1Cgn), CD40−/− (Cd40tm1Kik), CD40L−/− (Cd40lgtm1Imx), and KitW-sh/W-sh mice were purchased from The Jackson Laboratory, housed in a specific pathogen–free animal facility at Konkuk University (Seoul, Korea) and fed with a sterilized diet and autoclaved water before being used for experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Konkuk University.

Preparation and adoptive transfer of B cell subsets

Splenic B cells were presorted with CD19 microbeads (Miltenyi Biotec). Then CD5+ or CD5 B cells were isolated with a FACSAria flow cytometer (BD Biosciences). For in vivo adoptive transfer of B cell subsets, the isolated B cells were transferred intravenously [2 × 106 cells/0.2 ml of phosphate-buffered saline (PBS)] into recipient mice. Three days after the adoptive transfer of cells, PSA was induced in the recipient mice.

Induction of PSA or PCA

Mice were primed with 3 μg of DNP-specific IgE (SPE-7; Sigma) by intravenous injection. On the next day, the mice were injected intravenously with 250 μg of DNP-BSA (Sigma) in 200 μl of PBS or as indicated in the figure legends. Rectal temperatures of the mice were measured every 10 min for 1 hour and 30 min after they were injected with antigen. For the histamine assay, the mice were euthanized with CO2 30 min after they were injected with antigen, and serum was obtained by cardiac puncture. The concentration of histamine in the serum was measured by ELISA according to the manufacturer’s instructions (Beckman Coulter). PCA was induced as described previously (43). All experiments with mice were performed three times, with five mice for each condition used per experiment.

Flow cytometric analysis

Single-cell suspensions were isolated from the spleen, inguinal lymph node, peritoneal cavity, and blood. To detect intracellular IL-10 in B cells from each site, isolated B cells were cultured with medium alone or with medium containing BMMCs, IgE-treated BMMCs, IgE- and antigen-treated BMMCs, or CD40L (1 μg/ml; R&D Systems) for 24 hours or the times indicated in the figure legends, and phorbol 12-myristate 13-acetate (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and brefeldin A (3 μg/ml; eBioscience) were added during the last 5 hours of incubation. Before cell surface markers were stained, Fcγ receptors were blocked with anti-CD16 and anti-CD32 monoclonal antibodies (2.4G2, BD Biosciences), and conjugated and dead cells were excluded from the analysis on the basis of forward and side light scatter parameters and staining with Fixable Viability Dye (eBioscience). Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) and then were incubated with anti–IL-10 monoclonal antibody (JES5-16E3, eBioscience) at 4°C for 30 min. The antibodies against cell surface proteins were as follows: anti-CD1d (1B1), anti-CD4 (RM4-5), anti-CD5 (53–7.3), anti-CD11b (M1/70), anti-CD19 (eBio1D3), anti-CD21/CD35 (eBioBD9), anti-CD23 (B3B4), anti-CD25 (PC61.5), anti-CD40 (HM40-3), anti-CD86 (GL1), anti-B220 (RA3-6B2), anti-IgD (11–26), anti-IgM (eB121-15 F9), anti–c-Kit (2B8), and anti-CD40L (MR1), which were purchased from eBioscience, and anti-FcεRI (anti-IgE, R35-72), which was purchased from BD Biosciences. To detect FcεRI subunits and intracellular Syk, fixed BMMCs were stained with antibodies against FcεRIα (G-14), FcεRIβ (N-18), FcεRIγ (H-5), and Syk (N-19), which were obtained from Santa Cruz Biotechnology, and with anti-STAT3 antibody (M59-50), which was obtained from BD Biosciences. To evaluate the extent of phosphorylation of Syk or STAT3, BMMCs were primed with DNP-specific IgE (500 ng/ml) and cultured with CD5+ B cells for 24 hours before being stimulated with DNP-BSA (100 ng/ml) for 7 min (for pSyk) or 15 min (for pSTAT3). The BMMCs were immediately fixed and permeabilized and then were stained with anti-CD19 (eBio1D3), anti–c-Kit (2B8, eBioscience), and either anti-ZAP70(Tyr319)/Syk(Tyr352) (17A/P-ZAP70) or anti-STAT3 (Tyr705) (4/P-STAT3) antibodies (BD Biosciences). Briefly, after CD19+ B cells were excluded from gated mast cells because of staining with an anti-CD19 monoclonal antibody, c-Kit+ cells that stained with anti-ZAP70(Tyr319)/Syk(Tyr352)+ or anti-STAT3 (Tyr705)+ were analyzed. Cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo version 10 software (TreeStar).

BMMC differentiation and transfection with STAT3-specific siRNA

BMMCs derived from C57BL/6 or BALB/c mice were cultured in RPMI 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; PeproTech Inc.). After 4 weeks, >98% of the cells were verified as BMMCs, as previously described (44). BMMCs (5 × 106 cells) were transfected with 100 nM STAT3-specific siRNA or scrambled siRNA with an Amaxa Nucleofector (Lonza Cologne AG) with program T-5 in Dulbecco’s modified Eagle’s medium with 20% FBS and 50 mM Hepes (pH 7.5). Cells were used within 48 hours of transfection.

Measurement of degranulation and release of cytokines

BMMCs were primed for 4 hours with DNP-specific IgE (500 ng/ml; Sigma). The IgE-primed BMMCs were then stimulated with antigen [DNP-BSA (100 ng/ml); Sigma] in Tyrode-BSA buffer [20 mM Hepes (pH 7.4), 135 mM NaCl, 5 mM potassium chloride, 1.8 mM calcium chloride, 1 mM 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. Degranulation was determined by measuring the release of the granule marker β-hexosaminidase as previously described (45). In some coculture experiments, BMMCs and B cell subsets were separated by 3.0-μm transwell membrane plates (Corning Life Sciences). Cells were stimulated with antigen for 24 hours (or the times indicated in the figure legends) in complete medium to measure the secretion of TNF-α, IL-4, and IL-10 with ELISA kits from Invitrogen (BioSource) or R&D Systems Inc.

Immunohistochemistry

Paraffin-embedded spleen sections were subjected to immunohistochemical analysis with specific antibodies and isotype controls according to a regular protocol. The signal was amplified with horseradish peroxidase– or alkaline phosphatase–conjugated streptavidin with a Vectastain Elite ABC kit (Vector). The sections were visualized with N-DAB or VectorRed, and then the mast cells were stained with toluidine blue.

Analysis of CD5+ or CD5 B cell–mast cell conjugation

IgE-primed or untreated BMMCs were stained with 1 μM CellTracker Green probe (BODIPY, Invitrogen), and isolated splenic CD5+ B cells were stained with 1 μM CellTracker Red probe (CMPTX, Invitrogen). The stained BMMCs (1 × 106 cells in 0.5 ml) were combined with 0.5 ml of stained CD5+ B cells (1 × 106 cells) at a cell/cell ratio of 1:1. The mixed cells were incubated with or without antigen (100 ng/ml) at 37°C for 45 min. Cells were then immediately fixed in 4% paraformaldehyde. Cell conjugates were determined by flow cytometric analysis with a FACSCalibur flow cytometer (Becton Dickinson).

Western blotting

After BMMCs were cocultured with wild-type or IL-10−/− CD5+ B cells for 24 hours, CD19 microbeads (Miltenyi Biotec) were used to purify BMMCs by negative selection according to the manufacturer’s instructions. The BMMCs were lysed in 100 μl of ice-cold lysis buffer containing a protease inhibitor cocktail tablet. The cell lysates were subjected to Western blotting analysis according to a standard protocol. Antibodies against FcεRγ and actin were purchased from Upstate Biotechnology; antibodies against FcεRα, FcεRβ, Lyn, Fyn, Fgr, and Syk were obtained from Santa Cruz Biotechnology; and antibody against the phosphorylated form of Syk (pSyk) was purchased from Cell Signaling Technology.

Statistical analysis

Data were expressed as means ± SEM. Statistical analysis was performed by one-way analysis of variance and Dunnett’s test. Statistical significance (*P < 0.05 and **P < 0.01) was determined with SigmaStat software (Systat Software Inc).

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/8/368/ra28/DC1

Fig. S1. Analysis of the population of IL-10–producing CD5+ B cells involved in PSA.

Fig. S2. Characterization of CD5+ B cells in IL-10−/− mice and of IL-10–producing B cells stimulated by mast cells.

Fig. S3. Analysis of the production of IL-10 by B cells, mast cells, and various tissue-derived CD5+ B cells.

Fig. S4. The CD40-CD40L interaction is critical for mast cell–mediated IL-10 production by B cells.

Fig. S5. IL-10 reduces the abundances of several critical signaling molecules in mast cells.

REFERENCES AND NOTES

Funding: This work was supported by the National Research Foundation of Korea (NRF) grant [Ministry of Science, ICT, and Future Planning (MSIP), No. 2012R1A2A1A03670516] and in part by an NRF grant (MSIP, NRF-2013R1A4A1069575) funded by the Korean government. M.A.B. was supported by the Intramural Program of the National Heart, Lung, and Blood Institute, NIH. Author contributions: W.S.C. and Y.M.K. designed the experiments, analyzed the data, and wrote the paper; Hyuk S.K. and A.-R.K. performed most of the experiments; D.K.K. and G.H.J. collected and analyzed flow cytometry data; H.W.K. and Y.H.P. performed in vivo experiments; J.S.Y. and Hyung S.K. performed all experiments for cell signaling analysis; B.K. and Y.M.P. contributed reagents and knockout animals and designed the experiments; and Y.M.K. and M.A.B. provided intellectual input and wrote the paper. Competing interests: The authors declare that they have no competing interests.
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