Insulin-like Growth Factor-2 Enhances Functions of Antigen (Ag)-specific Regulatory B Cells*

Background: Antigen-specific B cells (AgsBCs) are important in immune regulation. Results: AgsBCs express IGF2 receptor. IGF2 can drive AgsBCs to produce IL-10 and differentiate to regulatory B cells. Conclusion: IGF2 can enhance the immune suppressor function of AgsBCs. Significance: IGF2 has the potential to enhance immunotherapy of allergic disorders. Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10+ OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.


Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and
OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10 ؉ OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.
After exposure to antigens (Ags), 4 Ag-specific B cells (AgsBCs) may remain in a quiescent state to be memory B cells (1) or differentiate into plasma cells to produce antibodies (2). Regulatory B cells (Bregs) are a subpopulation of AgsBCs. Production of interleukin-10 (IL-10) upon activation is one of the features of Bregs (3). However, factors inducing AgsBCs to differentiate into Ag-specific Bregs are less clear.
The insulin-like growth factor (IGF) family, including the two major members IGF1 and IGF2, is a subtype of the growth factor family, and the sequence of IGF is highly similar to insulin (4). IGF1 is mainly secreted by the liver, plays an important role in normal physiology (5), and is involved in the pathogenesis of cancer (6). Similar to IGF1, IGF2 is also secreted by the liver. As a hormone, after secretion, IGF2 is absorbed into the bloodstream to enter the circulation. In contrast to IGF1, which mainly functions in adult life, IGF2 is regarded as a primary growth factor involved in the early development of the body (7).
Two types of receptors of IGF have been characterized: the IGF1 receptor (IGF1R) and IGF2R. Both IGF1 and IGF2 can bind IGF2R to regulate physiological functions. IGF2 can bind IGF2R on the cell surface to form clathrin-coated vesicles and helps the internalization of the IGF2-IGF2R complexes (5).
Published data indicate that dendritic cells and T cells express IGF2R (9). Whether Bregs express IGF2R has not been reported yet. Because IGF2 facilitates the development of stem cells at early stages (10,11), we hypothesize that IGF2 plays a role in the regulation of Breg functions. The hypothesis is corroborated by the fact that IGF2 and the B cell receptor (BCR) share a common signal transduction pathway, the mitogen-activated protein kinase (MAPK) pathway (12,13). In carrying out this study, we observed that ovalbumin (OVA)-specific B cells (OVAsBCs) expressed IGF2R and that exposure to IGF2 significantly enhanced the functions of Bregs.
Mice-Male BALB/c mice (6 -8 weeks of age) were purchased from the Guangzhou Experimental Animal Center (Guangzhou, China). IL-10-deficient mice and B6 mice were purchased from the Xian Experimental Animal Center (Xian, China). Mice were maintained in a pathogen-free environment. The experimental procedures were approved by the Animal Care Committee at Shenzhen University.
OVA Tolerization of Mice-BALB/c mice were gavage-fed with OVA (5 mg/mouse) daily for 7 days. The mice were sacrificed on day 8. The spleen and small intestine were excised. Following our established procedures (14,15), spleen cells and lamina propria mononuclear cells (LPMCs) were isolated for use in further experiments.
Construction of an Ag-specific Tetramer-To isolate the OVAsBCs, a tetramer was constructed following reported procedures (16) with a minor modification. The biotinylated OVA was incubated with magnetic particle-conjugated streptavidin for 30 min at room temperature. Unconjugated reagents less than 10 kDa were filtered through a filter tube by centrifugation. The OVA tetramers were collected for OVAsBC isolation.
OVAsBC Isolation-LPMCs were isolated following our established procedures (14,17), and OVA tetramer was added to the cells at a concentration of 2 g/ml. The cells were incubated at room temperature for 30 min and then passed through the columns in the magnetic apparatus provided by Miltenyi Biotech. Cells were collected, washed with acidic phosphatebuffered saline (PBS) (pH 3) to remove the bound OVA on the cell surface, and transferred to RPMI 1640 medium for further experiments.
Morphological Assay of OVAsBCs-The isolated OVAsBCs were stained with an anti-OVA antibody and followed by stain-ing with a FITC-labeled secondary antibody. The stained cells were observed with a confocal microscope.
OVAsBC Proliferation Assay-The isolated OVAsBCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and cultured in the presence of OVA (the specific Ag; 1 g/ml) and/or IGF2 (200 ng/ml) for 3 days. The frequency of CFSE ϩ cells was assessed by flow cytometry using the CFSE dilution assay.
Cell Culture-Cells were cultured at 37°C in a 5% CO 2 environment in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/streptomycin. The medium was changed every 3 days. Cell viability was assessed by the trypan blue exclusion assay and was greater than 96% before using for further experiments. A portion of dendritic cells was cultured with LPS at 100 ng/ml and used as activated control dendritic cells.
Flow Cytometry-Cells were fixed with 2% paraformaldehyde for 2 h (in the case of intracellular staining, 0.1% Triton X-100 was added to the fixative). After blocking with 1% bovine serum albumin (BSA) for 30 min, the cells were incubated with the fluorescence-labeled antibodies for 1 h at room temperature. After washing with PBS, the cells were analyzed by flow cytometry.
Quantitative Real Time RT-PCR-Total RNA was extracted from the collected cells with TRIzol reagents. The cDNA was synthesized with a reverse transcription kit. Quantitative PCR was performed using the MiniOpticon PCR system. The 2 Ϫ⌬⌬Ct method was used to calculate the results. The primers using in the experiments include: IGF1R: forward, aagtcctgcagctggtgtat; reverse, tccattccgcacagtacact; IL-10: forward, ggtgagaagctgaagaccct; reverse, tgtctaggtcctggagtcca; and ␤-actin: forward, gtgggaatgggtcagaagga; reverse, tcatcttttcacggttggcc. The reverse transcription was performed at 50°C for 30 min, the denaturation was carried out for 2 min at 94°C, 35 cycles (94°C for 30 s, 55°C for 45 s, and 68°C for 45 s) were run for amplification, and the elongation time was 7 min at 68°C. The results of the target gene expression were normalized to the percentage of the internal control (␤-actin).
Western Blotting-Total proteins were extracted from the collected cells, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 30 min and then incubated with the primary antibodies (50 -100 ng/ml) followed by incubation with the secondary antibodies. The membrane was washed with a mixture of Tris-buffered saline and Tween 20 after incubation with antibodies. The immune complex on the membrane was developed by enhanced luminol-based chemiluminescence (ECL). The results were photographed using the UVP BioSpectrum Imaging System (Upland, CA).
ELISA-The levels of OVA-specific IgE, IL-4, IL-10, and IL-13 were determined by ELISA with purchased reagent kits following the manufacturers' instructions.
Gene Silencing-The STAT5 and IGF1 genes were knocked down in AgsBCs by RNA interference with reagent kits following the manufacturer's instructions. The effect of gene silencing was assessed by Western blotting. The results of gene silencing are presented in Figs. 3 and 6, respectively.

Creating a Mouse Model of Intestinal Allergic Inflammation-
Following reported procedures (18), we developed a mouse model of intestinal allergic inflammation. Briefly, BALB/c mice were gavage-fed with OVA (1 mg/mouse) and cholera toxin (20 g/mouse) in 0.3 ml OF saline once a week for 4 weeks. The mice were challenged with OVA (5 mg/mouse) via gavage on week 5 and sacrificed THE next day.
Assessment of Intestinal Allergic Inflammation-Following our established procedures (14,17), we assessed the intestinal allergic inflammation by examining the serum levels of OVAspecific IgE, IL-4, and IL-13; counting the numbers of mast cells and eosinophils in jejunal sections; and evaluating the Ag-specific CD4 ϩ T cell proliferation by the CFSE dilution assay following our established procedures (14,15). After oral challenge with the specific Ags, diarrhea and core temperature were also recorded (14,15).
Assessment of IL-10 Promoter Methylation-OVAsBCs were prepared, and using the Wizard genomic DNA purification kit, the genomic DNA was purified from the OVAsBCs. Methylation analyses were performed by bisulfite conversion of genomic DNA using the EpiXplore Methyl Detection kit. The primer sequences of the IL-10 promoter (NCBI accession number AF121965) used in the present study are as follows: (i) methylation primer: forward, CGTATTGATGTAGGAAGG-ATAGTTC; reverse, CAATTTAAATAAAAAAAACATT-CGC; and (ii) demethylation primer: forward, TGTATTGAT-GTAGGAAGGATAGTTTG; reverse, AATTTAAATAA-AAAAAACATTCACC.
Inhibition of DNA Methyltransferase in OVAsBCs-To inhibit DNA methyltransferase, a portion of OVAsBCs was treated with 5-azacytidine, a DNA methyltransferase inhibitor, at a concentration of 1 M for 72 h.
Statistics-The data are presented as mean Ϯ S.D. Differences between two groups were determined by non-paired Student t test or analysis of variance if more than two groups or by non-parametric Mann-Whitney test. p Ͻ 0.05 was set as the significance criterion.

RESULTS
OVAsBCs Express High Levels of IGF2R-We treated BALB/c mice with OVA daily for 7 days to make the mice tolerant to OVA. CD19 ϩ CD20 ϩ B cells were isolated from the small intestine, and the OVAsBCs were further isolated using an OVA tetramer. The remaining CD19 ϩ CD20 ϩ B cells were designated as "NsBCs" in contrast to the OVAsBCs. The purity of isolated OVAsBCs was greater than 99% as demonstrated by confocal imaging (Fig. 1, A and B). To determine whether the isolated B cells were OVA-specific, the cells were labeled with CFSE and cultured in the presence of OVA for 3 days. As shown by flow cytometry assay, more than 90% of cells proliferated. The results indicate that the cells are OVAsBCs (Fig. 1, C-F).
The expression of IGF1R and IGF2R on NsBCs and OVAsBCs was analyzed by quantitative real time RT-PCR and Western blotting. The results showed that both IGF1R and IGF2R were detected in both NsBCs and OVAsBCs. The expression of IGF2R was much higher in OVAsBCs than NsBCs. Only modest expression of IGF1R was detected in both NsBCs and OVAsBCs (Fig. 2, A-C). Next we analyzed the frequency of IGF2R expression on B cells by flow cytometry. The results showed that 27% of OVAsBCs expressed IGF2R, whereas only 3% of NsBCs expressed IGF2R (Fig. 2, D-F). Because the levels of costimulatory molecules are critical in tolerogenic cells, we next assessed the expression of CD80 in these B cells. The results showed that the frequency of CD80 ϩ cells was 23.5% in OVAsBCs and 49.4% in NsBCs (73.3% CD80 ϩ cells were detected in LPS-treated NsBCs) (Fig. 2, G-J).
IGF2 Enhances the Expression of IL-10 in OVAsBCs-IL-10 is one of the chief mediators of the suppressor functions of Bregs (20). We next tested whether IGF2 could ligate the IGF2R to activate OVAsBCs to differentiate into Bregs by enhancing the expression of IL-10. To this end, OVAsBCs were isolated from the small intestine of OVA tolerant BALB/c mice. The effect of IGF2 on enhancing IL-10 expression in OVAsBCs was observed. As assessed by flow cytometry, 20.5% of freshly isolated OVAsBCs expressed IL-10. IL-10 expression was markedly increased after stimulation with IGF2 in the culture in the presence of the specific Ag OVA, and the increase was in an IGF2 dose-dependent manner. The frequency of IL-10 ϩ OVAsBCs was 92.3% when the IGF2 dose reached 200 ng/ml (Fig. 3, A-E). Exposure to OVA alone did not significantly alter the frequency of IL-10 ϩ OVAsBCs (Fig. 3F). To confirm that the increase in IL-10 in OVAsBCs was mediated by IGF2R, we treated IGF2R-null OVAsBCs with OVA and IGF2, and indeed, the increase in IL-10 was abolished (Fig. 3, G and H). Because the MAPK/ERK pathway is downstream of BCR and IGF2R activation, we treated OVAsBCs with inhibitors of ERK or MAPK prior to exposure to OVA/IGF2 and found that the expression of IL-10 was abolished (Fig. 3, I and J). Pretreatment with Btk (an inhibitor of BCR signal) also blocked the expres-sion of IL-10 induced by OVA/IGF2 (Fig. 3K). In addition, treating naïve B cells with both OVA and IGF2 did not alter the expression of IL-10 (Fig. 3, L and M). The IGF2R gene silencing results are shown by Fig. 3N. We also observed the release of IL-10 from the OVAsBCs by assessing the expression of IL-10 at both the mRNA (Fig. 3O) and protein levels (Fig. 3P), which were in parallel to the frequency of IL-10 ϩ OVAsBCs in Fig. 3, A-E. The results indicate that IGF2 can enhance the production of IL-10 by OVAsBCs. According to the notion that IL-10 ϩ B cells have an immune suppressor function (20), the IGF2treated, IL-10-expressing OVAsBCs may be Bregs.
Based on published data that IL-10-expressing B cells also express other cytokines (21,22), we further analyzed the phenotypes of IL-10 ϩ OVAsBCs. Positive staining of CD5, CD38, CD1d, TIM1, CD23, and CD27 was observed in 56 -78% of OVAsBCs. Furthermore, about 14% of OVAsBCs expressed IFN-␥, the signature cytokine of Th1. A very low frequency (1.3%) of CD24-positive OVAsBCs was also detected (Fig. 4,  A-H). We also assessed the phenotypes of IL-10 ϩ B cells from naïve mice by flow cytometry. The results showed a similar tendency but lower frequency of the B cell phenotypes compared with those of OVAsBCs except that more CD24 ϩ IL-10 ϩ B cells (14.2%) were observed (Fig. 4, J-R).
Exposure to IGF2 Enhances OVAsBC Proliferation-Because IGF2 can augment hematopoietic cell differentiation and pro- liferation (23), we postulated that IGF2 also facilitates the OVAsBC proliferation. Thus, we next investigated the role of IGF2 in facilitating OVAsBC proliferation. The results showed that exposure to specific Ag OVA slightly induced OVAsBC proliferation as compared with the saline control group (Fig. 5,  A, B, and E). Exposure to both IGF2 and OVA significantly enhanced the OVAsBC proliferation (Fig. 5, C and E), whereas there was not much of an increase when OVAsBCs were exposed to IGF2 alone (Fig. 5, D and E).
To understand whether such a phenomenon also occurred in vivo, the OVAsBCs were labeled with CFSE and adoptively transferred to naïve BALB/c mice at 10,000 OVAsBCs/mouse. The mice were then gavage-fed with OVA and/or IGF2 (intraperitoneal) daily for 3 days. The CFSE ϩ cells in 100,000 LPMCs were counted per mouse by flow cytometry. The results showed that there were 2.55 Ϯ 0.58% CFSE ϩ cells in saline-treated mice on the next day after the injection. Three days later, we counted CFSE ϩ cells in 100,000 LPMCs of mice treated with CFSElabeled OVAsBCs by flow cytometry. The results showed that the frequency of CFSE ϩ cells was 2.98% in the saline group (Fig. 5, F and K). There were 5.64% CFSE ϩ cells detected in the OVA group (Fig. 5, G and K), 13.9% CFSE ϩ cells in the OVA/ IGF2 group (Fig. 5, H and K), and 2.85% CFSE ϩ cells in the IGF2 group (Fig. 5, I and K). The results also showed that treatment with a BCR signal blocker, Btk inhibitor, abolished the OVA/ IGF2-induced CFSE ϩ cell proliferation (Fig. 5, J and K). The results indicate that exposure to specific Ags can induce OVAsBC proliferation in vivo that can be further enhanced by IGF2.
IGF2 Induces IL-10 Promoter Demethylation in B Cells-We next looked for further insight into the mechanism by which IGF2 regulates Breg functions. IL-10 expression is one of the signature features of Bregs. Previous reports indicate that STAT5 is a critical component in the signal transduction pathway of IL-10 gene transcription (24). IGF2 also can activate STAT5 (24). In separate experiments, we cultured OVAsBCs in the presence of the specific Ag OVA and/or IGF2 for 72 h in the presence or absence of 5-azacytidine. The cells were then analyzed by methylation-specific PCR and Western blotting. The results showed that the IL-10 protein levels (Fig. 6, A and B) were associated with the IL-10 promoter DNA demethylation levels (Fig. 6, C and D). Treatment with 5-azacytidine abolished the OVA/IGF2-increased IL-10 protein in OVAsBCs (Fig. 6B). Both methylated and unmethylated IL-10 promoter DNA was detected in medium-treated OVAsBCs. The levels of demethylated IL-10 promoter DNA were significantly increased in OVAsBCs treated with both OVA and IGF2. Treatment with either OVA or IGF2 alone showed levels of IL-10 promoter DNA methylation in OVAsBCs similar to those of OVAsBCs treated with medium alone. In the STAT5-null OVAsBCs (Fig.  6E), however, the methylation of the IL-10 promoter was not altered (Fig. 6, A-D). The results indicate that the concurrent exposure to both specific Ags and IGF2 can induce IL-10 promoter DNA demethylation.
IGF2 Enhances the Effect of ASIT-Finally, we tested the role of IGF2 in strengthening OVAsBC functions in vivo. OVAsBCs play a critical role in the ASIT (25). Based on the data of Figs. 2 and 3, we postulated that administration of IGF2 may strengthen the therapeutic effect of ASIT. Following published procedures (18), we developed a mouse model of intestinal allergic inflammation with OVA as the specific Ag. The allergic mice were treated with ASIT and/or IGF2. The allergic inflammation in the small intestine was assessed with the parameters and procedures we reported previously (15,17). As compared with naïve control group, mice sensitized to OVA showed high serum levels of OVA-specific IgE, low levels of IgG4 (Fig. 7A), high levels of IL-4 and IL-13 (Fig. 7B), infiltration of mast cells and eosinophils in the intestinal mucosa (Fig. 7C), high proliferation of Ag-specific CD4 ϩ T cells in response to specific Ag stimulation in the culture (Fig. 7D), a drop in the core temperature (Fig. 7E), and diarrhea (Fig. 7F). After treatment with ASIT and IGF2, the allergic inflammation in the intestine was significantly attenuated. Treatment with ASIT alone resulted in a lesser inhibitory effect on the allergic inflammation than administration of both ASIT and IGF2. Treatment with IGF2 alone did not result in an appreciable improvement of the allergic inflammation. It is noteworthy that the serum levels of OVA-specific IgG4, the so-called "blocking antibody," were markedly increased in mice treated with both IGF2 and OVA as compared with those treated with either IGF2 or OVA alone. To test the role of IL-10 in the inhibitory effect on the allergic inflammation, IL-10-deficient mice were sensitized to OVA and treated with the same procedures of the counterpart BALB/c mice. The results showed that treatment with both ASIT and IGF2 did not attenuate the allergic inflammation in the intestine of the OVA-sensitized IL-10-deficient mice.

DISCUSSION
ASIT is the only specific therapeutic remedy for allergic diseases recommended by the World Health Organization (26), and it is extensively used in the allergy clinic. However, the therapeutic efficiency of ASIT could be improved. The present study identified a new subpopulation of OVAsBCs that expresses high levels of IGF2R. Exposure to IGF2 can significantly enhance the efficiency of ASIT in the inhibition of allergic inflammation of the intestine under a mechanism of inducing the IGF2R-bearing OVAsBCs to proliferate and to release IL-10. It has been proposed that the secretion of IL-10 is one of the signature identifications of Bregs (27,28). Published data indicate that IL-10 ϩ Bregs can be induced by TIM1 ligation with the low affinity anti-TIM1 mAb RMT1-10 (29). Our data are in line with these reports by showing that more than half of the IL-10 ϩ OVAsBCs express TIM1. CD40 activation can increase the  IL-10 secretion and Breg differentiation evidenced by ectopic B cell expression of CD40 ligand (CD154) in transgenic mice that induces increased CD40 signaling and increased IL-10-secreting B cell numbers (30). Activation of Toll-like receptor 4 or 9 also can increase the induction of IL-10-secreting B cells in response to stimulation of LPS or CpG (31). Our data have added novel information to the research of Bregs by showing that IGF2 also promotes an increase in the number of IL-10secreting Bregs. It is noteworthy that IGF2 alone does not have such a function; it has to collaborate with the activation of BCR to induce the IL-10-secreting Bregs. Such a feature is in line with previous reports in which the activation of BCR is also required in TIM1 activation-induced Breg differentiation (29,32).
The present data show that IGF2 can enhance the Ag-driving OVAsBC proliferation. In the process of isolation, the plasma cells were excluded. Thus, the isolated OVAsBCs are Ag-specific B memory cells and those derived from activated B memory cells. In other words, these cells are still plastic. The bearing of IGF2R confers these OVAsBCs to respond to IGF2. The data show two important features of IGF2R ϩ OVAsBCs. Upon exposure to the specific Ag OVA and IGF2, the cells release IL-10 and proliferate. Previous reports suggest that IL-10-producing B cells are Bregs (33,34), and Bregs play an important role in the therapeutic effect of ASIT (35,36). Thus, this subpopulation of OVAsBCs could be Bregs. Our data indicate that this subpopulation of OVAsBCs has a potential effect on enhancing the therapeutic effect of ASIT.
Although the data show that treatment with IGF2 and the specific Ag (OVA) vaccine significantly increases the release of IL-10, OVAsBC proliferation, and inhibition of allergic inflammation, using IGF2 alone did not show such an effect. The results suggest that the effect of IGF2 on inducing those changes is via enhancing the activation of BCR by the specific Ag OVA. This inference is supported by treatment of the OVAsBCs with a BCR signal inhibitor (the Btk inhibitor) that abolished the OVA/IGF2-induced IL-10 release and OVAsBC proliferation. This is in line with previous reports. Di Paolo et al. (37) report that using Btk inhibitors can inhibit autoantibody release from B cells and ameliorate the symptoms of arthritis. Honigberg et al. (38) also found that Btk inhibitors can block B cell activation. The underlying mechanism of the present data could be that both IGF2 and BCR share a common signal transduction pathway. By strengthening the same signal transduction pathway, IGF2 has the potential to amplify the specific Ag-induced BCR activation. The subsequent results of the present study support the reasoning that pretreatment with the ERK inhibitor or MAPK inhibitor abolished the OVA/IGF2induced OVAsBC activation.
We also observed lower levels of the costimulatory molecule CD80 in the IGF2R ϩ OVAsBCs. Low levels of costimulatory molecules on Ag-presenting cells are regarded as one of the major tolerogenic features. We observed previously that dendritic cells with lower levels of CD80 had tolerogenic features in the generation of regulatory T cells (14,17). Vogt et al. (39) found that B cells expressing low levels of CD80 had tolerogenic features.
The in vivo experimental data strengthen the novel findings we observed in the in vitro experiments. By using an egg-derived Ag, OVA, we created a food allergy mouse model. The allergic mice were treated with ASIT in an up-dosing approach to mimic the ASIT used in the allergy clinic. However, the specific allergen OVA vaccine-treated allergic mice did not show satisfactory inhibition of the allergic inflammation in the intestine. The results are in line with previous studies such as Leonard et al. (19) demonstrating that ASIT only shows some improvements in the allergic inflammation. Administration of both OVA and IGF2 nearly abolished the allergic inflammation in the intestine as shown by the present data. The underlying mechanism may be that using both OVA and IGF2 significantly enhances the production of IgG4, an inhibitory antibody induced by ASIT (8).
In summary, the present study revealed that a subpopulation of OVAsBCs expressed IGF2R. IGF2 ligated the IGF2R to facil- itate the expression of IL-10 in the OVAsBC and to enhance cell proliferation. Administration of both specific Ag vaccine and IGF2 efficiently inhibited allergic inflammation in the intestine. The results indicate that it is worthwhile to further investigate the therapeutic potential of IGF2 in the treatment of allergic disorders.