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Originally published online as doi:10.1189/jlb.1007710 on March 19, 2008

Published online before print March 19, 2008
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(Journal of Leukocyte Biology. 2008;83:1451-1458.)
© 2008 by Society for Leukocyte Biology

A dual role of activin A in regulating immunoglobulin production of B cells

Kenji Ogawa*,1, Masayuki Funaba{dagger} and Masafumi Tsujimoto*

* Laboratory of Cellular Biochemistry, RIKEN, Wako, Saitama, Japan; and
{dagger} Laboratory of Nutrition, Azabu University School of Veterinary Medicine, Sagamihara, Kanagawa, Japan

1Correspondence: Laboratory of Cellular Biochemistry, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: kkogawa{at}riken.jp


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ABSTRACT
 
Here, we report that activin A has a dual role in regulating Ig production of murine B cells. Activated B cells secrete activin activity by increasing activin A and decreasing follistatin expression. B cells also express type I and type II activin receptors, suggesting that they are targets of activin. Pretreatment of naïve B cells with activin A and subsequent activation by LPS resulted in increased cell growth and IgG production. In contrast, no significant effect was observed when activin A was added to naïve B cells simultaneously with LPS, indicating that activin A acts on resting but not activated B cells. In addition, activin A did not induce B cells to produce IgE, even when added prior to activation; however, in vivo antigen-specific IgE production was reduced significantly by neutralization of circulating activin A. These findings indicate that activin A plays an important role in Th2-mediated immune responses by enhancing antibody production through two distinct modes: acts directly on resting B cells to elicit full functions of activated B cells and acts indirectly on activated B cells through modulation of other immune cells.

Key Words: class-switch recombination • mouse • TGF-β


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INTRODUCTION
 
B cells are important effector cells in Th2 cell-mediated humoral immune responses. Upon activation, resting B cells proliferate and differentiate into Ig-secreting plasma cells [1 ]. In addition to antigens, various cytokines are involved in this process. Th1 cytokines are involved in cell-mediated immunity and Ig class-switching to IgG2a. In contrast, Th2 cytokines are involved in humoral immune responses and Ig class-switching to IgG1 and IgE [2 , 3 ].

Activin A, a member of the TGF-β superfamily, is a local regulator of cell growth and differentiation [4 ]. Our previous study showed that activin A is produced in Th2 but not Th1 cells upon activation [5 ]. The activin βA proximal promoter contains a binding site for c-Maf, a Th2-specific transcriptional factor, at close proximity to a NFAT-binding site, and both factors are implicated in activin βA transcription in Th2 cells [5 ]. Cooperative regulation of the activin βA gene by NFAT, pre-existing, and c-Maf is consistent with the transcriptional regulation of a representative Th2 cytokine IL-4 [6 ]. These findings suggested that activin A has a role in Th2-mediated immune responses.

Activin A induces the expression of matrix metalloproteinase-2 (MMP-2) in peritoneal macrophages [7 ] and increases the migration and gene expression of mast cell-specific protease-1 in mast cell progenitors [8 ]. Mast cells are known to be critical effector cells of Th2-induced immune responses such as allergic inflammation and immediate hypersensitivity [9 ]. Macrophages play important roles in Th1- and Th2-mediated immune responses. Classical activation of macrophages with Th1 cytokines results in free-radical release and increased cytokine secretion, implicated as essential signaling components of a successful response to infection by intracellular bacteria and viruses [10 , 11 ]. On the other hand, the alternative activation of macrophages with Th2 cytokines is required for defense against extracellular pathogens and parasites [12 ]. Like other Th2 cytokines, treatment of macrophages with activin A markedly induced the expression of arginase-1 and decreased IFN-{gamma}-induced expression of inducible NO synthase, indicating that activin A is involved in the alternative activation of macrophages [5 ].

In this study, we hypothesized that activin A mediates humoral immunity by stimulating B cells to produce antibody, as do other Th2 cytokines. To address this hypothesis, we investigated the effects of activin A on Ig production in murine B cells. As cytokines are also produced by activated B cells, some of which act as autocrine or paracrine factors, we first examined the expression of activin A in murine B cells.


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MATERIALS AND METHODS
 
Reagents
Recombinant human/murine/rat activin A, human TGF-β1, murine IL-4, and anti-activin A mAb were obtained from R&D Systems (Minneapolis, MN, USA). The National Hormone and Pituitary Program (Rockville, MD, USA) provided the recombinant human follistatin-288. mAb against mouse CD4, CD8, and Thy1.2 were purified from culture supernatants of the hybridomas GK-1.5, HO-2.2, and HO-13-4 (American Type Culture Collection, Manassas, VA, USA), respectively. LPS from Salmonella minnesota Re 595 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Low-Tox guinea pig complement was from Cedarlane Laboratories Ltd. (Ontario, Canada). The ELISA kit for TGF-β1 (TGFβ1 Emax ImmunoAssay System) was obtained from Promega (Madison, WI, USA).

Animals
Female, specific pathogen-free BALB/c mice were obtained from SLC (Shizuoka, Japan) and maintained at the animal facilities of RIKEN (Saitama, Japan). Mice were used at 8–12 weeks of age. All animal experiments were carried out in accordance with the guidelines for animal experiments in RIKEN. All efforts were made to minimize the suffering and the number of animals used.

Cell isolation and culture
For RT-PCR, murine B cells were isolated from RBC-depleted spleen cells by incubation with Dynabeads mouse pan B (B220) beads (Dynal, Lake Success, NY, USA). For immunization experiments, mice were immunized i.p. with OVA (100 µg) in CFA (Difco, Detroit, MI, USA) and boosted i.p. with OVA (100 µg) at 14 days after priming. Three days after boosting, B cells were isolated from the spleen by the magnetic beads method. For in vitro culture of B cells, spleen cells depleted of RBC were treated with anti-Thy 1.2, anti-CD4 plus anti-CD8 mAb, and guinea pig complement to remove T cells. The cells were depleted of adherent cells by adherence to plastic Petri dishes. For certain experiments, T cell-depleted spleen cells were fractionated further into high-density (resting) and low-density (activated) cells by discontinuous gradient composed of 50%, 60%, and 70% Percoll. After centrifugation at 2300 g for 12 min at 4°C, the cells banding at the 70%/60% and 60%/50% interfaces were collected as high-density and low-density B cells, respectively. The preparation was confirmed to contain B220-positive cells by more than 95% in immunocytochemical analysis and did not proliferate significantly in cultures in the presence of 5 µg/ml Con A. Naïve B cells were isolated from mouse spleens by negative selection using a mouse B cell isolation kit (Miltenyi Biotech, Auburn, CA, USA) in a MACS preparation column. Cells were resuspended at 5 x 105 cells/ml in RPMI-1640 medium supplemented with 2 mM glutamine, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated (56°C, 30 min) FBS.

Bioassay for activin activity
Activin activity in culture supernatant was assayed by the erythroid differentiation assay using mouse erythroleukemia F5-5.fl cells (RIKEN Cell Bank, Tsukuba, Japan) as described previously [13 ]. In the erythroid differentiation assay, F5-5.fl cells were differentiated into hemoglobin-positive cells in the presence of activin A, activin AB, or activin B but not TGF-β1, bone morphogenetic protein-4, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IFN-{gamma}, and TNF-{alpha} [13 , 14 ]. To examine whether erythroid differentiation activity of the supernatant was a result of activin A, samples were incubated with 400 ng/ml recombinant human follistatin-288 or 1 µg/ml anti-human activin A neutralizing mAb.

RT-PCR and quantitative RT-PCR
Total RNA isolation, cDNA synthesis, and competitive PCR were conducted as described previously [5 , 7 ]. Oligonucleotides in primer sets for activin βA, βB, follistatin, activin receptors [ActRII activin-like kinase-4 (ALK4), ActRII, and ActRIIB], and G3PDH were used as described previously [5 , 7 ]. RT-PCR of germline transcripts (GLT) and postswitch transcripts (PST) for IgA ({alpha}) and IgG2b ({gamma}2b) was performed using the following sets of primers: for {alpha}GLT, I{alpha}-forward (5'-GGTACCATCTGGACTCCTCT-3') and C{alpha}-reverse (5'-CCAGGTCACATTCATCGTGC-3'); for {alpha}PST, Iµ-forward (5'-TGCTGGTTGGTGGTTGAGAG-3') and C{alpha}-reverse; for {gamma}2bGLT, I{gamma}2b-forward (5'- CTCACACACAGAAGAATGGAC-3') and C{gamma}2b-reverse (5'-TGCAGGTGACGGTCTGACTT-3'); for {gamma}2bPST, Iµ-forward and C{gamma}2b-reverse.

Western blotting
For Western blotting, B cells were cultured for 6 days with or without LPS (50 µg/ml) in serum-free S-Clone SF-O2 medium (Sanko Junyaku, Tokyo, Japan). The supernatant was subjected to SDS-PAGE under nonreducing conditions, blotted to Immobilon-P (Millipore, Bedford, MA, USA), and immunostained with anti-activin A mAb. Bands were visualized using peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and ECL plus reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).

MTT assay
For MTT assays, B cell cultures were incubated for 4 h with MTT at a final concentration of 1 mg/ml. The medium was then removed, and cells were lysed in 2-propanol containing 0.04 M HCl. Absorbance at 570 nm (A570 nm) was measured using a microplate reader.

ELISA of Ig
Ig levels in the supernatants and sera were determined by ELISA. To determine total Ig isotype levels, microtiter plates were coated with goat anti-mouse Ig isotype-specific antibodies (Southern Biotechnology Associates, Birmingham, AL, USA). To detect antigen-specific Ig, plates were coated with OVA. Plates were incubated with serially diluted samples for 2.5 h at room temperature. Bound Ig was detected by biotin-labeled, isotype-specific antibodies (Southern Biotechnology Associates) and streptavidin-HRP (Prozyme, San Leandro, CA, USA) with 3,3',5,5'-tetramethylbenzidine (KPL, Gaithersburg, MD, USA) as a substrate. The peroxidase reaction was stopped by adding 2 N HCl, and absorbance was determined at a wavelength of 450 nm.

Statistical analysis
Data are presented as the mean + SD. Comparisons between groups were conducted by Student’s t-test.


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RESULTS
 
Activin production in B cells with their activation
To elucidate the role of activins in B cells, we first examined the effect of LPS, a polyclonal activator for B cells, on the production of activin in B cells. Activin activity in the culture supernatant of whole spleen cells, unfractionated B cells, high-density B cells, and low-density B cells was augmented in response to stimulation by LPS (Fig. 1A ). This activity was neutralized by the addition of excess follistatin-288 or anti-activin A neutralizing mAb (Fig. 1B) . Western blot analysis revealed that polyclonal activation by LPS induced the production of activin A in B cells (Fig. 1C) . RT-PCR of splenic B cell RNA yielded the activin βA subunit and follistatin PCR products but not products of the activin βB subunit (Fig. 1D) . These results suggested that B cells expressed activin A, a homodimer of the inhibin βA subunit. To determine whether the effect of LPS on activin activity in B cells was a result of changes at the mRNA level, quantitative RT-PCR was used [5 , 7 ]. In response to stimulation by LPS, activin βA mRNA levels were augmented, whereas follistatin mRNA levels were decreased in cultured B cells (Fig. 1E) . We next examined whether the activation of B cells regulated the expression of activin in vivo. To this end, mice were immunized with OVA in CFA, and activin βA subunit and follistatin mRNAs in B cells were measured by quantitative RT-PCR. Consistent with the results of cultured B cells stimulated by LPS in vitro, activin βA and follistatin mRNA levels were increased and decreased, respectively, after immunization (Fig. 1F) .


Figure 1
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  		Figure 1. Production of activin A in B cells upon activation. (A) Activin production from whole spleen cells (WSP), unfractionated B cells (UF-B), high-density B cells (HD-B), and low-density B cells (LD-B) in response to LPS stimulation. Cells were cultured for 6 days with LPS (50 µg/ml), and the supernatants were assayed for activin. (B) Neutralization effect of the addition of excess follistatin or anti-activin A neutralizing mAb on recombinant human activin A or culture media. Erythroid differentiation activity in the supernatant of LPS-stimulated, unfractionated B cells was abolished by excess follistatin-288 (Fs288; 400 ng/ml) or anti-activin A mAb (mAb; 1 µg/ml) as well as that of recombinant human activin A. (C) Western blotting of activin A in the supernatants of unfractionated B cells incubated with no stimulus (None) or 50 µg/ml LPS for 6 days. Immunoreactive activin A was detected in the culture supernatant as a 25-kDa band only when cells were stimulated with LPS. (D) RT-PCR products of activin subunits, follistatin, and G3PDH from B cells. Positive control: mouse ovary; negative control: no RT control of B cells (noRT). (E) Activin βA and follistatin mRNA levels in B cells activated for 4 days with LPS were compared with those in freshly prepared B cells (Fresh) by competitive RT-PCR relative to G3PDH mRNA level. (F) Changes in activin βA and follistatin mRNA levels in B cells after immunization. BALB/c mice were immunized with OVA in CFA and boosted with OVA at 14 days after priming. Three days after boosting, B cells were isolated, and mRNA levels were measured by competitive RT-PCR. Data are expressed as the mean + SD. Cont, Control; Immun, immunized.

Expression of activin receptors in B cells
Upon antigenic stimulation, B cells proliferate and differentiate into effector cells that produce antigen-specific antibodies. Activated B cells also have immunoregulatory functions that are partly mediated through their cytokine production [15 ]. B cells may be targets of cytokines, some of which act as autocrine factors to induce activation, proliferation, apoptosis, and Ig class-switch [16 , 17 ]. To determine whether B cells are targets of activin, the mRNA expression of activin receptors was analyzed in B cells. RT-PCR of mouse spleen (whole spleen cells) and B cell mRNAs yielded type I and type II activin receptor products of the expected size as shown in that of mouse ovary mRNA (Fig. 2A ), suggesting that B cells could be targets of activin. Analysis of mRNA levels revealed that the expression of activin receptors was decreased with the activation of B cells (Fig. 2B) . Receptor mRNA levels in B cells were also decreased after immunization in vivo, although the effect was less intense than LPS-induced activation of B cells (Fig. 2B) .


Figure 2
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  		Figure 2. Changes in mRNA levels of activin receptors in B cells upon activation. (A) mRNA expression of type I and type II receptors for activin in mouse B cells was compared with that in the ovary as a positive control. Negative control: no RT sample of B cells. (B) Changes in mRNA levels of activin receptors in B cells activated by LPS in vitro and by immunization with OVA in vivo. For in vitro activation, B cells were cultured with 50 µg/ml LPS for 4 days. For in vivo experiments, mice were immunized with OVA as described in Figure 1F . Total RNA was isolated and subjected to competitive RT-PCR. Data are expressed as the mean + SD.

Activin A does not affect the proliferation and Ig production of LPS-activated B cells
Each member of the TGF-β superfamily initiates its cellular action by binding to a characteristic combination of type I and type II receptors, both of which are needed for signaling [18 ]. B cells express mRNA encoding type I and type II activin receptors, suggesting that activin produced by B cells is an autocrine regulator of B cell function. Activins have overlapping biological activities with TGF-β1 [19 ], a prototype of TGF-β superfamily proteins. TGF-β is known to inhibit many immune cell functions, including B cell proliferation [20 ]. On the other hand, TGF-β induces class-switch recombination to IgA and IgG2b in B cells [21 , 22 ]; therefore, we next determined if activin A has an effect on proliferation and Ig production similar to TGF-β1.

As shown in Figure 3A , exogenous activin A did not show any significant effect on the proliferation of B cells at the tested concentrations, and TGF-β1 clearly inhibited B cell proliferation in a dose-dependent manner. As expected, TGF-β1 significantly enhanced IgA and suppressed IgM production by LPS-activated B cells (Fig. 3B) . TGF-β1 also enhanced LPS-induced IgG2b secretion in B cells (Fig. 3C) ; however, activin A did not have any significant effects on Ig production (Fig. 3B and 3C) , in contrast to TGF-β1.


Figure 3
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  		Figure 3. Effects of exogenous activin A and TGF-β1 on B cell function. (A) Effects of activin A and TGF-β1 on B cell proliferation. Naïve splenic B cells (5x104) were cultured for 6 days with LPS (50 µg/ml) in the presence of different doses of activin A (left; 2–125 ng/ml) and TGF-β1 (right; 0.4–25 ng/ml). Cell proliferation was measured by MTT assay and expressed as mean A570 nm + SD. *, P < 0.01, compared with no-activin/TGF-β control. (B and C) Effects of activin A and TGF-β1 on Ig secretion. Naïve B cells (5x105) were cultured for 6 days with LPS (50 µg/ml) in the presence of activin A (Act; 125 ng/ml) and TGF-β1 (Tβ1; 5 ng/ml). Culture supernatants were serially diluted and tested for total IgA and IgM (B) or IgG subclasses (C) by ELISA and expressed as mean A450 nm + SD at the same dilutions. *, P < 0.01, compared with no-activin/TGF-β control.

Pretreatment of naïve B cells with activin A results in significantly increased IgG production
The expression of mRNA coding three activin receptors in B cells was decreased by activation in vitro and by immunization in vivo (Fig. 2B) . Two possible explanations of these findings are: The expression of activin receptors was down-regulated as a result of activin action on B cells, or the expression of activin receptors was decreased in activated B cells to respond less well to activin. The above results (Fig. 3) showed that when activin A was added to naïve B cells simultaneously with LPS, no significant effect was observed. Furthermore, neutralization of activin A with anti-activin A mAb did not significantly affect LPS-induced proliferation and Ig secretion in B cells (data not shown), suggesting that the first assumption is unlikely. To test the second assumption, we next examined the effects of pretreatment of naïve B cells with activin A on LPS-induced proliferation and Ig secretion.

Pretreatment of naïve B cells with activin A for 6 h prior to LPS stimulation resulted in a significant increase in cell proliferation (Fig. 4A ). Furthermore, pretreatment with activin A significantly increased IgG (all subclasses) but not IgA and IgM production of LPS-stimulated B cells (Fig. 4B and 4C) . The results indicate that resting but not activated B cells may be targets of activin A.


Figure 4
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  		Figure 4. Effect of pretreatment of naïve B cells with activin A on LPS-induced proliferation and Ig secretion. (A) Effect of pretreatment of naïve B cells with activin A on B cell proliferation. Naïve, splenic B cells (5x104) were treated with or without activin A (125 ng/ml) for 6 h prior to stimulation with the indicated concentrations of LPS (2–50 µg/ml) for 6 days. Cell proliferation was measured by MTT assay and expressed as mean A570 nm + SD. (B and C) Effects of pretreatment of naïve B cells with activin A on Ig secretion. Naïve B cells (5x105) were pretreated with or without activin A (125 ng/ml) for 6 h and then stimulated with LPS (50 µg/ml) for 6 days. Culture supernatants were serially diluted and tested for total IgG, IgA, and IgM (B) or IgG subclasses (C) by ELISA and expressed as mean A450 nm + SD at the same dilutions. * and **, P < 0.01 and P < 0.001, compared with no-activin control, respectively.

Pretreatment of naïve B cells with activin A, however, did not influence the effect of TGF-β1 on the proliferation (Fig. 5A ) and class-switch to IgA and IgG2b in LPS-stimulated B cells (Fig. 5B and 5C) . Thus, activin A and TGF-β1 may act on naive B cells by different mechanisms. The results indicate that function of activin A in B cells is distinct from that of TGF-β.


Figure 5
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  		Figure 5. Pretreatment of naïve B cells with activin A does not affect TGF-β- or IL-4-mediated Ig class-switch in B cells. (A) Effect of pretreatment of naïve B cells with activin A on TGF-β-mediated growth arrest in B cells. Naïve, splenic B cells (5x104) were treated with or without activin A (125 ng/ml) for 6 h prior to stimulation with the indicated concentrations of LPS (2–50 µg/ml) in the presence or absence of TGF-β1 (5 ng/ml) for 6 days. Cell proliferation was measured by MTT assay and expressed as mean A570 nm + SD. (B) Effects of pretreatment of naïve B cells with activin A on TGF-β-induced Ig secretion. Naïve B cells were pretreated for 6 h with activin A (125 ng/ml) and then stimulated with LPS (50 µg/ml) for 6 days in the presence or absence of TGF-β (5 ng/ml). Culture supernatants were serially diluted and tested for total IgG, IgA, and IgM by ELISA and expressed as mean A450 nm + SD at the same dilutions. ns, Not significant. (C) Effects of pretreatment of naïve B cells with activin A on the TGF-β-mediated class-switch recombination. Naïve B cells pretreated with activin A (125 ng/ml) for 6 h were stimulated with LPS (50 µg/ml) with or without TGF-β1 (5 ng/ml) for 48 h. Total RNA was isolated and subjected to RT-PCR of GLT and PST for IgA ({alpha}) and IgG2b ({gamma}2b). (D) Enhancement of IgE and IgG1 secretion in B cells by IL-4. Naïve, splenic B cells (5x104) were cultured for 6 days with LPS (50 µg/ml) in the presence of different concentrations of IL-4 (0–100 U/ml). Culture supernatants were assayed for total IgE (left) and IgG1 (right) by ELISA and expressed as mean A450 nm + SD at the same dilutions. * and **, P < 0.01 and P < 0.001, compared with no-IL-4 control, respectively. (E) Effects of pretreatment of naïve B cells with activin A on IL-4 induced IgE and IgG1 production in B cells. Naïve B cells pretreated with or without activin A (125 ng/ml) for 6 h were cultured with LPS in the presence of IL-4 (25 U/ml) for 6 days (right). Total IgE and IgG1 in the culture supernatant were measured by ELISA and expressed as mean A450 nm + SD at the same dilution.

Involvement of activin A in Th2-type immune responses
Our previous study has shown that activin A is produced in Th2 cells upon activation [5 ], suggesting that activin A has a role in Th2-mediated immune responses. As IgE production is a characteristic feature of Th2-mediated immune responses, it was expected that activin A also induces IgE production; however, pretreatment of naïve B cells with activin A did not induce IgE production (data not shown). Furthermore, pretreatment with activin A did not affect IL-4-induced IgE production by LPS-stimulated B cells (Fig. 5D) ; thus, we concluded that activin A does not directly induce B cells to produce IgE.

Our previous in vitro studies revealed that activin A has an effect on other immune cells involved in Th2-mediated immune responses. Activin A induces the expression of MMP-2 in peritoneal macrophages [7 ] and increases the migration and gene expression of mast cell-specific protease-1 in mast cell progenitors [8 ]. Furthermore, activin A promotes the differentiation of macrophages toward the M-2 phenotype, similar to other Th2 cytokines such as IL-4 and IL-13 [5 ]. Thus, we considered the possibility that activin A promotes IgE production indirectly through activation of other immune cells. To address this possibility, we examined the effect of activin A neutralization in vivo on antigen-specific antibody production in mice immunized with OVA (Fig. 6A ). As shown in Figure 6B , neutralization of activin A resulted in a significant decrease in serum antigen-specific IgE. Total serum IgE levels in mice treated with anti-activin A mAb also tended to be lower than those in control mice, although this was not statistically significant (Fig. 6B) . These results indicate that activin A is involved in Th2-mediated immune responses through the promotion of antigen-specific IgE production. Neutralization of activin A also resulted in a significant decrease in serum IL-4 levels (Fig. 5C) . On the other hand, serum OVA-specific IgG subclasses and IgM levels were slightly, but not significantly, decreased by neutralization of activin A (Fig. 6D) .


Figure 6
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  		Figure 6. Involvement of activin A in Th2-type immune responses. (A) Schematic illustration of the immunization and treatment protocol. (B) Effect of neutralization of endogenous activin A on the antigen-specific IgE response. BALB/c mice were immunized with OVA and treated with anti-activin A mAb or isotype control IgG1, as shown in A. Serum levels of OVA-specific and total IgE were analyzed by ELISA and expressed as A450 nm at the same dilution. Each dot represents an individual mouse. Horizontal lines represent group means. (C) Neutralization of circulating activin A results in decreased serum IL-4 levels, which in mice, were treated as described in A and were measured by ELISA and expressed as the mean + SD. (D) Effect of neutralization of endogenous activin A on the antigen-specific IgM and IgG response. Serum OVA-specific IgM and IgG levels in mice treated as described in A were measured by ELISA and expressed as A450 nm at the same dilution.


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DISCUSSION
 
B cells play a critical role in Th2-mediated immune responses as antibody-producing cells. The process by which resting B cells differentiate into antibody-secreting cells is a complex, multistep process. Not only antigens but also various cytokines are involved in the regulation of B cell activation and differentiation [1 ]. Th2 cytokines provide help for humoral (antibody) immune responses by stimulating B cells to produce neutralizing and opsonizing antibodies. Our previous study showed that activin A is produced in Th2 cells and indicated that activin A has a role in Th2-mediated immune responses [5 ]. This finding prompted us to investigate the expression and function of activin A in murine B cells.

Activin A expression was induced in B cells in response to their activation. This is consistent with previous studies showing the expression of activin A, but not activin AB and activin B, in peritoneal macrophages [7 ], mast cells [8 ], and Th2 cells [5 ]. Activin A could be a major activin in immune cells. In the present study, we also detected follistatin gene transcripts in B cells. Follistatin is an activin-binding protein that neutralizes activin activities in many biological systems [23 , 24 ]. Interestingly, the expression of follistatin was inversely related to that of activin βA during B cell activation. The increased net activin activity in the culture supernatant of LPS-stimulated B cells can be explained by a combination of increased activin A production and decreased follistatin production. These results also suggest the involvement of follistatin in the regulation of activin function in the immune system.

Our results indicate that activin A acts on B cells to increase IgG production. One of the important implications is that the effect was observed only when naïve B cells were pretreated with activin A prior to LPS stimulation. This finding indicates that activin A acts on resting B cells but not activated B cells. Consistently, the expression of activin receptors was decreased in activated B cells; thus, the unresponsiveness of activated B cells to activin A would result from decreases in the expression of receptors for activin.

Some reports showed that activin induced growth arrest and apoptosis in B cell-derived cell lines [25 ] and B cell hybridoma [26 ]. In contrast to previous findings, our results show no evidence of apoptosis in cultured B cells from mouse spleen (data not shown). Although activin A inihibits B cell generation from marrow stem cells [27 ], our data showed that activin A is involved in stimulation of proliferation of mature B cells and antibody secretion, suggesting the stage-dependent activities of activin A on the B cell lineage.

In the present study, neutralization of circulating activin A resulted in a decreased, antigen-specific IgE response. Based on these results, we propose a model for the role of activin A in antibody production (Fig. 7 ). Activin A produced by activated B cells acts in part directly on resting B cells to enhance IgG production. Activin A is also involved in antigen-specific IgE production, presumably through the activation of other immune cells. Neutralization of activin A also resulted in a significant decrease in serum IL-4 levels in OVA-immunized mice. Thus, it may be mediated, at least in part, by regulating the production of IL-4. In addition to activated B cells, activin A is produced by other immune cells associated with Th2 immune responses, such as macrophages [7 ], mast cells [8 ], and Th2 cells [5 ]. These cells may also regulate B cell function through activin A production. In our preliminary experiments, however, IgE secretion from B cells was not found, even when they were cocultured with macrophages pretreated with activin A (data not shown). More complex mechanisms should be involved in activin A regulation of IgE antibody production in vivo.


Figure 7
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  		Figure 7. Schematic representation of a proposed model for involvement of activin A in antibody responses. Activin A produced by activated B cells acts in part directly on resting B cells to promote IgG production. On the other hand, activin A promotes IgE production indirectly through activation of other immune cells.

A previous study showed that secretion of activin A increased in the airway of mice after OVA sensitization followed by antigen challenge [28 ]. Activin A in bronchoalveolar lavage fluid from OVA-sensitized mice was also elevated after antigen challenge [29 ]. Activin βA mRNA was highly induced in murine bone marrow mast cells after stimulation by IgE receptor cross-linking [28 ]. In a human study, the serum level of activin A was increased in patients with asthma, and T cells from these patients exhibited an enhanced level of activin A mRNA [30 ]. Furthermore, increased expression of activin A was found only in Th2 cells, but not in Th1 cells, indicating that activin A production is associated with Th2-type immune responses. Taken together with our current findings, it seems conclusive that activin A plays an important role in Th2-mediated immune responses by enhancing antibody production.

Activin A is produced by a wide variety of tissues, and its diverse activities are found in neural and endocrine tissues [4 ]. However, the in vivo role of activin A in immune response is not yet fully understood, as activin A-deficient mice exhibit craniofacial defects and die shortly after birth [31 , 32 ]. In contrast, the immune-suppressive function of TGF-β is illustrated by in vivo studies. Targeted disruption of TGF-β1 in mice resulted in severe multifocal inflammation, indicating a role of TGF-β in immune suppression [33 ]. In fact, TGF-β inhibits B cell proliferation and induces apoptosis in immature and resting B cells [34 ]. On the other hand, TGF-β also positively regulates B cell responses by inducing class-switching to IgA and IgG2b [34 ]. In most in vitro studies, activin has overlapping biological activities with TGF-β [19 ]. This is partly a result of the fact that activin and TGF-β use the same proteins (Smad2 and/or Smad3) in signal transduction [35 ]. It would therefore be expected that activin A has similar effects to TGF-β on B cells; however, in the present study, activin A did not induce Ig class-switching to IgA and IgG2b in B cells. Thus, the function of activin A in B cells is quite different from that of TGF-β, suggesting that these two structurally related proteins have different roles in the immune system.

We have previously shown that activin A induces the expression of MMP-2 in peritoneal macrophages [7 ]. As TGF-βs are activated by MMPs [36 37 38 ], it is possible that activin A plays a role in the activation of latent TGF-β1 to its active form. However, in our experiments, active TGF-β1 was not detected by ELISA in the culture supernatant of B cells, even when they were treated with activin A (data not shown). Therefore, we concluded that activin A does not activate latent TGF-β1.

In our experiments, the expression of Smad7 was induced rapidly in B cells by activin A and TGF-β1 (data not shown). Smad7 is a TGF-β- and activin-inducible gene, and its transcription is activated by TGF-β/activin-specific Smads, Smad2 and Smad3 [39 ]. The effects of activin A on naïve B cells are mediated, at least in part, by a receptor and Smad-dependent pathway and partly overlap those of TGF-β1 in B cells. However, the effects of activin A on proliferation and class-switch recombination of B cells are distinct from those of TGF-β1. So far, there are no known differences between signaling pathways for activin and TGF-β. B cells may provide a good model system to study the molecular bases of functional differences between activin and TGF-β.


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ACKNOWLEDGEMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research (18580300) from Japan Society for the Promotion of Science and a grant for Chemical Biology Research Program from RIKEN. We thank the National Hormone and Pituitary Program for providing human recombinant follistatin.

Received October 26, 2007; revised February 5, 2008; accepted February 18, 2008.


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