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Activin A stimulates IgA expression in mouse B cells
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Biochemical and Biophysical Research Communications 366 (2008) 574–578 www.elsevier.com/locate/ybbrc
Activin A stimulates IgA expression in mouse B cells Hwa-Joung Lee a, Goo-Young Seo a, Hyun-A Kim a, Pyeung-Hyeun Kim a
a,b,*
Department of Molecular Bioscience, School of Bioscience & Biotechnology, Kangwon National University, Chunchon 200-701, Republic of Korea b Vascular System Research Center, Kangwon National University, Chunchon 200-701, Republic of Korea Received 19 November 2007 Available online 21 December 2007
Abstract In this study, a potential role for activin A in mouse immunoglobulin (Ig) regulation was investigated. We observed that activin A increased IgA secretion in B lymphoma cells. In contrast, little effect was observed on IgM and IgG2b secretion. Activin A also significantly increased surface IgA expression and Ig germ-line a transcript (GLTa) levels. In parallel, activin A increased GLTa and postswitch transcripts a (PSTa) expression in normal B cells, which was augmented by IL-5. An increase in IgA production by surface IgA negative B cells by activin A was apparent. Finally, the increase of IgA secretion by activin A was blocked by an activin receptor inhibitor (SB431542). The increase of GLTa by activin A was augmented by Smad3/4 overexpression and abolished by Smad3 dominant negative overexpression. These results suggest that activin A induces IgA isotype switching via Smad3/4-mediated germ line a transcription. 2007 Elsevier Inc. All rights reserved. Keywords: Activin A; TGF-b; IgA; Class switching; Smad
Activins, transforming growth factor-b (TGF-b superfamily members, are protein dimers composed of b subunits linked by a disulfide bond, that include activin A (bAbA), activin AB (bAbB), and activin B (bBbB) [1]. Activins were originally identified as inducers of follicle stimulating hormone release from the anterior pituitary gland [2,3]. Activin A is also expressed in bone marrow cells, monocytes, macrophages, mast cells, and Th2 cells [4–8]. A wide variety of functions for activins have been described, including roles in wound repair, fibrosis, and inflammatory disease [9,10]. Nevertheless, the function of activins in B lymphocytes is virtually unknown. TGF-b1 is known to induce IgA expression in B lymphocytes [11–15]. Ig class switching allows the recombined variable region gene segment (VDJ) to be expressed with a new downstream heavy chain constant region (CH). CSR is directed to a particular CH gene based on cytokine stimula-
tion-mediated induction of germ-line (GL) CH gene transcription before switch recombination at the same CH gene [16]. It is now clear that isotype switching requires GL transcription through target S regions [16,17] and expression of activation-induced cytidine deaminase (AID) [18]. TGF-b1 induces both GLa transcription and subsequent switching to IgA [19] and IgG2b [20]. It has been shown that Smad3/ 4 and Runx3 mediate TGFb1-induced GLa promoter activity [21–23]. Furthermore, we have shown that TGFb1induced GLa transcription results in IgA secretion [24]. Since activins have overlapping biological activities with TGF-b [25], and activins and TGF-bs use the same proteins (Smad2 and/or Smad3) in signal transduction [1], we were interested in the role of activin A in the regulation of Ig expression in mouse B cells. We found that activin A selectively modulates mouse B cells to express IgA, but not other isotypes, such as IgG2b.
* Corresponding author. Address: Department of Molecular Bioscience, School of Bioscience & Biotechnology, Kangwon National University, Chunchon 200-701, Republic of Korea. Fax: +82 33 241 4627. E-mail address: [email protected] (P.-H. Kim).
Materials and methods
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.008
Animals. BALB/c mice were purchased from Orient Co. Ltd. (Gyeonggi-do, Korea) and maintained in an animal environmental control
H.-J. Lee et al. / Biochemical and Biophysical Research Communications 366 (2008) 574–578
mouse IgA (Becton Dickinson, San Jose, CA) was added to the cell suspension and placed at 4 C for 30 min. Cells were washed with HBSS three times and resuspended in PBS-1% formalin. Cytofluorometric analysis was carried out using a FACScan (Becton Dickinson, Mountain View, CA). RNA preparation and RT-PCR. RNA preparation, reverse transcription, and PCR were performed as described previously [23]. Primers were synthesized by Bioneer Corp. (Seoul, Korea), including: GLTa sense, 5 0 CTACC ATAGG GAAGA TAGCC T-3 0 and antisense, 5 0 -TAATC GTGAA TCAGG CAG-3 0 (product size, 206 bp); PSTa sense, 5 0 -CTCTG GCCCT GCTTA TTGTT G-3 0 (product size, 267 bp); b-actin sense, 5 0 CATGT TTGAG ACCTT CAACA CCCC-3 0 and antisense, 5 0 -GCCAT CTCCT GCTCG AAGTC TAG-3 0 (product size, 320 bp). All reagents for RT-PCR were purchased from Promega (Madison, WI). PCR reactions for b-actin were performed in parallel in order to normalize cDNA concentrations within each set of samples. Aliquots of the PCR products were resolved by electrophoresis on 0.2% agarose gels.
chamber (Myung Jin Inst. Co., Seoul, Korea). Animals were fed Purina Laboratory Rodent Chow 5001 ad libitum. Eight- to twelve-week-old mice were used in this study. Animal care followed institutional guidelines set forth by Kangwon National University. B cell preparations and cell culture. The murine B lymphoma cell line, CH12F3-2A was provided by Dr. T. Honjo (Osaka University, Japan) [26]. These cells were cultured at 37 C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% FBS, 50 lM 2-mercaptoethanol, 5 mM Hepes, penicillin (100 U/ml)/streptomycin (100 lg/ml). Mouse spleen B cell suspensions were prepared as described before [27]. For the preparation of surface IgA negative (sIgA) B cells, cells (5 ml of 5 · 107 cells/ml) were added to petri dishes precoated with goat anti-mouse IgA (7 lg/ml) and incubated for 60 min at 4 C. This step was repeated once after harvesting unattached cells. Based on cytofluorometric analysis, this procedure resulted in greater than 95% depletion of sIgA+ cells. Expression plasmids and transfection. Genes encoding Smad3 [28] and Smad4 [29] subcloned into Flag–pcDNA3 [30] were provided by Dr. M. Kawabata (The Cancer Institute, Tokyo, Japan). The dominant-negative Smad3 expression plasmid (Smad3D407E) [31] was provided by Dr. M. Kato (The Cancer Institute, Tokyo, Japan). Transfection was performed by electroporation with a Gene Pulser II (Bio-Rad, USA) as described [23]. Isotype-specific ELISA. Enzyme linked immuno-sorbent assays (ELISAs) were performed as described previously [23]. Reaction products were measured at 405 nm with an ELISA reader (VERSAMAX reader, Molecular Devices, Sunnyvale, CA). Flow cytometry analysis. Cultured cells were washed with Hanks’ Balanced Salt Solution (HBSS) and resuspended in DMEM, 5% FBS, 0.1% NaN3 at a density of 1 · 106 cells/ml. FITC-conjugated goat anti-
Results and discussion TGF-b1 enhances IgA and IgG2b production by murine B cells [11,15,27] and activins have overlapping biological activities with TGF-b [25]. Therein, we asked whether activin A affects Ig production in mouse B cells. Activin A increased IgA secretion by 2-fold in CH12F3A-2A B lymphoma cells (Fig. 1A); not shown here, TGF-b1 was more potent than activin A (8-fold). In contrast, activin A little affected IgM and IgG2b secretion. Subsequent FACS analysis showed that activin A also increased surface IgA
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Surface IgA Fig. 1. Activin A increases IgA expression in B lymphoma cells. In (A), CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ml) and activin A (5 ng/ml). After 3 days of culture, supernatants were collected and secretion of IgA, IgM, and IgG2b was determined by ELISA. Data are means of triplicate cultures ± SEM. In (B), Purified sIgA CH12F3A-2A B lymphoma cells were cultured with LPS (12.5 lg/ml), TGF-b1 (1 ng/ml), and activin A (5 ng/ml) for two days. Cells were then collected and stained with FITC-labeled goat anti-mouse IgA.
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expression on the cells. Though, the induction of IgA by activin A was less than that for TGF-b1 (Fig. 1B). These results reveal that activin A can selectively modulate IgA response in mouse B cells. CSR requires germ line transcription through target S regions [16,17]. In addition, TGF-b1 induces expression of GLa transcripts (GLTa) and isotype switching to IgA [19]. Therefore, it was necessary to determine whether activin A stimulates B cells to express GLTa. As shown in Fig. 2, activin A increased the expression of GLTa in the presence of LPS, suggesting that this cytokine possibly causes IgA isotype switching. Once switching to IgA occurs, the GLl promoter becomes associated with the Ca gene and continues to be active, generating transcripts termed a post-switch transcripts (PSTa [18,32]. Thus, expression of PSTa as well as GLTa can be utilized as indicators of active IgA switching (Fig. 2, left panel). On the other hand, IgA isotype expression was shown to be selectively induced by TGF-b1 and further augmented by the addition of either IL-2 or IL-5 to cultures of LPS-activated murine spleen B cells [11,13,27]. Therefore, we assessed the effect of activin A and IL-5 on expression of GLTa and PSTa in normal spleen B cells and observed that GLTa expression was increased by activin A and further enhanced in the presence of IL-5 (Fig. 2C). This accompanied an increase in the expression of PSTa. These data show that activin A modulates B cell IgA switching in normal mouse B cells, particularly when B cell growth factors, like IL-5, are available. Since activin A upregulates GLa transcription in normal B cells, it was important to ask whether activin A could
selectively promote IgA secretion in LPS-activated normal sIgA B cells. Activin A treatment results in a 2-fold increase in IgA secretion, but little or no increase in IgM, IgG2b or IgG1 secretion (Fig. 3). The activin A-stimulatory effect on IgA secretion was further augmented by IL-5. In contrast, IL-5 resulted in little to marginal effect on secretion of IgM, IgG2b or IgG1. These results indicate that activin A selectively increases IgA secretion by normal spleen B cells. Such an activity for activin A may be physiologically relevant since activin A functions as a Th2 cytokine [8]. We have previously demonstrated that Smad3 and Smad4 mediate TGFb1-induced IgA expression [23]. Activin A is known to use ALK4 (activin type I receptor), Smad2 and/or Smad3 in signal transduction [33]. Thus, we explored the possibility that activin A may mediate GLTa expression through Smad3 (Fig. 4). First, SB431542 (an inhibitor of ALK4, ALK5, and ALK7) abrogated the increase of IgA secretion by activin A, suggesting that ALK4 mediates activin A-induced IgA expression. Moreover, activin A treatment increased GLTa expression, and this effect was augmented by overexpressed Smad3/4. Finally, DN-Smad3 completely abolished activin A-induced GLTa expression. These results imply that activin A induces GLTa expression through Smad3 and 4, leading to IgA isotype switching. In conclusion, activin A, a member of TGF-b superfamily, selectively increases IgA isotype expression in mouse B cells, but not the expression of other isotypes. This property of activin A reveals that it is more specific than TGF-b1 in terms of IgA isotype switching since the latter
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Fig. 2. Effect of activin A and IL-5 on levels of germ line a transcripts. In (A), a diagram of DNA recombination events occurring during switching to IgA is shown. In (B), CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ml) and activin A (5 ng/ml) for 24 h. In (C), mouse spleen B cells were cultured with LPS (12.5 lg/ml), activin A (5 ng/ml), and IL-5 (5 ng/ml) for two days. Levels of transcripts were measured by RT-PCR. GLT, germline transcripts; PST, post-switch transcripts.
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Fig. 3. Effect of activin A and IL-5 on Ig secretion by mouse spleen sIgA B cells. Mouse spleen sIgA B cells were stimulated with LPS (12.5 lg/ml), IL-5 (5 ng/ml), and activin A (5 ng/ml). Supernatants were harvested after seven days of culture. Ig secretions were determined by ELISA. Data are means of triplicate cultures ± SEM.
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Fig. 4. Activin A induces IgA expression through ALK4 and Smad3. In (A), sIgA CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ ml), activin A (5 ng/ml), and 10 lM of SB431542. Supernatants were collected after three days of culture and secretion of IgA was determined by ELISA. Data are means ± SEM of triplicate cultures. In (B), CH12F3-2A B lymphoma cells were transfected with expression plasmids for Smad3, Smad4, DNSmad3 or pcDNA3 (10 lg of each). They were then cultured with LPS (12.5 lg/ml) and activin A (5 ng/ml), and after 24 h, levels of endogenous GLTa and PSTa transcripts were measured by RT-PCR.
causes both IgA and IgG2b switching in mouse B cells. We note that the present report does not identify any co-activators that cooperate with Smad3/4 in activin A-induced GLTa expression. Previously, we have demonstrated that Runx3 and p300 cooperate with Smad3/4 in the expression of TGFb1-induced IgA and IgG2b isotypes [24,34]. In this context, it will be interesting to evaluate potential cofactors that interact with Smad3/4 under the influence of activin A. Ultimately, such studies will provide insights into how activin A influences IgA isotype switching in mouse B cells. Acknowledgments This work was supported by a Vascular System Research Center grant from the Korea Science & Engineering
Foundation to P.-H.K. and by the second stage of the Brain Korea 21 program. This work was partially carried out in facility the Institute of Bioscience and Biotechnology at Kangwon National University. References [1] J. Massague, TGF-beta signal transduction, Annu. Rev. Biochem. 67 (1998) 753–791. [2] N. Ling, S.Y. Ying, N. Ueno, S. Shimasaki, F. Esch, M. Hotta, R. Guillemin, Pituitary FSH is released by a heterodimer of the betasubunits from the two forms of inhibin, Nature 321 (1986) 779– 782. [3] W. Vale, J. Rivier, J. Vaughan, R. McClintock, A. Corrigan, W. Woo, D. Karr, J. Spiess, Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid, Nature 321 (1986) 776–779.
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[4] S. Luisi, P. Florio, F.M. Reis, F. Petraglia, Expression and secretion of activin A: possible physiological and clinical implications, Eur. J. Endocrinol. 145 (2001) 225–236. [5] M. Abe, Y. Shintani, Y. Eto, K. Harada, M. Kosaka, T. Matsumoto, Potent induction of activin A secretion from monocytes and bone marrow stromal fibroblasts by cognate interaction with activated T cells, J. Leukoc. Biol. 72 (2002) 347–352. [6] K. Ogawa, M. Funaba, L.S. Mathews, T. Mizutani, Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages, J. Immunol. 165 (2000) 2997– 3003. [7] M. Funaba, T. Ikeda, K. Ogawa, M. Murakami, M. Abe, Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration, J. Leukoc. Biol. 73 (2003) 793–801. [8] K. Ogawa, M. Funaba, Y. Chen, M. Tsujimoto, Activin A functions as a Th2 cytokine in the promotion of the alternative activation of macrophages, J. Immunol. 177 (2006) 6787–6794. [9] G.P. Risbridger, J.F. Schmitt, D.M. Robertson, Activins and inhibins in endocrine and other tumors, Endocr. Rev. 22 (2001) 836–858. [10] S. Werner, C. Alzheimer, Roles of activin in tissue repair, fibrosis, and inflammatory disease, Cytokine Growth Factor Rev. 17 (2006) 157– 171. [11] R.L. Coffman, D.A. Lebman, B. Shrader, Transforming growth factor beta specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes, J. Exp. Med. 170 (1989) 1039– 1044. [12] P.H. Kim, M.F. Kagnoff, Transforming growth factor beta 1 increases IgA isotype switching at the clonal level, J. Immunol. 145 (1990) 3773–3778. [13] E. Sonoda, R. Matsumoto, Y. Hitoshi, T. Ishii, M. Sugimoto, S. Araki, A. Tominaga, N. Yamaguchi, K. Takatsu, Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production, J. Exp. Med. 170 (1989) 1415–1420. [14] H. Spieker-Polet, P.C. Yam, Z. Arbieva, S.K. Zhai, K.L. Knight, In vitro induction of the expression of multiple IgA isotype genes in rabbit B cells by TGF-beta and IL-2, J. Immunol. 162 (1999) 5380– 5388. [15] P. van Vlasselaer, J. Punnonen, J.E. de Vries, Transforming growth factor-beta directs IgA switching in human B cells, J. Immunol. 148 (1992) 2062–2067. [16] J. Stavnezer, Molecular processes that regulate class switching, Curr. Top. Microbiol. Immunol. 245 (2000) 127–168. [17] J. Chaudhuri, F.W. Alt, Class-switch recombination: interplay of transcription, DNA deamination and DNA repair, Nat. Rev. Immunol. 4 (2004) 541–552. [18] M. Muramatsu, K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, T. Honjo, Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme, Cell 102 (2000) 553–563. [19] D.A. Lebman, F.D. Lee, R.L. Coffman, Mechanism for transforming growth factor beta and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures, J. Immunol. 144 (1990) 952–959.
[20] T.M. McIntyre, D.R. Klinman, P. Rothman, M. Lugo, J.R. Dasch, J.J. Mond, C.M. Snapper, Transforming growth factor beta 1 selectivity stimulates immunoglobulin G2b secretion by lipopolysaccharide-activated murine B cells, J. Exp. Med. 177 (1993) 1031–1037. [21] M.J. Shi, J. Stavnezer, CBF alpha3 (AML2) is induced by TGF-beta1 to bind and activate the mouse germline Ig alpha promoter, J. Immunol. 161 (1998) 6751–6760. [22] J. Hanai, L.F. Chen, T. Kanno, N. Ohtani-Fujita, W.Y. Kim, W.H. Guo, T. Imamura, Y. Ishidou, M. Fukuchi, M.J. Shi, J. Stavnezer, M. Kawabata, K. Miyazono, Y. Ito, Interaction and functional cooperation of PEBP2/CBF with Smads. Synergistic induction of the immunoglobulin germline Calpha promoter, J. Biol. Chem. 274 (1999) 31577–31582. [23] S.R. Park, J.H. Lee, P.H. Kim, Smad3 and Smad4 mediate transforming growth factor-beta1-induced IgA expression in murine B lymphocytes, Eur. J. Immunol. 31 (2001) 1706–1715. [24] S.R. Park, E.K. Lee, B.C. Kim, P.H. Kim, p300 cooperates with Smad3/4 and Runx3 in TGFbeta1-induced IgA isotype expression, Eur. J. Immunol. 33 (2003) 3386–3392. [25] J.J. Lebrun, Y. Chen, W. Vale, Receptor serine kinases and signaling by activins and inhibins, in: T. Aono, H. Sugino, W. Vale (Eds.), Inhibin, Activins and Flollistatin: Regulatory Functions in System and Cell Biology, Springer-Verlag, New York, 1997, pp. 1–20. [26] M. Nakamura, S. Kondo, M. Sugai, M. Nazarea, S. Imamura, T. Honjo, High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells, Int. Immunol. 8 (1996) 193–201. [27] P.H. Kim, M.F. Kagnoff, Transforming growth factor-beta 1 is a costimulator for IgA production, J. Immunol. 144 (1990) 3411–3416. [28] J.J. Zhang, U. Vinkemeier, W. Gu, D. Chakravarti, C.M. Horvath, J.E. Darnell Jr., Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling, Proc. Natl. Acad. Sci. USA 93 (1996) 15092–15096. [29] S.A. Hahn, M. Schutte, A.T. Hoque, C.A. Moskaluk, L.T. da Costa, E. Rozenblum, C.L. Weinstein, A. Fischer, C.J. Yeo, R.H. Hruban, S.E. Kern, DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1, Science 271 (1996) 350–353. [30] T. Imamura, M. Takase, A. Nishihara, E. Oeda, J. Hanai, M. Kawabata, K. Miyazono, Smad6 inhibits signalling by the TGF-beta superfamily, Nature 389 (1997) 622–626. [31] D. Goto, K. Yagi, H. Inoue, I. Iwamoto, M. Kawabata, K. Miyazono, M. Kato, A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF-beta signals, FEBS Lett. 430 (1998) 201–204. [32] S.C. Li, P.B. Rothman, J. Zhang, C. Chan, D. Hirsh, F.W. Alt, Expression of I mu-C gamma hybrid germline transcripts subsequent to immunoglobulin heavy chain class switching, Int. Immunol. 6 (1994) 491–497. [33] J. Massague, Y.G. Chen, Controlling TGF-beta signaling, Genes Dev. 14 (2000) 627–644. [34] S.R. Park, G.Y. Seo, A.J. Choi, J. Stavnezer, P.H. Kim, Analysis of transforming growth factor-beta1-induced Ig germ-line gamma2b transcription and its implication for IgA isotype switching, Eur. J. Immunol. 35 (2005) 946–956.
Biochemical and Biophysical Research Communications 366 (2008) 574–578 www.elsevier.com/locate/ybbrc
Activin A stimulates IgA expression in mouse B cells Hwa-Joung Lee a, Goo-Young Seo a, Hyun-A Kim a, Pyeung-Hyeun Kim a
a,b,*
Department of Molecular Bioscience, School of Bioscience & Biotechnology, Kangwon National University, Chunchon 200-701, Republic of Korea b Vascular System Research Center, Kangwon National University, Chunchon 200-701, Republic of Korea Received 19 November 2007 Available online 21 December 2007
Abstract In this study, a potential role for activin A in mouse immunoglobulin (Ig) regulation was investigated. We observed that activin A increased IgA secretion in B lymphoma cells. In contrast, little effect was observed on IgM and IgG2b secretion. Activin A also significantly increased surface IgA expression and Ig germ-line a transcript (GLTa) levels. In parallel, activin A increased GLTa and postswitch transcripts a (PSTa) expression in normal B cells, which was augmented by IL-5. An increase in IgA production by surface IgA negative B cells by activin A was apparent. Finally, the increase of IgA secretion by activin A was blocked by an activin receptor inhibitor (SB431542). The increase of GLTa by activin A was augmented by Smad3/4 overexpression and abolished by Smad3 dominant negative overexpression. These results suggest that activin A induces IgA isotype switching via Smad3/4-mediated germ line a transcription. 2007 Elsevier Inc. All rights reserved. Keywords: Activin A; TGF-b; IgA; Class switching; Smad
Activins, transforming growth factor-b (TGF-b superfamily members, are protein dimers composed of b subunits linked by a disulfide bond, that include activin A (bAbA), activin AB (bAbB), and activin B (bBbB) [1]. Activins were originally identified as inducers of follicle stimulating hormone release from the anterior pituitary gland [2,3]. Activin A is also expressed in bone marrow cells, monocytes, macrophages, mast cells, and Th2 cells [4–8]. A wide variety of functions for activins have been described, including roles in wound repair, fibrosis, and inflammatory disease [9,10]. Nevertheless, the function of activins in B lymphocytes is virtually unknown. TGF-b1 is known to induce IgA expression in B lymphocytes [11–15]. Ig class switching allows the recombined variable region gene segment (VDJ) to be expressed with a new downstream heavy chain constant region (CH). CSR is directed to a particular CH gene based on cytokine stimula-
tion-mediated induction of germ-line (GL) CH gene transcription before switch recombination at the same CH gene [16]. It is now clear that isotype switching requires GL transcription through target S regions [16,17] and expression of activation-induced cytidine deaminase (AID) [18]. TGF-b1 induces both GLa transcription and subsequent switching to IgA [19] and IgG2b [20]. It has been shown that Smad3/ 4 and Runx3 mediate TGFb1-induced GLa promoter activity [21–23]. Furthermore, we have shown that TGFb1induced GLa transcription results in IgA secretion [24]. Since activins have overlapping biological activities with TGF-b [25], and activins and TGF-bs use the same proteins (Smad2 and/or Smad3) in signal transduction [1], we were interested in the role of activin A in the regulation of Ig expression in mouse B cells. We found that activin A selectively modulates mouse B cells to express IgA, but not other isotypes, such as IgG2b.
* Corresponding author. Address: Department of Molecular Bioscience, School of Bioscience & Biotechnology, Kangwon National University, Chunchon 200-701, Republic of Korea. Fax: +82 33 241 4627. E-mail address: [email protected] (P.-H. Kim).
Materials and methods
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.008
Animals. BALB/c mice were purchased from Orient Co. Ltd. (Gyeonggi-do, Korea) and maintained in an animal environmental control
H.-J. Lee et al. / Biochemical and Biophysical Research Communications 366 (2008) 574–578
mouse IgA (Becton Dickinson, San Jose, CA) was added to the cell suspension and placed at 4 C for 30 min. Cells were washed with HBSS three times and resuspended in PBS-1% formalin. Cytofluorometric analysis was carried out using a FACScan (Becton Dickinson, Mountain View, CA). RNA preparation and RT-PCR. RNA preparation, reverse transcription, and PCR were performed as described previously [23]. Primers were synthesized by Bioneer Corp. (Seoul, Korea), including: GLTa sense, 5 0 CTACC ATAGG GAAGA TAGCC T-3 0 and antisense, 5 0 -TAATC GTGAA TCAGG CAG-3 0 (product size, 206 bp); PSTa sense, 5 0 -CTCTG GCCCT GCTTA TTGTT G-3 0 (product size, 267 bp); b-actin sense, 5 0 CATGT TTGAG ACCTT CAACA CCCC-3 0 and antisense, 5 0 -GCCAT CTCCT GCTCG AAGTC TAG-3 0 (product size, 320 bp). All reagents for RT-PCR were purchased from Promega (Madison, WI). PCR reactions for b-actin were performed in parallel in order to normalize cDNA concentrations within each set of samples. Aliquots of the PCR products were resolved by electrophoresis on 0.2% agarose gels.
chamber (Myung Jin Inst. Co., Seoul, Korea). Animals were fed Purina Laboratory Rodent Chow 5001 ad libitum. Eight- to twelve-week-old mice were used in this study. Animal care followed institutional guidelines set forth by Kangwon National University. B cell preparations and cell culture. The murine B lymphoma cell line, CH12F3-2A was provided by Dr. T. Honjo (Osaka University, Japan) [26]. These cells were cultured at 37 C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% FBS, 50 lM 2-mercaptoethanol, 5 mM Hepes, penicillin (100 U/ml)/streptomycin (100 lg/ml). Mouse spleen B cell suspensions were prepared as described before [27]. For the preparation of surface IgA negative (sIgA) B cells, cells (5 ml of 5 · 107 cells/ml) were added to petri dishes precoated with goat anti-mouse IgA (7 lg/ml) and incubated for 60 min at 4 C. This step was repeated once after harvesting unattached cells. Based on cytofluorometric analysis, this procedure resulted in greater than 95% depletion of sIgA+ cells. Expression plasmids and transfection. Genes encoding Smad3 [28] and Smad4 [29] subcloned into Flag–pcDNA3 [30] were provided by Dr. M. Kawabata (The Cancer Institute, Tokyo, Japan). The dominant-negative Smad3 expression plasmid (Smad3D407E) [31] was provided by Dr. M. Kato (The Cancer Institute, Tokyo, Japan). Transfection was performed by electroporation with a Gene Pulser II (Bio-Rad, USA) as described [23]. Isotype-specific ELISA. Enzyme linked immuno-sorbent assays (ELISAs) were performed as described previously [23]. Reaction products were measured at 405 nm with an ELISA reader (VERSAMAX reader, Molecular Devices, Sunnyvale, CA). Flow cytometry analysis. Cultured cells were washed with Hanks’ Balanced Salt Solution (HBSS) and resuspended in DMEM, 5% FBS, 0.1% NaN3 at a density of 1 · 106 cells/ml. FITC-conjugated goat anti-
Results and discussion TGF-b1 enhances IgA and IgG2b production by murine B cells [11,15,27] and activins have overlapping biological activities with TGF-b [25]. Therein, we asked whether activin A affects Ig production in mouse B cells. Activin A increased IgA secretion by 2-fold in CH12F3A-2A B lymphoma cells (Fig. 1A); not shown here, TGF-b1 was more potent than activin A (8-fold). In contrast, activin A little affected IgM and IgG2b secretion. Subsequent FACS analysis showed that activin A also increased surface IgA
160
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20 0
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5 0 102
103
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Surface IgA Fig. 1. Activin A increases IgA expression in B lymphoma cells. In (A), CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ml) and activin A (5 ng/ml). After 3 days of culture, supernatants were collected and secretion of IgA, IgM, and IgG2b was determined by ELISA. Data are means of triplicate cultures ± SEM. In (B), Purified sIgA CH12F3A-2A B lymphoma cells were cultured with LPS (12.5 lg/ml), TGF-b1 (1 ng/ml), and activin A (5 ng/ml) for two days. Cells were then collected and stained with FITC-labeled goat anti-mouse IgA.
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expression on the cells. Though, the induction of IgA by activin A was less than that for TGF-b1 (Fig. 1B). These results reveal that activin A can selectively modulate IgA response in mouse B cells. CSR requires germ line transcription through target S regions [16,17]. In addition, TGF-b1 induces expression of GLa transcripts (GLTa) and isotype switching to IgA [19]. Therefore, it was necessary to determine whether activin A stimulates B cells to express GLTa. As shown in Fig. 2, activin A increased the expression of GLTa in the presence of LPS, suggesting that this cytokine possibly causes IgA isotype switching. Once switching to IgA occurs, the GLl promoter becomes associated with the Ca gene and continues to be active, generating transcripts termed a post-switch transcripts (PSTa [18,32]. Thus, expression of PSTa as well as GLTa can be utilized as indicators of active IgA switching (Fig. 2, left panel). On the other hand, IgA isotype expression was shown to be selectively induced by TGF-b1 and further augmented by the addition of either IL-2 or IL-5 to cultures of LPS-activated murine spleen B cells [11,13,27]. Therefore, we assessed the effect of activin A and IL-5 on expression of GLTa and PSTa in normal spleen B cells and observed that GLTa expression was increased by activin A and further enhanced in the presence of IL-5 (Fig. 2C). This accompanied an increase in the expression of PSTa. These data show that activin A modulates B cell IgA switching in normal mouse B cells, particularly when B cell growth factors, like IL-5, are available. Since activin A upregulates GLa transcription in normal B cells, it was important to ask whether activin A could
selectively promote IgA secretion in LPS-activated normal sIgA B cells. Activin A treatment results in a 2-fold increase in IgA secretion, but little or no increase in IgM, IgG2b or IgG1 secretion (Fig. 3). The activin A-stimulatory effect on IgA secretion was further augmented by IL-5. In contrast, IL-5 resulted in little to marginal effect on secretion of IgM, IgG2b or IgG1. These results indicate that activin A selectively increases IgA secretion by normal spleen B cells. Such an activity for activin A may be physiologically relevant since activin A functions as a Th2 cytokine [8]. We have previously demonstrated that Smad3 and Smad4 mediate TGFb1-induced IgA expression [23]. Activin A is known to use ALK4 (activin type I receptor), Smad2 and/or Smad3 in signal transduction [33]. Thus, we explored the possibility that activin A may mediate GLTa expression through Smad3 (Fig. 4). First, SB431542 (an inhibitor of ALK4, ALK5, and ALK7) abrogated the increase of IgA secretion by activin A, suggesting that ALK4 mediates activin A-induced IgA expression. Moreover, activin A treatment increased GLTa expression, and this effect was augmented by overexpressed Smad3/4. Finally, DN-Smad3 completely abolished activin A-induced GLTa expression. These results imply that activin A induces GLTa expression through Smad3 and 4, leading to IgA isotype switching. In conclusion, activin A, a member of TGF-b superfamily, selectively increases IgA isotype expression in mouse B cells, but not the expression of other isotypes. This property of activin A reveals that it is more specific than TGF-b1 in terms of IgA isotype switching since the latter
LPS activin A
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Mouse Ig heavy chain gene VDJ
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Iμ Sμ/Sα Cα
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Fig. 2. Effect of activin A and IL-5 on levels of germ line a transcripts. In (A), a diagram of DNA recombination events occurring during switching to IgA is shown. In (B), CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ml) and activin A (5 ng/ml) for 24 h. In (C), mouse spleen B cells were cultured with LPS (12.5 lg/ml), activin A (5 ng/ml), and IL-5 (5 ng/ml) for two days. Levels of transcripts were measured by RT-PCR. GLT, germline transcripts; PST, post-switch transcripts.
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Fig. 3. Effect of activin A and IL-5 on Ig secretion by mouse spleen sIgA B cells. Mouse spleen sIgA B cells were stimulated with LPS (12.5 lg/ml), IL-5 (5 ng/ml), and activin A (5 ng/ml). Supernatants were harvested after seven days of culture. Ig secretions were determined by ELISA. Data are means of triplicate cultures ± SEM.
300
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activin A Smad3/4 DN-Smad3
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GLTα 100 50
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Fig. 4. Activin A induces IgA expression through ALK4 and Smad3. In (A), sIgA CH12F3A-2A B lymphoma cells were stimulated with LPS (12.5 lg/ ml), activin A (5 ng/ml), and 10 lM of SB431542. Supernatants were collected after three days of culture and secretion of IgA was determined by ELISA. Data are means ± SEM of triplicate cultures. In (B), CH12F3-2A B lymphoma cells were transfected with expression plasmids for Smad3, Smad4, DNSmad3 or pcDNA3 (10 lg of each). They were then cultured with LPS (12.5 lg/ml) and activin A (5 ng/ml), and after 24 h, levels of endogenous GLTa and PSTa transcripts were measured by RT-PCR.
causes both IgA and IgG2b switching in mouse B cells. We note that the present report does not identify any co-activators that cooperate with Smad3/4 in activin A-induced GLTa expression. Previously, we have demonstrated that Runx3 and p300 cooperate with Smad3/4 in the expression of TGFb1-induced IgA and IgG2b isotypes [24,34]. In this context, it will be interesting to evaluate potential cofactors that interact with Smad3/4 under the influence of activin A. Ultimately, such studies will provide insights into how activin A influences IgA isotype switching in mouse B cells. Acknowledgments This work was supported by a Vascular System Research Center grant from the Korea Science & Engineering
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