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Originally published online as doi:10.1189/jlb.0708450 on January 23, 2009

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(Journal of Leukocyte Biology. 2009;85:744-750.)
© 2009 Society for Leukocyte Biology

IL-21 ensures TGF-β1-induced IgA isotype expression in mouse Peyer’s patches

Goo-Young Seo*, Jeehee Youn{dagger} and Pyeung-Hyeun Kim*,{ddagger},1

* Department of Molecular Bioscience, School of Bioscience and Biotechnology, and
{ddagger} Vascular System Research Center, Kangwon National University, Chuncheon, Korea; and
{dagger} Department of Anatomy and Cell Biology, Institute of Biomedical Science, Hanyang University, Seoul, Korea

1 Correspondence: Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea. E-mail: phkim{at}kangwon.ac.kr

ABSTRACT

It is well established that TGF-β1 induces IgA and IgG2b class-switching recombination in murine B cells. In the present study, we assessed the activity of IL-21 along with TGF-β1 in Ig synthesis by murine spleen B cells. IL-21 showed antiproliferative activity on LPS-activated splenic B cells, comparable with that of TGF-β1. IL-21 alone had little effect on IgA secretion and decreased other isotypes. Likewise, IL-21 also did not alter the TGF-β1-induced IgA synthesis and concurrently diminished the syntheses of IgM and IgG2a, which were repressed by TGF-β1. Unexpectedly, IL-21 inhibited the TGF-β1-induced IgG2b production. This IL-21 effect was examined using B cells from IL-21R knockout mice, where the IgA production profile was paralleled by that seen in wild-type B cells. However, the inhibitory effect of IL-21 on TGF-β1-induced IgG2b synthesis was not seen in the IL-21R–/– mouse, suggesting that IL-21 causes TGF-β1-stimulated B cells to decrease IgG2b synthesis. Expression patterns of Ig germ-line {alpha}(GL{alpha})/GL{gamma}2b transcripts under the influence of TGF-β1 and IL-21 were paralleled by IgA/IgG2b secretion. This was also observed in the activities of GL{alpha} and GL{gamma}2b promoters. These results indicate that IL-21 decreases IgG2b secretion mainly through inhibition of GL{gamma}2b transcription and is ultimately associated with selective IgA secretion induced by TGF-β1. Our results showed that IL-21 was expressed in greater magnitude in Peyer’s patches (PP) than in spleen. These results suggest that IL-21 has an important effect on selective IgA+ B cell commitment in PP.

Key Words: class switch • promoter • germ line transcript

INTRODUCTION

IgA is the most abundant Ig isotype in mucosal secretions [1 , 2 ] and plays a critical role in mucosal immunity [3 ]. IgA serves as a first line of defense in mucosa by inhibiting adhesion of microorganisms, removing immune complexes, and neutralizing intracellular viruses [4 ]. Notably, the IgA class-switch recombination (CSR) mainly takes place in Peyer’s patches (PP), and then, IgA+ B cells migrate through the lymph and blood circulation and eventually home to the lamina propria of the intestine [5 ]. It is not clear why IgA CSR takes place mostly in PP but not in other lymphoid organs.

Ig CSR allows expression of the recombined variable region gene segment [V(D)J] with a new downstream heavy-chain constant region (CH) gene. CSR is directed to a particular CH gene by cytokines that induce transcription from germ-line (GL) CH genes before switch recombination to the same CH gene [6 ]. It is now clear that CSR requires GL transcription through target S regions [6 , 7 ] and expression of activation-induced cytidine deaminase [8 ]. TGF-β1 induces GL{alpha} transcription and subsequent CSR to IgA [9 ] and IgG2b [10 ].

IL-21, a potent immunomodulatory four-{alpha}-helical-bundle type I cytokine, is produced by NK T cells (NKT) [11 ], CD4+ T cells [12 ], and TH17 cells [13 , 14 ]. IL-21R consists of the IL-21R and the common cytokine receptor {gamma}-chain [12 , 15 ]. IL-21R is expressed on T cells, B cells, NK cells, dendritic cells, and macrophages [12 , 16 , 17 ]. Like other type I cytokines, IL-21 signals via the Jak-STAT pathway [18 ] and induces pleiotropic effects on the proliferation and differentiation of B cells into plasma cells [19 , 20 ]. IL-21 can enhance the proliferative response of human and murine B cells stimulated with anti-CD40 antibody, and it inhibits B cell proliferation in response to LPS [16 ] or anti-IgM plus IL-4 [12 ]. Thus, IL-21 has different effects on proliferation according to stimulation conditions.

IL-21 is known to regulate Ig expression in mice and humans. IL-21 alone has little effect on IgE and IgG1 production in normal mouse B cells, but it inhibits IL-4-induced IgE production and GL{epsilon} transcripts [21 ]. Reduced levels of serum IgG1 and increased levels of IgE have been shown in IL-21R knockout (KO) mice [22 ]. Thus, one of the regulatory functions of IL-21 is to down-regulate IgE expression in mice. However, in human B cells, IL-21 has positive and negative effects on IgE production, depending on stimulation conditions [23 ]. In addition, it acts as a switch factor for the production of IgG1and IgG3 by human B cells stimulated with anti-CD40 mAb [24 ]. Similarly, IL-21 induces IgG3 production in naive cord blood B cells stimulated with anti-CD40 but not with anti-IgM [20 ]. Based on these observations, the effects of IL-21 on Ig expression seem to be quite complicated, with different effects resulting from different stimulatory signals.

Little is known about the effects of IL-21 on IgA expression in the mouse. In the present study, we investigated the effects of IL-21 and TGF-β1 on Ig synthesis in mouse splenic B cells. We found that IL-21 has minor effects on TGF-β1-induced IgA expression, and it decreases TGF-β1-induced IgG2b expression. In addition, IL-21 was expressed quite strongly in the PP CD4+ T cell population. These results suggest that in cooperation with TGF-β1, CD4+ T cell-derived IL-21 may cause IgM+ B cells to switch to IgA+ B cells exclusively in PP.

MATERIALS AND METHODS

Animals
BALB/c mice and C57BL/6 mice were purchased from the Orient Co. Ltd. (Gyeonggi-do, Korea). Dr. Warren J. Leonard (National Heart, Lung, and Blood Institute, Bethesda, MD, USA) provided IL-21R–/– mice [22 ]. All animals were maintained in an animal environmental control chamber (Myung Jin Inst. Co., Seoul, Korea). Animals were fed Purina Laboratory Rodent Chow 5001 ad libitum. Mice that were 8–12 weeks of age were used in this study. Animal care was performed in accordance with the institutional guidelines set forth by Kangwon National University (Chuncheon, Korea).

B cell preparations and cell culture
Murine splenic B cell suspensions were prepared as described previously [25 ]. Briefly, cell suspensions were washed in PBS and treated with 0.83% ammonium chloride to lyse RBC. T cells were depleted from the cell suspensions by treatment with a cocktail of anti-Thy1.2, anti-Lyt2.2, and anti-L3T4 mAb and low-tox rabbit complement (Cedarlane, Ontario, Canada). This resulted in an ~60% loss in total spleen cells, and B cells comprised more than 90% of the residual population, as assessed by surface Ig (sIg) using flow cytometry. Cells were then washed with HBSS three times and suspended in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10% FBS, 50 µM 2-ME, 5 mM HEPES, and penicillin (100 U/ml)/streptomycin (100 µg/ml). For the preparation of sIgA-negative (sIgA–) B cells, T cell-depleted, splenic B cells were incubated with petri dishes precoated with goat anti-mouse IgA (4 µg/ml) for 70 min at 4°C. This step was repeated once more after harvesting unattached cells. Based on cytofluorometric analysis, this procedure resulted in >95% depletion of sIgA+ cells.

A total of 2 x 105 cells/well was cultured in flat-bottomed, 96-well, tissue-culture plates (SPL, Korea) in a volume of 200 µl complete medium with added LPS (12.5 µg/ml; Escherichia coli 0127:B8; Sigma Chemical Co.) in the presence or absence of murine IL-21 (5 ng/ml; R&D Systems, Minneapolis, MN, USA) and/or TGF-β1 (0.2 ng/ml; R&D Systems).

Preparation of PP cell population
PP cells were prepared as described previously [26 , 27 ]. Mice were killed by cervical dislocation, and PPs were aseptically removed and transferred to a beaker containing cold PBS. PPs were washed vigorously in RPMI 1640, supplemented with protease (Sigma Chemical Co.) and DNase (Promega, Madison, WI, USA) for 30 min at 37°C. The medium containing dissociated cells was collected by aspiration, and fresh enzyme solution was added immediately to the remaining tissue, which was incubated again for 30 min. This procedure was repeated four times. Next, the harvested lymphoid cells were washed twice with HBSS, and the cell suspension was pooled and cultured in complete medium.

Isotype-specific ELISA
ELISAs were performed as described previously [28 ]. The reaction products were measured at 405 nm with an ELISA reader (VERSAMAX reader, Molecular Devices, Sunnyvale, CA, USA). For the detection of antibody retained in the gut, fecal pellets were diluted in PBS, centrifuged at 10,000 g for 10 min, and supernatants collected.

RNA preparation and RT-PCR
RNA preparation, RT, and PCR were performed as described previously [28 ]. PCR primers were synthesized by Bioneer Corp. (Seoul, Korea): GL{alpha} transcripts (GLT{alpha}) sense, 5'-CAA GAA GGA GAA GGT GAT TCA G-3', and antisense, 5'-GAG CTG GTG GGA GTG TCA GTG-3'; GLT{gamma}2b sense, 5'-GGG AGA GCA CTG GGC CTT-3', and antisense, 5'-AGT CAC TGA CTC AGG GAA-3'; GLT{epsilon} sense, 5'-ACT AGA GAT TCA CAA CG-3', and antisense, 5'-AGC GAT GAA TGG AGT AGC-3'; {alpha} post-switch transcript (PST{alpha}) sense, 5'-GAG CTG GTG GGA GTG TCA GTG-3', and antisense, 5'-CTC TGG CCC TGC TTA TTG TTG-3'; PST{gamma}2b sense, 5'-ACC TGG GAA TGT ATG GTT GTG GCT T-3', and antisense, 5'-AGT CAC TGA CTC AGG GAA-3'; PST{epsilon} sense, 5'-CTC GGT GGC TTT GAA GGA AC-3', and antisense, 5'-AGC GAT GAA TGG AGT AGC-3'; IL-21 sense, 5'-CCC TTG TCT GTC TGG TAG TCA TC-3', and antisense, 5'-ATC ACA GGA AGG GCA TTT AGC-3'; β-actin sense, 5'-CATGT TTGAG ACCTT CAACA CCCC-3', and antisense, 5'-GCCAT CTCCT GCTCG AAGTC TAG-3'. All reagents for RT-PCR were purchased from Promega. PCR reactions for β-actin were performed in parallel to normalize cDNA concentrations within each set of samples. Aliquots of the PCR products were resolved by electrophoresis on 2% agarose gels.

Expression plasmids and transfection
GL{gamma}2b-Luciferase (Luc) was constructed in our laboratory [29 ]. Dr. Janet Stavnezer (University of Massachusetts Medical School, Worcester, MA, USA) provided GL{alpha}-Luc [30 ]. CH12F3.2A B lymphoma cells (provided by Dr. Tasuku Honjo, Osaka University, Japan [31 ]) were transfected by electroporation with a Gene Pulser II (Bio-Rad, Hercules, CA, USA) as described previously [28 ].

Intracellular cytokine staining and flow cytometry analysis
Cells were washed and resuspended in staining buffer 1 x PBS, 1% FBS, and 0.05% NaN3. Surface staining was performed on ice with PerCP-rat anti-mouse CD4 antibody (BD Biosciences, San Jose, CA, USA) for 30 min. Intracellular cytokine staining was then performed according to the manufacturer’s protocol (eBioscience, San Diego, CA, USA). Goat IgG anti-mouse IL-21 antibody and FITC-conjugated anti-goat IgG were obtained from R&D Systems and Southern Biotech (Birmingham, AL, USA), respectively. Cytofluorometric analysis was carried out using a FACSCalibur (BD Biosciences).

Labeling with CFSE
Isolated splenic B cells were labeled with a CFSE kit (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions, and added with LPS, murine IL-21, and TGF-β1. Dilution of CFSE was measured by counting 30,000 viable cells with a FACSCalibur.

Statistical analysis
Statistical differences between experimental groups were determined by ANOVA, and values of P < 0.05 by unpaired two-tailed Student’s t-test were considered significant.

RESULTS

IL-21 inhibits LPS-stimulated B cell growth
LPS is generally used for nonspecific stimulation of mouse B cells. We first tested the effect of IL-21 on LPS-stimulated splenic B cells. LPS supported cell viability up to Day 4 in culture (Fig. 1A ), and this increase was inhibited substantially by IL-21 addition. The antiproliferative effect of IL-21 was analogous to that of TGF-β1, as shown in our earlier work [32 ]. We tested the effects of IL-21 and TGF-β1 on B cell growth. TGF-β1 markedly inhibited the viability and proliferation of LPS-stimulated splenic B cells, and IL-21 strengthened the inhibitory activity of TGF-β1 (Fig. 1) .


Figure 1
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  		Figure 1. Effect of IL-21 and TGF-β1 on the growth kinetics of splenic B cells. (A) Normal splenic B cells (2x105) were cultured with LPS (12.5 µg/ml), TGF-β1 (0.2 ng/ml), and IL-21 (5 ng/ml). Numbers of viable cells were enumerated by trypan blue exclusion. Data are means ± SEM (vertical bars) of triplicate culture wells. (B) CFSE-labeled, splenic B cells (2x106) were cultured as in A. B cell proliferation was assessed after 72 h analyzing the dilution of CFSE in the same number of viable cells. Dotted line indicates CFSE-labeled B cells at Day 0.

Effect of IL-21 on TGF-β1-induced IgA expression
TGF-β1 is a potent IgA- and IgG2b-stimulating cytokine in the mouse [10 , 33 ]. Although IL-21 has been shown to regulate Ig production in the mouse and human [21 , 22 , 24 ], it is not known if it can affect IgA expression, particularly TGF-β1-induced IgA and IgG2b expression by mouse B cells. We investigated the effects of IL-21 along with TGF-β1 in Ig synthesis by mouse splenic B cells. As shown in Figure 2 , IL-21 did not increase IgA secretion. Under the same conditions, IL-21 decreased IgG2b, IgM, and IgG2a synthesis. Interestingly, IL-21 enabled B cells to maintain TGF-β1-stimulated IgA synthesis. In contrast, IL-21 further diminished the syntheses of other isotypes that were repressed by TGF-β1, including TGF-β1-induced IgG2b production.


Figure 2
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  		Figure 2. Effect of IL-21 and TGF-β1 on Ig secretion by splenic B cells. Normal, splenic B cells (2x105) from BALB/c mice were cultured with LPS (12.5 µg/ml), IL-21 (5 ng/ml), and TGF-β1 (0.2 ng/ml). After 7 days of culture, supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data are means of triplicate samples ± SEM. *, P < 0.05; **, P < 0.01.

To confirm that IL-21 is actually involved in TGF-β1-induced IgA synthesis, we performed these experiments using splenic B cells from IL-21R KO mice. As shown in Figure 3 , the effects of IL-21 and TGF-β1 on the patterns of IgA synthesis were quite similar between IL-21R+/+ and IL-21R–/– mice. However, a decrease in TGF-β1-induced IgG2b synthesis by IL-21 in the IL-21R+/+ mouse was not seen in the IL-21R–/– mouse. These results indicate that IL-21 has little effect on TGF-β1-induced IgA expression. Instead, it actively modulates TGF-β1-stimulated B cells, leading to a decrease of IgG2b synthesis. Herein, it was necessary to investigate IgA and IgG2b production in the gut of IL-21R+/+ and IL-21R–/– mice. We determined the amounts of Igs retained in the fecal pellets, where large amounts of IgA were detected ({approx}15 µg/ml) but with no significant difference between both mice. Similarly, there was no significant difference in the amount of fecal IgG2b either (not more than 30 ng/ml). Thus, we failed to see diminishing IgG2b production in the gut of the IL-21R–/– mouse. This phenomenon seems to occur, as such a small amount of IgG2b is secreted to the lumen intrinsically.


Figure 3
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  		Figure 3. Effect of IL-21 and TGF-β1 on IgA and IgG2b secretion by splenic B cells from the IL-21R–/– mouse. Splenic B cells (2x105) from C57BL/6 wild-type (WT) or IL-21R–/– mice were cultured with LPS (12.5 µg/ml), IL-21 (5 ng/ml), and TGF-β1 (0.2 ng/ml). After 7 days of culture, supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data are means of triplicate samples ± SEM. *, P < 0.05; **, P < 0.01.

Effect of IL-21 on Ig isotype CSR
Thus far, our data reveal that IL-21 has antiproliferative activity but also affects Ig production. It was therefore important to determine whether IL-21 is involved in Ig CSR, particularly in IgA and IgG2b CSR, which requires GLT through target S regions [6 , 7 ]. As shown in the diagram in Figure 4A , once CSR to IgA occurs, the GLµ promoter becomes associated with the C{alpha} gene and continues to be active and generates transcripts, PST{alpha} [8 , 34 ]. Furthermore, the DNA sequences between Sµ and S{alpha} are looped out of the chromosome as switch circles during CSR, and another type of transcript, a circle transcript (CT) consisting of the I{alpha} exon spliced to the Cµ exon (CT{alpha}), is transcribed from the switch circle from the active I{alpha} promoter [35 ]. Thus, expression of PST{alpha} and CT{alpha}as well as GLT{alpha} can be used as indicators of active IgA CSR.


Figure 4
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  		Figure 4. Effect of IL-21 and TGF-β1 on the levels of Ig GLT in splenic B cells. (A) Diagram of DNA recombination occurring during switching to IgA. Rectangles and ovals represent exons and S regions, respectively. RNA transcripts are indicated beneath the DNA diagrams. (B) Culture conditions were the same as described in Figure 2 . After 2 days of culture, total RNA was isolated, and levels of endogenous GLTs and PSTs were measured by RT-PCR.

We and others [9 , 10 , 28 , 29 ] have shown that TGF-β1 induces levels of IgA and IgG2b CSR-predictive transcripts. We first determined the effects of IL-21 and TGF-β1 on GLT and PST expression by splenic B cells. As shown in Figure 4B , IL-21 alone did not increase GLT{alpha} and PST{alpha} expression and decreased GLT{gamma}2b and PST{gamma}2b expression. In addition, IL-21 little-affected the GLT{alpha} and PST{alpha} expression, which was increased by TGF-β1 and concurrently down-regulated TGF-β1-induced GLT{gamma}2b and PST{gamma}2b expression. This was paralleled by patterns of the IgA and IgG2b secretion as shown in Figure 2 . On the other hand, IL-21 alone and IL-21 and TGF-β1 substantially inhibited GLT{epsilon} and PST{epsilon} expression, as reported previously [21 ]. Taken together, these results suggest that IL-21 does not possess IgA isotype-switching activity but has inhibitory activity on the CSR of IgG2b and IgE.

To delineate roles of IL-21 in IgA and IgG2b regulation more precisely, we examined expression at the GLT and Ig secretion levels using a sorted sIgA– B cell population. Under the influence of IL-21 and TGF-β1, overall expression patterns of IgA/IgG2b and GLT{alpha}/GLT{gamma}2b were similar to those seen in the whole splenic B cell population (Fig. 5A and B ). These results prompted us to investigate the GL{alpha} and GL{gamma}2b promoter activities in the presence of IL-21 and TGF-β1. IL-21 mainly functions at the GL{gamma}2b transcriptional level, affecting GL{gamma}2b promoter activity. As predicted, IL-21 decreased TGF-β1-inducible GL{gamma}2b promoter activity (Fig. 5C) . Again, it had little effect on the TGF-β1-inducible GL{alpha} promoter activity. Taken together, these results indicate that IL-21 decreases IgG2b secretion mainly through inhibition of GL{gamma}2b transcription. Furthermore, we postulate that IL-21 can force TGF-β1-stimulated B cells to secrete only IgA isotype.


Figure 5
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  		Figure 5. Effect of IL-21 and TGF-β1 on IgA and IgG2b expression. Normal sIgA– B cells from BALB/c mice were cultured with LPS (12.5 µg/ml), IL-21 (5 ng/ml), and TGF-β1 (0.2 ng/ml). (A) After 7 days of culture, supernatants were collected, and Ig production was determined by isotype-specific ELISA. Data are means of triplicate samples ± SEM. (B) After 2 days of culture, total RNA was isolated, and levels of endogenous GLT{alpha} and GLT{gamma}2b were measured by RT-PCR. (C) Effects of IL-21 and TGF-β1 on Ig GL promoter activity. CH12F3.2A B lymphoma cells were transfected with 15 µg GL{alpha}-Luc or GL{gamma}2b-Luc reporters. TGF-β1 (1 ng/ml) and IL-21 (5 ng/ml) were added, and luciferase activity was determined 16 h later. Transfection efficiency was normalized to β-galactosidase activities. Data represent the average luciferase activities from three independent transfections with SEM (bars). *, P < 0.05; **, P < 0.01. RLA, relative luciferase activity.

Significance of the inhibitory activity of IL-21 in mouse PP
IgA is the major Ig isotype produced in the gastrointestinal tract, and PP is the most important mucosa-associated lymphoid tissue, where IgA B cells are committed [36 ]. Although TGF-β1 is considered a physiological mediator that causes IgA CSR [37 , 38 ], it also influences IgG2b CSR, which does not occur in PP. We hypothesize that IL-21 may contribute to IgA CSR in PP. To address this possibility, we compared IL-21 expression between cells derived from PP and spleen. We found that IL-21 transcript levels were much higher in PP than in spleen (Fig. 6A ). Subsequent FACS analysis showed that IL-21 expression by CD4+-gated T cells isolated from PP is also higher than in spleen cells (Fig. 6B) . Therefore, our results suggest that high levels of IL-21 derived from CD4+ T cells may play a key role in the dominant commitment of IgA B cells in PP.


Figure 6
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  		Figure 6. IL-21 is strongly expressed in PP. (A) Transcriptional levels of IL-21 in PP and spleen (SP) were measured by RT-PCR. (B) Intracellular staining of IL-21 with CD4+-gated T cells isolated from PP and spleen. Dotted line indicates isotype control. (C) Proposed mechanisms underlying TGF-β1/IL-21-regulated IgA and IgG2b isotype expression in PP.

DISCUSSION

In the present study, we demonstrate that IL-21 inhibits TGF-β1-induced IgG2b expression and has no effect on TGF-β1-induced IgA expression. Consequently, the IgA isotype is produced selectively under the influence of TGF-β1 and IL-21. These findings may provide insights into why PP predominantly generate IgA+ B cells. Nearly four decades have passed since PP were acknowledged as the main site for generating IgA+ B cells [36 ]. However, despite the enormous amount of knowledge that has been accumulated over the years, researchers continue to ask the basic question: What is the mechanism of IgA class-switching in PP? It is generally accepted that TGF-β1 is the most potent inducer of IgA CSR in mouse and human [33 , 39 ], although the cell type that supplies TGF-β1 to B cells has not been identified definitely. TH, T regulatory cells, TH3, and macrophages have been proposed to be candidates in the context of IgA B cell commitment [40 41 42 43 ]. However, even if the physiological sources of TGF-β1 are identified, another important issue still remains. IgG2b+ B cells do not appear in PP, despite the fact that TGF-β1 induces IgA CSR as well as IgG2b CSR [10 , 33 ]. Our observations that IL-21 reduces IgG2b expression provide an important clue as to why IgA+ B cells prevail solely in PP.

In the present study, we found that IL-21 actually inhibits GLT{gamma}2b transcription, which is paralleled by a decrease of IgG2b secretion. This is a novel finding concerning the effect of IL-21 on Ig expression. It has been shown previously that IL-21 down-regulates IgE production from IL-4-stimulated B cells by inhibiting GLT{epsilon} expression [21 ]. We also found that IL-21 decreases GLT{epsilon} expression. Therefore, IL-21 can inhibit expression of at least two different GLTs, indicating that the inhibitory activity of IL-21 on GLT{gamma}2b expression is not specific. We note that the present study does not address the specific mechanism by which IL-21 exerts its inhibitory activity. In this regard, there are reports suggesting the possibility that Stat molecules may mediate such inhibitory activity of IL-21. Thus, IL-21 activates Stat1, Stat3, and Stat5 in mouse splenic B cells [21 ]. Stat1 represses TGF-β-induced, Smad-mediated transcription by competing for a limited pool of the p300/CREB-binding protein coactivator [44 ]. In addition, we noticed that there are putative Stat-binding elements (atcctGGAA) within the GL{gamma}2b promoter region but not within the GL{alpha} promoter. Until now, specific factors that can inhibit GL{epsilon} promoter activity have not been discovered either. We are currently examining the possible involvement of Stat molecules in GL{gamma}2b and GL{epsilon} promoter activities.

IL-21 is a multifunctional cytokine that regulates the proliferation and differentiation of B cells [18 ]. We found that IL-21 exerts antiproliferative activity on LPS-activated B cells, similar to that of TGF-β1. We have demonstrated previously that TGF-β1 actually increases IgA isotype-switching at the clonal level [32 ]. At the same time, the antiproliferative activity of TGF-β1 also facilitated IgA CSR, leading to increased IgA production [32 ]. Therefore, although the mechanisms by which TGF-β1 and IL-21 act to alter cell proliferation are different, slowing of cell proliferation by IL-21 may influence TGF-β1-induced IgA expression through a kind of negative selection. This notion is supported by the fact that other isotypes such as IgM, IgG2a, and IgG2b are all decreased by IL-21 (Fig. 2) .

We found that PP CD4+ T cells express IL-21 more strongly than splenic CD4+ T cells. Thus, the next question would be which subpopulation of T cells provides IL-21 to B cells in PP. In this regard, it has been shown recently that NKT and TH17 cells are potent producers of IL-21 [11 , 13 , 14 ]. Based on the transcriptional levels of V{alpha}14J{alpha}281 (a marker of NKT), there was no difference in NKT cells in PP and spleen (data not shown). In contrast, the transcriptional levels of IL-17 (a marker of TH17) in PP were much higher than in spleen (data not shown). Therefore, it is plausible that IL-21 produced from TH17 is involved in selective IgA+ B cell commitment in PP. It remains to be determined whether TH17 cells actually secrete IL-21 in PP.

In conclusion, the mechanisms by which IL-21 contributes to TGF-β1-induced IgA expression are at least twofold: through antiproliferative activity and inhibitory activity on GLT{gamma}2b expression. Presently, we do not distinguish the relative importance of the two activities. In addition, as IL-21 has pleiotropic effects regarding the proliferation and differentiation of B cells, all should be considered to define its distinct role in IgA B cell differentiation. We raise here the possibility that IL-21 derived from CD4+ T cells may play a key role in IgA+ B cell commitment in PP. Possible pathways through which TGF-β1 and IL-21 regulate IgA+ B cell commitment in PP are proposed in Figure 6C . To prove this model definitely, further experiments are needed to show which cell types in PP are major sources of TGF-β and IL-21 in the context of IgA+ B cell commitment.

ACKNOWLEDGEMENTS

This work was supported by a Korea Research Foundation grant to P-H. K. (KRF-2007-313-C00566) and by the second stage of the Brain Korea 21 program. It was carried out in the facilities of the Vascular System Research Center and Institute of Bioscience and Biotechnology of Kangwon National University.

Received July 30, 2008; revised December 8, 2008; accepted January 5, 2009.

REFERENCES

    1
  1. Hanson, L. A. (1961) Comparative immunological studies of the immune globulins of human milk and of blood serum Int. Arch. Allergy Appl. Immunol. 18,241-267[Medline]
    2. 2
  2. Tomasi, T. B., Jr, Tan, E. M., Solomon, A., Prendergast, R. A. (1965) Characteristics of an immune system common to certain external secretions J. Exp. Med. 121,101-124[Abstract/Free Full Text]
    3. 3
  3. Van Egmond, M., Damen, C. A., van Spriel, A. B., Vidarsson, G., van Garderen, E., van de Winkel, J. G. (2001) IgA and the IgA Fc receptor Trends Immunol. 22,205-211[CrossRef][Medline]
    4. 4
  4. Brandtzaeg, P. (2007) Induction of secretory immunity and memory at mucosal surfaces Vaccine 25,5467-5484[CrossRef][Medline]
    5. 5
  5. Fagarasan, S., Honjo, T. (2003) Intestinal IgA synthesis: regulation of front-line body defences Nat. Rev. Immunol. 3,63-72[CrossRef][Medline]
    6. 6
  6. Stavnezer, J. (2000) Molecular processes that regulate class switching Curr. Top. Microbiol. Immunol. 245,127-168[Medline]
    7. 7
  7. Chaudhuri, J., Alt, F. W. (2004) Class-switch recombination: interplay of transcription, DNA deamination and DNA repair Nat. Rev. Immunol. 4,541-552[CrossRef][Medline]
    8. 8
  8. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., Honjo, T. (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme Cell 102,553-563[CrossRef][Medline]
    9. 9
  9. Lebman, D. A., Lee, F. D., Coffman, R. L. (1990) Mechanism for transforming growth factor β and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures J. Immunol. 144,952-959[Abstract]
    10. 10
  10. McIntyre, T. M., Klinman, D. R., Rothman, P., Lugo, M., Dasch, J. R., Mond, J. J., Snapper, C. M. (1993) Transforming growth factor β 1 selectivity stimulates immunoglobulin G2b secretion by lipopolysaccharide-activated murine B cells J. Exp. Med. 177,1031-1037[Abstract/Free Full Text]
    11. 11
  11. Coquet, J. M., Kyparissoudis, K., Pellicci, D. G., Besra, G., Berzins, S. P., Smyth, M. J., Godfrey, D. I. (2007) IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production J. Immunol. 178,2827-2834[Abstract/Free Full Text]
    12. 12
  12. Parrish-Novak, J., Dillon, S. R., Nelson, A., Hammond, A., Sprecher, C., Gross, J. A., Johnston, J., Madden, K., Xu, W., West, J., Schrader, S., Burkhead, S., Heipel, M., Brandt, C., Kuijper, J. L., Kramer, J., Conklin, D., Presnell, S. R., Berry, J., Shiota, F., Bort, S., Hambly, K., Mudri, S., Clegg, C., Moore, M., Grant, F. J., Lofton-Day, C., Gilbert, T., Rayond, F., Ching, A., Yao, L., Smith, D., Webster, P., Whitmore, T., Maurer, M., Kaushansky, K., Holly, R. D., Foster, D. (2000) Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function Nature 408,57-63[CrossRef][Medline]
    13. 13
  13. Nurieva, R., Yang, X. O., Martinez, G., Zhang, Y., Panopoulos, A. D., Ma, L., Schluns, K., Tian, Q., Watowich, S. S., Jetten, A. M., Dong, C. (2007) Essential autocrine regulation by IL-21 in the generation of inflammatory T cells Nature 448,480-483[CrossRef][Medline]
    14. 14
  14. Korn, T., Bettelli, E., Gao, W., Awasthi, A., Jager, A., Strom, T. B., Oukka, M., Kuchroo, V. K. (2007) IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells Nature 448,484-487[CrossRef][Medline]
    15. 15
  15. Ozaki, K., Kikly, K., Michalovich, D., Young, P. R., Leonard, W. J. (2000) Cloning of a type I cytokine receptor most related to the IL-2 receptor β chain Proc. Natl. Acad. Sci. USA 97,11439-11444[Abstract/Free Full Text]
    16. 16
  16. Jin, H., Carrio, R., Yu, A., Malek, T. R. (2004) Distinct activation signals determine whether IL-21 induces B cell costimulation, growth arrest, or Bim-dependent apoptosis J. Immunol. 173,657-665[Abstract/Free Full Text]
    17. 17
  17. Brandt, K., Bulfone-Paus, S., Foster, D. C., Ruckert, R. (2003) Interleukin-21 inhibits dendritic cell activation and maturation Blood 102,4090-4098[Abstract/Free Full Text]
    18. 18
  18. Spolski, R., Leonard, W. J. (2008) Interleukin-21: basic biology and implications for cancer and autoimmunity Annu. Rev. Immunol. 26,57-79[CrossRef][Medline]
    19. 19
  19. Ozaki, K., Spolski, R., Ettinger, R., Kim, H. P., Wang, G., Qi, C. F., Hwu, P., Shaffer, D. J., Akilesh, S., Roopenian, D. C., Morse, H. C., III, Lipsky, P. E., Leonard, W. J. (2004) Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6 J. Immunol. 173,5361-5371[Abstract/Free Full Text]
    20. 20
  20. Ettinger, R., Sims, G. P., Fairhurst, A. M., Robbins, R., da Silva, Y. S., Spolski, R., Leonard, W. J., Lipsky, P. E. (2005) IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells J. Immunol. 175,7867-7879[Abstract/Free Full Text]
    21. 21
  21. Suto, A., Nakajima, H., Hirose, K., Suzuki, K., Kagami, S., Seto, Y., Hoshimoto, A., Saito, Y., Foster, D. C., Iwamoto, I. (2002) Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line C({epsilon}) transcription of IL-4-stimulated B cells Blood 100,4565-4573[Abstract/Free Full Text]
    22. 22
  22. Ozaki, K., Spolski, R., Feng, C. G., Qi, C. F., Cheng, J., Sher, A., Morse, H. C., III, Liu, C., Schwartzberg, P. L., Leonard, W. J. (2002) A critical role for IL-21 in regulating immunoglobulin production Science 298,1630-1634[Abstract/Free Full Text]
    23. 23
  23. Wood, N., Bourque, K., Donaldson, D. D., Collins, M., Vercelli, D., Goldman, S. J., Kasaian, M. T. (2004) IL-21 effects on human IgE production in response to IL-4 or IL-13 Cell. Immunol. 231,133-145[CrossRef][Medline]
    24. 24
  24. Pene, J., Gauchat, J. F., Lecart, S., Drouet, E., Guglielmi, P., Boulay, V., Delwail, A., Foster, D., Lecron, J. C., Yssel, H. (2004) Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells J. Immunol. 172,5154-5157[Abstract/Free Full Text]
    25. 25
  25. Kim, P. H., Kagnoff, M. F. (1990) Transforming growth factor-β 1 is a costimulator for IgA production J. Immunol. 144,3411-3416[Abstract]
    26. 26
  26. Frangakis, M. V., Koopman, W. J., Kiyono, H., Michalek, S. M., McGhee, J. R. (1982) An enzymatic method for preparation of dissociated murine Peyer’s patch cells enriched for macrophages J. Immunol. Methods 48,33-44[CrossRef][Medline]
    27. 27
  27. Yasui, H., Mike, A., Ohwaki, M. (1989) Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer’s patches after oral administration J. Dairy Sci. 72,30-35[Medline]
    28. 28
  28. Park, S. R., Lee, J. H., Kim, P. H. (2001) Smad3 and Smad4 mediate transforming growth factor-β1-induced IgA expression in murine B lymphocytes Eur. J. Immunol. 31,1706-1715[CrossRef][Medline]
    29. 29
  29. Park, S. R., Seo, G. Y., Choi, A. J., Stavnezer, J., Kim, P. H. (2005) Analysis of transforming growth factor-β1-induced Ig germ-line {gamma}2b transcription and its implication for IgA isotype switching Eur. J. Immunol. 35,946-956[CrossRef][Medline]
    30. 30
  30. Shi, M. J., Stavnezer, J. (1998) CBF {alpha}3 (AML2) is induced by TGF-β1 to bind and activate the mouse germline Ig {alpha} promoter J. Immunol. 161,6751-6760[Abstract/Free Full Text]
    31. 31
  31. Nakamura, M., Kondo, S., Sugai, M., Nazarea, M., Imamura, S., Honjo, T. (1996) High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells Int. Immunol. 8,193-201[Abstract/Free Full Text]
    32. 32
  32. Kim, P. H., Kagnoff, M. F. (1990) Transforming growth factor β 1 increases IgA isotype switching at the clonal level J. Immunol. 145,3773-3778[Abstract]
    33. 33
  33. Coffman, R. L., Lebman, D. A., Shrader, B. (1989) Transforming growth factor β specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes J. Exp. Med. 170,1039-1044[Abstract/Free Full Text]
    34. 34
  34. Li, S. C., Rothman, P. B., Zhang, J., Chan, C., Hirsh, D., Alt, F. W. (1994) Expression of I µ-C {gamma} hybrid germline transcripts subsequent to immunoglobulin heavy chain class switching Int. Immunol. 6,491-497[Abstract/Free Full Text]
    35. 35
  35. Kinoshita, K., Harigai, M., Fagarasan, S., Muramatsu, M., Honjo, T. (2001) A hallmark of active class switch recombination: transcripts directed by I promoters on looped-out circular DNAs Proc. Natl. Acad. Sci. USA 98,12620-12623[Abstract/Free Full Text]
    36. 36
  36. Craig, S. W., Cebra, J. J. (1971) Peyer’s patches: an enriched source of precursors for IgA-producing immunocytes in the rabbit J. Exp. Med. 134,188-200[Abstract]
    37. 37
  37. Cazac, B. B., Roes, J. (2000) TGF-β receptor controls B cell responsiveness and induction of IgA in vivo Immunity 13,443-451[CrossRef][Medline]
    38. 38
  38. Van Ginkel, F. W., Wahl, S. M., Kearney, J. F., Kweon, M. N., Fujihashi, K., Burrows, P. D., Kiyono, H., McGhee, J. R. (1999) Partial IgA-deficiency with increased Th2-type cytokines in TGF-β 1 knockout mice J. Immunol. 163,1951-1957[Abstract/Free Full Text]
    39. 39
  39. Van Vlasselaer, P., Punnonen, J., de Vries, J. E. (1992) Transforming growth factor-β directs IgA switching in human B cells J. Immunol. 148,2062-2067[Abstract]
    40. 40
  40. Assoian, R. K., Fleurdelys, B. E., Stevenson, H. C., Miller, P. J., Madtes, D. K., Raines, E. W., Ross, R., Sporn, M. B. (1987) Expression and secretion of type β transforming growth factor by activated human macrophages Proc. Natl. Acad. Sci. USA 84,6020-6024[Abstract/Free Full Text]
    41. 41
  41. Kehrl, J. H., Wakefield, L. M., Roberts, A. B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M. B., Fauci, A. S. (1986) Production of transforming growth factor β by human T lymphocytes and its potential role in the regulation of T cell growth J. Exp. Med. 163,1037-1050[Abstract/Free Full Text]
    42. 42
  42. Chen, Y., Kuchroo, V. K., Inobe, J., Hafler, D. A., Weiner, H. L. (1994) Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis Science 265,1237-1240[Abstract/Free Full Text]
    43. 43
  43. Nakamura, K., Kitani, A., Strober, W. (2001) Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor β J. Exp. Med. 194,629-644[Abstract/Free Full Text]
    44. 44
  44. Ghosh, A. K., Yuan, W., Mori, Y., Chen, S., Varga, J. (2001) Antagonistic regulation of type I collagen gene expression by interferon-{gamma} and transforming growth factor-β. Integration at the level of p300/CBP transcriptional coactivators J. Biol. Chem. 276,11041-11048[Abstract/Free Full Text]




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