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Originally published online as doi:10.1189/jlb.0206096 on August 30, 2006

Published online before print August 30, 2006
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(Journal of Leukocyte Biology. 2006;80:1416-1423.)
© 2006 by Society for Leukocyte Biology

Redundant and unique regulation of activated mouse B lymphocytes by IL-4 and IL-21

Haoli Jin1 and Thomas R. Malek2

Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, Florida, USA

2 Correspondence: Department of Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA. E-mail: tmalek{at}med.miami.edu


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ABSTRACT
 
IL-21 distinctively regulates B cell growth and death, and it redundantly functions with IL-4 for IgG production. B cells likely encounter IL-4 and IL-21 in vivo, as both are secreted by activated T cells. Therefore, the action of both these cytokines was investigated during activation of B cells. IL-21 or the combination of IL-4 and IL-21 inhibited proliferation by purified mouse B cells to LPS or CpG DNA, whereas these cytokines enhanced proliferation after engaging the BCR or CD40. Although B cell subsets expressed somewhat varied levels of the IL-21 receptor, LPS-stimulated follicular and marginal B cell subsets were also dominantly susceptible to IL-21-induced growth arrest and cell death. After activation of B cells with CD40 and LPS, IL-4 and IL-21 distinctively regulated the expression of CD23, CD44, and CD138, and they cooperatively promoted IgG1 class-switching and synthesis. These findings support a model in which the presence of IL-4 and IL-21 inhibits B cells activated by polyclonal innate signals, and they promote B cell expansion and differentiation during T cell-dependent antibody responses, although the individual responses to IL-4 and IL-21 do not always overlap.

Key Words: apoptosis • costimulation


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INTRODUCTION
 
IL-21 is the newest {gamma}c-dependent cytokine family member that is produced by activated CD4+ T cells [1 2 3 ]. The IL-21 receptor (IL-21R) is readily detected on mature T and B cells with higher levels on activated T, B, and NK cells [4 ]. Correspondingly, IL-21 regulates the function of T, B, and NK cells in vitro [1 2 3 ]. IL-21R knockout mice exhibit normal lymphocyte development and are otherwise outwardly normal [5 , 6 ]. However, upon immunization of IL-21R–/– mice, the degree of CD8+ T cell expansion is reduced, and a lower level of IgG1 and a higher amount of IgE are produced when compared with normal mice [6 , 7 ]. IL-21R/IL-4 double-deficient mice are essentially unable to produce IgG and IgE antibodies, indicting that these two cytokines redundantly function for Ig production [6 ]. The overlapping biological effect of these two cytokines is not surprising, as the gene loci for IL-21R and IL-4R receptors are tightly linked, and these cytokines and their receptors share common features in the gene and protein structures [8 ]. However, the action of IL-21 and IL-4 is not entirely overlapping, as IgE production requires IL-4, and IL-21 down-regulates IgE [9 10 11 ].

In concordance with the importance of IL-21 for antibody responses in vivo, the highest level of IL-21R is detected on activated B lymphocytes, and it is easy to demonstrate IL-21-regulated B cell responses in vitro. However, the role of IL-21 for B cells is complex, as it induces proapoptotic or costimulatory responses [4 , 12 ]. IL-21 induces growth arrest and Bim-dependent apoptosis for B cells activated with innate signals such as LPS or CpG DNA, whereas costimulation and prosurvival are dominant for B cells stimulated through BCR and Th signals [4 ]. Thus, the outcome of IL-21-dependent signaling for B cells appears highly context-dependent. Conversely, IL-4 is typically considered as a costimulatory cytokine with a prosurvival function.

Follicular (FO) and marginal zone (MZ) B cells are anatomically, phenotypically, and functionally distinct subsets of mature B cells. MZ B cells typically provide the immediate immune defense against blood-borne pathogens, whereas FO B cells generate antigen-specific, T cell-dependent, high-affinity antibody responses [13 ]. The distinctive responsiveness to antigen and costimulatory signals by FO and MZ B cells raises the question of whether these B cell subsets are similarly regulated by IL-21. Furthermore, during a T cell-dependent antibody response, B cells are expected to receive signals from multiple cytokines secreted by Th cells, and two of these must be IL-4 and IL-21 by virtue of their redundant function for Ig production in vivo. The net effect, therefore, of the combined action of IL-4 and IL-21 on B cell responses is especially important, as these cytokines generate overlapping but not identical responses in vitro and in vivo. With these issues in mind, in this report, we compared the effects of IL-4 versus IL-21, including the combined action of these cytokines, on responses of mouse B cells in vitro and investigated the expression and function of the IL-21R on FO and MZ B cells.


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MATERIALS AND METHODS
 
Mice
C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and used at 2–4 months of age, according to the guidelines established by the Animal Care Committee at the University of Miami (FL).

Cell purification and culture
CD19+ splenic B cells were purified as described previously [4 ]. To purify FO and MZ B cells, positively selected CD19+ B cells were stained with fluorescent-conjugated antibodies to CD21, CD23, and B220. These cells were washed twice with HBSS followed by high-speed sorting for B220+ CD21int CD23+ and B220+ CD21hi CD23– cells representing FO and MZ B cells, respectively, using a Becton Dickinson Vantage SE sorter and CellQuest software (Becton Dickinson, Mountain View, CA).

Purified B cells were cultured in complete RPMI-1640 medium containing 5% FCS, glutamine (30 µg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2-ME (5x10–5 M) as described [14 ]. Cells were placed in 96-well, flat-bottom plates (1x105/well) for [3H]-thymidine incorporation and apoptosis assays or in 24-well, flat-bottom plates (1x106/well) for FACS analyses. These cells were stimulated with LPS (1 µg/ml; Escherichia coli 026:B6, Sigma Chemical Co., St. Louis, MO), anti-CD40 mAb (HM40-3, 1 µg/ml, BD PharMingen, San Diego, CA), mouse soluble CD40 ligand (sCD40L; 0.5-1 µg/ml, Peprotech Inc., Rocky Hill, NJ), the F(ab)'2 fragment of the anti-µ mAb (1 µg/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA), or CpG-containing nuclease-resistant phosphorothioate oligodeoxynucleotides 1826 (CpG ODN-1826; 0.3 µM; 5'-CCATGACGTTCCTGACGTT-3') in the absence or presence of mouse IL-21 (30 ng/ml, R&D Systems, Minneapolis, MN) and/or IL-4 (1ng/ml, Peprotech, Rocky Hill, NJ). At the indicated time, cell proliferation was measured by [3H] thymidine incorporation as described previously [14 ].

Flow cytometry
FITC-conjugated mAb to CD23 (B3B4) and CD44 (Pgp-1), PE-conjugated mAb to CD138 (281-2), PE-streptavidin, biotinylated mAb to mouse IgG1 (A85-1), and 7-amino-actinomycin D (7AAD) were purchased from BD PharMingen. A rat antimouse IL-21R mAb (4A9) was prepared by our laboratory [4 ]. Surface IgG1 or IL-21R expression was assessed by a two-step staining protocol that consisted of incubation with biotinlyated anti-IgG1 or biotinlyated 4A9 followed by PE-streptavidin. As a control, this staining was blocked specifically by preincubation of the cells with unlabeled antimouse IgG1 or IL-21R. Multicolor FACS analysis for cell-surface marker expression was determined as described previously [15 ] using a Becton Dickinson LSR analyzer and CellQuest software. Typically, 100,000 viable cells were analyzed based on forward- versus side-scatter gating. Cell death was determined using propidium iodine (Sigma Chemical Co.) followed by FACS analysis as described previously [4 ].

IgG1 ELISA
Enhanced protein-binding ELISA plates (Nalge Nunc International, Rochester, NY) were coated with purified antimouse IgG1 (A85-3, 1 µg/ml, BD PharMingen) overnight, blocked with 10% FCS for 30 min, incubated with a serial dilution of purified IgG1 or culture supernatants for 1 h, washed with PBS containing 0.05% Tween-20, and incubated with HRP anti-IgG1 (x56, 1:5000 dilution, BD PharMingen) for 30 min. After washing, color was developed by incubation with 3-ethylbenzthiazoline-6-sulfonic acid (Sigma Chemical Co.), the OD at 414 nm was determined, and the IgG1 levels were calculated by comparison with a standard curve.


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RESULTS
 
Relationship between IL-4 and IL-21 for survival and proliferation of activated B cells
The failed production of IgG by IL-4/IL-21R double-knockout mice demonstrates that these two cytokines play a critical overlapping role in humoral immune responses [6 ]. However, the distinct regulation of IgE by IL-4 and IL-21 illustrates that these two cytokines also exhibit a unique capacity to regulate a B cell response. To determine whether the direct action of IL-4 and IL-21 on B cells leads to distinct functional responses, we first investigated the survival and proliferation of CD19+-purified B cells from C57BL/6 mice after their activation in the presence or absence of these two cytokines (Fig. 1 ). As previously shown [4 ], IL-21 substantially inhibited the proliferation and increased cell death for B cells stimulated with LPS or CpG DNA. IL-4 did not substantially affect LPS-stimulated B proliferation or death but somewhat decreased proliferation to CpG without significantly affecting cell death (Fig. 1A and 1B) . In cocultures containing IL-21 and IL-4 and LPS or CpG, the proliferative and apoptotic responses resembled that induced by IL-21 alone.


Figure 1
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  		Figure 1. IL-21 and IL-4 suppress B cell response to LPS or CpG DNA but primarily promote B cell response to anti-CD40 or anti-IgM. Purified CD19+ B cells were stimulated by LPS (A), CpG DNA (B), anti-CD40 (C), sCD40L (D), or anti-IgM (E) in the presence or absence of IL-21 and/or IL-4. Proliferation and cell death were assayed 48 h later. Data shown are derived from the mean ± SD of three to four separate experiments. *, P < 0.05, nonpaired Student’s t-test when comparing cultures containing IL-4 and/or IL-21 versus those cultures lacking these cytokines.

Although the results shown are limited to analysis at a single time-point, previous time course and dose response experiments indicate that these are the optimal culture conditions to evaluate proliferation and apoptosis of purified mouse B cells [4 ]. Furthermore, IL-21 and/or IL-4 similarly inhibited proliferation (Fig. 2A ) and cell death (Fig. 2B) over a range of concentrations of LPS or CpG. Thus, the inhibition of proliferation and induction of apoptosis were not a result of overstimulation. Dose-response studies indicated that IL-21 dominantly inhibited LPS-induced proliferation over a large range of IL-4 levels (Fig. 3A ). Therefore, IL-21 is much more effective than IL-4 to down-regulate B cell responses to innate signals such as LPS and CpG DNA, and this inhibitory effect is the dominant response in the presence of both cytokines.


Figure 2
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  		Figure 2. Sensitivity of TLR-mediated B cell activation to inhibition by IL-4 and IL-21. Purified B cells were cultured with various concentrations of LPS or CpG and IL-4 and/or IL-21 as described in Figure 1 . Proliferation (A) or cell death (B) was assayed 48 h after culture initiation.


Figure 3
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  		Figure 3. Dose-response relationship of IL-4 and IL-21 regulation of B cell proliferation. Purified CD19+ B cells were stimulated by LPS (A) or anti-CD40 (B) in the presence of the indicated concentration of cytokines. Proliferation was assessed 48 h later. Data are representative of three experiments.

For B cells that were engaged through the BCR using anti-IgM or CD40 using sCD40L, IL-21 slightly enhanced these proliferative responses, and IL-4 exhibited a much greater costimulatory effect (Fig. 1C 1D 1E ; ref. [4 ]), although the magnitude of IL-4-dependent costimulation varied between these groups. In the presence of IL-4 and IL-21 at 48 h, although proliferation in response to anti-CD40 or anti-IgM was enhanced, these responses were lower than that in the presence of IL-4 alone, whereas both cytokines induced a slightly higher response in the presence of CD40L. Dose-response experiments indicated that costimulation of anti-CD40 B cell proliferation was dependent on the concentration of IL-4 and that IL-21 proportionally inhibited this response (Fig. 3B) . As for cell survival, IL-21 or IL-21 plus IL-4 significantly induced cell death for anti-CD40- or sCD40L-stimulated B cells (Fig. 1C and 1D) . This pattern of B cell apoptosis in the presence of these cytokines resembles that seen for LPS- and CpG-activated B cells (Fig. 1A and 1B) . By contrast, IL-21 and/or IL-4 enhanced the survival of anti-IgM-stimulated B cells (Fig. 1E) , which may reflect the high B cell death in the presence of only anti-IgM. Nevertheless, IL-4 alone or IL-4 and IL-21 together generate signals that costimulate B cell proliferation after activation through BCR or CD40.

IL-21R expression on B cell subsets
The above functional experiments were confined to test the effects of IL-21 on unfractionated, purified B cells. We next wished to examine IL-21R expression and function on peripheral B cell subsets. With respect to B-lineage cells, the IL-21R is first detected at a low level on pre-B cells. Upon maturation, essentially all peripheral B cells express relatively high levels of IL-21R, which are increased after B cell activation [4 ]. The earliest developing B cells in the periphery are IgMhigh IgD– cells and are designated as transitional 1 (T1) B cells. These B lymphocytes expressed a low level of IL-21R (Fig. 4A ). However, upon maturation to T2 (IgMhigh IgDhigh) B cells, IL-21R expression increased to levels comparable with that detected on mature B cells from the bone marrow (data not shown) and FO B cells in the spleen (Fig. 4A) . Although using antibodies specific for surface IgM and IgD did not distinguish some T1 cells from MZ B cells, FO B cells expressed a higher level of the IL-21R than the population comprising T1 and MZ B cells (Fig. 4A) .


Figure 4
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  		Figure 4. IL-21R expression on splenic B cell subsets. Multicolor FACS staining for IL-21R expression on unfractionated C57BL/6 spleen (A) or purified splenic CD19+ B cells (B). Specific (thick lines) and control (thin lines) staining of IL-21R is shown in the histograms. (A) The gating for immature, transitional and mature B cell subsets was based on IgD/IgM expression and is indicated on the dot plots. (B) The gating for B cell subsets was based on CD23/CD21 expression and is indicated on the dot plots. The level of IL-21R was determined by subtracting the mean fluorescent intensity (MFI) for control staining of each sample from the MFI for 4A9 staining. Data shown are derived from the mean ± SD of three to four independent experiments.

To further compare IL-21R expression by FO and MZ B cells, splenic CD19+ B cells were purified and stained for expression of CD23 and CD21 to identify T1, T2, FO, and MZ B cells (Fig. 4B) . In accordance with the previous result, on average, there was approximately a twofold lower MFI that corresponds to a lower level of expression for IL-21R on T1 and MZ B cells when compared with T2 and FO B cells (Fig. 4B) , although there is some overlap in IL-21R expression by cells within all these subsets. Nevertheless, these data demonstrate that the IL-21R is regulated during peripheral B cell development with distinctive levels on mature B cell subsets.

IL-21 and IL-4 responsiveness by FO and MZ B cells
As FO and MZ B cells are subsets of B cells with distinct functional capabilities, we examined the whether IL-21 similarly regulated their growth and death in the context of LPS or anti-CD40 activation. FO and MZ B cells were purified by cell sorting from the spleen of C57BL/6 mice, such that both populations were typically 98% pure (data not shown). As expected, MZ B cells exhibited relatively higher proliferative responses to LPS or anti-CD40 than FO B cells. However, cell death for LPS or anti-CD40-stimulated MZ B cells was somewhat higher than that for LPS or anti-CD40-stimulated FO B cells (Fig. 5A and 5B ). Nevertheless, the proliferation of LPS-stimulated FO and MZ B cells was inhibited substantially by IL-21, which was accompanied by enhanced apoptosis (Fig. 5 A). In contrast, IL-21 did not costimulate the proliferation by MZ B cells to anti-CD40, whereas this cytokine costimulated proliferation by total CD19+ or FO B cells to anti-CD40 (Fig. 5B) . During costimulation with anti-CD40, IL-21 slightly increased the percentage of dead cells for all cell populations (Fig. 5B) . Thus, with respect to stimulation by LPS, FO and MZ B cells were largely regulated in a similar manner by IL-21, whereas MZ B cells appeared refractory to IL-21 costimulation of the anti-CD40-proliferative response.


Figure 5
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  		Figure 5. FO and MZ B cell response to IL-21. FO and MZ B cells were purified by a positive selection for CD19+ splenocytes, followed by high-speed sorting for B220+ CD21int CD23+ and B220+ CD21hi CD23– cells. Purified CD19+, FO, and MZ B cells were stimulated with LPS (A) or anti-CD40 (B) in the presence or absence of IL-21. Proliferation and cell death were assessed 48 h later. Data shown are derived from one representative experiment of three.

Relationship between IL-4 and IL-21 in regulating B cell maturation and Ig production
B cell maturation and cytokine-dependent Ig production are commonly assessed in vitro by the coculture of purified B lymphocytes with anti-CD40, LPS, and the cytokine(s) of interest. This mode of B cell stimulation was used to further evaluate the relationship of IL-4 and IL-21 during B cell activation. Purified splenic B cells were cultured, as IL-4 and IL-21 similarly regulated the proliferation of B cell subsets. For B cells activated by LPS and CD40, IL-21 did not inhibit proliferation (Fig. 6A ), which markedly contrasts with B cells solely activated by LPS (Fig. 1A) . It is interesting that in this mode of B cell stimulation, IL-4 dominantly blocked this proliferative response by ~50% (Fig. 6A) . This inhibition by IL-4 was noted each day of a time course experiment, indicating that this block was not simply a result of overstimulation of the B cells (Fig. 6B) . Thus, in the presence of LPS and anti-CD40, IL-21 and IL-4 differentially regulated B cell proliferation.


Figure 6
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  		Figure 6. IL-4 inhibits B cell proliferation by anti-CD40 plus LPS. Purified CD19+ B cells were stimulated by anti-CD40 and LPS in the presence or absence of IL-21 and/or IL-4. (A) Proliferation was assessed 48 h later, and data shown were derived from the mean ± SD of three to four separate experiments. *, P < 0.05, nonpaired Student’s t-test when comparing cultures containing cytokines and cultures containing no cytokine. (B) Time course analysis by assessing proliferation daily for 3 days.

During an extended culture period with LPS and anti-CD40, the effect of IL-4 and IL-21 on the expression of several surface markers, which are known to be regulated during activated B cell differentiation, was examined (Fig. 7 and ref. [16 ]). For the cell-surface molecules examined, the expression of CD23 and CD44 was differentially regulated by IL-21 and IL-4 (Fig. 7A) , whereas the expression of IgD, IgM, CD19, CD21, B220, and MHC Class II was not changed significantly (data not shown). CD23 and CD44 expression on activated B cells was consistently reduced in the presence of IL-21 but enhanced with IL-4 during the course of observation (Fig. 7A and 7B) . In the presence of both cytokines, the expression of CD23 and CD44 was antagonized when compared with cells only stimulated with IL-4. With the exception for IL-21 at 24 h when more dead cells were seen, IL-21 or IL-4 did not significantly change the survival rate during the 6-day culture (Fig. 7B) . This transient effect on IL-21-induced cell death probably reflects the subsequent growth of the surviving B cells. Nevertheless, the reduced expression of CD23 and CD44 on B cells was not the consequence of IL-21-induced apoptosis, as this result was also seen when using B cells from Bcl-2 transgenic mice, which were shown previously to resist IL-21-induced cell death (data not shown and ref [4 ]).


Figure 7
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  		Figure 7. IL-21 and IL-4 exert opposing effects on CD23 and CD44 expression. Purified B cells from C57BL/6 mice were stimulated with LPS and anti-CD40 in the presence or absence of IL-21 and/or IL-4. Cells were then assessed for the expression of CD23 and CD44 by FACS analysis on viable 7AAD– cells. (A) Expression of CD23 and CD44 at 48 h was shown in the representative histograms. (B) Expression levels of CD23 and CD44 on 7AAD–-cultured B cells during a 6-day culture are shown as the MFI. Data are the mean ± SD of three to six experiments.

CD138, also designated Sydecan-1, is a glycoprotein whose expression is induced as activated B cells differentiate into plasma cells [16 ]. The expression of CD138 was induced by LPS and anti-CD40 at early time-points (Days 1 and 2) on a small fraction of B cells but declined thereafter (Fig. 8B ). During the first 4 days in culture, IL-4 and IL-21 did not affect CD138 expression (Fig. 8B) . However, after 5 days in culture, in the presence of IL-21, there was a twofold increase in the proportion of B cells that expressed CD138 (Fig. 8A) , and these cells also expressed a higher level of CD138 (Fig. 8B) . Although IL-4 did not affect CD138 expression, coculture of B cells with LPS, CD40, IL-4, and IL-21 resulted in fewer B cells that expressed CD138 at a lower level when compared with cultures only containing IL-21. Thus, IL-21 induced a high level of CD138 on some activated B cells at a late phase of the response, which was antagonized by IL-4.


Figure 8
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  		Figure 8. IL-21 and IL-4 regulate the expression of CD138. Purified B cells were stimulated with LPS and anti-CD40 in the presence or absence of IL-21and/or IL-4. The expression of CD138 was assessed by FACS analysis on viable 7AAD– cells. (A) The percentages of CD138-positive cells at Day 5 culture in one of three independent experiments were shown in the representative histograms. (B) The line graph shows the total CD138 MFI from all viable 7AAD– B cells at indicated time-points and represents the mean ± SD of three to six independent experiments.

IgG1 is a dominant IgG isotype in secondary antibody responses, and class-switching to this isotype depends on IL-4 [17 ]. Given the critical role for IL-4 and IL-21 in controlling Ig production in vivo [6 ], we also examined the relative contribution of these two cytokines for IgG1 production by LPS and anti-CD40-stimulated B cells in vitro (Fig. 9 ). IgG1-expressing B cells were not detected for freshly isolated B cells (data not shown). LPS and anti-CD40 stimulation induced minimal cell-surface IgG1 expression (Fig. 9A and 9B) . In contrast, after 5 days in culture, IL-21 and IL-4 increased the number of IgG1-expressing cells approximately three- and fivefold, respectively, when compared with cultures containing only LPS and anti-CD40 (Fig. 9A) . The combined effect of IL-21 and IL-4 led to approximately a ninefold increase in the number of IgG1-expressing cells. The surface level of IgG1 for the IgG1+ cells was lower for cells cultured with IL-21 than with IL-4 or IL-4 and IL-21 (Fig. 9B) . Nevertheless, the level of secreted IgG1 was similar in the presence of IL-4 or IL-21, and the highest levels of IgG1 secreted in cultures contained both cytokines (Fig. 9C) . Therefore, although IL-4 and IL-21 differentially affect the expression of several cell-surface markers and the proliferative response, both promote IgG1 production by activated B cells.


Figure 9
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  		Figure 9. IL-21 and IL-4 synergistically promote IgG1 class-switching and secretion. Purified B cells were stimulated by LPS and anti-CD40 in the presence or absence of IL-21 and/or IL-4. (A and B) The levels of surface IgG1 were assessed by FACS analysis on viable 7AAD– cells. (A) The percentages of IgG1-positive cells on Day 5 of culture from one of three independent experiments were shown in the representative histograms. (B) The line graph shows the total IgG1 MFI from all viable 7AAD– B cells at indicated time-points and was derived from the mean ± SD of three to six experiments. (C) The level of IgG1 secreted after 6 days in culture was measured by ELISA. The bar graph represents mean ± SD of four independent experiments.


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DISCUSSION
 
Examination of mice deficient in responsiveness to IL-4 and IL-21 clearly demonstrates an essential and redundant role of these cytokines for production of IgG [6 ]. This observation in conjunction with the finding that IL-4 and IL-21 are activated T cell cytokines [10 , 18 , 19 ] indicates that it is highly likely that during physiological T cell-dependent Ig production, activated B cells will encounter IL-4 and IL-21. However, based on their distinct capacity to regulate IgE secretion [6 , 11 ], it is also clear that all activities of IL-4 and IL-21 do not completely overlap. Therefore, we have placed some effort to investigate the combined effect of both these cytokines on B cell growth, death, and differentiation into IgG production cells. A model summarizing our main findings is shown in Figure 10 . With respect to regulation of B cell growth and death in the presence of IL-4 and IL-21, proliferation was inhibited, and apoptosis was favored for B cells stimulated with innate signals such as LPS or CpG DNA. As shown previously [4 ], inhibition of proliferation and apoptosis by IL-21 are two distinct events, as IL-21 inhibited B cell proliferation, although these cells were resistant to apoptosis as a result of lack of Bim or overexpression of Bcl-2. In contrast, the combined activity of IL-4 and IL-21 enhanced B cell proliferation after stimulation through CD40 or the BCR and increased cell survival after stimulation through the BCR.


Figure 10
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  		Figure 10. Summary of IL-21 and IL-4 control of B cell activation. The typical dominant effect of IL-21 or IL-4 is represented by the bold lines with arrow.

IL-4 has been shown to protect T and B cells from spontaneous and Fas-induced apoptosis [17 , 20 21 22 ]. This survival activity of IL-4 was not sufficient to interfere with the negative regulatory activity of IL-21 for B cells, nonspecifically activated by innate signals or ligation of CD40. Thus, in these cases, the inhibitory activity is unique to IL-21 and dominates even in the presence of IL-4. Conversely, for B cells stimulated solely through the BCR or CD40, it is IL-4 that primarily supported B cell proliferation. In the current study, where C57BL/6 mice were used exclusively, costimulation by IL-21 was marginal. However, when BALB/c B cells are cultured with anti-CD40 or anti-IgM, IL-21 costimulates much stronger proliferative responses [4 ]. Although the proliferation by B cells stimulated through IL-4 and BCR or CD40 decreased after adding IL-21, the net response remained higher than B cells stimulated through BCR or CD40 alone. These observations suggest that these cytokines promote T cell–dependent B cell responses, whereas they inhibit TLR-mediated response. However, the finding that IL-4 alone optimally inhibited B cell proliferation to anti-CD40 and LPS suggests a more complex relationship between these cytokines and regulation of activated B lymphocytes. One possibility is that this dominant response by IL-4 might reflect a mechanism to inhibit highly activated B cells. Alternatively, this block in proliferation might reflect a necessary step as B cell differentiate into antibody-producing cells, as cell-cycle arrest normally precedes B cell differentiation [16 ].

Further evaluation of cell-surface marker changes and IgG1 production in response to IL-4 and IL-21 supports this latter notion. Importantly, that we show that IL-4 and IL-21 function alone or together to stimulate LPS- and anti-CD40-activated B cells to undergo IgG1 class-switching and secretion, with optimal IgG1 levels in the presence of both cytokines. Thus, during this context of B cell activation, IL-4 and IL-21 function redundantly and dominantly to support maturation into IgG-secreting cells. However, during such B cell maturation, several surface markers were differentially regulated by IL-4 and IL-21. The present and previous reports showed that IL-4 increases the expression of CD44, which is a hyaluronan-binding protein that is important for lymphocyte migration, extravasation, and activation [23 24 25 ]. Memory T and B lymphocytes express high level of CD44 [24 , 26 ], and IL-4 has been implicated as a factor for memory B cell differentiation [16 , 24 ]. In comparison with IL-4, however, IL-21 slightly decreased CD44 expression, and it induced more effectively the expression of the plasma cell marker CD138. Moreover, IL-4 seems to induce generation of IgG1 "memory" cells, with high-surface IgG1, and IL-21 is required to finish differentiation into "plasma cells," as reflected by the low surface IgG1 expression (Fig. 9) . This finding agrees with the recent observation that IL-21 transgenic mice exhibited increased numbers of post-switch IgM–IgD– B cells, which were positive for IgG and CD138 [27 ], and IL-21 promotes differentiation of human plasma cells from naïve and memory B cells upon IgM and anti-CD40 cross-linking, which can be antagonized by IL-4 [28 ]. Taken together, we favor a model in which IL-21 dominantly functions to minimize the expansion and survival of B cells solely stimulated by innate signal such as LPS or CpG DNA. However, for those B cells that receive more complex activation by signaling through multiple pathways, including the BCR, Toll receptors and CD40, IL-4, and IL-21 promote their survival, expansion, and possibly subsequent cell-cycle arrest, leading to differentiation into IgG-secreting B cells. Although either cytokine leads to IgG-secreting cells, the distinctive regulation of surface proteins by IL-4 and IL-21 during B cell maturation suggests that their overall functional status is likely not equivalent.

The mature B2 cell population consists of FO and MZ B cells, which are distinguished by different expression level of several cell-surface markers [13 ]. MZ B cells express higher levels of activation markers including CD80, CD86, CD40, and CD44 and a lower level of CD62L [29 , 30 ]. In vitro, MZ B cells respond more rapidly and vigorously to stimulation with LPS, anti-IgM, and CD40L than FO B cells [29 , 30 ]. However, they are more sensitive to BCR-induced apoptosis [13 ]. In vivo, MZ cells also exhibit a rapid response to T-independent and T-dependent antigens [31 , 32 ]. The distinctive responsiveness to antigen and costimulatory signals by FO and MZ B cells led us to examine whether these B cell subsets are similarly regulated by IL-21. We found that the IL-21R is somewhat distinctively expressed on peripheral B cells, with higher expression on T2 and FO B cells than T1 and MZ B cells. One common property of FO and MZ B cells is that IL-21 negatively regulates their response to LPS, such that proliferation was inhibited markedly, and cell death was increased. This activity of IL-21 may limit the expansion of MZ and FO B cells to polyclonal activation by innate signals and may be important to prevent activation of self-reactive B cells. This negative mechanism may be especially critical for MZ B cells, as this B cell subset is enriched for self-reactive clones [13 ] and predominately functions to rapidly generate antibody responses to bacterial-derived LPS. It is likely that as a high-affinity T cell-dependent antibody response develops, Th2-derived IL-21 will suppress the immediate response by MZ cells.

CD23 (Fc{epsilon}RII) is the low-affinity IgE receptor that is often increased in cells from allergic patients [33 34 35 ]. CD23 is thought to mediate the internalization of the IgE-bound allergens, which augments antigen presentation and T cell responses, leading to higher IgE production [36 ]. We found that IL-21 decreased, and IL-4 increased the expression of CD23. This is consistent with the opposing function of these two cytokines in regulating IgE and airway hyper-reactivity [6 , 11 ]. Unfortunately, in the 6-day B cell culture system we used, we were not able to measure IgE production, possibly as a result of the limited amount of IgE in the cell culture. The down-regulation of CD23 by IL-21 occurs even after blocking IL-21-mediated cell death by using Bcl-2 transgenic B cells. The negative regulation of allergic response by IL-21 represents another means by which this cytokine down-regulates unwanted B cell responses. Therefore, IL-21 might prove to be generally useful to suppress allergic responses. The clinical relevance of IL-21 in other types of atopy,such as allergic dermatitis, rhinitis, and conjunctivitis, warrants further investigation.


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ACKNOWLEDGEMENTS
 
This research was supported by National Institutes of Health R01AI40114 and an American Heart Association predoctoral fellowship (H. J.). We thank Aixin Yu for technical assistance.


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FOOTNOTES
 
1 Current address: Division of Immunology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA. Back

Received February 13, 2006; revised June 28, 2006; accepted July 14, 2006.


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REFERENCES
 
    1
  1. Leonard, W. J., Spolski, R. (2005) Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation Nat. Rev. Immunol. 5,688-698[CrossRef][Medline]
    2. 2
  2. Mehta, D. S., Wurster, A. L., Grusby, M. J. (2004) Biology of IL-21 and the IL-21 receptor Immunol. Rev. 202,84-95[CrossRef][Medline]
    3. 3
  3. Collins, M., Whitters, M. J., Young, D. A. (2003) IL-21 and IL-21 receptor—a new cytokine pathway modulates innate and adaptive immunity Immunol. Res. 28,131-140[CrossRef][Medline]
    4. 4
  4. Jin, H. L., Carrio, R., Yu, A. X., 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]
    5. 5
  5. Kasaian, M. T., Whitters, M. J., Carter, L. L., Lowe, L. D., Jussif, J. M., Deng, B. J., Johnson, K. A., Witek, J. S., Senices, M., Konz, R. F., Wurster, A. L., Donaldson, D. D., Collins, M., Young, D. A., Grusby, M J. (2002) IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity Immunity 16,559-569[CrossRef][Medline]
    6. 6
  6. Ozaki, K., Spolski, R., Feng, C. G., Qi, C. F., Cheng, J., Sher, A., Morse, H. C., Liu, C. Y., 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]
    7. 7
  7. Zeng, R., Spolski, R., Finkelstein, S. E., Oh, S. K., Kovanen, P. E., Hinrichs, C. S., Pise-Masison, C. A., Radonovich, M. F., Brady, J. N., Restifo, N. P., Berzofsky, J. A., Leonard, W. J. (2005) Synergy of IL-21 and IL-15 in regulating CD8(+) T cell expansion and function J. Exp. Med. 201,139-148[Abstract/Free Full Text]
    8. 8
  8. Parrish-Novak, J., Dillon, S. R., Nelson, A., Hammond, A., Sprecher, C., Gross, J. A., Johnston, J., Madden, K., Xu, W. F., West, J., et al (2000) Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function Nature 408,57-63[CrossRef][Medline]
    9. 9
  9. Finkelman, F. D., Holmes, J., Katona, I. M., Urban, J. F., Beckmann, M. P., Park, L. S., Schooley, K. A., Coffman, R. L., Mosmann, T. R., Paul, W. E. (1990) Lymphokine control of in vivo immunoglobulin isotype selection Annu. Rev. Immunol. 8,303-333[CrossRef][Medline]
    10. 10
  10. Kuhn, R., Rajewsky, K., Muller, W. (1991) Generation and analysis of interleukin-4-deficient mice Science 254,707-710[Abstract/Free Full Text]
    11. 11
  11. 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]
    12. 12
  12. Mehta, D. S., Wurster, A. L., Whitters, M. J., Young, D. A., Collins, M., Grusby, M. J. (2003) IL-21 induces the apoptosis of resting and activated primary B cells J. Immunol. 170,4111-4118[Abstract/Free Full Text]
    13. 13
  13. Martin, F., Kearney, J. F. (2002) Marginal-zone B cells Nat. Rev. Immunol. 2,323-335[CrossRef][Medline]
    14. 14
  14. Malek, T. R., Porter, B. O., Codias, E. K., Scibelli, P., Yu, A. X. (2000) Normal lymphoid homeostasis and lack of lethal autoimmunity in mice containing mature T cells with severely impaired IL-2 receptors J. Immunol. 164,2905-2914[Abstract/Free Full Text]
    15. 15
  15. Malek, T. R., Robb, R. J., Shevach, E. M. (1983) Identification and initial characterization of a rat monoclonal-antibody reactive with the murine interleukin-2 receptor ligand complex Proc. Natl. Acad. Sci. USA. 80,5694-5698[Abstract/Free Full Text]
    16. 16
  16. Henderson, A., Calame, K. (1998) Transcriptional regulation during B cell development Annu. Rev. Immunol. 16,163-200[CrossRef][Medline]
    17. 17
  17. Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J., Paul, W. E. (1999) The IL-4 receptor: signaling mechanisms and biologic functions Annu. Rev. Immunol. 17,701-738[CrossRef][Medline]
    18. 18
  18. Wurster, A. L., Rodgers, V. L., Satoskar, A. R., Whitters, M. J., Young, D. A., Collins, M., Grusby, M. J. (2002) Interleukin 21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naive Th cells into interferon {gamma}-producing Th1 cells J. Exp. Med. 196,969-977[Abstract/Free Full Text]
    19. 19
  19. Mehta, D. S., Wurster, A. L., Weinmann, A. S., Grusby, M. J. (2005) NFATc2 and T-bet contribute to T-helper-cell-subset-specific regulation of IL-21 expression Proc. Natl. Acad. Sci. USA 102,2016-2021[Abstract/Free Full Text]
    20. 20
  20. Foote, L.C., Howard, R.G., MarshakRothstein, A., Rothstein, T.L. (1996) IL-4 induces Fas resistance in B cells J. Immunol. 157,2749-2753[Abstract]
    21. 21
  21. Vella, A., Teague, T. K., Ihle, J., Kappler, J., Marrack, P. (1997) Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: Stat6 is probably not required for the effect of IL-4 J. Exp. Med. 186,325-330[Abstract/Free Full Text]
    22. 22
  22. Wurster, A. L., Rodgers, V. L., White, M. F., Rothstein, T. L., Grusby, M. J. (2002) Interleukin-4-mediated protection of primary B cells from apoptosis through Stat6-dependent up-regulation of Bcl-xL J. Biol. Chem. 277,27169-27175[Abstract/Free Full Text]
    23. 23
  23. Haynes, B. F., Telen, M. J., Hale, L. P., Denning, S. M. (1989) Cd44–a molecule involved in leukocyte adherence and T-cell activation Immunol. Today 10,423-428[CrossRef][Medline]
    24. 24
  24. Choe, J., Kim, H. S., Armitage, R. J., Choi, Y. S. (1997) The functional role of B cell antigen receptor stimulation and IL-4 in the generation of human memory B cells from germinal center B cells J. Immunol. 159,3757-3766[Abstract]
    25. 25
  25. Siegelman, M. H., DeGrendele, H. C., Estess, P. (1999) Activation and interaction of CD44 and hyaluronan in immunological systems J. Leukoc. Biol. 66,315-321[Abstract]
    26. 26
  26. Sprent, J., Surh, C. D. (2002) T cell memory Annu. Rev. Immunol. 20,551-579[CrossRef][Medline]
    27. 27
  27. 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., 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-61 J. Immunol. 173,5361-5371[Abstract/Free Full Text]
    28. 28
  28. 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]
    29. 29
  29. Oliver, A. M., Martin, F., Gartland, G. L., Carter, R. H., Kearney, J. F. (1997) Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses Eur. J. Immunol. 27,2366-2374[Medline]
    30. 30
  30. Oliver, A. M., Martin, F., Kearney, J. F. (1999) IgM(high)CD21(high) lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells J. Immunol. 162,7198-7207[Abstract/Free Full Text]
    31. 31
  31. Martin, F., Oliver, A. M., Kearney, J. F. (2001) Marginal zone and B1B cells unite in the early response against T-independent blood-borne particulate antigens Immunity 14,617-629[CrossRef][Medline]
    32. 32
  32. Song, H., Cerny, J. (2003) Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen J. Exp. Med. 198,1923-1935[Abstract/Free Full Text]
    33. 33
  33. Sanchez-Guerrero, I., Albaladejo, M. D., Garciaalonso, A. M., Muro, M., Hernandez, J., Alvarez, M. R. (1994) Soluble Cd23 (Scd23) serum levels and lymphocyte subpopulations in peripheral-blood in rhinitis and extrinsic and intrinsic asthma Allergy 49,587-592[Medline]
    34. 34
  34. Park, C. S., Ra, D. J., Lee, S. M., Jeong, S. W., Uh, S., Kim, H. T., Kim, Y. H. (1996) Interleukin-4 and low-affinity receptor for IgE on B cells in peripheral blood of patients with atopic bronchial asthma J. Allergy Clin. Immunol. 97,1121-1128[CrossRef][Medline]
    35. 35
  35. Horiguchi, S., Okamoto, Y., Chazono, H., Sakurai, D., Kobayashi, K. (2005) Expression of membrane-bound CD23 in nasal mucosal B cells from patients with perennial allergic rhinitis Ann. Allergy Asthma Immunol. 94,286-291[Medline]
    36. 36
  36. Fujiwara, H., Kikutani, H., Suematsu, S., Naka, T., Yoshida, K., Tanaka, T., Suemura, M., Matsumoto, N., Kojima, S., Kishimoto, T., Yoshida, N. (1994) The absence of IgE antibody-mediated augmentation of immune-responses in Cd23-deficient mice Proc. Natl. Acad. Sci. USA 91,6835-6839[Abstract/Free Full Text]



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