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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-04-1115.
IMMUNOBIOLOGY
From the Department of Internal Medicine II, Chiba
University School of Medicine, Chiba, Japan; and
Department of Cytokine Biology, ZymoGenetics Inc, Seattle, WA.
Interleukin 21 (IL-21) has recently been identified as a
multifunctional cytokine that induces the proliferation of T cells and
B cells and differentiation of natural killer cells. To determine whether IL-21 regulates IL-4-mediated immune responses, we examined the effect of IL-21 on antigen-specific IgE production in mice. We also
examined the effect of IL-21 on IL-4-induced IgE production from B
cells and antigen-induced T-helper 2 (Th2) cell
differentiation. The in vivo injection of IL-21 prevented
antigen-specific IgE but not IgG2a production on immunization. IL-21
did not affect Th2 cell differentiation or IL-4 production
from CD4+ T cells but directly inhibited IL-4-induced IgE
production from B cells at single-cell levels. Moreover, IL-21
inhibited IL-4-induced germ line C Immunoglobulin E (IgE) plays a crucial role
in the pathogenesis of allergic diseases, in which there is a
polarization of T-lymphocyte responses toward T-helper 2 (Th2) cells that produces cytokines such as interleukin 4 (IL-4) and IL-5.1-4 During the exposure to antigen
stimulation, B cells are activated and switched from expressing IgM to
other immunoglobulin isotypes through the mechanism of switch
recombination.5,6 Gene-targeting experiments indicate that
switch recombination requires the synthesis of specific classes of germ
line transcripts,7,8 which are regulated by promoters
located upstream of each switch region.6 In conjunction with signals that activate B cells, such as CD40 ligand (CD40L), cross-linking of B-cell receptors, or lipopolysaccharide (LPS) stimulation, specific cytokines regulate the activities of germ line
promoters; that is, IL-4, interferon It has recently been shown that IL-21 is a multifunctional cytokine
that induces the proliferation of T cells and B cells and
differentiation of natural killer (NK) cells.9 IL-21 is a
4-helix bundle type I cytokine with a significant homology to IL-2,
IL-15, and IL-4.9 Production of IL-21 is restricted to activated CD4+ T cells9 and the best
stimulation for CD4+ T cells to produce IL-21 is the
combination of anti-CD3 and anti-CD28.9 The secreted IL-21
enhances IL-2- and IL-15-induced proliferation of T
cells.9 Thus, IL-21 may function as an auto-growth factor for T cells like IL-2. On the other hand, IL-21 regulates B-cell proliferation either positively or negatively, depending on the costimuli encountered by the B cells.9 This observation
suggests that signaling through IL-21 receptor may function as a fine
regulator of B-cell function during immune responses.
IL-21 receptor belongs to the class I cytokine receptor family
and exhibits amino acid sequence similarity to IL-2 receptor To determine whether IL-21 regulates IL-4-mediated immune responses,
we examined the effect of IL-21 on antigen-specific IgE production in
vivo. We also examined the effect of IL-21 on IL-4-induced IgE
production from B cells and antigen-induced Th2 cell
differentiation in vitro. We found that IL-21 down-regulated
IL-4-dependent IgE production from B cells but did not affect
Th2 cell differentiation. We also found that IL-21
inhibited IL-4-induced germ line C Mice and cytokines
Effect of IL-21 on antigen-specific IgE, IgG1, and IgG2a
production
Antigen-induced airway inflammation BALB/c mice were immunized with OVA as described above. Fourteen days after the second immunization, the sensitized mice were challenged with inhaled OVA (50 mg/mL in 0.9% saline) for 20 minutes as described previously.15 As a control, 0.9% saline alone was administered. To examine the effect of IL-21 on antigen-induced eosinophil and lymphocyte recruitment into the airways, the mice were injected with IL-21 (0.5 µg/mouse) twice a week during the immunization period as well as 2 hours before the inhaled OVA challenge and 22 hours after the challenge. At 36 hours after the inhaled OVA challenge, bronchoalveolar lavage was performed with 3 mL PBS and differential cell counts were examined on cytospin cell preparations stained with Wright-Giemsa solution.Antigen-induced cytokine production in splenocyte culture Splenocytes (1 × 106/mL) from DO10+ mice were stimulated with OVA323-339 peptide (50 µM) in the presence or absence of IL-21 (10 ng/mL) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 µM -mercaptoethanol, 2 mM L-glutamine, and antibiotics
(complete RPMI 1640 medium) in triplicate in a 96-well microtiter plate
at 37°C for 72 hours. As a control, cells were cultured with
nonstimulating OVA323-334 peptide (50 µM). The amount of IL-4, IL-5,
and interferon  (IFN-) in the culture supernatant was measured by
the enzyme immunoassay using murine IL-4, IL-5, and IFN- ELISA kits
(Pharmingen, San Diego, CA). The assays were performed in duplicate
according to the manufacturer's instruction. The detection limits of
these assays were 10 pg/mL IL-4 and IL-5, and 50 pg/mL
IFN-.
Antigen-induced T-cell differentiation Splenocytes from DO10+ mice were stimulated with OVA323-339 peptide (50 µM) in the presence of IL-21 (10 ng/mL) or sIL-21R (20 µg/mL) in a 24-well microtiter plate at 37°C for 48 hours. Where indicated, IL-12 (7.5 ng/mL) was added to polarize toward Th1 cells (Th1 condition) and IL-4 (7.5 ng/mL) was added to polarize toward Th2 cells (Th2 condition). Cells were washed with PBS and cultured for another 3 days in Th0 (nonpolarizing), Th1, or Th2 condition in the presence of IL-2 (5 ng/mL). Intracellular cytokine analyses for IL-4 versus IFN- were performed
as described previously.16
B-cell purification from splenocytes Splenocytes from BALB/c mice or from Stat6 / mice
or the littermate wild-type (WT) mice were incubated with a mixture of
fluorescein isothiocyanate (FITC)-labeled antibodies to CD4 (RM4-5;
Pharmingen), CD8 (53.6.7; Pharmingen), pan NK (DX5; Pharmingen), and
CD11b (M1/70; Pharmingen) for 20 minutes at 4°C, washed 3 times with PBS, and incubated with anti-FITC magnetic microbeads (Miltenyi Biotec,
Sunnyvale, CA) for 20 minutes at 4°C. After washing with PBS, cells
were passed through the magnetic-activated cell sorting (MACS)
separation CS column (Miltenyi Biotec) according to the manufacturer's
instructions and cells in the flow-through were collected by
centrifugation. These cells were more than 95% pure B220+
cells by fluorescence-activated cell sorting (FACS) analysis.
IgE, IgG1, and IgG2a production from cultured splenic B cells Splenic B cells (1 × 106/mL) from BALB/c mice were stimulated with lipopolysaccharide (LPS, 10 µg/mL; Sigma) in the presence of IL-4 (50 ng/mL) or IL-21 (0-20 ng/mL) or both in the complete RPMI 1640 medium in a 48-well microtiter plate at 37°C for 7 days. The amount of IgE, IgG1, and IgG2a in the supernatant was measured by ELISA according to the manufacturer's instruction (Pharmingen).Membrane IgE+ B cells with cell division Membrane IgE+ cells were detected with cell division as described previously.17,18 Briefly, purified splenic B cells from Stat6/ mice or littermate WT mice were
incubated with 5-(and-6)-carboxyfluorescein diacetate,
succinimidyl ester (CFSE, 10 µM; Molecular Probes, Eugene, OR) in PBS
at 37°C for 10 minutes and then washed with RPMI 1640 medium.
CFSE-labeled B cells were cultured at 37°C for 6 days with LPS (10 µg/mL) in the presence of IL-4 (50 ng/mL) and indicated amounts of
IL-21 (0-20 ng/mL). Where indicated, sIL-21R (20 µg/mL) was added to
the culture. Cells were harvested and Fc receptors were blocked with
anti-CD16/32 antibody (2.4G2; Pharmingen) prior to staining. Cells were
incubated with biotin-conjugated anti-IgE antibody (R35-72; Pharmingen)
at 4°C for 30 minutes, washed with PBS, and then visualized with
streptavidin allophycocyanin (APC; Pharmingen) on FACS. In
agreement with the previous report,19 acid treatment (pH
4.0) did not affect the percentage of IgE+ cells (data not
shown), suggesting that the IgE+ cells were not due to the
binding of produced IgE to Fc receptors. Analogously, membrane
IgG1+ cells and membrane IgG2a+ cells were
detected by FACS using anti-IgG1 antibody (A85-1; Pharmingen) and
anti-IgG2a antibody (R19-15; Pharmingen), respectively.
RT-PCR for germ line C  ,
C1, and C2a transcripts was performed as described elsewhere.20,21 RT-PCR for -actin mRNA was also
performed to control the sample-to-sample variation in RNA isolation
and integrity, RNA input, and reverse transcription. All PCR
amplifications were performed at least 3 times with multiple sets of
experimental RNAs.
Immunoblotting Whole cell extracts were prepared and immunoblottings were performed as described previously.16 The following antisera were used: antiphospho-Stat1 (Tyr701; New England Biolabs, Beverly, MA), antiphospho-Stat3 (Tyr705; New England Biolabs), antiphospho-Stat5 (Tyr694; New England Biolabs), antiphospho-Stat6 (Tyr641; New England Biolabs), antimouse Stat6 (M20; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-Bcl-6 (N3; Santa Cruz Biotechnology).Electrophoretic mobility shift assays After splenic B cells were stimulated with LPS (10 µg/mL) or IL-21 (10 ng/mL) or both at 37°C for 30 minutes, nuclear extracts were prepared as described elsewhere.22 A nuclear factor- B (NF-B) consensus
double-stranded oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Promega Biotech, Madison, WI) was labeled with [-32P]
adenosine triphosphate (ATP). DNA-binding reaction was performed at
room temperature for 20 minutes using 5 µg nuclear extracts according
to the manufacturer's instruction (Promega Biotech). For DNA
competition experiments, a 50-fold molar excess of unlabeled competitor
oligonucleotide relative to the labeled probe was incubated in the
binding mixture for 20 minutes before addition of the
32P-labeled probe. The binding reaction mixtures were
electrophoresed on 6% retardation gels (Invitrogen, Carlsbad, CA) and
followed by autoradiography.
Luciferase assay Stat6-dependent reporter plasmid, TPU474,23 was a kind gift from Dr U. Schindler (Tularik, CA). The germ line C
promoter plasmid, -162Luc,24 which contains the segment
extending from  163 to +53 relative to the first RNA initiation site
of germ line C, was a kind gift from Dr J. Stavnezer (University of
Massachusetts Medical School, Boston). Another germ line C
promoter plasmid, -623Luc, which contains the segment extending from
623 to +53 of germ line C, was constructed as described
previously.24 M12.4.5 cells (a kind gift from Dr T. Tokuhisa, Chiba University, Japan) were mixed with 20 µg of either
TPU474, -162Luc, or -623Luc in the presence of 200 ng
pRL-TK (Promega Biotech) and 5 µg Stat6-expression plasmid
(pcDNA3 Stat6) in 800 µL serum-free RPMI 1640 medium and electroporated at 960 µF/300V. After cells were cultured at 37°C for 12 hours in the complete RPMI 1640 medium, aliquoted cells were
left untreated or treated for another 12 hours with IL-4 (20 ng/mL) or
IL-21 (10 ng/mL) or both. The luciferase activity was measured
by dual luciferase assay system (Promega Biotech) according to the
manufacturer's instruction. Firefly luciferase activity of TPU474,
-162Luc, or -623Luc was normalized by Renilla luciferase activity
of pRL-TK. All values were obtained from experiments carried out in
triplicate and repeated at least 4 times.
Decay of germ line C
transcripts and -actin was determined by RT-PCR as described above.
Consistent with a previous report,25 no significant loss
of cell viability was observed by the trypan blue exclusion method
after the incubation.
Data analysis Data are summarized as mean ± SD. The statistical analysis of the results was performed by the unpaired t test. P < .05 was considered significant.
IL-21 inhibits antigen-specific IgE production and antigen-induced eosinophil recruitment into the airways It has recently been shown that IL-21 induces the proliferation of T cells and B cells and differentiation of NK cells.9 However, the physiologic role of IL-21 remains elusive. Therefore, we first determined the effect of IL-21 on antigen-specific immunoglobulin production on antigen immunization in mice. BALB/c mice were immunized intraperitoneally twice with OVA and given recombinant IL-21 (0.5 µg/mouse) intraperitoneally twice a week during the immunization. Two weeks after the second immunization, sera were collected and the amounts of anti-OVA IgE, IgG1, and IgG2a antibodies were evaluated. As shown in Figure 1A, IL-21 significantly decreased anti-OVA IgE production (n = 7 mice in each group, P < .005). IL-21 also decreased anti-OVA IgG1 production (P < .01). In contrast, the amount of antigen-specific IgG2a was not significantly affected by IL-21 (Figure 1A). The number of leukocytes and erythrocytes in peripheral blood as well as the number of B cells and T cells in spleen were not significantly affected by the injection of IL-21, either (data not shown).
We also examined the effect of IL-21 on antigen-induced eosinophil recruitment into the airways of sensitized mice. Interestingly, IL-21 significantly decreased antigen-induced eosinophil recruitment into the airways of sensitized mice (PBS 24.9 ± 4.2 versus IL-21 9.3 ± 2.0 × 104 eosinophils/mouse, n = 5 mice in each group, P < .01; Figure 1B). In contrast, IL-21 did not affect antigen-induced lymphocyte recruitment into the airways (Figure 1B). IL-21 does not affect IL-4-induced Th2 cell differentiation It is well known that antigen-specific IgE production from B cells depends on IL-4 from Th2 cells.4,5,26,27 We therefore attempted to determine whether IL-21 inhibits Th2 cell differentiation. Splenocytes from OVA-specific TCR transgenic mice (DO10+ mice) were stimulated with antigenic OVA peptide (OVA323-339) for 3 days in the presence or absence of IL-21 (10 ng/mL) and cytokine levels in the supernatants were measured by ELISA. The production of Th2 cytokines (IL-4 and IL-5) as well as Th1 cytokine (IFN-) was not significantly altered by IL-21 (Figure
2A), although IL-21 receptor was
expressed in both Th1 cells and Th2 cells at mRNA levels
(data not shown).
We next examined the effect of IL-21 on the differentiation of Th2 cells at single-cell levels. Consistent with the data shown in Figure 2A, IL-21 did not significantly affect the T-helper cell differentiation in nonpolarizing (Th0) condition (Figure 2B). Moreover, IL-21 did not affect IL-4-induced Th2 cell differentiation or IL-12-induced Th1 cell differentiation (Figure 2B). Given that activated CD4+ T cells produce IL-21,9 endogenously produced IL-21 may be sufficient for optimal T-helper cell differentiation. Thus, we next examined the effect of sIL-21R, which neutralizes IL-21,9 on T-helper cell differentiation. However, the addition of sIL-21R (20 µg/mL) did not affect either Th1 cell or Th2 cell differentiation (Figure 2B), indicating that endogenously produced IL-21 also plays no significant role in the regulation of T-helper cell differentiation. IL-21 inhibits IL-4-induced IgE but not IgG1 production from splenic B cells Because IL-21 is not crucial for regulating Th2 cell differentiation (Figure 2), we then examined the effect of IL-21 on IgE production from purified splenic B cells. When splenic B cells from BALB/c mice were stimulated with LPS (10 µg/mL) plus IL-4 (50 ng/mL) for 7 days, IgE was readily detected in the supernatant of the culture (Figure 3A). IL-4 alone was not sufficient for the induction of IgE production from splenic B cells (data not shown). Interestingly, IL-21 (5-20 ng/mL) decreased IgE production from LPS/IL-4-stimulated B cells in a dose-dependent fashion (n = 5 experiments, P < .01-.001; Figure 3A). In contrast, IL-21 (20 ng/mL) enhanced IgG1 production from LPS/IL-4-stimulated B cells (n = 5 experiments, P < .01; Figure 3B), although IL-21 inhibited antigen-specific IgG1 production in vivo (Figure 1A). On the other hand, IL-21 did not affect IgG2a production from LPS-stimulated B cells or did not antagonize the inhibitory effect of IL-4 on IgG2a production from LPS-stimulated B cells (Figure 3C).
IL-21 inhibits IL-4-induced IgE but not IgG1 production at single-cell levels We next examined the effect of IL-21 on IgE production from B cells at single-cell levels. When splenic B cells from WT mice were cultured with LPS plus IL-4 for 6 days, membrane IgE-positive (mIgE+) B cells were significantly increased (Figure 4A). In contrast, IL-4 did not increase mIgE+ B cells in Stat6/ mice (Figure 4A),
indicating that Stat6 plays a crucial role in the generation of
mIgE+ B cells. Interestingly, IL-21 (5-20 ng/mL) dose
dependently decreased mIgE+ cells in LPS/IL-4-stimulated B
cells in WT mice (Figure 4A). The addition of sIL-21R, together with
IL-21, reversed the inhibitory effect of IL-21 on the generation of
mIgE+ cells in LPS/IL-4-stimulated B cells (Figure 4A). In
addition, IL-21 negligibly inhibited the cell division of
LPS/IL-4-stimulated B cells (Figure 4A), although a high concentration
of IL-21 (80 ng/mL) significantly suppressed cell division of
LPS/IL-4-stimulated B cells (data not shown). Therefore, these results
indicate that IL-21 inhibits IL-4-induced, Stat6-dependent IgE
production in B cells.
We also examined the effect of IL-21 on the generation of membrane IgG1
positive (mIgG1+) and membrane IgG2a positive
(mIgG2a+) B cells. IL-4 significantly increased
mIgG1+ B cells in WT mice but not in Stat6 IL-21 inhibits IL-4-induced germ line C genes and directs switching to IgE in LPS-stimulated
B cells.26,27 Thus, we next examined whether IL-21
inhibits IL-4-induced germ line C transcription. In agreement with
a previous report,20 germ line C transcripts were
detected when splenic B cells were stimulated with LPS plus IL-4 for 16 hours (Figure 5A). Interestingly, IL-21 (10 ng/mL) almost completely suppressed the IL-4-induced germ line
C transcription in LPS-stimulated B cells (Figure 5A), whereas IL-21
did not significantly affect IL-4-induced germ line C1 transcription in LPS-stimulated B cells or germ line C2a
transcription in LPS-stimulated B cells (Figure 5A). In addition, IL-21
did not significantly affect the number of B cells recovered in 16 hours of culture (data not shown). Taken together, these results indicate that IL-21 inhibits IgE production mainly through the inhibitory effect on germ line C transcription in B cells. However, because IL-21 slightly (but not statistically significantly) inhibited LPS/IL-4-mediated B-cell proliferation in 48 hours of culture (Figure
5B), it is possible that a small antiproliferative effect on B cells
may also contribute to the inhibition of IgE production by
IL-21.
IL-21 does not inhibit IL-4-induced Stat6 phosphorylation or
LPS-induced nuclear accumulation of NF-
transcription and IgE production.12,20,28,29 When splenic
B cells were stimulated with IL-4 (20 ng/mL) for 20 minutes, the
phosphorylated form of Stat6 was readily detected by antiphospho Stat6
antibody (Figure 6A). IL-21 (10 ng/mL)
did not induce the phosphorylation of Stat6 at all (Figure 6A).
Moreover, preincubation of B cells with IL-21 for 2 minutes before IL-4
stimulation did not inhibit IL-4-induced phosphorylation of Stat6
(Figure 6A). These results suggest that IL-4-mediated signaling is
normally transduced to the activation of Stat6 even in the presence
of IL-21.
The ligation of IL-21 receptor has been shown to result in the
activation of Stat1, Stat3, and Stat5.9-11 Because Stat
proteins regulate the expression of a variety of genes, including
negative regulators of cytokine signaling such as SOCS/CIS/SSI family
proteins,30 it was possible that IL-21 induced these genes
and thus inhibited the subsequent IL-4-mediated Stat6 activation.
Therefore, we examined IL-4-induced Stat6 phosphorylation in splenic B
cells that were pretreated with IL-21 for either 4 hours or 12 hours.
However, the pretreatment with IL-21 did not affect the subsequent
activation of Stat6 in IL-4-stimulated B cells (Figure 6A). In
addition, the duration of IL-4-induced phosphorylation of Stat6 was
not affected by the presence of IL-21 (Figure 6B). Consistent with these results, the expression levels of IL-4 receptor It has been shown that IL-4-induced IgE production is down-regulated
at the transcriptional level by Bcl-6,31 a site-specific transcriptional repressor belonging to the POZ/zinc-finger
family.32 An in vitro-defined binding site for Bcl-6
demonstrates a marked similarity to the consensus element recognized by
Stat633 and, in fact, Bcl-6 binds to Stat6 site of germ
line C We also examined the possibility that IL-21 inhibits LPS-mediated
NF- IL-21 does not inhibit Stat6-mediated transcription from core germ
line C promoter using a reporter assay. To perform the assay,
we first tried to identify a murine cell line that exhibited IL-21
responsiveness similar to splenic B cells. As shown in Figure 7A, when splenic B cells were stimulated
with IL-21 (10 ng/mL) for 20 minutes, strong phosphorylation of Stat1
and Stat3 and weak phosphorylation of Stat5 were observed (Figure 7A).
A similar phosphorylation pattern of Stat proteins was observed when
M12.4.5 cells and A20 cells were stimulated with IL-21 (Figure 7A). In contrast, IL-21 did not induce phosphorylation of Stat proteins in
Ba/F3 cells, suggesting that Ba/F3 cells may lack the expression of
IL-21 receptors (Figure 7A).
To examine the effect of IL-21 on the transcription from the core germ
line C IL-21 does not accelerate the decay of IL-4-induced germ line
C
transcripts in B cells (Figure 5), but IL-21 did not inhibit the
transcription from the core promoter of germ line C (Figure 7).
These data raised the possibility that IL-21 decreased the stability of
germ line C transcripts. To address this possibility, we examined the effect of IL-21 on the decay of germ line C transcripts in splenic B cells. After splenic B cells were stimulated with LPS plus
IL-4 for 15 hours, de novo transcription was inhibited by the addition
of actinomycin D (10 µg/mL). Cells were then incubated with or
without IL-21 (10 ng/mL) for 1, 3, or 5 hours and remaining germ line
C transcripts were measured by RT-PCR (Figure
8). Contrary to our expectation, IL-21
did not accelerate the decay of germ line C transcripts, indicating
that the inhibitory effect of IL-21 on germ line C transcription is
not due to an accelerated decay of germ line C transcripts.
In this study, we show a novel function of IL-21 in the regulation
of IgE production. The in vivo injection of IL-21 prevented antigen-specific IgE production on immunization (Figure 1). IL-21 did
not inhibit IL-4 production or Th2 cell differentiation
from CD4+ T cells (Figure 2) but directly inhibited
IL-4-induced IgE production from B cells at single-cell levels
(Figures 3 and 4). Moreover, IL-21 inhibited IL-4-induced germ line
C We show that IL-21 inhibits IL-4-induced germ line C In addition to Stat6, several other molecules have been demonstrated to
be involved in the regulation of germ line C Despite the inhibitory effect of IL-21 on germ line C It is well documented that IL-4 directs isotype switching in mouse B
cells not only to IgE but also to IgG1.5,6,26,27 However,
it has been shown that switching to IgG1 is at most partially reduced
in Stat6 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
transcription of IL-4-stimulated B
cells
Top
Abstract
Introduction
chain.
) or without (
) OVA at a 2-week interval. The mice were
injected intraperitoneally with IL-21 (0.5 µg/mouse) twice a week for
4 weeks, with the first injection of IL-21 being done at 4 hours before
the first immunization with OVA. As a control, the mice were injected
intraperitoneally with PBS. Two weeks after the second immunization,
the sera were collected and antigen-specific IgE, IgG1, and IgG2a were
evaluated. Data are means ± SD for 7 mice in each group. Single
and double asterisks indicate significantly different from the mean
value of the corresponding response in the control mice,
P < .005 and P < .01, respectively. (B)
IL-21 decreases antigen-induced eosinophil recruitment into the
airways. BALB/c mice were immunized with OVA and injected with IL-21 or
PBS (as a control) as described above. Two weeks after the second
immunization, mice were challenged with the inhaled OVA (
