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The Journal of Immunology, 2004, 172: 6158-6166.
Copyright © 2004 by The American Association of Immunologists

IL-4-Induced GATA-3 Expression Is a Time-Restricted Instruction Switch for Th2 Cell Differentiation1

Noriyasu Seki*,{dagger}, Mayumi Miyazaki*, Wataru Suzuki*, Katsuhiko Hayashi{ddagger}, Kazuhiko Arima§, Elmarie Myburgh||, Kenji Izuhara§,¶, Frank Brombacher|| and Masato Kubo2,*,#

* Research Institute for Biological Sciences, Tokyo University of Science, Yamazaki, Noda City, Chiba, Japan; {dagger} Research and Development Division, Mitsubishi Pharma, Aoba-ku, Yokohama, Japan; {ddagger} Department of Molecular Embryology, Research Institute Osaka Medical Center for Maternal and Child Health, Izumi-shi, Osaka, Japan; § Division of Medical Biochemistry, Department of Biomolecular Sciences, and Division of Medical Research, Center for Comprehensive Community Medicine, Saga Medical School, Saga, Japan; || Division of Immunology, University of Cape Town, Groote Schuur Hospital, Cape Town, South Africa; and # Signal/Network Team, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Suehiro-cho, Tsurumi, Yokohama, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An initial activation signal via the TCR in a restricted cytokine environment is critical for the onset of Th cell development. Cytokines regulate the expression of key transcriptional factors, T-bet and GATA-3, which instruct the direction of Th1 and Th2 differentiation, through changes in chromatin conformation. In this study, we investigated the kinetics of IL-4-mediated signaling in a transgenic mouse, expressing human IL-4R on a mouse IL-4{alpha}R-deficient background. These experiments, allowing induction with human IL-4 at defined times, demonstrated that an IL-4 signal was required at the early stage of TCR-mediated T cell activation for lineage commitment to Th2, along with structural changes in chromatin, which take place in the conserved noncoding sequence-1 and -2 within the IL-4 locus. At later times, however, IL-4 failed to promote efficient Th2 differentiation and decondensation of chromatin, even though GATA-3 was clearly induced in the nuclei by IL-4 stimulation. Moreover, IL-4-mediated Th2 instruction was independent from cell division mediated by initial TCR stimulation. The role of IL-4 signaling may have a time restriction during Th2 differentiation. In late stages of initial T cell activation, the chromatin structure of the IL-4 locus retains condensation state. These results demonstrate that IL-4-induced GATA-3 expression is time-restriction switch for Th2 differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Helper T cells exhibit cytokine expression patterns that divide them into at least two functionally distinct subsets. Th1 cells secrete IL-2, IFN-{gamma}, and TNF-{alpha}, which promote cellular immune responses against intracellular pathogens and viruses, mediate delayed-type hypersensitivity responses, and may lead to organ-specific autoimmune diseases. Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13, which promote humoral immune responses mainly against extracellular pathogens. IL-4 and IL-5 regulate the immune response via mast cells and eosinophils, especially in atopic and allergic conditions. The development of Th cells is determined by cytokines present at the early stages of T cell activation, upon encounter with Ag on APCs. The most critical role for the instruction of Th1 and Th2 development is played by cytokines such as IL-12 and IL-4, which act through the STAT4 pathway, or through the STAT6 signaling pathway, respectively (1, 2, 3, 4).

Differential cytokine production within Th1 and Th2 cells is controlled at the level of gene transcription. During differentiation, IL-4 transcription is amplified by TCR stimulation in the presence of IL-4. TCR stimulation regulates the expression of a trans activator of the IL-4 promoter, c-maf, and also regulates the activation of NF-AT and AP-1 family members (5, 6, 7). IL-4R signaling controls expression of GATA-3, a Th2-specific transcription factor that regulates lineage commitment to Th2 (8, 9). TCR-mediated signaling, together with CD28 stimulation, augments GATA-3 expression through activating NF-{kappa}B (10). GATA-3 protein induces further GATA-3 expression in an autocrine manner, leading to massive up-regulation of GATA-3 transcription (4, 11, 12). In the absence of IL-4-mediated STAT6 activation, ectopic overexpression of GATA-3 inhibits IL-12R{beta}2 expression (8) and chromatin remodeling at the IL-4 locus, as well as Th2 cytokine gene expression (12, 13, 14). Therefore, in Th2 differentiation, the role of the IL-4 signal is thought to be the initial induction of GATA-3.

Decondensation of chromatin is characterized by hyperacetylation of histones H3 and H4, as well as by increased accessibility to restriction enzymes, DNase I, and transcription factors (15, 16, 17). During Th2 differentiation, the chromatin structure in the IL-13/IL-4 locus changes, allowing transcription of the Th2 cytokine genes. In Th cells, TCR stimulation in the presence of IL-4 elicits a cluster of DNase I-hypersensitive sites (18, 19) and histone acetylation (20). Agarwal et al. (21, 22) have found six Th2-specific hypersensitive sites, named HS-I to -V and HS-Va, in the region spanning the IL-4 promoter to the KIF3 locus. Takemoto et al. (23) found additional sites, named HSS1 to HSS3, in the noncoding sequences between the IL-13 and IL-4 genes. The sequences of HSS1–3 and HS-Va are highly conserved between humans and mice, and are designated as conserved noncoding sequence-1 and -2, respectively (24). The deletion of these sequences either in a human YAC transgene or in the endogenous mouse locus reduces secretion of Th2 cytokines during restimulation (24, 25, 26). In both Th1 and Th2 lineages, core histone acetylation seems to occur within the first 48 h in the promoter, HS-Va, or conserved noncoding sequence-1 and -2. The IL-4 signal then further regulates Th2-specific acetylation, subsequently leading to chromatin remodeling (20, 27). HSS1–3, HS-II, intronic enhancer (IE),3 and HS-Va have all the consensus sequence for GATA-3 binding. Indeed, in Th2 cells, the HS-Va region is precipitated by Ab against GATA-3 (20, 22). This suggests that GATA-3 binding to conserved noncoding sequence-1 and -2 could regulate Th2-specific histone acetylation and chromatin remodeling. However, the mechanisms of how GATA-3 regulates these structural changes remain unclear.

IL-2 and IFN-{gamma} mRNA are induced within 6 h in G1 to S phase, while IL-4 mRNA is induced after 48 h at a time point when T cells have undergone more than three cell cycles. This indicates that cell division might be needed to induce Th2 cytokine transcription (28). However, other reports using a cell cycle blocker and IL-4 withdrawal to control early IL-4R signaling showed that IL-4 instructed IL-4 production in the first S phase (29, 30). The coordination of IL-4R and TCR signaling regulates IL-4 gene transcription (31, 32). Nevertheless, the individual roles of IL-4R signaling and TCR signaling remain unclear, because both occur around the same time, and their effects are experimentally difficult to separate.

To overcome this problem, we established a transgenic (Tg) mouse model, expressing human IL-4R{alpha} (hIL-4R{alpha}), under the control of an IE from the Ig H chain Eµ locus, to allow specific expression in lymphocytes only. The chimeric IL-4R molecule, composed of the hIL-4R{alpha} chain and the mouse common {gamma}-chain is responsive to hIL-4, therefore allowing us to control IL-4R signaling independent of endogenous IL-4 in lymphocytes only. In this study, we found that IL-4 signaling regulates the competence of effector cytokine production during a restricted phase of initial T cell activation, irrespective of progressive cell divisions. We discuss the importance of the timing of IL-4-mediated GATA-3 expression on Th2-specific chromatin remodeling and on the Th2 lineage commitment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Tg constructs for the hIL-4R were expressed under the control of an IE of Ig H chain locus (Eµ) promoter. A Tg line that expressed hIL-4R on T and B cells at similar level to endogenous mouse (m)IL-4R (hIL-4R{alpha}Tg) was selected for this study. The hIL-4R Tg mice were backcrossed to BALB/c genetic background for more than 10 generations. OVA-specific TCR (DO11.10) Tg mice on BALB/c background were kindly provided by K. Murphy (Washington University, St. Louis, MO). IL-4R{alpha}-deficient (mIL-4R{alpha} knockout (KO)) mice were generated on a BALB/c genetic background (33) and crossed with hIL-4R{alpha}Tg mice. BALB/c mice were purchased from Sankyo (Tokyo, Japan).

Preparation of Th cell

Single spleen cell suspensions were incubated with anti-CD8 mAb for 30 min on ice, and CD8+ cells and B cells were eliminated with rabbit anti-mouse Ig-coated dishes. The enriched CD4+ T cells were suspended in RPMI 1640 medium containing 10% (v/v) FCS, 10 mM HEPES-KOH, pH 7.4, 2 mM L-glutamine, and 50 µM 2-ME, and were stimulated with a combination of plate-coated anti-TCR mAb (H57-597) and soluble anti-CD28 mAb (PV-1), as previously described (34). For DO11.10 Tg T cells, cells were stimulated with 1 µM OVA323–339 and irradiated APCs. After 48 h, 30 U/ml IL-2 was added, and cells were cultured another 5 days. Cells from BALB/c and mIL-4R{alpha} KO mice were stimulated in the presence of anti-IL-4 mAb (11B11). Th2 development in hIL-4R{alpha} Tg was conducted with rhIL-4 (10 U/ml) (PeproTech, London, U.K.). Th1 and Th2 cells were prepared with the induction culture by the addition of either 10 U/ml rIL-12 and anti-IL-4 mAb (11B11) or 100 U/ml rIL-4 and anti-IL-12 mAb (C15.6 and C17.8; The Wister Institute, Philadelphia, PA), respectively.

Intracellular cytokine staining

The activated CD4+ T cells were restimulated with anti-TCR mAb for 6 h in the presence of 2 µM monensin (Sigma-Aldrich, St Louis, MO). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 3% BSA-PBS, cells were stained with anti-IFN-{gamma} (XMG1.2) FITC and anti-IL-4 (11B11) PE Abs (34). Flow cytometric analysis was conducted on a FACSort and analyzed by CellQuest software (BD Biosciences, San Diego, CA).

Proliferation assay

CD4+ T cells were stimulated with either mouse or hIL-4 in the presence of PMA for 48 h. The cells were pulsed with [3H]thymidine (0.5 µCi/well) for the last 12 h, and the incorporation of [3H]thymidine was measured by a beta counter.

Analysis of cell division and cytokine production. The enriched CD4+ T cells were labeled with CFSE (Molecular Probes, Eugene, OR) by incubating 1 x 107 cells/ml in PBS with 10 µM CFSE for 8 min at room temperature. The labeling process was stopped with the addition of one aliquot FCS and subsequent three washing steps. Cell divisions were analyzed by FACSort with intracellular cytokine staining after restimulation with anti-TCR mAb, as described above.

Northern blot analysis

Total cytoplasmic RNA was isolated from cells using a TRIzol reagent (Life Technologies, Rockville, MD). Two micrograms of RNA was separated on a 1% (w/v) agarose gel containing 2.2 M formaldehyde. Transfer of RNA onto a Hybond N membrane (Amersham Pharmacia Biotech, Piscataway, NJ), hybridization, and washing were performed according to the procedure supplied by the manufacturer (Roche). The probes used were digoxigenin-labeled antisense riboprobes transcribed from the cDNA template of IL-12R{beta}2, T-bet, GATA-3, and G3PDH. Signals were visualized using an alkaline phosphatase-conjugated, anti-digoxigenin Ab (Roche, Mannheim, Germany).

Separation of nuclear and cytoplasmic fraction and Western blot analysis

The nuclear and cytoplasmic fractions were separated by nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL). The nuclear and cytoplasmic protein (25 µg) were loaded on SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were blotted with anti-GATA-3 mAb (HG3-35) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-NF-AT1 mAb (4G6-G5) (Santa Cruz Biotechnology), anti-NF-AT2 mAb (7A6) (Alexis, San Diego, CA), and anti-c-maf rabbit serum (M-153) (Santa Cruz Biotechnology). The blots were visualized with HRP-conjugated goat anti-mouse or anti-rabbit Ig (DAKO, Glostrup, Denmark).

Restriction enzyme accessibility assay

Enriched CD4+ T cells (5 x 106 cells) were used for each condition. Nuclei were prepared, as described previously (20), and resuspended in buffer F (100 mM NaCl, 50 mM Tris (pH 8.0), 5 mM MgCl2, 1 mM EGTA, and 1 mM 2-ME). XhoI and HgaII (100 U; Toyobo, Osaka, Japan) were added and incubated at room temperature for 1 h. Purified DNA was digested with either EcoRI or HindIII, ethanol precipitated, and resuspended in TE buffer, and the concentration was measured at absorbance of 260 nm. The digested DNA (5 µg) was transferred onto a Hybond N membrane (Amersham Pharmacia Biotech), and blotted with digoxigenin-labeled appropriated probes.

Retrovirus infection

The protocol for retroviral infection has been previously described in detail (12). Briefly, the murine GATA-3 cDNA was inserted into pMX-GFP (green fluorescent protein) vector (pMX-GFP-GATA-3). To ensure that GATA-3 and GFP were translated bicistronically, an internal ribosomal entry site was ligated upstream of the GFP. The pMX-GFP-GATA-3 and pMX-GFP control plasmid were transfected into a packaging cell line, PLAT-E, using FuGENE6 (Roche), and, after incubation for 24~48 h, the culture supernatant was harvested and condensed as a viral stock. CD4+-enriched T cells were stimulated with anti-TCR and anti-CD28 mAbs and infected with a viral stock at the indicated time point after primary stimulation. The viral infected CD4+ T cells were restimulated with anti-TCR mAb for 6 h in the presence of 2 µM monensin, and subsequent intracellular staining was conducted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hIL-4R substitutes for the function of the mIL-4R

We established a series of Tg mouse lines expressing hIL-4R{alpha} chain under the control of the Ig H chain promoter, and selected lines in which T cells apparently expressed hIL-4R{alpha} chain for backcrossing to a BALB/c genetic background. As expected, T (Fig. 1A) and B (data not shown) cells from Tg mice, but not from control littermates, responded to hIL-4 in a proliferation assay. Furthermore, splenic CD4+ T cells from the Tg animals, stimulated with exogenous hIL-4 in the presence of anti-mIL-4 mAb, showed Th2 differentiation at levels comparable to those seen with endogenous IL-4 (Fig. 1B). These results suggest that hIL-4 is able to fully substitute for endogenous mIL-4, resulting in Th2 polarization.



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  		FIGURE 1. A, Enriched CD4+ T cells, obtained either from control BALB/c littermates (•) or Tg animals containing hIL-4R (hIL-4R Tg) ({circ}), were stimulated with hIL-4 in the presence of PMA (50 ng/ml) and anti-mIL-4 mAb for 48 h. The proliferative response was measured by the incorporation of [3H]thymidine. B, Th cell differentiation was induced with a combination of anti-TCR and anti-CD28 mAbs in the presence or absence of anti-mIL-4 mAb and/or hIL-4. After 7 days, cells were restimulated with anti-TCR mAb and stained for intracellular cytokine. C, CD4+ T cells from hIL-4R{alpha} Tg (1) and hIL-4R{alpha} Tg/mIL-4R KO (3) were stimulated with the combination of anti-TCR and anti-CD28 mAbs at time point 0. For CD4+ T cells from hIL-4R{alpha} Tg/DO11.10Tg mice (2) and hIL-4R{alpha} Tg/mIL-4R{alpha} KO/DO11.10 mice (4), cells were stimulated with OVA peptide in context of APCs at time point 0. For hIL-4R{alpha} Tg (1) and DO11.10/hIL-4R{alpha} Tg (2), endogenous IL-4 was neutralized with anti-mIL-4 mAb. hIL-4 (10 U/ml) was added at different time points before or after initial T cell activation (–12 and 0–72 h). For time point –12 h, after 7 days from time point 0, cells were restimulated with anti-TCR mAb, and intracellular cytokine staining was conducted. The numbers represent the percentage of either IL-4- or IFN-{gamma}-producing cells. The experiment was independently performed three times with similar results.

 
To study the kinetics of IL-4-mediated Th2 instruction, T cells from hIL-4R{alpha} Tg and DO11.10/hIL-4{alpha} Tg were stimulated with anti-TCR and anti-CD28 mAbs and OVA antigenic peptide in the presence of BALB/c APCs. To eliminate the effect of endogenous IL-4, cells were cultured in anti-IL-4 mAb. To exclude signaling via endogenous IL-4 completely, hIL-4R{alpha} Tg mice were also crossed with mIL-4R{alpha} KO mice on a BALB/c genetic background (hIL-4R{alpha} Tg/IL-4R{alpha} KO). Time 0 was the initial stimulation with either anti-TCR and anti-CD28 mAb, or OVA peptide, and 10 U/ml hIL-4 was added at different times (–12 to 72 h; Fig. 1C, top panels).

First, we defined the kinetic relationship between initial TCR signaling and IL-4 signaling. Before T cell activation though TCR, CD4+ T cells of hIL-4R{alpha} Tg were cultured in the presence of hIL-4 for 12 h, and then after hIL-4 withdrawal, cells were stimulated with either anti-TCR and anti-CD28 mAbs, or Ag. The IL-4-pretreated T cells failed to induce IL-4-producing cells upon restimulation with anti-TCR mAb (Fig. 1C). When T cells from hIL-4R{alpha} Tg, hIL-4R{alpha} Tg/DO11.10 double Tg, or hIL-4R{alpha}/mIL-4R{alpha} KO/DO11.10 mice were initially stimulated with TCR cross-linking or OVA in the absence of hIL-4, T cells differentiated no or few Th2 cells. Th2 cells first appeared when hIL-4 was added between 0 and 36 h after initial TCR stimulation (Fig. 1C). However, their frequency rapidly declined thereafter. At 60~72 h after initial TCR stimulation, the frequency of IL-4-producing Th2 cells was drastically reduced in both initial TCR cross-linking and antigenic stimulation (Fig. 1C). These results demonstrate that TCR activation is needed before IL-4 signaling to induce Th2 differentiation, and that the IL-4 signal is effective 0~48 h after initial TCR activation.

However, we cannot exclude the possibility that an unskewed population, which has remained at day 7, could subsequently differentiate into Th2 cells. To investigate this possibility, hIL-4R{alpha} Tg CD4+ T cells that had been treated with hIL-4 at 0, 36, or 72 h were constitutively restimulated with Ag and hIL-4 at weekly intervals, and their differentiation profile was determined after 1, 2, or 3 wk. In cells that had been treated with hIL-4 at 0 or 36 h, the frequency of Th2 cells increased during the 3-wk restimulation (Fig. 2). In contrast, in cells that had been treated with hIL-4 at 72 h, Th2 differentiation was impaired, and consecutive Ag and hIL-4 restimulation did not restore it (Fig. 2), although these T cells retained hIL-4 responsiveness in a proliferation assay (data not shown). These results suggest that CD4+ T cells lost their capability to differentiate into Th2 cells during initial TCR activation.



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  		FIGURE 2. CD4+ T cells from hIL-4R Tg were stimulated by anti-TCR and anti-CD28 mAbs in the presence of anti-mIL-4 mAb. hIL-4 was added 0, 36, and 72 h after initial activation. These precultured cells were repeatedly stimulated with anti-TCR and anti-CD28 mAbs in the presence of hIL-4 at weekly intervals. Cells were restimulated with anti-TCR mAb, and the cytokine production profile was assessed by intracellular cytokine staining. The experiment was independently performed three times with similar results.

 
Kinetics of Th2-specific chromatin remodeling at conserved noncoding sequence-1 and -2 on IL-4 locus was related to the requirement of the IL-4 signal

A conformational change in chromatin structure to allow access to the transcriptional factors is critical for cell lineage commitment (21, 23). To understand the behavior of chromatin at the IL-4 locus during Th2 differentiation, nuclear fractions were collected at different times after primary stimulation, and treated with restriction enzymes that cut in each regulatory region, the conserved noncoding sequence-1, conserved noncoding sequence-2, and IE (Fig. 3A). CD4+ T cells from DO11.10 Tg mice were stimulated with OVA in the presence of APCs, and 48 h later, Th2-specific alterations in the chromatin structure were observed in the conserved noncoding sequence-1 and -2 regions and IE (Fig. 3B).



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  		FIGURE 3. A, Position of conserved noncoding sequence-1, IE, and conserved noncoding sequence-2 region on the IL-4 locus (upper panel). Restriction enzyme site and probe for the detection of conserved noncoding sequence-1, IE, and conserved noncoding sequence-2 (lower left). HSS1~3 and HS I~V indicate DNase I-hypersensitive site. B, Nuclei were prepared from naive CD4+ T, Th1, and Th2 cells (5 x 106 cells) and digested with XhoI for conserved noncoding sequence-1, PstI for IE, or HgaI for conserved noncoding sequence-2. DNA was purified and digested with either EcoRI for conserved noncoding sequence-1 or HindIII for IE and conserved noncoding sequence-2 and analyzed by Southern blotting, using probes for conserved noncoding sequence-1 or conserved noncoding sequence-2. The arrow indicates XhoI, PstI, and HgaI sites on conserved noncoding sequence-1, IE, and conserved noncoding sequence-2. CD4+ T cells from DO11.10 Tg mice were stimulated with OVA peptide and APCs in Th1- and Th2-skewing condition. Nuclei were prepared at different time points after initial T cell activation and the restriction enzyme assay was conducted, as described in A. C, CD4+ T cells from hIL-4R/mIL-4R KO/DO11.10 mice were stimulated with OVA peptide in the context of APCs. hIL-4 was added at different time points, and cells were further cultured until 7 days after the initial activation. Nuclei were prepared from the precultured cells, and the restriction enzyme assay was conducted. The experiment was independently performed three times with similar results.

 
We next examined whether the requirement for an IL-4 signal correlated with the kinetics of Th2-specific chromatin remodeling at conserved noncoding sequence-1 and -2. CD4+ T cells from hIL-4R{alpha}/mIL-4R{alpha} KO/DO11.10 mice were stimulated with Ag, and hIL-4 was added at different time points. Seven days after the initial stimulation, chromatin structure was examined as above. When hIL-4 was given 0–48 h after initial stimulation, Th2-specific remodeling was seen in the conserved noncoding sequence-1 and -2 regions; but when hIL-4 was given 72 h after initial stimulation, remodeling was absent (Fig. 3C). However, the IE that mainly acts on mast cells showed distinct profile in the remodeling. Decondensation was observed even when hIL-4 was given 72 h after initial stimulation (Fig. 3C). These results indicated that the requirement for IL-4 signaling for the competence to secrete IL-4 in restimulation strictly corresponds with the alteration of the chromatin structure at conserved noncoding sequence-1 and -2 regions.

Influence of the IL-4 signal on the expression of transcriptional factors regulating IL-4 gene expression

GATA-3 is a master regulator controlling the lineage commitment of Th2 cells (12, 14), and its expression is tightly regulated by the IL-4-mediated STAT6 activation pathway (11). Therefore, selective expression of GATA-3 occurs in committed Th2 cells only. We examined the kinetics of GATA-3, T-bet, and IL-12R{beta}2 mRNA expression. CD4+ T cells from hIL-4R{alpha} Tg/mIL-4R{alpha} KO mice were stimulated with anti-TCR/CD28 mAbs. In the absence of hIL-4, T cells predominantly differentiated into Th1 cells and no GATA-3 expression was found. When hIL-4 was added at time 0, GATA-3 expression clearly appeared from 24 h after initial TCR/CD28 stimulation (Fig. 4A). These results demonstrate that initial GATA-3 expression was regulated by the IL-4 signal. In contrast, T-bet and IL-12R{beta}2 expression kinetics were not affected by the IL-4 signal (Fig. 4A).



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  		FIGURE 4. A, Enriched hIL-4R Tg/IL-4KO CD4+ T cells were stimulated with anti-TCR and anti-CD28 mAbs in the presence or absence of hIL-4. Cells were harvested at the indicated time points after the initial activation, and the expression of IL-12{beta}2, T-bet, GATA-3, and G3PDH mRNAs was measured by Northern blotting analysis. The number indicates the analyzed time points. B, Enriched hIL-4R Tg cells were stimulated with anti-TCR and anti-CD28 mAbs in the presence of anti-mIL-4 mAb. hIL-4 was added at time 0 or 36 h after initial activation, and protein expressions of NF-AT1, NF-AT2, GATA-3, and STAT6 in cytoplasmic (left) or nuclear (right) fraction were examined by Western blotting. The levels of the transcript were quantitated with densitometry and normalized with G3PDH.

 
Other transcriptional factors or coactivators, NF-AT and c-maf, are also involved in commitment to the Th2 lineage (35, 36, 37, 38). Thus, we examined IL-4 responsiveness on the expression of these transcriptional factors. The hIL-4 Tg CD4+ T cells expressed a small amount of NF-AT2, and no NF-AT1, c-maf, or GATA-3. Following TCR stimulation, expression of NF-AT2 increased rapidly for 48 h and then declined independently of IL-4. Similarly, NF-AT1 expression increased following TCR stimulation, however, in later time points (Fig. 4B). IL-4 delayed the induction of NF-AT1 expression in the cytoplasm, and c-maf expression was not detected in the nuclear fraction (data not shown). Furthermore, IL-4 made no difference to TCR-induced dephosphorylation of NF-AT1 or its translocation to the nucleus (Fig. 4B). Together, these results suggest that the IL-4 signal did not influence the kinetics of NF-AT1 and -2 expression, or of TCR-induced nuclear localization.

Administration of hIL-4 at 0 or 36 h after initial TCR stimulation rapidly induced GATA-3 protein in cytoplasm and nuclei (Fig. 4B), again confirming IL-4-dependent GATA-3 expression at early stages of initial activation. We next studied whether the hIL-4R-mediated signal was able to induce GATA-3 expression even later, as IL-4R failed to transduce the signal in Th1 cells. Thus, we analyzed expression of GATA-3 at 72 h, in which most T cells were differentiating into Th1 cells. We also examined the expression of growth-factor independent 1 (GFI-1), which was induced by IL-4-STAT6 signaling. Both GATA-3 mRNA and protein as well as GFI-1 mRNA clearly appeared at this late time point (Fig. 5, A and B).



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  		FIGURE 5. A, Enriched hIL-4R Tg cells were stimulated with anti-TCR/CD28 mAbs (time point 0) in the presence of anti-mIL-4 mAb. hIL-4 was added at 0 or 72 h, and cytoplasmic GATA-3 and STAT6 were assessed by Western blotting. The experiment was performed three times independently with similar results. B, Enriched hIL-4R Tg/mIL-4R KO CD4+ T cells were stimulated with anti-TCR and anti-CD28 mAbs. hIL-4 was added at 0, 36, and 72 h, and expression of T-bet, GATA-3, and G3PDH mRNA was analyzed by Northern blotting. Expression of GF-1 was analyzed by RT-PCR. C, TCR-activated CD4+ T cells from hIL-4R Tg/mIL-4R KO mice were stimulated with hIL-4 at 72 h. After 5 days, cells were restimulated with anti-TCR mAb, and intracellular cytokine staining was conducted. IFN-{gamma}-producing and nonproducing cells were sorted, and expression of GATA-3 was examined by Western blotting.

 
Next, we studied whether GATA-3 protein is only expressed in the residual uncommitted population of T cells. Subsequent GATA-3 expression in Th1-committed and uncommitted T cells was examined when hIL-4 was added at 72 h. At day 7, cells were restimulated with anti-TCR mAb, IFN-{gamma}-producing or nonproducing subpopulations were sorted, and their GATA-3 protein was analyzed by Western blotting. Both Th1-committed and uncommitted T cells expressed GATA-3 to a similar extent (Fig. 5C). These results indicate that IL-4 signaling through the hIL-4R induced GATA-3 expression, even in a Th1-skewing condition.

Timing of GATA-3 expression regulates the efficiency of Th2 development

Ectopic expression of GATA-3 can mimic the function of IL-4 signal, by inducing Th2 cytokines and Th2-specific chromatin remodeling, but our results address that timing may be critical. To further investigate this, we examined whether ectopic expression of GATA-3 late in the differentiation of CD4+ T cells could result in efficient development of Th2 cells. GATA-3 and GFP were coexpressed in T cells by a bicistronic retrovirus construct (pMX-GATA3-GFP), and the proportion of IL-4-producing GFP-positive population was assessed. The mIL-4R KO T cells were stimulated with anti-TCR/CD28 mAbs, and then infected with pMX-GATA3-GFP after 24, 36, or 60 h. Infection at 24 h resulted in 9% GATA-3- and IL-4-coproducing T cells, which corresponds to ~20% of the total GFP+ cells (Fig. 6). Infection at 60 h reduced the proportion of T cells producing IL-4, to ~7% of total GFP+ cells (2.4% GFP+IL-4+ from 33.1% GFP+ cells). These results confirm that the timing of GATA-3 expression is critical for determining lineage commitment. Nevertheless, the presence of some IL-4+/GFPhigh cells after infection at 60 h indicates that even at this late stage, high levels of GATA-3 expression can induce commitment to the Th2 lineage.



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  		FIGURE 6. CD4+ T cells from mIL-4R KO were stimulated with anti-TCR and anti-CD28 mAbs, and infected with the retrovirus vector, pMX-GATA3-IRES-GFP, at different time points. At day 7 after the initial activation, cytokine production was examined by intracellular staining. The experiment was performed three times independently with similar results.

 
Relationship between cell division and lineage-specific IL-4 gene expression

Previous reports concluded that lineage-specific IL-4 gene expression is regulated by the number of cell divisions. Thus, we studied the relationship between cell division and IL-4 signaling required for Th2 lineage commitment. CFSE-labeled hIL-4R{alpha} Tg/mIL-4R{alpha} KO/DO11.10 CD4+ T cells were stimulated with OVA peptide. Cell division was profiled in a time kinetic (Fig. 7, upper panel), and the proportion of IL-4-producing cells following restimulation with anti-TCR mAb was examined at day 7 (Fig. 7, lower panel). Consistent with previous observations (26), IL-4-producing cells appeared after five generations (Fig. 7, lower panel). At 36 h poststimulation, in which hIL-4 efficiently induced Th2 differentiation (Fig. 1C), most T cells remained at the stage before undergoing into first cell division, while at 72 h poststimulation, all cells entered into successive cell division (Fig. 7, upper panel). These results suggest that IL-4 signaling may be able to instruct Th2 lineage commitment before progression to cell division.



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  		FIGURE 7. CFSE-labeled DO11.10 CD4+ T cells were stimulated with OVA peptide in the presence of BALB/c APCs, and cell division was assessed by the intensity of green fluorescence at indicated time point after initial T cell stimulation (1st). The x-axis represents the intensity of green fluorescence, with progression of cell division moving right to left owing to dilution of intracellular dye. To examine IL-4 production ability in recall response, cells were restimulated with anti-TCR mAb (2nd) after 7 days, and IL-4-producing cells were detected, as described for Fig. 1. 0–9, Indicates the number of cell divisions. The experiment was performed three times independently with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the relevance of IL-4 signaling for the regulation of Th2 differentiation. We established a Tg mouse model allowing us to separate IL-4 signaling from TCR-mediated stimulation. IL-4-mediated signaling regulates chromatin structure at the IL-4 locus and thereby influences the competence to secrete effector cytokines during Th2 differentiation. In this study, we show evidence that the initial 48 h is a critical period for Th2 commitment. This commitment depended on IL-4-induced GATA-3 expression, as we demonstrated that Th2 cells differentiated from naive T cells only, when GATA-3 was expressed at appropriate times. Furthermore, we found that IL-4-mediated lineage commitment was independent of cell division.

During lineage commitment, TCR and IL-4 signaling act synergistically on Th2-specific alterations of chromatin structure, by hyperacetylation of core histones H3 and H4 (20). TCR stimulation causes the formation of BAG or Brm associated factor (BAF) complexes with the nuclear matrix, leading to decondensation of heterochromatin (39). However, the regulatory regions of the IL-4 locus are acetylated equally in the first 24 h in both Th1- and Th2-skewing conditions (20), suggesting that initial TCR signaling promotes the decondensation of heterochromatin and the early increase of histone acetylation. Th2-specific decondensation in the regulatory regions of the IL-4 locus became visible only in a time window between 36 and 48 h (Fig. 3C). These observations suggest that if the chromatin remains condensed for long times, it eventually becomes incapable of Th2-type condensation. The major role of IL-4 signaling is to induce GATA-3 expression, and the induced GATA-3 then initiates the Th2-specific chromatin remodeling, subsequently leading to competence to secrete IL-4 upon restimulation (12). Therefore, the timing of GATA-3 expression may be critical for permissive chromatin alteration. This notion is supported by evidence that the timing of Th2-specific chromatin remodeling in the conserved noncoding sequence-1 and -2 regions matches the kinetics of the IL-4-mediated GATA-3 expression.

Previous work has examined whether cell division was necessary for the instruction of IL-4 production upon restimulation (28). It has been reported that T cells have to complete a certain number of cell cycles to become competent to secrete relatively high amounts of IL-4 upon restimulation. We confirmed this as five cell divisions were needed until substantial IL-4 production upon restimulation (Fig. 7, lower panel). However, the previous report did not clearly indicate the timing of IL-4 signaling during Th2 differentiation (28), because it ignored the possible involvement of IL-4 secreted from naive T cells. Richter et al. (29) argued that cell division is not critical for instruction into IL-4-producing cells upon restimulation, because use of L-mimosine, which blocks the cell cycle before S phase, did not affect IL-4-induced Th2 differentiation. Furthermore, they showed that coordination of TCR and IL-4R signaling is necessary for the recall production of IL-4, and that this coordination is able to control Th2 commitment for at least 1 day. Their results resemble our data using hIL-4R Tg mice, as IL-4 was able to induce a substantial number of Th2 cells, even before the cells undergo cell division. Taken together, our data suggest that the timing of GATA-3 expression, rather than cell division, is the important factor in the acquisition of competence to secrete IL-4.

The recent discovery of GFI-1 provides an alternative explanation for the role of IL-4 signaling, namely that IL-4 simply selects a subpopulation that responds to the IL-4-STAT6 signaling, rather than instructing the entire population into Th2 differentiation (4, 40). Previous reports have demonstrated that IL-4R signaling is selectively impaired in Th1-committed cells (41, 42, 43). This selective defect causes the selective expansion of IL-4-responding Th2 cells. However, in this study, we showed that the IL-4 signal introduced through hIL-4R is able to induce GATA-3 and GFI-1 expression, even in Th1-committed cells. In these cells, the expressed GATA-3 and GFI-1 expression dose not promote Th2 differentiation, suggesting that IL-4 signaling may act by instruction, rather than selection. However, further investigation will be required to clarify the role of IL-4 signaling on Th2 lineage commitment.


   Acknowledgments
 
We are very grateful to Tomomi Sekiguchi for technical assistance.


   Footnotes
 
1 This work was supported by Grant in Aid for Scientific Research and Grant in Aid for Scientific Research on Priority Areas of the Ministry of Education, Culture, Sports, Science, and Technology (Japan). F.B. is a Welcome Trust fellow for medical research in South Africa. Back

2 Address correspondence and reprint requests to Dr. Masato Kubo, Division of Immunobiology, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda City, Chiba 278, Japan. E-mail address: raysolfc{at}rs.noda.tus.ac.jp Back

3 Abbreviations used in this paper: IE, intronic enhancer; GFI, growth-factor independent; GFP, green fluorescent protein; hIL, human IL; KO, knockout; mIL, mouse IL; Tg, transgenic. Back

Received for publication October 28, 2003. Accepted for publication February 27, 2004.


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 Introduction
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 Discussion
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