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The Journal of Immunology, 2003, 171: 3627-3635.
Copyright © 2003 by The American Association of Immunologists

Identification of Novel IL-4/Stat6-Regulated Genes in T Lymphocytes1

Zhi Chen2,*,{dagger}, Riikka Lund*,{ddagger}, Tero Aittokallio§, Minna Kosonen*, Olli Nevalainen§ and Riitta Lahesmaa*

* Turku Centre for Biotechnology, {dagger} Drug Discovery Graduate School, {ddagger} Turku Graduate School of Biomedical Sciences, and § Turku Centre for Computer Science, University of Turku and Åbo Akademi University, Turku, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-4, primarily produced by T cells, mast cells, and basophiles, is a cytokine which has pleiotropic effects on the immune system. IL-4 induces T cells to differentiate to Th2 cells and activated B lymphocytes to proliferate and to synthesize IgE and IgG1. IL-4 is particularly important for the development and perpetuation of asthma and allergy. Stat6 is the protein activated by signal transduction through the IL-4R, and studies with knockout mice demonstrate that Stat6 is critical for a number of IL-4-mediated functions including Th2 development and production of IgE. In the present study, novel IL-4- and Stat6-regulated genes were discovered by using Stat6-/- mice and Affymetrix oligonucleotide arrays. Genes regulated by IL-4 were identified by comparing the gene expression profile of the wild-type T cells induced to polarize to the Th2 direction (CD3/CD28 activation + IL-4) to gene expression profile of the cells induced to proliferate (CD3/CD28 activation alone). Stat6-regulated genes were identified by comparing the cells isolated from the wild-type and Stat6-/- mice; in this experiment the cells were induced to differentiate to the Th2 direction (CD3/CD28 activation + IL-4). Our study demonstrates that a number a novel genes are regulated by IL-4 through Stat6-dependent and -independent pathways. Moreover, elucidation of kinetics of gene expression at early stages of cell differentiation reveals several genes regulated rapidly during the process, suggesting their importance for the differentiation process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Naive CD4+ T lymphocytes can differentiate into two subsets with distinct cytokine profiles and functions, Th1 and Th2. IL-12 and IL-4 are key cytokines promoting Th1 and Th2 development, respectively. Th1 cells secrete IFN-{gamma}, TNF-{alpha}, and IL-2, cytokines important for cellular immunity. Th2 cells produce IL-4, IL-5, and IL-13, which promote further Th2 development and help B cells to produce IgE and promote humoral immunity and allergic responses. The imbalance of the two Th subsets in humans is associated with the development of autoimmune and allergic diseases (1, 2, 3).

Th2 differentiation is induced by IL-4 signaling mediated through Stat6. Binding of IL-4 to its receptor (IL-4R) results in tyrosine phosphorylation of Janus kinase (Jak)3-1 and Jak-3, which further leads to the tyrosine phosphorylation of the IL-4R{alpha} chain. After binding to the phosphotyrosine docking site on IL-4R through the Src homology 2 domain, Stat6 is phosphorylated by Jak kinases. Phosphorylated Stat6, released from IL-4R, forms a homodimer and translocates to the nucleus where it binds to a specific DNA sequence and triggers the transcription of its target genes (4).

All Stats have similar DNA binding motifs, which are called IFN-{gamma}-activated sequences (4, 5). For Stat6, the specific binding sequence is TTC-N4-GAA (6, 7). Ehret et al. (8) studied the binding specificities of Stat proteins and demonstrated that Stat6 also binds well to N3 sites. These specific DNA binding sites have been found in IL-4 and IL-4R promoters (9, 10, 11, 12), the CD23 promoter (13, 14, 15, 16), and the I{epsilon} promoter (Ig, H chain germline {epsilon}) (17, 18, 19, 20). A number of genes have been demonstrated to be Stat6-mediated targets of IL-4 in various cell types. These include 12/15-lipoxygenase (21, 22), lymphotoxin {alpha} (23), angiotensinogen (24), C{gamma}1 (25), C{gamma}3 (26, 27), eotaxin (28), IL-1 receptor antagonist (IL-1ra) (29, 30), {beta}3 integrin (31, 32), MHC class II (9, 15, 16, 33), P-selectin (34), and Fig1 (35, 36). In addition to these, a number of new Stat6 target genes were recently described in B cells (37).

In T lymphocytes, plenty of data has demonstrated that Gata3 is the downstream gene of Stat6 (1). A study exploiting Stat6-deficient mice has shown that Cdk inhibitor p27kip1, dysregulated in Stat6-deficient lymphocytes, affects T cell proliferation (38). Recently, Zhu et al. (39) reported that a growth factor independent-1 (Gfi-1) was induced by IL-4 and that this induction is Stat6-dependent. However, to our knowledge a large scale screening of Stat6 target genes in T cells has not been reported. The present study was undertaken to elucidate IL-4-inducible genes and Stat6-regulated genes in murine CD4+ T cells by using Stat6-deficient mice and Affymetrix oligonucleotide array technology. As a result, numerous novel IL-4- or Stat6-regulated genes were identified.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, reagents, and cell culture

Stat6-deficient mice and control wild-type BALB/cJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Splenic mononuclear cells were isolated with Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden). CD4+ T lymphocytes were further purified by magnetic CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were activated with plate-bound anti-mouse CD3 (clone 500A2, 1.26 µg/ml; BD PharMingen, San Diego, CA) and soluble anti-mouse CD28 (500 ng/ml; BD PharMingen). Thereafter, the cells were cultured in IMDM containing 10% FCS, nonessential amino acids, and 2-ME (all from Life Technologies, Paisley, Scotland). Recombinant mouse IL-12 (10 ng/ml; R&D Systems Europe, Abingdon, U.K.) and anti-IL-4 (11B11, 10 µg/ml; BD PharMingen) were added in cultures set for Th1 polarization. In the cultures with cells driven to Th2 direction, recombinant mouse IL-4 (10 ng/ml; BD PharMingen) anti-IL-12, and anti-IFN-{gamma} (both are 10 µg/ml; BD PharMingen) were added. CD4+ T cells pooled from different mice were cultured in conditions favoring Th0 (with CD3/CD28 activation and anti-IL-4, anti-IL-12, and anti-IFN-{gamma}) or in conditions promoting Th1 or Th2 differentiation. Cells were harvested at 0, 2, 6, 24, and 48 h.

Affymetrix oligonucleotide array analysis

Total RNA was extracted by TRIzol (Life Technologies) and was further purified with an RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized by a Superscript II kit (Life Technologies) using T7-(dT)24 as primer. Biotin-labeled cRNA was prepared by in vitro transcription reaction using a BioArray HighYield RNA Transcript Labeling kit (Enzo Diagnostics, Farmingdale, NY) based on the manufacturer’s protocol. The cRNA was purified, fragmented, and hybridized to Affymetrix MG-U74A GeneChips (Santa Clara, CA). Chips were stained and scanned according to Affymetrix protocols.

The data analysis was performed according to instructions and recommendations provided by Affymetrix. The Affymetrix statistical data analysis software, Affymetrix Microarray Suite (version 5.0), was used for the processing of results. For each gene, the levels in gene expression of cells treated in different ways were compared. The comparisons were based on a statistical analysis of probe sets consisting of 16 oligos recognizing different portions of the target gene. As recommended by Affymetrix, the probe sets were excluded if the detection call for both target and reference was absent; or if the change call gave no change (NC) in comparison analysis or if the signal log ratio between target and reference was between -1 and 1. Signal log ratio is used to describe the change between a target and reference array. The change is expressed as log2 ratio. Therefore, signal log ratio of 1 equals a fold change of 2. To identify the Stat6 target genes, only the genes that fulfilled the filtering criteria in at least two of three to five biological repeats were considered significant. Because there were five independent cell cultures to study IL-4-inducible genes at 48 h, a t test was also used to compare the signals between target (CD3/CD28 activation + IL-4) and reference (CD3/CD28 activation) groups. In statistical testing, the error risk p < 0.05 was considered significant. Kinetic data for 2, 6, and 24 h was studied from one culture.

Real-time quantitative PCR (TaqMan) detection

The principle of TaqMan detection has been described previously (40, 41). Primers and probes for TaqMan detection were designed by Primer Express software (Applied Biosystems, Foster City, CA) and made by MedProbe (Oslo, Norway). The sequences for the primers and probes are listed in Table I. Samples from three independent cultures were measured in duplicate in two separate runs. The SD of individual measurements had to be <0.5. Cycle threshold (CT) value means the number of PCR cycles required for the detection of fluorescence signal to exceed a fixed threshold. The relative levels of gene expression of target mRNA were normalized against elongation factor (EF)1{alpha}:


Here, {Delta}CT2 represents the average of {Delta}CT from not treated (time point 0 h) wild-type (wt) or Stat6-/- CD4+ T cells and {Delta}CT1 represents the average of {Delta}CT from a given time point. With this calculation, basically {Delta}{Delta}CT equals to log2 ratio, therefore value 1 of {Delta}{Delta}CT corresponds to a 2-fold change. Genes which showed over a 2-fold change in more than one biological repeat were considered significant. The experiments were performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems).


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  		Table I. TaqMan primers and probes

 
Immunofluorescence staining and FACS analysis

CD4+ T cells from the wt and Stat6 -/- mice were activated and primed to polarize to Th2 direction. At 48 h, samples were collected and stained as described elsewhere. Briefly, cells were washed with PBS and then stained with FITC-conjugated rat anti-mouse Ly-6A/E mAb (BD Biosciences, San Diego, CA) or with isotype control Ab at 4°C for 20 min. Cells were washed twice and then analyzed by FACScan with CellQuest software (BD Biosciences, San Jose, CA). At least 10,000 cells were analyzed per each sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4-regulated genes

To identify genes regulated by IL-4, CD4+ T lymphocytes from the wt BALB/cJ mice polarized to Th2 direction (CD3/CD28 activation + IL-4) were compared with the CD4+ T cells activated with CD3/CD28 alone. As shown in Fig. 1A, from five independent cell cultures, 117 probe sets (116 known genes) were regulated by IL-4, 26 of them up-regulated and 91 down-regulated. The differentially expressed genes were divided into six groups, genes belonging to or involved in 1) immune response and related, 2) transcription regulation and DNA binding, 3) enzymes and inhibitors, 4) apoptosis and cell proliferation, 5) cell surface and structural proteins, and 6) miscellaneous. The genes regulated by IL-4 in T cells included a panel of genes previously reported to be IL-4-induced, including IL-4, IL-1Rl1 (T1/ST2, ST2L) (42), Nfil3 (36, 37), Gfi1 (39), Hipk2 (37), Casp6 (37), Gata3 (1), and Protein S (43). In addition to these, there were 73 genes (marked with # in Fig. 1) that at least to our knowledge have not been previously reported to be regulated by IL-4 early in polarizing CD4+ T cells.



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  		FIGURE 1. A, IL-4-inducible genes in murine CD4+ T cells. CD4+ T cells isolated from the wt mice were activated with plate-bound anti-CD3 and soluble anti-CD28. IL-12 and anti-IL-4 were added to cells induced to polarize to Th1, whereas IL-4, anti-IL-12, and anti-IFN-{gamma} were added to cells induced to polarize to Th2 (as described in Materials and Methods). The data is based on five independent cell cultures (48 h) analyzed by Affymetrix MG-U74 GeneChips. Data analysis was done as described in Materials and Methods. IL-4-inducible genes were identified by comparing the results obtained using wt CD4+ T cells induced to Th2 direction (CD3/CD28 activation + IL-4) to the results obtained with cells activated by CD3/CD28 only. {blacksquare}, The genes up-regulated by IL-4; {square}, the genes down-regulated by IL-4. *, Genes induced by IL-4 and regulated via Stat6. #, The genes that to our knowledge are for the first time in this paper described as IL-4-regulated genes in CD4+ T cells. B, Stat6-regulated genes in murine CD4+ T cells. CD4+ T cells isolated from the wt or Stat6-/- mice were activated and cultured as described in Materials and Methods. The data are based on three independent cell cultures. Data analysis was done as described in Materials and Methods. Stat6-regulated genes were identified by comparing IL-4-induced cells (CD3/CD28 activation + IL-4) isolated from Stat6-/- mice to those isolated from the wt mice. {blacksquare}, Genes up-regulated; {square}, those down-regulated when Stat6 is deficient. *, Genes induced by IL-4 and regulated via Stat6. #, The genes that to our knowledge are for the first time in this paper described as IL-4-regulated genes in CD4+ T cells. C, Genes induced by IL-4 and regulated by Stat6 in murine T lymphocytes. The yellow circle indicates the number of IL-4-inducible genes, whereas the blue one indicates the number of Stat6-regulated genes. Genes induced by IL-4 and regulated via Stat6 are in green. The expressed sequence tags were not included in the numbers. D, IL-4-inducible and Stat6-regulated genes. Summary of the 20 genes identified as induced by IL-4 and regulated via Stat6. , How genes are expressed in Stat6-/- cells as compared with wt cells; both cell types were induced to polarize to Th2 direction. The genes induced by IL-4 (by comparing IL-4 + activation vs activation alone) in wt and Stat6-/- CD4+ T cells cultured for Th2 polarizing are shown as {blacksquare} and {square}, respectively.

 


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  		FIGURE 1A. Continued.

 
Stat6-regulated genes

As T cells from Stat6-/- mice are deficient in their ability to polarize to Th2 direction (9, 15, 16), we next wanted to identify the genes regulated by Stat6. We isolated both Stat6-/- and wt CD4+ T cells and activated and cultured the cells for 48 h in Th2 polarizing conditions. At 48 h, cells were harvested; RNA was isolated, labeled, and the Affymetrix experiments were conducted. Stat6-regulated genes were identified by comparing the Affymetrix data obtained from IL-4-induced (CD3/CD28 activation + IL-4) cells isolated from Stat6-/- and wt cells. As shown in Fig. 1B, 37 probe sets (36 known genes) were differentially expressed. Seventeen (including Stat6) of 37 probe sets were down-regulated in Stat6-deficient mice, whereas 20 of the 37 genes were up-regulated. Within these 37 genes, 20 were induced by IL-4 (marked with * in Fig. 1). The genes included some known Stat6 target genes, such as IL-4 and Gata3. Interestingly, Hipk2 and Nfil3, recently reported to be Stat6 target genes in B cells (37), appeared to be regulated by Stat6 also in CD4+ T cells.

It was of special interest that compared with their expression in the wt cells, a set of genes was expressed at a higher level in the cells deficient for Stat6 when the cells were cultured in Th2 polarizing conditions. Within this set of genes there were many known to be expressed primarily in Th1 cells or to be induced by IL-12/IFN-{gamma} (including IL-18R1, CCR5, Gzmb, Ly6a, and Txk).

Twenty of 37 Stat6-regulated genes were induced by IL-4, whereas only 20 of 117 IL-4-regulated genes were also regulated via Stat6 (Fig. 1C). Hipk2, Pros1, Gata3, Gp49a, Gpm6b, Zfp118, Nfil3, and Prnp belonged to the genes that were up-regulated by IL-4 and this up-regulation was prevented in Stat6-deficient mice. In contrast, Mov10, Ppicap, Trim30, Isg15, Gzmb, Tgtp, Serpinb6, Ifi203, Adam19, Ifi205, IL-18R1, and Isg20 belonged to another group whose expression was down-regulated by IL-4. Interestingly, the IL-4-induced down-regulation of these genes was blocked in Stat6-/- mice (Fig. 1D). To examine the possibility that the difference in gene expression derived from the developmental effects due to Stat6 deficiency, the comparison of gene expression profiles of CD4+ T cells (Thp cells) between wt and Stat6-/- mice was done. From three repetitions, there were only 10 genes that were differentially expressed, including Lzp-s, Pira1, Scyb2, and Stat6. Therefore, it is likely that signaling pathways other than those regulated by Stat6 are important for the regulation of gene expression by IL-4 (4).

Kinetic study of IL-4- and Stat6-regulated genes

To find out the direct and secondary effects of IL-4, we next studied the early kinetics of gene expression focusing on the genes regulated by IL-4 and Stat6. Fig. 2A shows only the genes regulated by IL-4 at more than one time point. At the early time points (2 and 6 h), two novel IL-4-regulated genes, Polg and Prnp, were identified. Also genes previously known to be regulated by IL-4 were observed; these included, e.g., Atf3 (44), Bcl2, and Cish (37). IL-4ra (10), Prnp, Gata3, and Pros1 were induced by IL-4 at both early (2 and 6 h) and late (24 and 48 h) time points. Some genes were induced by IL-4 mostly at late time points, e.g., IL-4, Casp6, and Ahr (Fig. 2, A and C). Most of the genes previously described as Th1 marker genes or IFN-inducible genes, such as IFN-{gamma}, Ifi205, Ifi203, Isg20, IL-18r1, Ifi204, Ifit1, and IL-12Rb2 were regulated at later time points i.e., at 24 or 48 h (Fig. 2, A and B).



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  		FIGURE 2. Kinetic study of IL-4-inducible genes and Stat6-regulated genes. CD4+ T cells were activated and cultured as in Fig. 1. RNA was isolated from cells harvested at 2, 6, 24, and 48 h and Affymetrix hybridization and data analysis were conducted as described in Materials and Methods. A, Kinetics of IL-4-inducible gene expression. In this study, we present only data on the genes whose expression was influenced by IL-4 in at least two or more time points. B, Kinetics of IL-4-down-regulated genes at 48 h. C, Kinetics of IL-4-up-regulated genes at 48 h. The genes were identified by comparing IL-4-induced wt cells (CD3/CD28 activation + IL-4) to those activated by CD3/CD28 alone. D, Kinetics of Stat6-regulated genes. Here, we present only data on the genes whose expression was influenced by Stat6 in at least two or more time points. E, Kinetics of Stat6-regulated genes at 48 h. Stat6-regulated genes were identified by comparing IL-4-induced cells (CD3/CD28 activation + IL-4) isolated from the Stat6-/- mice to those isolated from the wt mice. Red indicates genes up-regulated and green indicates genes down-regulated by Stat6. The genes were grouped into six categories: 1, genes involved in immune response and related; 2, genes involved in transcriptional regulation and DNA binding; 3, enzymes and inhibitors; 4, genes related to apoptosis and cell proliferation; 5, cell surface and structure proteins; 6, miscellaneous.

 
Based on this study, among the IL-4-inducible genes, there are 12 genes regulated by Stat6 at more than one time point. As shown in Fig. 2, D and E, these genes are IL-4, Prnp, IL-4ra, Fig1, Hipk2, Gata3, Zfp118, Nfil3, Pros1, Casp6, IL-18R1, and Ifi203. From early time points, i.e., 2 and 6 h, up to the later time points, i.e., 24 and 48 h, only 6 of the 12, IL-4, Prnp, IL-4ra, Pros1, Casp6 and IL-18r1, remained differentially expressed. Again, we found it very interesting that there were many genes that were up-regulated in Stat6-deficient T cells cultured in Th2 polarizing conditions ascompared with wt cells. Many of the genes that belong to this category are genes induced by IFN.

TaqMan and FACS analysis confirms the microarray results

To verify the findings obtained by the microarrays, selected genes were further studied by real-time quantitative RT-PCR. Based on Affymetrix results, Hipk2, Nfil3, Zfp118, and Atf3 were induced by IL-4 and were regulated by Stat6. In contrast, IL-4-induced inhibition of Ifi203 gene expression was mediated through Stat6. Moreover, at 48 h of culture under Th2 condition, Txk was expressed at a higher level in Stat6-deficient cells compared with wt cells.

To measure gene expression, CD4+ T cells were isolated from the wild-type and Stat6-deficient mice and three independent in vitro T cell polarization cultures were established. Kinetics of gene expression for the selected panel of genes was measured using TaqMan. The differences of gene expression which showed over a 2-fold change in more than one independent cell cultures were considered significant and were shown as "*" (differences in IL-12- or IL-4-treated cells) and "#" (differences induced by IL-4 comparing wt and Stat6-/- cells) in Fig. 3A.



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  		FIGURE 3. Verification of microarray results. A, A panel of genes was selected based on the microarray results to be verified by quantitative RT-PCR analysis. CD4+ T cells were isolated from the wt or Stat6-/- mice and were activated and cultured for indicated time points as described in Materials and Methods. Data shown is the average {Delta}{Delta}CT of three independent cell cultures. *, A significant difference in gene expression between CD4+ T cells polarized to Th1 or Th2 direction. #, A significant difference between wt and Stat6-/- CD4+ T cells polarized to Th2 direction. The primers and probes used to detect Hipk2, Nfil3, Zfp118, Atf3, Ifi203, and Txk are listed in Table I. B, Ly6a protein expression is regulated by Stat6. CD4+ T cells were activated and induced to polarize to Th2 direction. At 48 h, cells were harvested and stained with anti-Ly6A/E-FITC or isotype-specific control Ab. All the samples were analyzed with FACScan and at least 10,000 cells were analyzed for each sample. Representative results from three independent experiments are shown.

 
In CD4+ T cells isolated from the wt mice, the expression of Hipk2 was rapidly (at 2 h) induced by IL-4. The difference in Hipk2 gene expression between the cells induced to polarize to Th1 or Th2 direction reached its peak at 24 h and the difference remained until 48 h. Stat6 deficiency clearly inhibited the IL-4-induced up-regulation of Hipk2 and this effect was observed at all the time points studied. Therefore, we conclude that Hipk2 is most likely a primary target gene of Stat6. This conclusion is further confirmed by results obtained from cells treated with cycloheximide (CHX). The Hipk2 expression was induced by IL-4 also in CHX-treated cells, indicating that the inhibitor of new protein synthesis did not influence the results (data not shown).

A similar mode of regulation of gene expression was also seen for Nfil3 and Zfp118. Atf3 was also rapidly induced by IL-4 and already at 2 h this gene was differentially expressed in cells driven to Th1 or Th2 direction. The difference in gene expression during the early stages of differentiation vanished at 48 h. Moreover, the data indicates that IL-4-induced regulation of Atf3 is mediated through Stat6. In contrast, Ifi203 belongs to the group of genes whose expression was down-regulated by IL-4. In the absence of Stat6, the expression of Ifi203 was 14-fold higher (at 48 h) compared with the level observed in the cells isolated from the wt mice. The mode of regulation of Ifi203 suggested it to be a secondary target for Stat6. This conclusion was further supported by the data obtained using CHX-treated cells (data not shown).

Txk has been previously described as a Th1 cell-specific transcription factor, which regulates IFN-{gamma} gene transcription (45). Our Affymetrix data showed that its expression level was higher in Stat6-deficient cells compared with wt cells. As shown in Fig. 3A, in wt CD4+ T cells, Txk was significantly down-regulated by IL-4 and to a lesser extent by IL-12 during the first day of polarization. The Txk expression returned to the level similar to what it was at time 0 h in cells cultured in Th1 condition. In contrast, the expression of Txk remained down-regulated in cells cultured in Th2 condition. As a result, at 48 h, the expression of Txk in cells cultured in Th1 condition was significantly higher than that in cells cultured in Th2 condition. In Stat6-/- cells, the down-regulation of Txk expression in Th2 polarizing condition was prevented. At 24 and 48 h, the expression levels of Txk were significantly higher in Stat6-/- cells than in wt cells polarized to Th2 direction.

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation (46). The expression of Ly-6A/E is up-regulated in normal murine T and B cells by IFN-{gamma} (47). We next wanted to verify our findings on the regulation of Ly-6A/E by IL-4 and Stat6 at the protein level. The Ly6a protein on IL-4-polarized cells was stained with FITC-conjugated rat anti-mouse Ly-6A/E mAb and its expression was detected by FACScan. As expected based on Affymetrix results, at 48 h the protein expression of Ly6a was higher in Stat6-deficient IL-4-induced cells as compared with cells isolated from wt mice (Fig. 3B). This confirmed that Stat6 has a role in regulating IL-4-induced down-regulation of Ly6a.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we describe a number of genes that are regulated by IL-4 and Stat6 in T cells at early stages of their differentiation to Th2 cells. At 48 h after the induction of polarization, 73 novel IL-4-inducible genes and 18 novel Stat6-regulated genes were identified. Twenty genes identified both as IL-4-inducible and Stat6-regulated are summarized in Table II. The expression of selected genes identified by Affymetrix was confirmed by real-time quantitative PCR and FACS detection. To our knowledge, this is the first reported large-scale study addressing the role of IL-4/Stat6 in the early kinetics of gene expression in polarizing CD4+ T cells.


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  		Table II. IL-4/Stat6-regulated genes

 
We found it interesting that there were several genes that were expressed at a higher level in Stat6-deficient cells as compared with their expression in the wt cells when the cells were cultured in Th2 polarizing conditions. Many of these genes have previously been described to be IFN-induced, (such as Isg20, Ifi205, Ifi203, Isg15, and Isg20), or characteristic for Th1 cells. It has been reported that IL-4 down-regulates IL-12R and IL-18R expression (48, 49) and inhibits the production of IFN-{alpha}{beta} and IL-18 in response to virus infections in macrophages (50). However, the molecular mechanisms behind this negative regulatory effect of IL-4 have not been well-characterized. Previous studies have shown that Stat6 mainly binds to TTC-N4-GAA motif, but it can also bind to N3 sites, which are the preferred sites for Stat1 and Stat5 (8). Furthermore, it has been previously demonstrated that IL-4 can inhibit IFN-{gamma}-inducible gene expression by Stat6 competing with Stat1 for site occupancy or by interacting with some undefined transcriptional coactivator. Stat6 transcriptional activation domain appears to be essential for the inhibition of IFN-{gamma}-inducible gene expression (51, 52, 53). In conclusion, for a set of genes described in this study, Stat6 is required for the down-regulation of their gene expression.

Recently Schroder et al. (37) published a study of Stat6-regulated genes in B cells. Compared with our results, there are some genes regulated by Stat6 both in T and B cells. Such genes include Hipk2, Nfil3, Cish, Casp6, and Tgtp. The fact that these genes are regulated by Stat6 in both types of lymphocytes makes them particularly interesting for studies aiming at understanding the pathways leading to key factors mediating allergic inflammation, namely Th2 differentiation and IgE production.

One of the most interesting among these for further studies is Hipk2, a nuclear serine/threonine kinase that belongs to the family of corepressors for homeodomain transcription factors (54). Hipk2 is modified by a small ubiquitin-related modifier 1 and the modification correlates with its localization to nuclear speckles (55). It has been shown that Hipk2 can phosphorylate p53 at Ser46 and can cooperate with p53 in the activation of p53-dependent transcription and apoptosis (56, 57). Moreover, it has been demonstrated that Hipk2 can associate with TNFR1-associated death domain protein in the absence of death factor receptors (CD95 and TNF-R1) (58). Based on our data, Hipk2 is clearly an IL-4-inducible gene regulated via Stat6.

Another gene of interest is Nfil3 (also called E4 promoter-binding protein (E4bp4)). It belongs to the basic region/leucine zipper transcription factor superfamily. Nfil3 was initially identified by screening a {lambda}gt11 cDNA library of PHA-stimulated human T cells with a probe containing regulatory sequences of the IL-3 promoter. Nfil3 was identical to adenovirus E4BP4. It can specifically bind to regulatory sequences both in the adenovirus E4 promoter and human IFN-{gamma} promoter (59). It is induced by the IL-3 signaling pathway and plays an important role in promoting cell survival activity of IL-3 (60). Previous study has shown that Nfil3 is one of the genes induced by IL-4 treatment in B cells (36). It has also been reported that Gata1 and Gata2 proteins can bind to E4bp4 promoter and this binding is required for E4bp4 gene transcription (61). Our data indicate that Nfil3 is both induced by IL-4 and regulated by Stat6 in T cells. Further work is required to characterize the precise role of IL-4/Stat6-regulated genes for T cell differentiation or the IL-4 signaling pathway.

Chtanova et al. (62) reported a study on differential gene expression in CD4+ and CD8+ type 1 and type 2 T cells. Both the mouse strain and the cell culture conditions used in that study differ from those used in our experiments. Moreover, their study elucidated T cells at their later stages of differentiation, whereas ours focused on the early stages. Despite these differences in the experimental design, genes such as IL-4, Gata3, Nfil3, IL-12rb2, IFN-{gamma}, IL-18r, Gp49a, Gp49b, Pim2, and Crabp2 were found to be IL-4-regulated in both studies.

Our study indicates that there are more IL-4-inducible genes than Stat6-regulated genes at 48 h of culture. The most likely explanation is that these genes are regulated by IL-4 via Stat6-independent pathways. Moreover, at this time point (48 h), gene expression is already affected by the primary target genes of Stat6.

In conclusion, this study revealed a number of new early immediate genes regulated by IL-4 and Stat6. Studies in progress will further elucidate their role and importance in mediating the biological effects of this signaling pathway so important in the development of allergic inflammation.


   Acknowledgments
 
We gratefully acknowledge Marju Niskala, Miina Miller, and Arja Reinikainen for their technical assistance in this work. We thank Elizabeth Carpelan for language reviewing of the manuscript.


   Footnotes
 
1 This work was supported by the Academy of Finland and Turku University Hospital Research Fund. Back

2 Address correspondence and reprint requests to Dr. Zhi Chen, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, P. O. Box 123, FIN-20520, Turku, Finland. E-mail address: zhi.chen{at}btk.utu.fi Back

3 Abbreviations used in this paper: Jak, Janus kinase; IL-1ra, IL-1 receptor antagonist; Gfi, growth factor-independent; CT, cycle threshold; EF, elongation factor; wt, wild type; CHX, cycloheximide. Back

Received for publication February 19, 2003. Accepted for publication July 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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