IL-6 Plays a Unique Role in Initiating c-Maf Expression during Early Stage of CD4 T Cell Activation
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免疫学杂志 2005年第5期
Abstract
The transcription factor c-Maf plays a critical and selective role in IL-4 gene transcription. Little is known about the mechanism that guides c-Maf regulation during early T cell activation. We report that IL-6 but not IL-4 or other cytokines, rapidly up-regulates c-Maf transcription, as early as 3 h after TCR activation in naive CD4+ T cells. c-Maf induction requires both IL-6- and TCR-initiated signals, and is independent of IL-4/Stat6 signals. Cyclosporin A and FK506, which target calcineurin and thereby inhibit TCR-mediated Ca2+ signal pathways, block IL-6-mediated c-Maf expression. We show that Stat3 binds the c-maf promoter in CD4 T cells after IL-6 stimulation, and also transactivates the c-maf promoter in reporter gene assays. IL-6 induces similar c-Maf expression in protein kinase C-deficient CD4+ T cells. Furthermore, IL-6 enhances IL-4 gene expression very early after TCR activation in both wild-type and Stat6-deficient CD4+ T cells. Our findings suggest that IL-6 plays a unique role in initiating c-Maf expression after TCR engagement, and may subsequently regulate early IL-4 production and Th2 commitment.
Introduction
The immune system protects the host from a vast array of pathogens by evolving diverse effector mechanisms, which are optimally effective for the elimination of different types of infections. CD4+ T cells play a critical role in determining the outcome of any given antigenic stimulation. Two major subsets of Th cells have been characterized, and these cells direct ongoing immune responses through the secretion of different patterns of cytokines (1, 2). It is known that although the presence of IL-4 at the time of CD4 T cell activation is pivotal to Th2 differentiation, the cellular and molecular mechanisms that guide early IL-4 production remain less understood. Several innate immune cells, such as NK, T cells, NK1.1+ T cells, and mast cells, also produce IL-4 (3, 4) but none appear essential for Th2 development, as Th2 cells can differentiate from naive T cells independently of IL-4 produced by non-T cells or T cells. These findings suggested that non-IL-4 signals may also play important roles in directing Th2 development, and that naive T cells are capable of autonomous IL-4 production.
c-Maf was the first Th2-specific transcriptional factor identified (5). Overexpression of c-Maf using a transgenic approach showed the critical role of c-Maf in controlling IL-4 production in vivo, and ectopic expression of c-Maf in mature Th1 cells inhibits IFN- production and attenuates Th1 differentiation (6). Research on c-maf-deficient mice showed that CD4+ T cells and NK T cells from c-maf–/– mice were markedly deficient in IL-4 but not other Th2 cytokine (IL-5, IL-6, IL-10, or IL-13) production, and demonstrated that c-Maf has a critical and selective function in IL-4 gene transcription in vivo (7). In naive T cells, c-Maf is expressed at very low levels and little is known about how it is regulated. IL-4/Stat6 signals have been implicated in c-Maf up-regulation, and ectopic expression of activated Stat6 positively regulates c-Maf expression (8, 9). Recent studies in ICOS–/– CD4 T cells or with B7h-deficient APCs used to activate T cells have also suggested that interactions between ICOS and its ligand B7h are important in regulating c-Maf expression, possibly through IL-4 gene regulation (10, 11). GATA-3 (12), another Th2 specific transcription factor, has also been shown to augment c-Maf expression when overexpressed in CD4+ T cells (13). In addition, one report shows that a truncated c-maf inducing protein, Tc-mip, is able to up-regulate c-Maf expression when overexpressed in Jurkat T cells (14). Although these experiments measured c-Maf induction in later phases of T cell activation, the pathways and controlling elements directing initial c-Maf expression in early stages of T cell activation have not been reported.
Produced by both professional APCs (B cells, macrophages, and dendritic cells) and nonprofessional APCs (epithelial, endothelial, and some tumor cells), IL-6 plays important roles in T cell-mediated immune responses. It is a cofactor for T cell proliferation and prevents cell death (15, 16, 17). IL-6 also regulates polarization of naive CD4 T cells toward the Th2 phenotype. It inhibits Th1 differentiation by inducing suppressor of cytokine signaling (SOCS)3 1, and promotes Th2 differentiation by up-regulating NFAT1 expression (18, 19, 20). In the present work, we investigated the transcriptional mechanisms that regulate IL-6-mediated IL-4 gene expression. We found IL-6 to be unique in rapidly inducing c-Maf expression after TCR activation. c-Maf regulation by IL-6 is IL-4/Stat6 signaling-independent, but TCR-mediated Ca2+ signaling-dependent. Our data suggest a novel role for IL-6 in directing c-Maf initiation and IL-4 gene regulation.
Materials and Methods
Mice, cytokines, Abs, and other reagents
BALB/c, C57BL/6, B10.BR, OT-2 transgenic mice specific for OVA323–339, IL-4 knockout (BALB/c background), Stat6 knockout (BALB/c background), and IL-6 knockout (C57BL/6 background) were purchased from The Jackson Laboratory, and AND TCR transgenic mice specific for cytochrome c-I-Ak (21) were provided by Dr. S. M. Hedrick (University of California, San Diego, La Jolla, CA). Protein kinase C (PKC) mice (22) were provided by Dr. D. R. Littman (New York University Medical Center, New York, NY). Recombinant cytokines IL-4, IL-6, IL-10 were purchased from BD Pharmingen; IL-11, oncosatin M, and leptin were purchased from R&D Systems. Anti-IL-4, anti-CD3, anti-CD28, anti-CD4, and anti-B220 mAbs, and PE-labeled anti-IL-6R and anti-IL-10R were purchased from BD Pharmingen. Recombinant mouse soluble CD40L was purchased from PeproTech. Cyclosporin A (CsA), PMA, ionomycin, and LPS were purchased from Sigma-Aldrich. FK506 was purchased from A. G. Scientific. Anti-c-Maf (catalog no. sc-7866) was purchased from Santa Cruz Biotechnology. Anti-Stat1, Stat3, and anti-phosphorylated Stat3 were purchased from Cell Signaling Technology. CD11c microbeads were purchased from Miltenyi Biotec. Dynabeads mouse pan B (B220) beads were purchased from Dynal Biotech. Plasmid expressing a constitutively active Stat3 (Stat3C) was kindly provided by Dr. J. Darnell (The Rockefeller University, New York, NY). Promoterless luciferase reporter plasmid, phRL-null, and Renilla luciferase assay kit were purchased from Promega. The mouse fibroblast cell line C3H10T1/2 was obtained from the American Type Culture Collection. Chromatin immunoprecipitation (ChIP) assay kit was purchased from Upstate Biotechnology.
CD4 T cell, B cell, and DC purification and activation
Splenocytes from various strains of mice were purified and B cells were depleted by using Dynabeads mouse pan B, and then CD4+ T cells were purified by cell sorting by MoFlo (Cytomation) after staining with FITC anti-CD4. After sorting, cell purity is 99%. Cells were seeded into 96-well round-bottom plates coated with anti-CD3 mAb (5 μg/ml) at 2 x 104 cell/well, were then further activated with soluble anti-CD28 mAb (1 μg/ml), with or without IL-6 (20 ng/ml) or other cytokines (20 ng/ml). In APC-dependent assays, pigeon cytochrome c (PCC) peptides (provided by Dr. B. Murphy, Mount Sinai School of Medicine, New York, NY) and OVA peptides (OVA323–339) purchased from American Peptide at 0.5 μg/ml or 5 μg/ml were added. B cells were purified by cell sorting after staining with FITC anti-B220 from B10.BR splenocytes (after sorting, cell purity is 96%), and treated with mitomycin C (50 μg/ml; Sigma-Aldrich), for 50 min at 37°C. Dendritic cells were purified from B10.BR mice or C57BL/6 by CD11c microbeads per the manufacturer’s manual. Following cell sorting, the purity is above 98%. For dendritic cell maturation and activation, cells were cultured at 1 x 106/ml in 24-well plates, and stimulated with 500 ng/ml CD40L and 1 μg/ml LPS for 48 h.
ELISA and proliferation assays
ELISA for IL-4, IFN-, or IL-6 was performed using the OptEIA mouse IL-4, IFN-, or IL-6 set (BD Pharmingen) according to the manufacturer’s protocol. Absorbance was measured by a Spectra Max 250 (Molecular Devices) plate reader. For proliferative assays, purified CD4+ T cells (2 x 104) and mitomycin C (50 μg/ml) treated B cells (1 x 104) were incubated with PCC peptides (0.5 or 5 μg/ml) in a round-bottom 96-well plate with various cytokines, and then pulsed with 1 μCi [3H]thymidine for the last 16 h of a 3 day culture.
Western blot analysis
Protein extracts were prepared from various groups and concentrations were determined by BCA assay (Pierce) as previously described (23). Proteins were then separated by 16% (for c-Maf) or 7.5% (for Stat3) SDS-polyacrylamide and transferred to polyvinylidene difluoride membranes to probe with anti-c-Maf (1/200 dilution), anti-phosphorylated Stat3 (1/1000 dilution), anti-Stat3 (1/1000 dilution) subsequently incubated with HRP-conjugated anti-rabbit IgG and detected by the ECL system (Amersham).
RT-PCR
Total RNA was isolated from cultured cells using TRIzol following the manufacturer’s instructions (Invitrogen Life Technologies). Reverse transcription was conducted using Omniscript Reverse Transcriptase (Qiagen) and random primers as per protocol. Gene-specific primers for PCR amplification are: c-Maf, 5'-ACT GAA CCG CAG CTG CGC GGG GTC AG-3' and 5'-CTT CTC GTA TTT CTC CTT GTA GGC GTC C-3' to generate a 223-bp product; GATA-3, 5'-GAA GGC ATC CAG ACC CGA AAC-3' and 5'-ACC CAT GGC GGT GAC CAT GC-3'; growth factor-independent (Gfi)-1, 5'-GGA AGC ACA GAA CAC AGG CTC-3' and 5'-CTG CTA CAA GAG GAG GCA TCA-3'; SOCS1, 5'-CAG GTG GCA GCC GAC AAT GCG ATC-3' and 5'-CGT AGT GCT CCA GCA GCT CGA AAA-3' to generate a 450-bp product; SOCS3, 5'-CCG GCT AGC ATG GTC ACC CAC AGC AAG-3' and 5'-TTT GGA TCC TTA AAG TGG AGC ATC ATA-3' to generate a 690-bp product; GAPDH, 5'-TGA AGG TCG GTG TGA ACG GAT-3' and 5'-CAG GGG GGC TAA GCA GTT GGT-3' to generate a 376-bp product. The samples were amplified for 30 cycles at 94°C (1 min), 57°C (2 min), and 72°C (3 min). Five microliters of each sample was separated by 2% agarose gel electroporesis and DNA was visualized by ethidium bromide staining.
Real-time RT-PCR
Total RNA was isolated using TRIzol, and treated with DNase I (Invitrogen Life Technologies) to avoid DNA contamination; cDNA was prepared using Omniscript Reverse Transcriptase (Qiagen) with random primers. The primers are: c-Maf, AGC AGT TGG TGA CCA TGT CG and TGG AGA TCT CCT GCT TGA GG; IL-4, GAA GCC CTA CAG ACG AGC TCA and ACA GGA GAA GGG ACG CCAT; GATA-3, GAA GGC ATC CAG ACC CGA AAC and ACC CAT GGC GGT GAC CAT GC; cyclophilin A, AGG GTG GTG ACT TTA CAC GC and ATC CAG CCA TTC AGT CTT GG; GAPDH, TGA ACG GGA AGC TCA CTG G and TCC ACC ACC CTG TTG GTG TA; SOCS3, CCT TCA GCT CCA AAA GCG AG and GCT CTC CTG CAG CTT GCG. Each reaction was performed in the LightCycler system (Roche) with the SYBER green PCR kit (Qiagen). All experiments were done at least three separate times, and expression of specific genes was normalized and expressed as a percentage relative to housekeeping genes (cyclophilin A or GAPDH).
Chromatin immunoprecipitation
ChIP was performed according to the manufacturer’s protocol (Upstate Biotechnology). Briefly, cells were treated with anti-CD3 and anti-CD28 mAbs, with or without IL-6 for 1 h, cross-linked with formaldehyde at a final concentration of 1%, lysed and sonicated to shear DNA. After immunoprecipitation with anti-Stat3 or anti-Stat1 at 4°C overnight, Ab-DNA complexes were eluted and cross-links reversed by incubating at 65°C for 4 h with 5 M NaCl, and the DNA was then recovered by the QIAQuick PCR purification kit (Qiagen). Real-time PCR was then used to quantify the c-maf promoter sequences as described earlier; the primers used are: TTT CTA TAC TAT TAT GCT AAT CGC TGC CGC and CGA GAA GAG TTT AAA GCA ATT GCT GAG TTT.
Luciferase reporter assay
The c-maf promoter was amplified from BALB/c mouse genomic DNA by PCR with primers carrying restriction sites for XhoI and EcoRI 5'-AAC TCG AGC CGC AAG GGT TAA GCA AAC CTT GT-3' and 5'-CCG AAT TCA AGA TGA AAA GAG ATT TTA AAG CC-3', and cloned into the promoterless luciferase plasmid vector phRL-null, subsequently named as phRL-c-maf. Transient transfection experiments were performed with calcium phosphate. The plasmids Stat3C, null, c-maf, Stat3C combined with null, and Stat3C combined with c-maf were used at 2 μg each. For dose responses, null or c-maf luciferase constructs were used at 0.5 μg, along with different doses of Stat3C from 0 to 3 μg. Twenty-four hours after transfection, luciferase activity was measured with a Renilla Luciferase assay kit according to the manufacturer’s protocol and normalized to background activity from phRL-null.
Results
c-Maf plays an early role in IL-6-mediated IL-4 production
Studies by Rincon and colleagues (18, 19, 20) have been instrumental in elucidating the role of IL-6 in promoting IL-4 production and Th2 differentiation. As shown in Fig. 1A, IL-6 promotes IL-4 production in naive CD4+ T cells stimulated for 3 days with anti-CD3 and anti-CD28 mAbs. Anti-IL-6, but not anti-IL-10 mAbs, abrogated IL-6-driven IL-4 production, demonstrating that this effect is IL-6-specific and not due to a secondary cytokine such as IL-10. To investigate the molecular mechanisms of IL-6-driven IL-4 production, we examined whether IL-6 affects the expression of the two most critical Th2-specific transcription factors, c-Maf and GATA-3. RT-PCR was performed on BALB/c CD4+ T cells stimulated with anti-CD3 plus anti-CD28 mAbs for 3 days. Fig. 2B shows that at the end of the culture period both c-Maf and GATA-3 are up-regulated substantially in the IL-6 treatment group. Treating without IL-6 or with IL-10, another Th2 cytokine that activates Stat3 signaling similar to IL-6 and has been implicated in Th2 differentiation does not result in as much c-Maf or GATA-3 expression. These data suggest that IL-6-driven IL-4 and Th2 differentiation may involve mechanisms through c-Maf and GATA-3 up-regulation. It has been previously reported that Gfi-1, a Stat6-dependent transcriptional repressor, is also involved in Th2 cell expansion and proliferation (24). To determine whether Gfi-1 is regulated in IL-6-driven Th2 differentiation, we examined Gfi-1 expression. Unlike c-Maf or GATA-3, there is little effect of IL-6 on Gfi-1 expression (Fig. 1B), suggesting that Gfi-1 may not play a role in the early stages of IL-6-driven Th2 differentiation.
FIGURE 1. IL-6 promotes c-Maf, GATA-3, and IL-4 expression in CD4+ T cells. A total of 1 x 106 cells/ml were stimulated in plates coated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs, with or without IL-6 (20 ng/ml). A, IL-4 production after 72 h of stimulation. B, RT-PCR of c-Maf, GATA-3, Gfi-1 after 72 h of stimulation. C, Real-time RT-PCR of c-Maf expression over time. Expression of c-Maf was normalized and expressed as a percentage relative to cyclophilin A. D, Real-time RT-PCR of GATA-3 expression over time.
FIGURE 2. IL-6 is unique in rapidly inducing c-Maf expression. A, Real-time RT-PCR of c-Maf expression after 1, 3, and 24 h of stimulation. B, Real-time RT-PCR of GATA-3 expression after 1, 3, and 24 h of stimulation. C, Real-time RT-PCR of c-Maf expression after 1, 3, and 24 h of stimulation by IL-4 or IL-6. D, Western blot analysis of c-Maf expression after 24 h of stimulation with IL-4 or IL-6. E, C57BL/6 CD4 T cells were stimulated with plate-coated anti-CD3 plus anti-CD28 mAbs for 3 h along with the indicated cytokines (20 ng/ml) and c-Maf expression was measured by real-time RT-PCR. All experiments were repeated at least three separate times.
To further elucidate the roles of c-Maf and GATA-3 in IL-6-driven IL-4 production, we examined c-Maf and GATA-3 expression in CD4+ T cells at earlier time points of activation by real-time RT-PCR. We found that IL-6 is able to induce both c-Maf and GATA-3 gene expression at days 2 and 3 of culture following anti-CD3 plus anti-CD28 mAbs stimulation (Fig. 1, C and D). In contrast, in CD4+ T cells stimulated for 24 h, only c-Maf but not GATA-3 is induced by IL-6. These results suggested that although both c-Maf and GATA-3 might be important for IL-6-driven IL-4 production and Th2 differentiation, c-Maf and GATA-3 may play different roles in the process. c-Maf may play a more direct role in IL-6-initiated early IL-4 production, whereas GATA-3 may play a secondary role in regulating and maintaining IL-6-driven Th2 differentiation at later time points of stimulation.
IL-6 but not IL-4 nor other cytokines rapidly induces c-Maf expression
To investigate further the potential mechanisms of IL-6-mediated c-Maf expression, we next analyzed earlier kinetics of c-Maf expression in CD4+ T cells after stimulation. CD4+ T cells from BALB/c mice were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-6, harvested after 1, 3, or 24 h, and quantitative real-time RT-PCR was used to measure the cells c-Maf and GATA-3 expression. As shown in Fig. 2A, IL-6 is able to induce c-Maf expression as early as 3 h after stimulation (Fig. 2A). On the contrary, there is no GATA-3 induction within the 24 h period of stimulation (Fig. 2B), further indicating the earlier and more direct role of c-Maf in IL-6-mediated IL-4 expression and Th2 differentiation.
It is well-documented that IL-4 plays a central role in Th2 differentiation, and constitutively active Stat6 has been shown to up-regulate c-Maf expression (8, 9). To address the question of whether IL-6-driven c-Maf induction is secondary to IL-4 effects, such as IL-4 production or contamination in the culture resulting in activating Stat6 signals, we treated CD4+ T cells with anti-CD3 plus anti-CD28 mAbs along with IL-6 or IL-4, and then performed real-time RT-PCR after 3 h of stimulation. As shown in Fig. 2C, IL-6 but not IL-4 rapidly induces c-Maf expression, suggesting a direct and unique role of IL-6 in up-regulating Th2-specific transcription factor c-Maf expression. GAPDH instead of cyclophilin A was used as the housekeeping gene in this experiment, and IL-6 induced c-Maf in a similar fashion, thereby excluding the possibility that the increased c-Maf expression is due to possible effects of IL-6 on cyclophilin A expression (Fig. 2C). Western blot analysis was used to measure c-Maf protein 24 h after stimulation. Fig. 2D shows that IL-6, but not IL-4, induces c-Maf protein expression, paralleling the transcriptional results obtained by real-time RT-PCR. Together, these results demonstrate that IL-6-induced c-Maf expression is rapid and specific.
It is known that CD4+ T cells from BALB/c are likely biased to a Th2 phenotype, whereas CD4+ T cells on the C57BL/6 background are biased to Th1 development. To test whether IL-6-induced c-Maf expression is BALB/c intrinsic, CD4+ T cells from C57BL/6 were isolated and stimulated with anti-CD3 plus anti-CD28 mAbs, combined with IL-6. Fig. 2E shows that IL-6 induces c-Maf expression in CD4+ T cells on the C57BL/6 background, indicating that IL-6-mediated c-Maf expression is not strain limited. Besides IL-4 and IL-6, the Th2 cytokines IL-5, IL-10, and IL-13 have been implicated in Th2 responses. To investigate whether these cytokines affect acute c-Maf induction, CD4+ T cells were also stimulated with anti-CD3 plus anti-CD28 mAbs along with IL-5, IL-6, IL-10, or IL-13. As shown in Fig. 2E, IL-6 but not IL-5, IL-10, or IL-13 induces c-Maf expression, further demonstrating the unique role of IL-6 in initiating c-Maf expression upon TCR activation.
IL-6 induces c-Maf expression in CD4 T cells upon Ag-specific stimulation
CD4+ T cells produce very little IL-6 at early stages of activation, and the main sources of IL-6 are from APCs. We next examined the effects of IL-6 on T cells stimulated by specific Ag. CD4+ T cells were purified from AND TCR transgenic mice specific for cytochrome c presented by I-Ak (21). PCC peptides at 0.5 or 5 μg/ml purified B cells from B10.BR mice as APCs, and IL-6 was added to AND T cell cultures. As shown in Fig. 3A, only groups cultured with peptide, APCs, and responder T cells for 3 days have proliferative responses, demonstrating the antigenic specific response of this assay. c-Maf expression was also measured by real-time RT-PCR after 3 days, and the results demonstrate that stimulation with IL-6 induces c-Maf expression (Fig. 3B), in a fashion similar to cells stimulated with anti-CD3 plus anti-CD28 mAbs. We next examined IL-6-mediated c-Maf expression when purified CD11c+ dendritic cells were used as APCs. As shown in Fig. 3C, real-time RT-PCR for c-Maf expression in AND CD4+ T cells was measured after 3 h of stimulation, demonstrating that c-Maf is also induced in cultures including CD4+ T cells, IL-6, and dendritic cells presenting PCC peptide. These results indicate that IL-6 induces c-Maf expression in T cells after Ag-specific TCR activation, using B cell or dendritic cell as APC, as well as in APC-independent culture conditions with direct stimulation through mAbs.
FIGURE 3. IL-6 induces c-Maf expression after Ag-specific stimulation. CD4+ T cells were purified from AND TCR transgenic mice, and purified B cells or dendritic cells from B10.BR mice were used as APC. A, PCC peptides induce specific proliferative responses; purified B cells were used as APC. B, Real-time RT-PCR of c-Maf expression after specific peptide stimulation for 3 days, purified B cells were used as APC. C, Real-time RT-PCR of c-Maf expression after specific peptide stimulation for 3 h, purified CD11c+ dendritic cells were used as APC.
Dendritic cells promote c-Maf expression by IL-6
To test whether IL-6 mediates c-Maf expression at physiological levels, we isolated CD4+ T cells from BALB/c mice and evaluated their dose-response to IL-6 during c-Maf induction upon stimulation with anti-CD3 and anti-CD28 mAbs. As shown in Fig. 4A, IL-6 is able to induce c-Maf expression at a dose of 0.03 ng/ml, and achieve a plateau at around 1 ng/ml. The requirement for only a low concentration of IL-6 for inducing c-Maf expression suggests that IL-6-mediated c-Maf expression could occur under physiological conditions.
FIGURE 4. Dendritic cells mediate c-Maf expression by IL-6 production. A, Real-time RT-PCR of c-Maf expression induced by various doses of IL-6 on purified cells from BALB/c CD4+ T cells stimulated for 3 h with anti-CD3 and anti-CD28 mAbs. B, Real-time RT-PCR of c-Maf expression on purified CD4+ T cells from AND or OT-2 transgenic mice. Cells were stimulated for 3 h by anti-CD3 and anti-CD28 mAbs combined with 48 h culture supernatants from dendritic cells stimulated by soluble CD40L (500 ng/ml) and LPS (1 μg/ml). C, Real-time RT-PCR of c-Maf expression in AND or OT-2 CD4+ T cells after specific peptide stimulation for 12 h. Soluble CD40L and LPS activated CD11c+ dendritic cells were used as APC. D, Real-time RT-PCR of c-Maf expression in wild-type and IL-6-deficient CD4 T cells after stimulation with soluble anti-CD3 mAb (0.5 μg/ml) plus activated wild-type or IL-6-deficient DC11+ dendritic cells.
It has been well-documented that upon activation and maturation, dendritic cells produce IL-6 (25, 26). We then investigated whether IL-6 produced by dendritic cells is sufficient to induce c-Maf expression. Purified CD11c+ dendritic cells from both B10BR and C57B/6 mice were stimulated with soluble CD40L (500 ng/ml) combined with LPS (1 μg/ml) for 48 h, culture supernatants were harvested, and ELISA was used to confirm IL-6 production (at 1.5 ng/ml, data not shown). We then used the culture supernatant from these dendritic cells to stimulate CD4+ T cells purified from AND or OT-2 transgenic mice in combination with anti-CD3 and anti-CD28 mAbs. Fig. 4B showed that supernatants from dendritic cells stimulated by soluble CD40L and LPS are sufficient to induce c-Maf expression in CD4+ T cells, and anti-IL-6 mAbs completely abrogated c-Maf induction, demonstrating the specificity of IL-6 in regulating c-Maf expression.
Next, we examined the ability of activated dendritic cells themselves to regulate c-Maf expression. B10BR or C57BL/6 dendritic cells were activated with soluble CD40L and LPS for 48 h, thoroughly washed, and then cultured with CD4+ T cells from AND or OT-2 transgenic mice in combination with their respective specific antigenic peptides, PCC or OVA, for 12 h. As shown in Fig. 3C, after stimulation, both B10BR and C57BL/6 dendritic cells were able to induce c-Maf expression upon specific antigenic activation, and c-Maf induction was prevented by treatment with anti-IL-6 mAbs, further supporting the unique role of IL-6 in mediating c-Maf expression. Without addition of CD4+ T cells and peptides, there is no c-Maf expression, indicating that dendritic cells alone do not express c-Maf. To further examine how IL-6 deficiency affects c-Maf expression, we isolated dendritic cells from IL-6-deficient and wild-type B6 mice, activated them with LPS and CD40L for 48 h, and then cultured these dendritic cells with wild- type or IL-6-deficient CD4 T cells. As shown in Fig. 4D, wild-type dendritic cells, but not IL-6-deficient dendritic cells, induce c-Maf expression in both IL-6 knockout CD4 T cells and wild-type CD4 T cells, further indicating that IL-6 produced by DC plays an important role in c-Maf induction.
Both IL-6 and TCR induced signals are required to induce c-Maf expression
We further addressed the requirement of signals for IL-6-mediated c-Maf induction. As shown in Fig. 4A, without anti-CD3 mAb-mediated TCR activation, IL-6 failed to induce c-Maf expression, indicating that signals from IL-6 alone are not sufficient to induce c-Maf, and that TCR signals are essential for IL-6 to mediate c-Maf expression. To determine whether costimulatory signals are required for IL-6 to induce c-Maf, purified CD4+ T cells were stimulated with soluble anti-CD3 mAb only, plate-coated anti-CD3 mAb only, and plate-coated anti-CD3 plus anti-CD28 mAbs for 3 h. Fig. 5A shows that IL-6 induces c-Maf expression in CD4+ T cells stimulated with plate-coated or soluble anti-CD3 mAb alone, suggesting that costimulatory signals generated from anti-CD28 mAb are not essential for IL-6-mediated c-Maf expression. Standard RT-PCR confirmed the findings from real-time PCR; Fig. 5B shows IL-6 but not IL-10 induced early c-Maf expression, and that induction is dependent on both IL-6 and TCR signals, but independent of CD28 signaling. In the same experiment we measured the expression of Stat3 regulated SOCS molecules to assess cytokine stimulated signaling. The results indicated that IL-6 induced similar SOCS3 expression in all groups. In addition, the basal level of SOCS1 expression is increased by TCR activation, and IL-6 up-regulates SOCS1 expression in all groups (Fig. 5B). These results suggest that SOCS induction is unlikely to regulate IL-6-mediated c-Maf expression because IL-6 induces similar SOCS3 expression in groups treated with or without anti-CD3 mAbs, and the increased levels of SOCS1 in cells treated with anti-CD3 do not correspond to the levels of c-Maf expression (Fig. 5B). IL-10 fails to induce sustained SOCS1 or SOCS3 3 h after treatment (Fig. 5B), whereas it is able to induce SOCS1 and SOCS3 expression after 1 h of treatment, but significantly less compared with IL-6 (Ref. 23 and data not shown), suggesting that differential strength of signals (possibly Stat3 activation) may play a role in determining early c-Maf expression.
FIGURE 5. Signaling requirements for IL-6 to induce c-Maf expression. A, Real-time RT-PCR of c-Maf expression in CD4+ T cells stimulated for 3 h with or without TCR or CD28 activation. B, Conventional RT-PCR was performed in a separate experiment in which IL-6 and IL-10 were compared. SOCS1 and SOCS3 genes were assessed as measures of cytokine-stimulated signaling. Cells stimulated for 3 h with indicated mAbs (c, coated and s, soluble). C, Real-time RT-PCR for c-Maf expression 3 h after stimulation; CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without the indicated cytokines (300 ng/ml). D, Western blot analysis of Stat3 activation of various cytokines (20 ng/ml) after 30 min treatment. E, Flow cytometry for IL-6R and IL-10R expression on purified CD4 T cells. Isotope control (filled histogram), IL-6R expression (thick histogram), and IL-10R expression (dashed histogram) are shown.
Upon IL-6 binding to its receptor complex (IL-6R and gp130), two major signaling pathways are activated. Stat3 is recruited to gp130 through its Src homology protein 2 (SHP2) domain, and initiates the JAK-STAT signaling pathway. SHP2 is recruited to gp130, initiating the MAPK signaling cascade (27). To investigate whether other cytokines using Stat3 signaling pathways, or cytokines that share the common gp130 receptor, also induce c-Maf expression in CD4+ T cells after anti-CD3 and anti-CD28 mAb stimulation, we further examined the effects on c-Maf expression of IL-10, which activates Stat3, and IL-11, oncosatin M, and leptin that share the common signal transducer gp130 with IL-6. IL-10 is generally considered a Th2 cytokine, and IL-11 has been suggested to be able to promote Th2 differentiation (28). As shown in Fig. 5C, even under extremely high doses of cytokines (300 ng/ml), IL-10 and IL-11 induce some c-Maf expression, whereas oncosatin M and leptin induce no c-Maf expression (Fig. 5C), and prolonged incubation with IL-10, IL-11, oncosatin M or leptin did not improve c-Maf induction (data not shown). It is possible the reason IL-10, IL-11, oncosatin M, and leptin fail to induce significant c-Maf expression is due to insufficient cytokine receptor expression on the CD4+ T cell surface, and consequently insufficient Stat3 activation. Indeed, Western blotting for Stat3 activation revealed that IL-6 induced much stronger Stat3 activation compared with other cytokines (Fig. 5D), and flow cytometry measuring receptor expression showed that the IL-6R is expressed at much higher levels compared with the IL-10R on CD4 T cells (Fig. 5E). Together, these results imply that sustained IL-6-stimulated Stat3 activation may play an important role in up-regulating c-Maf induction. The reason IL-6 differs from other cytokines in regulating c-Maf expression might be due to the differential strength of Stat3 activation.
TCR-mediated Ca2+ signaling is required for IL-6 to induce c-Maf expression
PKC is predominantly expressed in T lymphocytes and has been implicated in the control of TCR/CD28-induced AP-1 and NF-B activation (22, 29). To determine whether TCR-mediated AP-1 and NF-B play a role in IL-6-mediated c-Maf expression, CD4+ T cells were purified from PKC knockout mice (22), and stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-6. Real-time RT-PCR was performed to measure c-Maf expression after 3 h of stimulation. As shown in Fig. 6A, IL-6 induces similar c-Maf expression in both wild-type and PKC-deficient CD4+ T cells, suggesting that IL-6-mediated c-Maf expression is independent of TCR-mediated AP-1 or NF-B signals.
FIGURE 6. TCR-mediated Ca2+ signals are required for IL-6 to induce c-Maf expression. A, IL-6 induces c-Maf expression in wild-type and PKC-deficient CD4+ T cells. B, CsA and FK506, but not RAPA, block IL-6-mediated c-Maf expression. C, PMA and ionomycin fail to generate sufficient TCR signals for IL-6 to induce c-Maf.
CsA and FK506 target calcineurin, thereby inhibiting TCR-mediated Ca2+ signal pathways. To test whether Ca2+ signaling pathways play a role in IL-6-mediated c-Maf expression, purified CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 mAbs in the presence of CsA or FK506. Real-time RT-PCR was then performed 3 h after stimulation to measure c-Maf expression. Fig. 6B shows that both CsA (10–7 M) and FK506 (10–7 M) block IL-6-induced c-Maf expression, indicating that Ca2+ pathway signaling is critical for IL-6 to induce c-Maf expression. Rapamycin (RAPA) shares the same target, FKBP12, with FK506, but binds to a distinct molecular site and inhibits phosphorylation of p70S6 kinase (30). RAPA (10–7 M) did not have any inhibitory effects on IL-6 mediated c-Maf expression (Fig. 6B), demonstrating the specific role of TCR-stimulated Ca2+ signaling pathways to induce c-Maf expression.
We next sought to investigate whether PMA, which activates PKC and the calcium ionophore ionomycin, alone or combination could generate the requisite TCR signals for IL-6 to mediate c-Maf expression. Fig. 6C shows that neither PMA (5 ng/ml) nor ionomycin (300 ng/ml), alone or in combination, induces c-Maf expression. Because it has been previously demonstrated that both PMA and ionomycin inhibited IL-6-mediated Stat3 activation (31, 32), the failure of c-Maf induction is possibly due to signals mediated by PMA and ionomycin that antagonize IL-6-mediated JAK-STAT signaling, further suggesting a critical role of Stat3 in IL-6-mediated c-Maf expression.
IL-6-induced c-Maf expression is independent of IL-4/Stat6 signaling
To address directly the role of IL-4/Stat6 signaling in IL-6-mediated c-Maf expression, we used CD4+ T cells from IL-4 and Stat6 knockout mice to further determine the dependence on these two molecules for IL-6-mediated c-Maf expression. CD4+ T cells from IL-4 knockout, Stat6 knockout or wild-type BALB/c mice were purified and stimulated with anti-CD3 and anti-CD28 mAbs for 3 h, with or without IL-6. As shown in Fig. 7A, there is no difference in c-Maf expression between wild-type cells and CD4+ T cells deficient in IL-4 or Stat6, demonstrating that IL-6-induced c-Maf expression is IL-4/Stat6 signal-independent. We also analyzed gene expression at later time points of T cell activation in these cells. Fig. 7B shows that after 3 days of stimulation IL-6 induces c-Maf expression in CD4+ T cells deficient in IL-4 or Stat6, but at reduced levels compared with wild type. These findings suggest that IL-4/Stat6 might be indirectly involved in stimulating optimal levels of c-Maf expression, perhaps through induction of GATA-3 because the expression of GATA-3 is crucial for the differentiation of Th2 cells. In fact, when the same cells were evaluated for the expression of GATA-3, contrary to the findings for c-Maf, there was little GATA-3 expression in IL-4 or Stat6-deficient CD4+ T cells treated with or without IL-6 (Fig. 7C), indicating that GATA-3 expression is dependent on IL-4/Stat6 signals.
FIGURE 7. IL-6-induced c-Maf expression and IL-4 expression in wild-type, IL-4-deficient, or Stat6-deficient CD4+ T cells stimulated by anti-CD3 plus anti-CD28. A, Real-time RT-PCR for c-Maf expression in wild-type, IL-4-deficient, or Stat6-deficient CD4+ T cells after 3 h of stimulation. B, Real-time RT-PCR for c-Maf expression after 3 days of stimulation. C, Real-time RT-PCR for GATA-3 expression after 3 days of stimulation. D, Real-time RT-PCR for IL-4 expression in BALB/c CD4+ T cells after 3, 6, 12, and 24 h of stimulation, with or without IL-4 or IL-6. E, Real-time RT-PCR for IL-4 expression in STAT6 knockout CD4+ T cells.
Because c-Maf is critical and selective in regulating IL-4 expression (5, 6, 7), we determined whether c-Maf induction by IL-6 could regulate early IL-4 gene transcription, by examining the effects of IL-4 or IL-6 on IL-4 gene expression during early stages of CD4+ T cell activation from 3 to 24 h. In agreement with previous findings that IL-4 gene expression is up-regulated upon TCR activation, even without cytokine treatment (33), the results show that before activation, CD4 T cells contained no detectable transcripts for IL-4 (data not shown), but these increased significantly after 3 h of anti-CD3 plus anti-CD28 mAb stimulation (Fig. 7D). Neither IL-4 nor IL-6 enhanced IL-4 gene expression at the 3 h time point, indicating that this early increase in IL-4 gene expression is cytokine independent. After 6 h of stimulation, IL-4 gene expression declines in all groups, however, cells treated with IL-6 had significantly higher IL-4 gene expression (3-fold) compared with cells in the control or IL-4 treatment groups. Similar results were also observed after 12 and 24 h of stimulation (Fig. 7D). These results clearly show that IL-6, but not IL-4, is able to play a positive role in regulating early IL-4 gene expression after TCR activation. Because c-Maf expression is sustained between 3 and 24 h (Fig. 2 and data not shown), and IL-6-driven IL-4 expression only after c-Maf is induced, this suggests IL-6-mediated c-Maf expression plays a role in mediating enhancement of IL-4 expression. We next performed similar experiments on highly purified CD4+ T cells from Stat6 knockout mice. As shown in Fig. 7E, IL-6 is able to sustain IL-4 gene expression at a higher level compared with groups treated with no cytokine or IL-4, but at a reduced level compared with wild-type BALB/c mice. Collectively, these results suggest that optimal levels of IL-4 expression are IL-4/Stat6-dependent; IL-6 is also able to regulate IL-4 expression in a IL-4/Stat6–independent fashion.
Stat3 binds and activates the c-maf promoter
Both the mouse and rat c-maf genes are composed of a single exon and have highly conserved promoters, there being, only three nucleotide differences within 200 bp of the 5' flanking region (34, 35). Structural analysis of the c-maf gene and promoter regulation during differentiation of lens epithelial cells to fiber cells show that Pax6, a master transcription factor for lens development, activates the c-maf promoter. The c-maf gene is also positively autoregulated by its own product, c-Maf, through two identified Maf recognition element binding sites (34, 35). Further examination of the c-maf promoter sequences shows that there are two class II IL-6 responsive elements (CTGGGA), which are potential Stat3 binding sites.
To further investigate whether Stat3 may bind to the c-maf promoter after IL-6 stimulation, purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 mAbs, with or without IL-6, and 45 min after stimulation ChIP assay was performed using anti-Stat3 and anti-Stat1 (as a control) for immunoprecipitation. Real-time PCR was used to quantitate the c-maf promoter sequences associated with Stat3 or Stat1. As shown Fig. 8A, anti-Stat3 precipitation enriches for the c-maf promoter by 4-fold compared with anti-Stat1, indicating that Stat3 binds to the c-maf promoter after IL-6 stimulation in vivo.
FIGURE 8. Stat3 binds and activates the c-maf promoter. A, ChIP assay for the c-maf promoter. Purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 mAbs, with or without IL-6 for 1 h, and anti-Stat3 and anti-Stat1 Abs were used for immunoprecipitation. Sequences of c-maf promoter precipitated by anti-Stat3 or anti-Stat1 were quantitated by real-time PCR. Data are expressed as the ratio of Stat3-associated c-maf promoter sequences to Stat1-associated c-maf promoter sequences. B, Activation of the c-maf promoter. Transient transfection of promoterless (null), c-maf promoter-driven (c-maf), Stat3C, Stat3C/null, or Stat3/c-maf at 2 μg each into C3H10T1/2 cells. Luciferase activity was measured 24 h posttransfection. C, Dose response to Stat3C for activation of the c-maf promoter. Cells were transfected with null or c-maf luciferase constructs (0.5 μg), along with different amounts of Stat3C, as indicated.
To determine whether Stat3 transactivates the c-maf promoter, the c-maf promoter was cloned into a luciferase reporter construct, and a series of transient transfection experiments was performed. The activation of the c-maf gene and its promoter regulation has been studied in a mouse embryonic fibroblast cell line C3H10T1/2. Both c-Maf itself and Pax6 activate the c-maf promoter (34, 35), indicating that there are the necessary components in C3H10T1/2 cells for studying c-maf promoter regulation. We cotransfected a promoterless luciferase construct (phRL-null), or the c-maf promoter-driven luciferase construct (phRL-c-maf), along with Stat3C, a construct expressing a constitutively active form of Stat3 (36), into C3H10T1/2 cells. Fig. 8B shows that Stat3C markedly enhances luciferase activity when cotransfected with the luciferase plasmid driven by the c-maf promoter. Dose response analysis to Stat3C further demonstrated that Stat3 transactivates the c-maf promoter (Fig. 8C). Together, these experiments suggest a critical role for IL-6-mediated Stat3 activation in activating the c-maf promoter and regulating c-Maf expression.
Discussion
The molecular mechanisms underlying the regulation of IL-4 gene expression in Th2 differentiation have been intensively studied. The transcription factors c-Maf, Stat6, and GATA-3 have all been demonstrated to play critical roles in regulating IL-4 expression and Th2 commitment, and c-Maf has been proven to have a more selective and direct effect on the IL-4 promoter to initiate IL-4 gene transcription (5, 7). Understanding the upstream factors that regulate c-Maf expression is critical for elucidating the mechanisms of IL-4 gene regulation and Th2 commitment. In this study, we showed that IL-6, but not IL-4 or other cytokines, is able to rapidly induce TCR-dependent c-Maf expression. IL-4 did not induce c-Maf induction within 24 h of stimulation, which suggests that IL-4/Stat6 signals do not have a direct impact on the c-Maf promoter to initiate c-Maf gene regulation. The observation that IL-6-induced c-Maf expression is independent of Stat6 further confirms this observation.
The functional IL-6R consists of two subunits: the -chain and the common signal transducing receptor gp130. IL-6 binds to the surface of IL-6R, leading to the dimerization of gp130/IL-6R, activation of JAK1, JAK2, and tyrosine kinase 2, and subsequent activation of two major signaling pathways: 1) Stat3 and 2) MAPK cascade through SHP2 (37). The MAPK signaling pathway has been elegantly elucidated by Flavell and colleagues (38, 39, 40) for its important role in promoting IFN- production and Th1 development, and therefore is unlikely to play a positive role in c-Maf/IL-4/Th2 induction. Stat3 has been shown to be important for Th2 effector responses (41), and in this study we demonstrated that Stat3 binds to the c-maf promoter in CD4 T cells after stimulation, and it also transactivates the c-maf promoter (Fig. 8).
Dendritic cells have been demonstrated to be initiators of immune responses, and play major roles in capturing, processing, and transporting of Ag to initiate Ag-specific immune responses of naive T lymphocytes. IL-6 has been shown to be produced by dendritic cells upon stimulation (25, 26), and its production is necessary for inhibiting IL-12-mediated Th1 differentiation (42). Our results showed that dendritic cells activated by soluble CD40L and LPS produce sufficient IL-6 to induce c-Maf expression, indicating that IL-6 might play an important role in regulating dendritic cell-mediated Th1/Th2 differentiation. Without Th1 cytokine-mediated (IL-12, IFN-) suppression, IL-6 induced early c-Maf could positively regulate IL-4 expression and subsequent Th2 differentiation.
Our results demonstrate that IL-6-mediated signals alone are not sufficient to induce c-Maf expression in CD4 T cells, but require TCR-mediated Ca2+ signals. The calcineurin inhibitors FK506 and CsA both block IL-6-mediated c-Maf induction. One family of critical regulators of gene transcription in response to TCR ligation is NFAT. Early studies have demonstrated that CsA blocked IL-6-mediated NFAT1 expression, and studies on NFAT1 knockout mice and transgenic mice expressing a mutant form of NFAT that acts as a dominant negative NFAT showed a requirement for NFAT1 for IL-6-mediated Th2 differentiation (20). We showed there is rapid induction of c-Maf expression after 3 h of stimulation and the induction is dependent on TCR-mediated Ca2+ signaling, demonstrating that IL-6-mediated c-Maf expression and NFAT1 activation share a common upstream Ca2+ signaling pathway. Sequence analysis of the c-maf promoter region (34, 35) reveals there are two potential NFAT TTTCC binding sites at –80 and –151 of the c-maf gene, suggesting a possible role for NFAT1 in regulating c-Maf expression. Whether NFAT1 is required for IL-6-mediated c-Maf expression in CD4 T cells is currently under investigation.
Recent evidence supports a two-stage model of Th differentiation. TCR activation leads to transient IL-4 and IFN- transcription within 1 h, peaks at 2 h, declines after 3 h, and this process is TCR dependent, but is independent of IL-4/Stat6 or IL-12/Stat4 signaling (33). Similar results are observed for histone acetylation at the regulatory regions of IL-4 and IFN- (43), indicating that TCR stimulation activates an immediate and global chromatin derepression in naive T cells to initiate localized histone modification with parallel gene expression. Continued culture with IL-4 or IL-12 is required to maintain increased histone acetylation. Without polarizing cytokines or key transcription factors, the early histone hyperacetylation that resulted from TCR activation is reduced to the low basal level (43). During the second stage, the induction and maintenance of high levels of T-bet or GATA-3 expression are necessary for Th1 or Th2 commitment, and T-bet or GATA-3 may also play roles in silencing the opposing
Th locus (33, 43). Our results showed that IL-6 combined with TCR activation leads to more sustained c-Maf expression, and significant IL-4 enhancement during early stages of CD4 cell activation. However, IL-6 does not change the trend of a decline in IL-4 transcription within 24 h of TCR activation, suggesting that sustained promoter availability might be a crucial factor in determining the final outcome of IL-4 expression. Due to the possible limited availability of the IL-4 promoter, c-Maf expression alone is then not sufficient to maintain high levels of IL-4 expression, which is consistent with previous observations that overexpressing c-Maf alone is not sufficient to initiate IL-4 expression in Th1 cells (6). It will be worth examining whether IL-6 affects chromatin structure and IL-4 promoter availability.
Earlier reports (18, 19, 20) and our current studies indicate that IL-6 plays an important role in promoting T cell IL-4 expression and Th2 differentiation. In contrast there are reports that suggest that IL-6 may play an important role in inhibiting Th2 effector cells. Infection with Giardia lamblia or Mycobacterium tuberculosis in IL-6-deficient mice resulted in elevated IL-4 production (44, 45), and IL-6 inhibits IL-4-mediated Th2 differentiation and aeroallergen-induced Th2 inflammation (46, 47). These disparate results suggest that the role played by IL-6 in regulating Th1/Th2 development is complex. One possible explanation may involve IL-6-stimulated Stat3 activation in diverse cell types. Although many cytokines, including the IL-6 family and other diverse family members such as G-CSF, IFN, epidermal growth factor, leptin, IL-2, and IL-10 all activate Stat3, IL-6 is both produced by many more cell types and is a much more potent activator for Stat3 compared with other ligands. As a result, IL-6-mediated Stat3 activation may account for a significant part of the overall Stat3 activation during immune responses. As demonstrated by tissue-specific disruption, Stat3 activation is important for T cell proliferation (48), fms-like tyrosine kinase 3 ligand-dependent dendritic cell differentiation (49), and innate immunity (50). Therefore, depending on the type of Ag encountered, other cytokines present, and the particular APCs used, IL-6-mediated Stat3 activation may play a divergent role that influence the outcome of the Th1/Th2 balance.
In conclusion, our results reveal a unique role of IL-6 in directing Th2-specific transcription factor c-Maf gene initiation, up-regulating the level of IL-4 transcription during early stages of TCR activation, and providing a novel mechanism for Th2 commitment regulation. The relevance of IL-6/gp130, and Stat3 involvement in c-Maf expression could explain why the initial sources of IL-4 have been difficult to define because many cells secret IL-6, and many cytokines use gp130 and/or Stat3 signaling pathways. During certain types of inflammation, or specific microenvironments, these cytokines might turn on the c-maf gene and generate IL-4/Stat6 independent IL-4 production.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Dongmei Chen, Minwei Mao, and Patricia A. Rebollo for their assistance in real-time PCR, and Dr. Littman for kindly providing PKC knockout mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant R01 AI44929.
2 Address correspondence and reprint requests to Dr. Yaozhong Ding, Department of Gene and Cell Medicine, Box 1496, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. E-mail address: yaozhong.ding{at}mssm.edu
3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; PCC, pigeon cytochrome c; CsA, cyclosporin A; Gfi-1, growth factor-independent 1; RAPA, rapamycin; PKC, protein kinase C; SHP2, Src homology protein 2.
Received for publication August 4, 2004. Accepted for publication December 14, 2004.
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The transcription factor c-Maf plays a critical and selective role in IL-4 gene transcription. Little is known about the mechanism that guides c-Maf regulation during early T cell activation. We report that IL-6 but not IL-4 or other cytokines, rapidly up-regulates c-Maf transcription, as early as 3 h after TCR activation in naive CD4+ T cells. c-Maf induction requires both IL-6- and TCR-initiated signals, and is independent of IL-4/Stat6 signals. Cyclosporin A and FK506, which target calcineurin and thereby inhibit TCR-mediated Ca2+ signal pathways, block IL-6-mediated c-Maf expression. We show that Stat3 binds the c-maf promoter in CD4 T cells after IL-6 stimulation, and also transactivates the c-maf promoter in reporter gene assays. IL-6 induces similar c-Maf expression in protein kinase C-deficient CD4+ T cells. Furthermore, IL-6 enhances IL-4 gene expression very early after TCR activation in both wild-type and Stat6-deficient CD4+ T cells. Our findings suggest that IL-6 plays a unique role in initiating c-Maf expression after TCR engagement, and may subsequently regulate early IL-4 production and Th2 commitment.
Introduction
The immune system protects the host from a vast array of pathogens by evolving diverse effector mechanisms, which are optimally effective for the elimination of different types of infections. CD4+ T cells play a critical role in determining the outcome of any given antigenic stimulation. Two major subsets of Th cells have been characterized, and these cells direct ongoing immune responses through the secretion of different patterns of cytokines (1, 2). It is known that although the presence of IL-4 at the time of CD4 T cell activation is pivotal to Th2 differentiation, the cellular and molecular mechanisms that guide early IL-4 production remain less understood. Several innate immune cells, such as NK, T cells, NK1.1+ T cells, and mast cells, also produce IL-4 (3, 4) but none appear essential for Th2 development, as Th2 cells can differentiate from naive T cells independently of IL-4 produced by non-T cells or T cells. These findings suggested that non-IL-4 signals may also play important roles in directing Th2 development, and that naive T cells are capable of autonomous IL-4 production.
c-Maf was the first Th2-specific transcriptional factor identified (5). Overexpression of c-Maf using a transgenic approach showed the critical role of c-Maf in controlling IL-4 production in vivo, and ectopic expression of c-Maf in mature Th1 cells inhibits IFN- production and attenuates Th1 differentiation (6). Research on c-maf-deficient mice showed that CD4+ T cells and NK T cells from c-maf–/– mice were markedly deficient in IL-4 but not other Th2 cytokine (IL-5, IL-6, IL-10, or IL-13) production, and demonstrated that c-Maf has a critical and selective function in IL-4 gene transcription in vivo (7). In naive T cells, c-Maf is expressed at very low levels and little is known about how it is regulated. IL-4/Stat6 signals have been implicated in c-Maf up-regulation, and ectopic expression of activated Stat6 positively regulates c-Maf expression (8, 9). Recent studies in ICOS–/– CD4 T cells or with B7h-deficient APCs used to activate T cells have also suggested that interactions between ICOS and its ligand B7h are important in regulating c-Maf expression, possibly through IL-4 gene regulation (10, 11). GATA-3 (12), another Th2 specific transcription factor, has also been shown to augment c-Maf expression when overexpressed in CD4+ T cells (13). In addition, one report shows that a truncated c-maf inducing protein, Tc-mip, is able to up-regulate c-Maf expression when overexpressed in Jurkat T cells (14). Although these experiments measured c-Maf induction in later phases of T cell activation, the pathways and controlling elements directing initial c-Maf expression in early stages of T cell activation have not been reported.
Produced by both professional APCs (B cells, macrophages, and dendritic cells) and nonprofessional APCs (epithelial, endothelial, and some tumor cells), IL-6 plays important roles in T cell-mediated immune responses. It is a cofactor for T cell proliferation and prevents cell death (15, 16, 17). IL-6 also regulates polarization of naive CD4 T cells toward the Th2 phenotype. It inhibits Th1 differentiation by inducing suppressor of cytokine signaling (SOCS)3 1, and promotes Th2 differentiation by up-regulating NFAT1 expression (18, 19, 20). In the present work, we investigated the transcriptional mechanisms that regulate IL-6-mediated IL-4 gene expression. We found IL-6 to be unique in rapidly inducing c-Maf expression after TCR activation. c-Maf regulation by IL-6 is IL-4/Stat6 signaling-independent, but TCR-mediated Ca2+ signaling-dependent. Our data suggest a novel role for IL-6 in directing c-Maf initiation and IL-4 gene regulation.
Materials and Methods
Mice, cytokines, Abs, and other reagents
BALB/c, C57BL/6, B10.BR, OT-2 transgenic mice specific for OVA323–339, IL-4 knockout (BALB/c background), Stat6 knockout (BALB/c background), and IL-6 knockout (C57BL/6 background) were purchased from The Jackson Laboratory, and AND TCR transgenic mice specific for cytochrome c-I-Ak (21) were provided by Dr. S. M. Hedrick (University of California, San Diego, La Jolla, CA). Protein kinase C (PKC) mice (22) were provided by Dr. D. R. Littman (New York University Medical Center, New York, NY). Recombinant cytokines IL-4, IL-6, IL-10 were purchased from BD Pharmingen; IL-11, oncosatin M, and leptin were purchased from R&D Systems. Anti-IL-4, anti-CD3, anti-CD28, anti-CD4, and anti-B220 mAbs, and PE-labeled anti-IL-6R and anti-IL-10R were purchased from BD Pharmingen. Recombinant mouse soluble CD40L was purchased from PeproTech. Cyclosporin A (CsA), PMA, ionomycin, and LPS were purchased from Sigma-Aldrich. FK506 was purchased from A. G. Scientific. Anti-c-Maf (catalog no. sc-7866) was purchased from Santa Cruz Biotechnology. Anti-Stat1, Stat3, and anti-phosphorylated Stat3 were purchased from Cell Signaling Technology. CD11c microbeads were purchased from Miltenyi Biotec. Dynabeads mouse pan B (B220) beads were purchased from Dynal Biotech. Plasmid expressing a constitutively active Stat3 (Stat3C) was kindly provided by Dr. J. Darnell (The Rockefeller University, New York, NY). Promoterless luciferase reporter plasmid, phRL-null, and Renilla luciferase assay kit were purchased from Promega. The mouse fibroblast cell line C3H10T1/2 was obtained from the American Type Culture Collection. Chromatin immunoprecipitation (ChIP) assay kit was purchased from Upstate Biotechnology.
CD4 T cell, B cell, and DC purification and activation
Splenocytes from various strains of mice were purified and B cells were depleted by using Dynabeads mouse pan B, and then CD4+ T cells were purified by cell sorting by MoFlo (Cytomation) after staining with FITC anti-CD4. After sorting, cell purity is 99%. Cells were seeded into 96-well round-bottom plates coated with anti-CD3 mAb (5 μg/ml) at 2 x 104 cell/well, were then further activated with soluble anti-CD28 mAb (1 μg/ml), with or without IL-6 (20 ng/ml) or other cytokines (20 ng/ml). In APC-dependent assays, pigeon cytochrome c (PCC) peptides (provided by Dr. B. Murphy, Mount Sinai School of Medicine, New York, NY) and OVA peptides (OVA323–339) purchased from American Peptide at 0.5 μg/ml or 5 μg/ml were added. B cells were purified by cell sorting after staining with FITC anti-B220 from B10.BR splenocytes (after sorting, cell purity is 96%), and treated with mitomycin C (50 μg/ml; Sigma-Aldrich), for 50 min at 37°C. Dendritic cells were purified from B10.BR mice or C57BL/6 by CD11c microbeads per the manufacturer’s manual. Following cell sorting, the purity is above 98%. For dendritic cell maturation and activation, cells were cultured at 1 x 106/ml in 24-well plates, and stimulated with 500 ng/ml CD40L and 1 μg/ml LPS for 48 h.
ELISA and proliferation assays
ELISA for IL-4, IFN-, or IL-6 was performed using the OptEIA mouse IL-4, IFN-, or IL-6 set (BD Pharmingen) according to the manufacturer’s protocol. Absorbance was measured by a Spectra Max 250 (Molecular Devices) plate reader. For proliferative assays, purified CD4+ T cells (2 x 104) and mitomycin C (50 μg/ml) treated B cells (1 x 104) were incubated with PCC peptides (0.5 or 5 μg/ml) in a round-bottom 96-well plate with various cytokines, and then pulsed with 1 μCi [3H]thymidine for the last 16 h of a 3 day culture.
Western blot analysis
Protein extracts were prepared from various groups and concentrations were determined by BCA assay (Pierce) as previously described (23). Proteins were then separated by 16% (for c-Maf) or 7.5% (for Stat3) SDS-polyacrylamide and transferred to polyvinylidene difluoride membranes to probe with anti-c-Maf (1/200 dilution), anti-phosphorylated Stat3 (1/1000 dilution), anti-Stat3 (1/1000 dilution) subsequently incubated with HRP-conjugated anti-rabbit IgG and detected by the ECL system (Amersham).
RT-PCR
Total RNA was isolated from cultured cells using TRIzol following the manufacturer’s instructions (Invitrogen Life Technologies). Reverse transcription was conducted using Omniscript Reverse Transcriptase (Qiagen) and random primers as per protocol. Gene-specific primers for PCR amplification are: c-Maf, 5'-ACT GAA CCG CAG CTG CGC GGG GTC AG-3' and 5'-CTT CTC GTA TTT CTC CTT GTA GGC GTC C-3' to generate a 223-bp product; GATA-3, 5'-GAA GGC ATC CAG ACC CGA AAC-3' and 5'-ACC CAT GGC GGT GAC CAT GC-3'; growth factor-independent (Gfi)-1, 5'-GGA AGC ACA GAA CAC AGG CTC-3' and 5'-CTG CTA CAA GAG GAG GCA TCA-3'; SOCS1, 5'-CAG GTG GCA GCC GAC AAT GCG ATC-3' and 5'-CGT AGT GCT CCA GCA GCT CGA AAA-3' to generate a 450-bp product; SOCS3, 5'-CCG GCT AGC ATG GTC ACC CAC AGC AAG-3' and 5'-TTT GGA TCC TTA AAG TGG AGC ATC ATA-3' to generate a 690-bp product; GAPDH, 5'-TGA AGG TCG GTG TGA ACG GAT-3' and 5'-CAG GGG GGC TAA GCA GTT GGT-3' to generate a 376-bp product. The samples were amplified for 30 cycles at 94°C (1 min), 57°C (2 min), and 72°C (3 min). Five microliters of each sample was separated by 2% agarose gel electroporesis and DNA was visualized by ethidium bromide staining.
Real-time RT-PCR
Total RNA was isolated using TRIzol, and treated with DNase I (Invitrogen Life Technologies) to avoid DNA contamination; cDNA was prepared using Omniscript Reverse Transcriptase (Qiagen) with random primers. The primers are: c-Maf, AGC AGT TGG TGA CCA TGT CG and TGG AGA TCT CCT GCT TGA GG; IL-4, GAA GCC CTA CAG ACG AGC TCA and ACA GGA GAA GGG ACG CCAT; GATA-3, GAA GGC ATC CAG ACC CGA AAC and ACC CAT GGC GGT GAC CAT GC; cyclophilin A, AGG GTG GTG ACT TTA CAC GC and ATC CAG CCA TTC AGT CTT GG; GAPDH, TGA ACG GGA AGC TCA CTG G and TCC ACC ACC CTG TTG GTG TA; SOCS3, CCT TCA GCT CCA AAA GCG AG and GCT CTC CTG CAG CTT GCG. Each reaction was performed in the LightCycler system (Roche) with the SYBER green PCR kit (Qiagen). All experiments were done at least three separate times, and expression of specific genes was normalized and expressed as a percentage relative to housekeeping genes (cyclophilin A or GAPDH).
Chromatin immunoprecipitation
ChIP was performed according to the manufacturer’s protocol (Upstate Biotechnology). Briefly, cells were treated with anti-CD3 and anti-CD28 mAbs, with or without IL-6 for 1 h, cross-linked with formaldehyde at a final concentration of 1%, lysed and sonicated to shear DNA. After immunoprecipitation with anti-Stat3 or anti-Stat1 at 4°C overnight, Ab-DNA complexes were eluted and cross-links reversed by incubating at 65°C for 4 h with 5 M NaCl, and the DNA was then recovered by the QIAQuick PCR purification kit (Qiagen). Real-time PCR was then used to quantify the c-maf promoter sequences as described earlier; the primers used are: TTT CTA TAC TAT TAT GCT AAT CGC TGC CGC and CGA GAA GAG TTT AAA GCA ATT GCT GAG TTT.
Luciferase reporter assay
The c-maf promoter was amplified from BALB/c mouse genomic DNA by PCR with primers carrying restriction sites for XhoI and EcoRI 5'-AAC TCG AGC CGC AAG GGT TAA GCA AAC CTT GT-3' and 5'-CCG AAT TCA AGA TGA AAA GAG ATT TTA AAG CC-3', and cloned into the promoterless luciferase plasmid vector phRL-null, subsequently named as phRL-c-maf. Transient transfection experiments were performed with calcium phosphate. The plasmids Stat3C, null, c-maf, Stat3C combined with null, and Stat3C combined with c-maf were used at 2 μg each. For dose responses, null or c-maf luciferase constructs were used at 0.5 μg, along with different doses of Stat3C from 0 to 3 μg. Twenty-four hours after transfection, luciferase activity was measured with a Renilla Luciferase assay kit according to the manufacturer’s protocol and normalized to background activity from phRL-null.
Results
c-Maf plays an early role in IL-6-mediated IL-4 production
Studies by Rincon and colleagues (18, 19, 20) have been instrumental in elucidating the role of IL-6 in promoting IL-4 production and Th2 differentiation. As shown in Fig. 1A, IL-6 promotes IL-4 production in naive CD4+ T cells stimulated for 3 days with anti-CD3 and anti-CD28 mAbs. Anti-IL-6, but not anti-IL-10 mAbs, abrogated IL-6-driven IL-4 production, demonstrating that this effect is IL-6-specific and not due to a secondary cytokine such as IL-10. To investigate the molecular mechanisms of IL-6-driven IL-4 production, we examined whether IL-6 affects the expression of the two most critical Th2-specific transcription factors, c-Maf and GATA-3. RT-PCR was performed on BALB/c CD4+ T cells stimulated with anti-CD3 plus anti-CD28 mAbs for 3 days. Fig. 2B shows that at the end of the culture period both c-Maf and GATA-3 are up-regulated substantially in the IL-6 treatment group. Treating without IL-6 or with IL-10, another Th2 cytokine that activates Stat3 signaling similar to IL-6 and has been implicated in Th2 differentiation does not result in as much c-Maf or GATA-3 expression. These data suggest that IL-6-driven IL-4 and Th2 differentiation may involve mechanisms through c-Maf and GATA-3 up-regulation. It has been previously reported that Gfi-1, a Stat6-dependent transcriptional repressor, is also involved in Th2 cell expansion and proliferation (24). To determine whether Gfi-1 is regulated in IL-6-driven Th2 differentiation, we examined Gfi-1 expression. Unlike c-Maf or GATA-3, there is little effect of IL-6 on Gfi-1 expression (Fig. 1B), suggesting that Gfi-1 may not play a role in the early stages of IL-6-driven Th2 differentiation.
FIGURE 1. IL-6 promotes c-Maf, GATA-3, and IL-4 expression in CD4+ T cells. A total of 1 x 106 cells/ml were stimulated in plates coated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs, with or without IL-6 (20 ng/ml). A, IL-4 production after 72 h of stimulation. B, RT-PCR of c-Maf, GATA-3, Gfi-1 after 72 h of stimulation. C, Real-time RT-PCR of c-Maf expression over time. Expression of c-Maf was normalized and expressed as a percentage relative to cyclophilin A. D, Real-time RT-PCR of GATA-3 expression over time.
FIGURE 2. IL-6 is unique in rapidly inducing c-Maf expression. A, Real-time RT-PCR of c-Maf expression after 1, 3, and 24 h of stimulation. B, Real-time RT-PCR of GATA-3 expression after 1, 3, and 24 h of stimulation. C, Real-time RT-PCR of c-Maf expression after 1, 3, and 24 h of stimulation by IL-4 or IL-6. D, Western blot analysis of c-Maf expression after 24 h of stimulation with IL-4 or IL-6. E, C57BL/6 CD4 T cells were stimulated with plate-coated anti-CD3 plus anti-CD28 mAbs for 3 h along with the indicated cytokines (20 ng/ml) and c-Maf expression was measured by real-time RT-PCR. All experiments were repeated at least three separate times.
To further elucidate the roles of c-Maf and GATA-3 in IL-6-driven IL-4 production, we examined c-Maf and GATA-3 expression in CD4+ T cells at earlier time points of activation by real-time RT-PCR. We found that IL-6 is able to induce both c-Maf and GATA-3 gene expression at days 2 and 3 of culture following anti-CD3 plus anti-CD28 mAbs stimulation (Fig. 1, C and D). In contrast, in CD4+ T cells stimulated for 24 h, only c-Maf but not GATA-3 is induced by IL-6. These results suggested that although both c-Maf and GATA-3 might be important for IL-6-driven IL-4 production and Th2 differentiation, c-Maf and GATA-3 may play different roles in the process. c-Maf may play a more direct role in IL-6-initiated early IL-4 production, whereas GATA-3 may play a secondary role in regulating and maintaining IL-6-driven Th2 differentiation at later time points of stimulation.
IL-6 but not IL-4 nor other cytokines rapidly induces c-Maf expression
To investigate further the potential mechanisms of IL-6-mediated c-Maf expression, we next analyzed earlier kinetics of c-Maf expression in CD4+ T cells after stimulation. CD4+ T cells from BALB/c mice were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-6, harvested after 1, 3, or 24 h, and quantitative real-time RT-PCR was used to measure the cells c-Maf and GATA-3 expression. As shown in Fig. 2A, IL-6 is able to induce c-Maf expression as early as 3 h after stimulation (Fig. 2A). On the contrary, there is no GATA-3 induction within the 24 h period of stimulation (Fig. 2B), further indicating the earlier and more direct role of c-Maf in IL-6-mediated IL-4 expression and Th2 differentiation.
It is well-documented that IL-4 plays a central role in Th2 differentiation, and constitutively active Stat6 has been shown to up-regulate c-Maf expression (8, 9). To address the question of whether IL-6-driven c-Maf induction is secondary to IL-4 effects, such as IL-4 production or contamination in the culture resulting in activating Stat6 signals, we treated CD4+ T cells with anti-CD3 plus anti-CD28 mAbs along with IL-6 or IL-4, and then performed real-time RT-PCR after 3 h of stimulation. As shown in Fig. 2C, IL-6 but not IL-4 rapidly induces c-Maf expression, suggesting a direct and unique role of IL-6 in up-regulating Th2-specific transcription factor c-Maf expression. GAPDH instead of cyclophilin A was used as the housekeeping gene in this experiment, and IL-6 induced c-Maf in a similar fashion, thereby excluding the possibility that the increased c-Maf expression is due to possible effects of IL-6 on cyclophilin A expression (Fig. 2C). Western blot analysis was used to measure c-Maf protein 24 h after stimulation. Fig. 2D shows that IL-6, but not IL-4, induces c-Maf protein expression, paralleling the transcriptional results obtained by real-time RT-PCR. Together, these results demonstrate that IL-6-induced c-Maf expression is rapid and specific.
It is known that CD4+ T cells from BALB/c are likely biased to a Th2 phenotype, whereas CD4+ T cells on the C57BL/6 background are biased to Th1 development. To test whether IL-6-induced c-Maf expression is BALB/c intrinsic, CD4+ T cells from C57BL/6 were isolated and stimulated with anti-CD3 plus anti-CD28 mAbs, combined with IL-6. Fig. 2E shows that IL-6 induces c-Maf expression in CD4+ T cells on the C57BL/6 background, indicating that IL-6-mediated c-Maf expression is not strain limited. Besides IL-4 and IL-6, the Th2 cytokines IL-5, IL-10, and IL-13 have been implicated in Th2 responses. To investigate whether these cytokines affect acute c-Maf induction, CD4+ T cells were also stimulated with anti-CD3 plus anti-CD28 mAbs along with IL-5, IL-6, IL-10, or IL-13. As shown in Fig. 2E, IL-6 but not IL-5, IL-10, or IL-13 induces c-Maf expression, further demonstrating the unique role of IL-6 in initiating c-Maf expression upon TCR activation.
IL-6 induces c-Maf expression in CD4 T cells upon Ag-specific stimulation
CD4+ T cells produce very little IL-6 at early stages of activation, and the main sources of IL-6 are from APCs. We next examined the effects of IL-6 on T cells stimulated by specific Ag. CD4+ T cells were purified from AND TCR transgenic mice specific for cytochrome c presented by I-Ak (21). PCC peptides at 0.5 or 5 μg/ml purified B cells from B10.BR mice as APCs, and IL-6 was added to AND T cell cultures. As shown in Fig. 3A, only groups cultured with peptide, APCs, and responder T cells for 3 days have proliferative responses, demonstrating the antigenic specific response of this assay. c-Maf expression was also measured by real-time RT-PCR after 3 days, and the results demonstrate that stimulation with IL-6 induces c-Maf expression (Fig. 3B), in a fashion similar to cells stimulated with anti-CD3 plus anti-CD28 mAbs. We next examined IL-6-mediated c-Maf expression when purified CD11c+ dendritic cells were used as APCs. As shown in Fig. 3C, real-time RT-PCR for c-Maf expression in AND CD4+ T cells was measured after 3 h of stimulation, demonstrating that c-Maf is also induced in cultures including CD4+ T cells, IL-6, and dendritic cells presenting PCC peptide. These results indicate that IL-6 induces c-Maf expression in T cells after Ag-specific TCR activation, using B cell or dendritic cell as APC, as well as in APC-independent culture conditions with direct stimulation through mAbs.
FIGURE 3. IL-6 induces c-Maf expression after Ag-specific stimulation. CD4+ T cells were purified from AND TCR transgenic mice, and purified B cells or dendritic cells from B10.BR mice were used as APC. A, PCC peptides induce specific proliferative responses; purified B cells were used as APC. B, Real-time RT-PCR of c-Maf expression after specific peptide stimulation for 3 days, purified B cells were used as APC. C, Real-time RT-PCR of c-Maf expression after specific peptide stimulation for 3 h, purified CD11c+ dendritic cells were used as APC.
Dendritic cells promote c-Maf expression by IL-6
To test whether IL-6 mediates c-Maf expression at physiological levels, we isolated CD4+ T cells from BALB/c mice and evaluated their dose-response to IL-6 during c-Maf induction upon stimulation with anti-CD3 and anti-CD28 mAbs. As shown in Fig. 4A, IL-6 is able to induce c-Maf expression at a dose of 0.03 ng/ml, and achieve a plateau at around 1 ng/ml. The requirement for only a low concentration of IL-6 for inducing c-Maf expression suggests that IL-6-mediated c-Maf expression could occur under physiological conditions.
FIGURE 4. Dendritic cells mediate c-Maf expression by IL-6 production. A, Real-time RT-PCR of c-Maf expression induced by various doses of IL-6 on purified cells from BALB/c CD4+ T cells stimulated for 3 h with anti-CD3 and anti-CD28 mAbs. B, Real-time RT-PCR of c-Maf expression on purified CD4+ T cells from AND or OT-2 transgenic mice. Cells were stimulated for 3 h by anti-CD3 and anti-CD28 mAbs combined with 48 h culture supernatants from dendritic cells stimulated by soluble CD40L (500 ng/ml) and LPS (1 μg/ml). C, Real-time RT-PCR of c-Maf expression in AND or OT-2 CD4+ T cells after specific peptide stimulation for 12 h. Soluble CD40L and LPS activated CD11c+ dendritic cells were used as APC. D, Real-time RT-PCR of c-Maf expression in wild-type and IL-6-deficient CD4 T cells after stimulation with soluble anti-CD3 mAb (0.5 μg/ml) plus activated wild-type or IL-6-deficient DC11+ dendritic cells.
It has been well-documented that upon activation and maturation, dendritic cells produce IL-6 (25, 26). We then investigated whether IL-6 produced by dendritic cells is sufficient to induce c-Maf expression. Purified CD11c+ dendritic cells from both B10BR and C57B/6 mice were stimulated with soluble CD40L (500 ng/ml) combined with LPS (1 μg/ml) for 48 h, culture supernatants were harvested, and ELISA was used to confirm IL-6 production (at 1.5 ng/ml, data not shown). We then used the culture supernatant from these dendritic cells to stimulate CD4+ T cells purified from AND or OT-2 transgenic mice in combination with anti-CD3 and anti-CD28 mAbs. Fig. 4B showed that supernatants from dendritic cells stimulated by soluble CD40L and LPS are sufficient to induce c-Maf expression in CD4+ T cells, and anti-IL-6 mAbs completely abrogated c-Maf induction, demonstrating the specificity of IL-6 in regulating c-Maf expression.
Next, we examined the ability of activated dendritic cells themselves to regulate c-Maf expression. B10BR or C57BL/6 dendritic cells were activated with soluble CD40L and LPS for 48 h, thoroughly washed, and then cultured with CD4+ T cells from AND or OT-2 transgenic mice in combination with their respective specific antigenic peptides, PCC or OVA, for 12 h. As shown in Fig. 3C, after stimulation, both B10BR and C57BL/6 dendritic cells were able to induce c-Maf expression upon specific antigenic activation, and c-Maf induction was prevented by treatment with anti-IL-6 mAbs, further supporting the unique role of IL-6 in mediating c-Maf expression. Without addition of CD4+ T cells and peptides, there is no c-Maf expression, indicating that dendritic cells alone do not express c-Maf. To further examine how IL-6 deficiency affects c-Maf expression, we isolated dendritic cells from IL-6-deficient and wild-type B6 mice, activated them with LPS and CD40L for 48 h, and then cultured these dendritic cells with wild- type or IL-6-deficient CD4 T cells. As shown in Fig. 4D, wild-type dendritic cells, but not IL-6-deficient dendritic cells, induce c-Maf expression in both IL-6 knockout CD4 T cells and wild-type CD4 T cells, further indicating that IL-6 produced by DC plays an important role in c-Maf induction.
Both IL-6 and TCR induced signals are required to induce c-Maf expression
We further addressed the requirement of signals for IL-6-mediated c-Maf induction. As shown in Fig. 4A, without anti-CD3 mAb-mediated TCR activation, IL-6 failed to induce c-Maf expression, indicating that signals from IL-6 alone are not sufficient to induce c-Maf, and that TCR signals are essential for IL-6 to mediate c-Maf expression. To determine whether costimulatory signals are required for IL-6 to induce c-Maf, purified CD4+ T cells were stimulated with soluble anti-CD3 mAb only, plate-coated anti-CD3 mAb only, and plate-coated anti-CD3 plus anti-CD28 mAbs for 3 h. Fig. 5A shows that IL-6 induces c-Maf expression in CD4+ T cells stimulated with plate-coated or soluble anti-CD3 mAb alone, suggesting that costimulatory signals generated from anti-CD28 mAb are not essential for IL-6-mediated c-Maf expression. Standard RT-PCR confirmed the findings from real-time PCR; Fig. 5B shows IL-6 but not IL-10 induced early c-Maf expression, and that induction is dependent on both IL-6 and TCR signals, but independent of CD28 signaling. In the same experiment we measured the expression of Stat3 regulated SOCS molecules to assess cytokine stimulated signaling. The results indicated that IL-6 induced similar SOCS3 expression in all groups. In addition, the basal level of SOCS1 expression is increased by TCR activation, and IL-6 up-regulates SOCS1 expression in all groups (Fig. 5B). These results suggest that SOCS induction is unlikely to regulate IL-6-mediated c-Maf expression because IL-6 induces similar SOCS3 expression in groups treated with or without anti-CD3 mAbs, and the increased levels of SOCS1 in cells treated with anti-CD3 do not correspond to the levels of c-Maf expression (Fig. 5B). IL-10 fails to induce sustained SOCS1 or SOCS3 3 h after treatment (Fig. 5B), whereas it is able to induce SOCS1 and SOCS3 expression after 1 h of treatment, but significantly less compared with IL-6 (Ref. 23 and data not shown), suggesting that differential strength of signals (possibly Stat3 activation) may play a role in determining early c-Maf expression.
FIGURE 5. Signaling requirements for IL-6 to induce c-Maf expression. A, Real-time RT-PCR of c-Maf expression in CD4+ T cells stimulated for 3 h with or without TCR or CD28 activation. B, Conventional RT-PCR was performed in a separate experiment in which IL-6 and IL-10 were compared. SOCS1 and SOCS3 genes were assessed as measures of cytokine-stimulated signaling. Cells stimulated for 3 h with indicated mAbs (c, coated and s, soluble). C, Real-time RT-PCR for c-Maf expression 3 h after stimulation; CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without the indicated cytokines (300 ng/ml). D, Western blot analysis of Stat3 activation of various cytokines (20 ng/ml) after 30 min treatment. E, Flow cytometry for IL-6R and IL-10R expression on purified CD4 T cells. Isotope control (filled histogram), IL-6R expression (thick histogram), and IL-10R expression (dashed histogram) are shown.
Upon IL-6 binding to its receptor complex (IL-6R and gp130), two major signaling pathways are activated. Stat3 is recruited to gp130 through its Src homology protein 2 (SHP2) domain, and initiates the JAK-STAT signaling pathway. SHP2 is recruited to gp130, initiating the MAPK signaling cascade (27). To investigate whether other cytokines using Stat3 signaling pathways, or cytokines that share the common gp130 receptor, also induce c-Maf expression in CD4+ T cells after anti-CD3 and anti-CD28 mAb stimulation, we further examined the effects on c-Maf expression of IL-10, which activates Stat3, and IL-11, oncosatin M, and leptin that share the common signal transducer gp130 with IL-6. IL-10 is generally considered a Th2 cytokine, and IL-11 has been suggested to be able to promote Th2 differentiation (28). As shown in Fig. 5C, even under extremely high doses of cytokines (300 ng/ml), IL-10 and IL-11 induce some c-Maf expression, whereas oncosatin M and leptin induce no c-Maf expression (Fig. 5C), and prolonged incubation with IL-10, IL-11, oncosatin M or leptin did not improve c-Maf induction (data not shown). It is possible the reason IL-10, IL-11, oncosatin M, and leptin fail to induce significant c-Maf expression is due to insufficient cytokine receptor expression on the CD4+ T cell surface, and consequently insufficient Stat3 activation. Indeed, Western blotting for Stat3 activation revealed that IL-6 induced much stronger Stat3 activation compared with other cytokines (Fig. 5D), and flow cytometry measuring receptor expression showed that the IL-6R is expressed at much higher levels compared with the IL-10R on CD4 T cells (Fig. 5E). Together, these results imply that sustained IL-6-stimulated Stat3 activation may play an important role in up-regulating c-Maf induction. The reason IL-6 differs from other cytokines in regulating c-Maf expression might be due to the differential strength of Stat3 activation.
TCR-mediated Ca2+ signaling is required for IL-6 to induce c-Maf expression
PKC is predominantly expressed in T lymphocytes and has been implicated in the control of TCR/CD28-induced AP-1 and NF-B activation (22, 29). To determine whether TCR-mediated AP-1 and NF-B play a role in IL-6-mediated c-Maf expression, CD4+ T cells were purified from PKC knockout mice (22), and stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-6. Real-time RT-PCR was performed to measure c-Maf expression after 3 h of stimulation. As shown in Fig. 6A, IL-6 induces similar c-Maf expression in both wild-type and PKC-deficient CD4+ T cells, suggesting that IL-6-mediated c-Maf expression is independent of TCR-mediated AP-1 or NF-B signals.
FIGURE 6. TCR-mediated Ca2+ signals are required for IL-6 to induce c-Maf expression. A, IL-6 induces c-Maf expression in wild-type and PKC-deficient CD4+ T cells. B, CsA and FK506, but not RAPA, block IL-6-mediated c-Maf expression. C, PMA and ionomycin fail to generate sufficient TCR signals for IL-6 to induce c-Maf.
CsA and FK506 target calcineurin, thereby inhibiting TCR-mediated Ca2+ signal pathways. To test whether Ca2+ signaling pathways play a role in IL-6-mediated c-Maf expression, purified CD4+ T cells were stimulated with anti-CD3 plus anti-CD28 mAbs in the presence of CsA or FK506. Real-time RT-PCR was then performed 3 h after stimulation to measure c-Maf expression. Fig. 6B shows that both CsA (10–7 M) and FK506 (10–7 M) block IL-6-induced c-Maf expression, indicating that Ca2+ pathway signaling is critical for IL-6 to induce c-Maf expression. Rapamycin (RAPA) shares the same target, FKBP12, with FK506, but binds to a distinct molecular site and inhibits phosphorylation of p70S6 kinase (30). RAPA (10–7 M) did not have any inhibitory effects on IL-6 mediated c-Maf expression (Fig. 6B), demonstrating the specific role of TCR-stimulated Ca2+ signaling pathways to induce c-Maf expression.
We next sought to investigate whether PMA, which activates PKC and the calcium ionophore ionomycin, alone or combination could generate the requisite TCR signals for IL-6 to mediate c-Maf expression. Fig. 6C shows that neither PMA (5 ng/ml) nor ionomycin (300 ng/ml), alone or in combination, induces c-Maf expression. Because it has been previously demonstrated that both PMA and ionomycin inhibited IL-6-mediated Stat3 activation (31, 32), the failure of c-Maf induction is possibly due to signals mediated by PMA and ionomycin that antagonize IL-6-mediated JAK-STAT signaling, further suggesting a critical role of Stat3 in IL-6-mediated c-Maf expression.
IL-6-induced c-Maf expression is independent of IL-4/Stat6 signaling
To address directly the role of IL-4/Stat6 signaling in IL-6-mediated c-Maf expression, we used CD4+ T cells from IL-4 and Stat6 knockout mice to further determine the dependence on these two molecules for IL-6-mediated c-Maf expression. CD4+ T cells from IL-4 knockout, Stat6 knockout or wild-type BALB/c mice were purified and stimulated with anti-CD3 and anti-CD28 mAbs for 3 h, with or without IL-6. As shown in Fig. 7A, there is no difference in c-Maf expression between wild-type cells and CD4+ T cells deficient in IL-4 or Stat6, demonstrating that IL-6-induced c-Maf expression is IL-4/Stat6 signal-independent. We also analyzed gene expression at later time points of T cell activation in these cells. Fig. 7B shows that after 3 days of stimulation IL-6 induces c-Maf expression in CD4+ T cells deficient in IL-4 or Stat6, but at reduced levels compared with wild type. These findings suggest that IL-4/Stat6 might be indirectly involved in stimulating optimal levels of c-Maf expression, perhaps through induction of GATA-3 because the expression of GATA-3 is crucial for the differentiation of Th2 cells. In fact, when the same cells were evaluated for the expression of GATA-3, contrary to the findings for c-Maf, there was little GATA-3 expression in IL-4 or Stat6-deficient CD4+ T cells treated with or without IL-6 (Fig. 7C), indicating that GATA-3 expression is dependent on IL-4/Stat6 signals.
FIGURE 7. IL-6-induced c-Maf expression and IL-4 expression in wild-type, IL-4-deficient, or Stat6-deficient CD4+ T cells stimulated by anti-CD3 plus anti-CD28. A, Real-time RT-PCR for c-Maf expression in wild-type, IL-4-deficient, or Stat6-deficient CD4+ T cells after 3 h of stimulation. B, Real-time RT-PCR for c-Maf expression after 3 days of stimulation. C, Real-time RT-PCR for GATA-3 expression after 3 days of stimulation. D, Real-time RT-PCR for IL-4 expression in BALB/c CD4+ T cells after 3, 6, 12, and 24 h of stimulation, with or without IL-4 or IL-6. E, Real-time RT-PCR for IL-4 expression in STAT6 knockout CD4+ T cells.
Because c-Maf is critical and selective in regulating IL-4 expression (5, 6, 7), we determined whether c-Maf induction by IL-6 could regulate early IL-4 gene transcription, by examining the effects of IL-4 or IL-6 on IL-4 gene expression during early stages of CD4+ T cell activation from 3 to 24 h. In agreement with previous findings that IL-4 gene expression is up-regulated upon TCR activation, even without cytokine treatment (33), the results show that before activation, CD4 T cells contained no detectable transcripts for IL-4 (data not shown), but these increased significantly after 3 h of anti-CD3 plus anti-CD28 mAb stimulation (Fig. 7D). Neither IL-4 nor IL-6 enhanced IL-4 gene expression at the 3 h time point, indicating that this early increase in IL-4 gene expression is cytokine independent. After 6 h of stimulation, IL-4 gene expression declines in all groups, however, cells treated with IL-6 had significantly higher IL-4 gene expression (3-fold) compared with cells in the control or IL-4 treatment groups. Similar results were also observed after 12 and 24 h of stimulation (Fig. 7D). These results clearly show that IL-6, but not IL-4, is able to play a positive role in regulating early IL-4 gene expression after TCR activation. Because c-Maf expression is sustained between 3 and 24 h (Fig. 2 and data not shown), and IL-6-driven IL-4 expression only after c-Maf is induced, this suggests IL-6-mediated c-Maf expression plays a role in mediating enhancement of IL-4 expression. We next performed similar experiments on highly purified CD4+ T cells from Stat6 knockout mice. As shown in Fig. 7E, IL-6 is able to sustain IL-4 gene expression at a higher level compared with groups treated with no cytokine or IL-4, but at a reduced level compared with wild-type BALB/c mice. Collectively, these results suggest that optimal levels of IL-4 expression are IL-4/Stat6-dependent; IL-6 is also able to regulate IL-4 expression in a IL-4/Stat6–independent fashion.
Stat3 binds and activates the c-maf promoter
Both the mouse and rat c-maf genes are composed of a single exon and have highly conserved promoters, there being, only three nucleotide differences within 200 bp of the 5' flanking region (34, 35). Structural analysis of the c-maf gene and promoter regulation during differentiation of lens epithelial cells to fiber cells show that Pax6, a master transcription factor for lens development, activates the c-maf promoter. The c-maf gene is also positively autoregulated by its own product, c-Maf, through two identified Maf recognition element binding sites (34, 35). Further examination of the c-maf promoter sequences shows that there are two class II IL-6 responsive elements (CTGGGA), which are potential Stat3 binding sites.
To further investigate whether Stat3 may bind to the c-maf promoter after IL-6 stimulation, purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 mAbs, with or without IL-6, and 45 min after stimulation ChIP assay was performed using anti-Stat3 and anti-Stat1 (as a control) for immunoprecipitation. Real-time PCR was used to quantitate the c-maf promoter sequences associated with Stat3 or Stat1. As shown Fig. 8A, anti-Stat3 precipitation enriches for the c-maf promoter by 4-fold compared with anti-Stat1, indicating that Stat3 binds to the c-maf promoter after IL-6 stimulation in vivo.
FIGURE 8. Stat3 binds and activates the c-maf promoter. A, ChIP assay for the c-maf promoter. Purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 mAbs, with or without IL-6 for 1 h, and anti-Stat3 and anti-Stat1 Abs were used for immunoprecipitation. Sequences of c-maf promoter precipitated by anti-Stat3 or anti-Stat1 were quantitated by real-time PCR. Data are expressed as the ratio of Stat3-associated c-maf promoter sequences to Stat1-associated c-maf promoter sequences. B, Activation of the c-maf promoter. Transient transfection of promoterless (null), c-maf promoter-driven (c-maf), Stat3C, Stat3C/null, or Stat3/c-maf at 2 μg each into C3H10T1/2 cells. Luciferase activity was measured 24 h posttransfection. C, Dose response to Stat3C for activation of the c-maf promoter. Cells were transfected with null or c-maf luciferase constructs (0.5 μg), along with different amounts of Stat3C, as indicated.
To determine whether Stat3 transactivates the c-maf promoter, the c-maf promoter was cloned into a luciferase reporter construct, and a series of transient transfection experiments was performed. The activation of the c-maf gene and its promoter regulation has been studied in a mouse embryonic fibroblast cell line C3H10T1/2. Both c-Maf itself and Pax6 activate the c-maf promoter (34, 35), indicating that there are the necessary components in C3H10T1/2 cells for studying c-maf promoter regulation. We cotransfected a promoterless luciferase construct (phRL-null), or the c-maf promoter-driven luciferase construct (phRL-c-maf), along with Stat3C, a construct expressing a constitutively active form of Stat3 (36), into C3H10T1/2 cells. Fig. 8B shows that Stat3C markedly enhances luciferase activity when cotransfected with the luciferase plasmid driven by the c-maf promoter. Dose response analysis to Stat3C further demonstrated that Stat3 transactivates the c-maf promoter (Fig. 8C). Together, these experiments suggest a critical role for IL-6-mediated Stat3 activation in activating the c-maf promoter and regulating c-Maf expression.
Discussion
The molecular mechanisms underlying the regulation of IL-4 gene expression in Th2 differentiation have been intensively studied. The transcription factors c-Maf, Stat6, and GATA-3 have all been demonstrated to play critical roles in regulating IL-4 expression and Th2 commitment, and c-Maf has been proven to have a more selective and direct effect on the IL-4 promoter to initiate IL-4 gene transcription (5, 7). Understanding the upstream factors that regulate c-Maf expression is critical for elucidating the mechanisms of IL-4 gene regulation and Th2 commitment. In this study, we showed that IL-6, but not IL-4 or other cytokines, is able to rapidly induce TCR-dependent c-Maf expression. IL-4 did not induce c-Maf induction within 24 h of stimulation, which suggests that IL-4/Stat6 signals do not have a direct impact on the c-Maf promoter to initiate c-Maf gene regulation. The observation that IL-6-induced c-Maf expression is independent of Stat6 further confirms this observation.
The functional IL-6R consists of two subunits: the -chain and the common signal transducing receptor gp130. IL-6 binds to the surface of IL-6R, leading to the dimerization of gp130/IL-6R, activation of JAK1, JAK2, and tyrosine kinase 2, and subsequent activation of two major signaling pathways: 1) Stat3 and 2) MAPK cascade through SHP2 (37). The MAPK signaling pathway has been elegantly elucidated by Flavell and colleagues (38, 39, 40) for its important role in promoting IFN- production and Th1 development, and therefore is unlikely to play a positive role in c-Maf/IL-4/Th2 induction. Stat3 has been shown to be important for Th2 effector responses (41), and in this study we demonstrated that Stat3 binds to the c-maf promoter in CD4 T cells after stimulation, and it also transactivates the c-maf promoter (Fig. 8).
Dendritic cells have been demonstrated to be initiators of immune responses, and play major roles in capturing, processing, and transporting of Ag to initiate Ag-specific immune responses of naive T lymphocytes. IL-6 has been shown to be produced by dendritic cells upon stimulation (25, 26), and its production is necessary for inhibiting IL-12-mediated Th1 differentiation (42). Our results showed that dendritic cells activated by soluble CD40L and LPS produce sufficient IL-6 to induce c-Maf expression, indicating that IL-6 might play an important role in regulating dendritic cell-mediated Th1/Th2 differentiation. Without Th1 cytokine-mediated (IL-12, IFN-) suppression, IL-6 induced early c-Maf could positively regulate IL-4 expression and subsequent Th2 differentiation.
Our results demonstrate that IL-6-mediated signals alone are not sufficient to induce c-Maf expression in CD4 T cells, but require TCR-mediated Ca2+ signals. The calcineurin inhibitors FK506 and CsA both block IL-6-mediated c-Maf induction. One family of critical regulators of gene transcription in response to TCR ligation is NFAT. Early studies have demonstrated that CsA blocked IL-6-mediated NFAT1 expression, and studies on NFAT1 knockout mice and transgenic mice expressing a mutant form of NFAT that acts as a dominant negative NFAT showed a requirement for NFAT1 for IL-6-mediated Th2 differentiation (20). We showed there is rapid induction of c-Maf expression after 3 h of stimulation and the induction is dependent on TCR-mediated Ca2+ signaling, demonstrating that IL-6-mediated c-Maf expression and NFAT1 activation share a common upstream Ca2+ signaling pathway. Sequence analysis of the c-maf promoter region (34, 35) reveals there are two potential NFAT TTTCC binding sites at –80 and –151 of the c-maf gene, suggesting a possible role for NFAT1 in regulating c-Maf expression. Whether NFAT1 is required for IL-6-mediated c-Maf expression in CD4 T cells is currently under investigation.
Recent evidence supports a two-stage model of Th differentiation. TCR activation leads to transient IL-4 and IFN- transcription within 1 h, peaks at 2 h, declines after 3 h, and this process is TCR dependent, but is independent of IL-4/Stat6 or IL-12/Stat4 signaling (33). Similar results are observed for histone acetylation at the regulatory regions of IL-4 and IFN- (43), indicating that TCR stimulation activates an immediate and global chromatin derepression in naive T cells to initiate localized histone modification with parallel gene expression. Continued culture with IL-4 or IL-12 is required to maintain increased histone acetylation. Without polarizing cytokines or key transcription factors, the early histone hyperacetylation that resulted from TCR activation is reduced to the low basal level (43). During the second stage, the induction and maintenance of high levels of T-bet or GATA-3 expression are necessary for Th1 or Th2 commitment, and T-bet or GATA-3 may also play roles in silencing the opposing
Th locus (33, 43). Our results showed that IL-6 combined with TCR activation leads to more sustained c-Maf expression, and significant IL-4 enhancement during early stages of CD4 cell activation. However, IL-6 does not change the trend of a decline in IL-4 transcription within 24 h of TCR activation, suggesting that sustained promoter availability might be a crucial factor in determining the final outcome of IL-4 expression. Due to the possible limited availability of the IL-4 promoter, c-Maf expression alone is then not sufficient to maintain high levels of IL-4 expression, which is consistent with previous observations that overexpressing c-Maf alone is not sufficient to initiate IL-4 expression in Th1 cells (6). It will be worth examining whether IL-6 affects chromatin structure and IL-4 promoter availability.
Earlier reports (18, 19, 20) and our current studies indicate that IL-6 plays an important role in promoting T cell IL-4 expression and Th2 differentiation. In contrast there are reports that suggest that IL-6 may play an important role in inhibiting Th2 effector cells. Infection with Giardia lamblia or Mycobacterium tuberculosis in IL-6-deficient mice resulted in elevated IL-4 production (44, 45), and IL-6 inhibits IL-4-mediated Th2 differentiation and aeroallergen-induced Th2 inflammation (46, 47). These disparate results suggest that the role played by IL-6 in regulating Th1/Th2 development is complex. One possible explanation may involve IL-6-stimulated Stat3 activation in diverse cell types. Although many cytokines, including the IL-6 family and other diverse family members such as G-CSF, IFN, epidermal growth factor, leptin, IL-2, and IL-10 all activate Stat3, IL-6 is both produced by many more cell types and is a much more potent activator for Stat3 compared with other ligands. As a result, IL-6-mediated Stat3 activation may account for a significant part of the overall Stat3 activation during immune responses. As demonstrated by tissue-specific disruption, Stat3 activation is important for T cell proliferation (48), fms-like tyrosine kinase 3 ligand-dependent dendritic cell differentiation (49), and innate immunity (50). Therefore, depending on the type of Ag encountered, other cytokines present, and the particular APCs used, IL-6-mediated Stat3 activation may play a divergent role that influence the outcome of the Th1/Th2 balance.
In conclusion, our results reveal a unique role of IL-6 in directing Th2-specific transcription factor c-Maf gene initiation, up-regulating the level of IL-4 transcription during early stages of TCR activation, and providing a novel mechanism for Th2 commitment regulation. The relevance of IL-6/gp130, and Stat3 involvement in c-Maf expression could explain why the initial sources of IL-4 have been difficult to define because many cells secret IL-6, and many cytokines use gp130 and/or Stat3 signaling pathways. During certain types of inflammation, or specific microenvironments, these cytokines might turn on the c-maf gene and generate IL-4/Stat6 independent IL-4 production.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Dongmei Chen, Minwei Mao, and Patricia A. Rebollo for their assistance in real-time PCR, and Dr. Littman for kindly providing PKC knockout mice.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant R01 AI44929.
2 Address correspondence and reprint requests to Dr. Yaozhong Ding, Department of Gene and Cell Medicine, Box 1496, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. E-mail address: yaozhong.ding{at}mssm.edu
3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; PCC, pigeon cytochrome c; CsA, cyclosporin A; Gfi-1, growth factor-independent 1; RAPA, rapamycin; PKC, protein kinase C; SHP2, Src homology protein 2.
Received for publication August 4, 2004. Accepted for publication December 14, 2004.
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