- Email: [email protected]
Regulation of GM-CSF expression by the transcription factor c-Maf
Regulation of GM-CSF expression by the transcription factor c-Maf Jane Gilmour, PhD, David J. Cousins, PhD, David F. Richards, MSc, Zahid Sattar, PhD, Tak H. Lee, MD, DSc, and Paul Lavender, PhD London, United Kingdom
Mechanisms of asthma and allergic inflammation
Background: Inflammation is a key feature of asthma and allergic disease. The proinflammatory cytokines IL-4, IL-5, and IL-13 are clustered on chromosome 5q with GM-CSF in close proximity, and each of these cytokines has been implicated in the pathogenesis of inflammatory disease. Although the expression of IL-4, IL-5, and IL-13 is coordinately regulated, the TH2-associated transcription factor c-Maf is thought to be involved only in the regulation of IL-4, the cytokine thought to be the main driver of TH2 differentiation. Objective: We sought to determine whether c-Maf influenced the expression of proinflammatory cytokines other than IL-4 in the Jurkat human T-cell line. Methods: RT-PCR, ELISA, and promoter-driven CAT assays were used to determine the effect of c-Maf overexpression on cytokine genes. A biotinylated oligo pulldown assay was used to demonstrate recruitment of c-Maf to the GM-CSF promoter. Results: We found that in addition to induction of IL-4, c-Maf could upregulate GM-CSF expression at both mRNA and protein levels, and that c-Maf could strongly activate the promoters of GM-CSF and IL-4 but not IL-5. Recruitment of c-Maf to the -33 to -97 bp region of the GM-CSF promoter was demonstrated. Conclusion: We propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. Clinical implications: These data suggest that c-Maf may be a therapeutic target affecting both IL-4 and GM-CSF. (J Allergy Clin Immunol 2007;120:56-63.) Key words: Human, TH1/TH2 cells, cytokines, transcription factors, gene regulation
Inflammation is a key feature of asthma and allergy, and TH2 cells have been implicated in these diseases. The TH2associated cytokine genes IL-4, IL-5, and IL-13 are located on human chromosome 5q in proximity to the GM-CSF gene (Fig 1, A). The proximity of IL-4, IL-5, and IL-13 From the Medical Research Council and Asthma UK Center in Allergic Mechanisms of Asthma, King’s College London. Supported by the Medical Research Council (G9536930), Asthma UK 00/53, and the Guy’s and St Thomas’ Charitable Foundation Wellcome Trust (068642/Z/02/Z). Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication September 15, 2006; revised March 2, 2007; accepted for publication March 14, 2007. Available online May 17, 2007. Reprint requests: Paul Lavender, PhD, MRC and Asthma UK Center in Allergic Mechanisms of Asthma, Department of Asthma, Allergy and Respiratory Science, King’s College London, 5th Floor Thomas Guy House, Guy’s Hospital, London SE1 9RT, United Kingdom. E-mail: paul. [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.03.033
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Abbreviations used bZIP: Basic leucine zipper domain CAT: Chloramphenicol acetyl transferase CBA: Cytometric bead array PDBu: Phorbol dibutyrate PMA: Phorbol 12-myristate 13-acetate qRT-PCR: Quantitative RT-PCR UK: United Kingdom
and their importance in asthma have led to speculation about coordinate mechanisms of regulation. Coordinate expression of IL-4, IL-5, and IL-13 was demonstrated in in vitro differentiated human TH2 cells,1 and a locus control region for these genes was recently identified.2 Although GM-CSF is not coordinately expressed with the TH2-associated cytokine genes,1 it has been implicated in the pathogenesis of inflammatory diseases such as asthma. In addition to a role in myeloid cell function, GM-CSF has been shown to promote eosinophil migration, differentiation, and survival.3-5 GM-CSF is essential for the differentiation and maturation of dendritic cells in vitro, either alone or in combination with other factors including IL-4.6,7 The TH2-associated proto-oncogene c-Maf is a member of the Maf family of transcription factors, which homodimerize or heterodimerize through a basic leucine zipper (bZIP) domain to facilitate DNA binding and regulation of target gene expression.8 The Maf family shares significant homology with the AP-1 and CREB/ATF families and may interact with a range of other transcription factors. In contrast with the transcription factor GATA3, which is thought to drive TH2 differentiation, c-Maf has been suggested to initiate production of IL-4 only and have no effect on IL-5 or IL-13.9 Overexpression of c-Maf in murine TH1 clones and the murine M12 B cell lymphoma line transactivated an IL-4 promoter construct and in combination with NFATp induced low levels of endogenous IL-4 from M12 cells.10 Splenocytes from mice overexpressing c-Maf under the control of the CD4 promoter and differentiated under nonskewing conditions preferentially develop a TH2 phenotype and demonstrate attenuated expression of IFN-g.11 Conversely, purified naive CD41 T cells and splenocytes from a mouse line lacking c-Maf demonstrated impaired production of IL-4 and an inability to polarize to a TH2 phenotype.9 More recently, Hausding et al12 observed increased lung eosinophilia in mice overexpressing c-Maf and attributed this
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FIG 1. c-Maf activates GM-CSF expression. A, Schematic diagram of the human chromosome 5q cytokine locus. Conventional RT-PCR (B) and quantitative real-time RT-PCR (C) of the chromosome 5q cytokine locus. D, qRT-PCR of Amaxa transfected CD41 cells; average data from 5 experiments. qRT-PCR was performed for each gene as described in Methods.
to c-Maf–induced upregulation of IL-5 production by lung CD41 T cells at an early stage of TH2 differentiation. Differentiation of T cells to a polarized TH1 or TH2 phenotype has been well characterized in murine systems. The extent to which human T-cell differentiation reflects that of the mouse has not been clearly defined. To investigate the role of c-Maf in the regulation of human cytokine genes, we studied the response of IL-4, IL-5, IL-2, and GM-CSF to overexpression of c-Maf in a human T-cell line, and also its effect on IL-4 and GM-CSF in human CD41 T cells. We analyzed DNA binding by c-Maf and studied the distribution of c-Maf, GATA3, and T-bet protein in in vitro differentiated human T cells.
METHODS Cells and culture conditions The human Jurkat T-cell line was grown in RPMI 1640 medium (Invitrogen, Paisley, United Kingdom [UK]) supplemented with 10% FCS (Sigma, Poole, UK), L-glutamine (2 mmol/L), penicillin (100 U/ mL), and streptomycin (100 mg/mL; all Invitrogen) at 378C, 5% CO2, in humidified air. HEK-293 cells were grown in Dulbecco’s modified Eagle medium (Invitrogen) supplemented as described above.
In vitro differentiation and intracellular cytokine staining of human TH1 and TH2 cells CD41 naive T cells were isolated from peripheral blood and differentiated into either TH1 or TH2 cells in vitro for 28 days. Cells were assessed for intracellular cytokine staining either before or after
activation for 4 hours with 5 ng/mL PMA (Sigma) and 500 ng/mL ionomycin (CN Biosciences, Nottingham, UK) as described previously.1
Protein isolation and Western blotting Cytoplasmic and nuclear protein extracts were isolated from resting and activated TH1 and TH2 cells on day 28 of differentiation using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Cramlington, UK) according to the manufacturer’s instructions. Samples were separated on 10% SDS-PAGE gels, and Western blots were performed and incubated with rabbit anti– c-Maf (M-153), mouse anti–T-bet (4B10), or mouse anti-GATA3 antisera (HG3-31; all Santa Cruz Biotechnology, Santa Cruz, Calif). Goat anti-Lamin B (C-20; Santa Cruz Biotechnology) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (6C5; Abcam, Cambridge, UK) were used as loading controls. The blots were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies—goat antimouse IgG (sc-2005), donkey antigoat IgG (sc-2020), or goat antirabbit IgG (sc-2004; Santa Cruz Biotechnology) and visualized with ECL reagent (GE Healthcare, Giles, UK).
Plasmids The plasmids containing fragments of the human GM-CSF promoter linked to CAT reporter genes pGM-259, pGM-194, and pGM-152 were described previously.13 The IL-4 promoter construct pIL-4 was described previously.14 Cloning strategies for other plasmids along with oligonucleotides used are described in this article’s Appendix E1 in the Online Repository at www.jacionline.org.
Transfections Transfections and CAT assays for Jurkat cells were performed as previously described.13 Cells were activated with 100 ng/mL phorbol
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dibutyrate (PDBu) and 1 mg/mL ionomycin (CN Biosciences) 10 minutes posttransfection where indicated. Cells were incubated for 20 hours at 378C, 5% CO2 in humidified air and harvested by centrifugation, and cell lysates were assayed for CAT activity. Amaxa transfection of primary human CD41 T cells was performed according to the manufacturer’s instructions.
ELISA
Mechanisms of asthma and allergic inflammation
GM-CSF and IL-5 were measured in 72-hour or 24-hour supernatants from transfected, activated Jurkat cells using commercially available matched antibody pairs (BD Biosciences, Oxford, UK) according to the manufacturer’s instructions and as previously described.15
Cytometric bead array The cytometric bead array (CBA) was performed according to the manufacturer’s instructions on supernatants harvested at 24 hours from transfected, activated Jurkat cells using a human TH1/TH2 Cytokine Kit (BD Biosciences). Samples were run on a BD FACSCalibur, and the levels of IL-2, IL-4, and IL-5 were quantified using BD Cytometric Bead Array software.
RNA isolation and RT-PCR Total RNA was isolated from Jurkat cells harvested 20 hours posttransfection using the Qiagen RNeasy minikit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. RNA was treated with RQ1 RNase free DNase (Promega, Southampton, UK) to remove residual genomic and plasmid DNA before being repurified using the RNeasy kit. RNA was reverse-transcribed with Revertaid Mouse Moloney Leukaemia Virus reverse-transcriptase enzyme (Fermentas, Vilnius, Lithuania) using 750 ng RNA in a 40-mL reaction with 0.4 mg random hexamers (GE Healthcare). RT-PCR was performed using the cDNA equivalent of 37.5 ng reversetranscribed RNA per reaction. The RT-PCR primers and conditions used are as previously described,1 or as in this article’s Appendix E2 in the Online Repository at www.jacionline.org. PCR products were analyzed by electrophoresis on 2.8% agarose gels run in glycine buffer (200 mmol/L glycine, 15 mmol/L NaOH, 2 mmol/L Na3EDTA).
Real-time qRT-PCR Real-time qRT-PCR was performed by using an ABI PRISM 7900 Sequence Detection System thermal cycler according to the manufacturer’s instructions (Applied Biosystems, Warrington, UK) with Real Time primer/probe sets purchased from Applied Biosystems: IL-4, Hs00174122_m1; GM-CSF, Hs00171266_m1; Kif3A, Hs00199901_m1; Rad50, Hs00194871_m1. SDS software was used to determine relative quantification of the target cDNA according to the 2-(DDct) method. The graphs shown (Fig 1, C and D) represent mean data from 3 experiments and show relative quantity for each gene using the pcDNA3 transfected sample as a calibrator.
Biotinylated oligo affinity pulldown To allow binding of protein to the oligonucleotides, 20 mg nuclear extract from c-Maf transfected 293 cells was incubated with 0.6 mmol/ L biotinylated oligonucleotides and 10 mg poly(dIdC)-poly(dIdC; GE Healthcare) in binding buffer (20 mmol/L TRIS pH 7.5; 150 mmol/L NaCl). Where competition was performed, the protein extract was preincubated with 5-fold molar excess of unbiotinylated oligonucleotides. Sense strand oligonucleotides were either 59 biotinylated, or unbiotinylated for competition studies. Antisense oligonucleotides were unbiotinylated. Oligonucleotide sequences are described in this article’s Appendix E3 in the Online Repository at www.jacionline.org.
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DNA-bound proteins were collected with streptavidin sepharose (GE Healthcare) for 1 hour, washed 4 times with PBS, and resuspended in sample loading buffer. Pulldown assays were performed with c-Maf transfected Jurkat nuclear extract as described with the exception that 1 mg instead of 10 mg poly(dIdC)-poly(dIdC) was used in the reactions. The oligonucleotides used as competitors in the Jurkat nuclear extract pulldown assays are described in the Plasmids section. The biotinylated oligonucleotide used was the 162/163 oligonucleotide. Western blots detected with anti–c-Maf antibody were performed as described above.
Statistical analysis Statistics were performed using the Student t test except where stated. Error bars are SEMs in all cases (*P < .05; **P < .01; ***P < .001).
RESULTS c-Maf upregulates GM-CSF and IL-4 mRNA To investigate its effect on T-cell cytokine expression, c-Maf was overexpressed in the Jurkat T-cell line, which does not produce measurable amounts of endogenous c-Maf. We examined the effect of overexpression of c-Maf on endogenous RNA of cytokine and noncytokine genes within the 5q locus using both conventional RT-PCR (Fig 1, B) and quantitative RT-PCR (qRT-PCR; Fig 1, C). Activation alone was not sufficient to induce IL-4 expression in Jurkat cells; however, overexpression of c-Maf in combination with activation initiated IL-4 expression (Fig 1, B, panel 2; compare lanes 4 and 2). Transcription of GM-CSF was initiated in response to activation of Jurkat cells (Fig 1, B, panel 1, lane 2) and was further increased in response to overexpression of c-Maf (Fig 1, B, panel 1; compare lanes 4 and 2). Overexpression of c-Maf without activation was not sufficient for expression of either GM-CSF or IL-4 (Fig 1, B, panels 1 and 2, lane 1) because CMV promoters are less active in hemopoietic cells, and stimulation is required for maximal expression.16 The levels of Rad50 mRNA and 18S rRNA were unchanged by activation or by overexpression of c-Maf (Fig 1, B, panels 3 and 5). Using qRT-PCR, a 3-fold increase in GM-CSF expression in c-Maf transfected Jurkat cells was demonstrated (Fig 1, C). Because only 40% of these cells were transfected as determined by fluorescence-activated cell sorting analysis (data not shown) after transient transfection of a GFP encoding vector, changes in mRNA levels observed will not fully reflect the effect of c-Maf at a single cell level. The difference in fold induction observed between IL-4 and GM-CSF is most likely a result of the existing background level of GM-CSF expression in activated Jurkat cells. Kif3A and Rad50 mRNA levels were unchanged by overexpression of c-Maf. IL-5 mRNA was undetectable in all samples (data not shown). Transient transfection of c-Maf into primary human CD41 cells showed a similar capacity to enhance both GM-CSF and IL-4 expression, causing an average 60% increase in GM-CSF expression and 120% increase in IL-4 mRNA (Fig 1, D). As with Jurkat cells, the transfection efficiency of CD41 cells was approximately 40%.
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Transactivation of the GM-CSF promoter by c-Maf c-Maf stimulates IL-4 expression through binding at the c-Maf response element (MARE) in the IL-4 proximal promoter.10 To investigate how c-Maf caused an increase in GM-CSF levels, we examined the effect of c-Maf on cytokine promoters driving a CAT reporter gene in transient transfection assays. Jurkat cells were transiently cotransfected with c-Maf and cytokine promoter reporter constructs and activated with PdBu and ionomycin. c-Maf specifically activated a 443-bp region of the human IL-4 promoter (pIL4) and a 194-bp region of the human GMCSF promoter (pGM-194) but had no effect on a 517-bp human IL-5 promoter construct (pIL-5) or the control empty expression vector pBLCAT3 (Fig 3, A). This suggests that c-Maf is acting at a site within the proximal 194 bp of the GM-CSF promoter. Comparison of the IL4 and GM-CSF promoter sequences failed to show any comparable MARE sites in the GM-CSF promoter (data not shown), suggesting that the mechanism of action of c-Maf on the GM-CSF promoter differs from that causing induction of IL-4 expression. Plasmids with sequential deletions of the GM-CSF promoter linked to a CAT reporter gene were then studied. These were cotransfected with c-Maf in Jurkat cells, and after overnight activation with PdBu and ionomycin, cells were harvested and CAT activity measured. CAT activity increased as the promoter fragment was shortened, with the highest level of CAT activity seen in the pGM-100 construct (Fig 3, B). This suggests that c-Maf is acting at a site contained within the proximal 100 bp of the GM-CSF promoter. c-Maf is recruited to the GM-CSF promoter The -100 bp region of the GM-CSF promoter does not contain an obvious MARE site; however, c-Maf is known to interact with several other protein families, so it could potentially heterodimerize with other transcription factors. To demonstrate recruitment of c-Maf to the GM-CSF promoter, we used a biotinylated oligonucleotide (oligo) affinity pulldown technique to capture bound c-Maf on a GM-CSF promoter oligo. The biotinylated oligo, termed 162/163, represented the sequence between -33 bp and -97 bp of the GM-CSF promoter and contains several
Mechanisms of asthma and allergic inflammation
c-Maf upregulates GM-CSF and IL-4 protein levels We examined cytokine levels in the supernatants from the transfected Jurkat cells described above by ELISA or CBA. Cells transfected with the c-Maf expression vector showed an average 3-fold increase in GM-CSF secretion (Fig 2, A), which mirrored the extent to which RNA was increased in the real-time qRT-PCR assay. IL-4 protein levels in the culture media were also increased in response to overexpression of c-Maf (Fig 2, B), whereas IL-2 levels were not significantly altered (Fig 2, C). IL-5 protein levels were undetectable with or without c-Maf overexpression when assayed by either ELISA or CBA (data not shown). Taken together, these data suggest that c-Maf can augment GM-CSF expression in Jurkat cells.
FIG 2. Increased GM-CSF and IL-4 production by c-Maf transfected Jurkat cells. Supernatants were taken from activated Jurkat cells transiently transfected with a plasmid containing c-Maf, the control plasmid pcDNA3, or no DNA. A, GM-CSF levels assayed by ELISA at 72 hours. B, IL-4 levels assayed by CBA at 24 hours. C, IL-2 levels assayed by CBA at 24 hours. Graphs represent mean data from 4, 6, and 6 separate experiments, respectively.
known binding sites for constitutive and activation responsive transcription factors, but lacks a consensus MARE (Fig 4, A). Nuclear extracts of HEK-293 cells overexpressing c-Maf were mixed with the biotinylated 162/ 163 oligo (Fig 4, B, lanes 1-4), a biotinylated IL-4 NFAT-MARE oligo (Fig 4, B, lanes 5-8), or a biotinylated IL-5 palindrome oligo (Fig 4, B, lane 9). The captured proteins were then run on a Western blot and tested for c-Maf immunoreactivity. Both the 162/163 oligo in lane 1 and the IL-4 NFAT-MARE oligo in lane 5 demonstrated pulldown of c-Maf. By contrast, an oligo representing a characterized palindromic element in the IL-5 promoter, used as a negative control (lane 9),13 did not interact with c-Maf protein. The interaction of c-Maf with the biotinylated 162/163 oligo could be competed with
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5-fold molar excess of an unbiotinylated 162/163 oligo (lane 2) or an unbiotinylated IL-4 NFAT-MARE oligo (lane 3) but not with the IL-5 palindrome oligo (lane 4). Similarly, for the biotinylated IL-4 NFAT-MARE sequence, there was self-competition with the IL-4 NFATMARE oligo (lane 7), and the 162/163 oligo (lane 6) competed for c-Maf to a greater extent than the IL-5 palindrome oligo (lane 8). This demonstrates recruitment of c-Maf to the GM-CSF promoter mapping to the region between -33 and -97 bp in HEK-293 cells overexpressing c-Maf. To define further the region of the GM-CSF promoter involved in c-Maf recruitment and to preclude the effect of cell-specific influences on c-Maf/DNA binding, a series of experiments were conducted using nuclear extracts from Jurkat cells overexpressing c-Maf. These experiments are described in this article’s Appendix E4 and results are shown in this article’s Fig E1 in the Online Repository at www.jacionline.org. These data suggest that c-Maf can be recruited to the GM-CSF promoter, but the lack of a consensus MARE site in the promoter and the lack of a tightly defined c-Maf binding site indicate a complex interaction mechanism. Interaction of c-Maf at the GM-CSF promoter may be as a result of tethering rather than direct DNA binding, and its recruitment may be stabilized by interaction with other DNA-binding proteins. These data are in keeping with previously reported modes of action of c-Maf.
Expression of c-Maf in human TH1 and TH2 cells In murine T-cell differentiation, c-Maf was selectively expressed in TH2 cells10 and would therefore not correlate with GM-CSF expression. If c-Maf plays a role in GMCSF expression, this would suggest a capacity for differential regulation of GM-CSF in murine TH1 and TH2 cells. To examine the role of c-Maf in GM-CSF expression in human primary cells, we used an in vitro differentiation assay to generate human TH1 and TH2 cells1 and to analyze the transcription factor and cytokine expression by these cells after 28 days in culture. In vitro differentiated TH1 and TH2 cells generated in this assay showed strong polarization toward either a TH1 or a TH2 phenotype as determined by multicolor intracellular cytokine staining for IL-5 and IFN-g (see this article’s Fig E2 in the Online Repository at www.jacionline.org). GM-CSF was expressed at high levels on activation in both TH1 and TH2 cells. Western blots were performed on nuclear and cytoplasmic extracts from both resting and activated TH1 and TH2 cells to assess transcription factor expression under these conditions (Fig 5, A). T-bet and GATA3 showed most abundant expression in TH1 and TH2 cells, respectively. c-Maf was expressed most strongly in resting TH2 cells (lane 7), and levels were substantially reduced after activation (lane 8). Surprisingly, the level of expression of c-Maf in activated TH2 cells was similar to that seen in resting and activated TH1 cells (lanes 3 and 4). Densitometry of western blots of c-Maf protein demonstrated a significant difference in the expression level of c-Maf between unactivated TH1 and TH2 cells and
FIG 3. Transactivation of the GM-CSF promoter by c-Maf. CAT activity was measured in extracts from activated Jurkat cells cotransfected with cytokine promoter constructs and either c-Maf or a control plasmid. Data are from 3 separate experiments and normalized to the sample cotransfected with c-Maf and pIL-4. *P < .05 and **P < .01 compared with the sample cotransfected with c-Maf and pBLCAT3. B, CAT activity was measured in extracts from activated Jurkat cells cotransfected with the GM-CSF promoter constructs and either a plasmid containing c-Maf or a control plasmid. Data are from 6 separate experiments and normalized to the sample cotransfected with c-Maf and pGM-100. **P < .01 compared with the sample cotransfected with c-Maf and pBLCAT3.
between unactivated and activated TH2 cells (Fig 5, B). The densitometry confirms the similarity in c-Maf protein levels between activated TH1 and activated TH2 cells. The protein expression profiles observed here strongly reflect the mRNA profiles previously documented for these transcription factors in human TH1 and TH2 cells.1
DISCUSSION In this study, we have shown that overexpression of c-Maf can increase expression of GM-CSF at both the mRNA and protein levels. We have used transient transfection assays to localize the effect of c-Maf to the GM-CSF proximal promoter and demonstrated recruitment of c-Maf to a region spanning -33 bp to -97 bp of the GMCSF proximal promoter. Furthermore, we have shown that both TH1 and TH2 cells express GM-CSF on activation
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FIG 4. c-Maf interacts with the GM-CSF proximal promoter. A, Sequence of the GM-CSF promoter region thought to be bound by c-Maf. Known binding sites are represented by black boxes. Oligos used as competitors in the affinity pulldown shown in Fig E1, A, and to construct the plasmids transfected in Fig E1 are represented by gray boxes. B, Biotinylated oligo affinity pulldown of the GM-CSF promoter in HEK-293 cells.
FIG 5. Analysis of 28-day in vitro differentiated human TH1 and TH2 cells. A, Western blot of nuclear (N) and cytoplasmic (C) extracts from TH1 and TH2 cells using the indicated primary antibodies and the relevant secondary antibodies. B, Densitometry of c-Maf protein expression. The graph represents mean data from 3 independent experiments normalized to the TH2 unstimulated sample. Data were analyzed by using a 2-way ANOVA test. NE, Nuclear extract.
and that c-Maf is found at similar levels in activated, in vitro differentiated human TH1 and TH2 cells. The Maf family of proteins can homodimerize or heterodimerize with other Maf proteins, other bZIP transcription factors, and a range of other non-bZIP factors.8
This property imparts on Maf proteins the ability to be recruited to a diverse range of DNA sequences and consequently be involved in regulation of genes that lack consensus Maf-response elements in their regulatory regions. Studies on the rat homologue of c-Maf (Maf-2)
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demonstrated altered specificity for DNA binding sites depending on its heterodimeric partner, with Maf-2 capable of interacting with c-Fos and to a lesser extent with Fos-B and Fra-2 but not with the Jun family of proteins.17 Interaction of c-Maf with c-Myb inhibited the cooperative activation of the CD13/APN gene by c-Myb and Ets-1 in human myeloid cell lines.18 Another Maf family member, MafB, synergistically transactivated the GPIIb promoter with GATA1 and Ets after cotransfection in K562 cells.19 No consensus MafB binding site was identified in the promoter, and the enhancement of transcriptional activity was presumed to be mediated by an interaction of MafB with GATA1 and Ets. c-Maf was found to inhibit IL-12 p40 gene expression in macrophages.20 Although a specific interaction domain could not be located in the promoter, overexpression of c-Maf affected the formation of several DNA-protein complexes as assessed by electrophoretic mobility shift assay. This suggested that c-Maf can impart an inhibitory activity without direct contact with DNA at the IL-12 proximal promoter and supports our suggestion that c-Maf may interact with multiple factors at more than 1 site in the GM-CSF proximal promoter to contribute to transcriptional activation. Polarization of CD41 T cells to a TH1 or TH2 lineage has been well characterized in mouse but less so in human T cells. In this study, we describe the protein expression patterns of the transcription factors T-bet, GATA3, and c-Maf in human TH1 and TH2 cells (Fig 5). We found that of the 3 factors, T-bet was the most lineage-restricted, with expression observed primarily in resting and activated TH1 cells, compatible with its role as a driving factor in TH1 differentiation and maintenance.21 Whether the low levels of T-bet observed in TH2 cells have a meaningful function is yet to be established, although it is unlikely to involve positive regulation of IFN-g because these cells are negative for IFN-g expression as determined by fluorescence-activated cell sorting. GATA3 showed strong expression in TH2 cells but also demonstrated low levels of expression in TH1 cells. This is unsurprising because in addition to its role in TH2 differentiation, GATA3 is also responsible for activation and tissue specific expression of the T-cell receptor a and b chains.22,23 Densitometric analysis of c-Maf Western blots demonstrated that the highest level of c-Maf protein was observed in resting TH2 cells, and that c-Maf was significantly downregulated in activated TH2 cells. The level of c-Maf expression in resting and activated TH1 cells was found to be similar to that seen in activated TH2 cells. The reason for this pattern of expression is currently unclear, but it was consistent with the pattern of c-Maf mRNA expression data that we have previously described.1 During T-cell differentiation, GATA3 is thought to induce chromatin remodeling at the 5q locus in TH2 cells and, in doing so, to increase accessibility of the IL-4 promoter to factors such as c-Maf, and therefore mediate its transcriptional regulation.24,25 The lack of sufficient GATA3 in TH1 cells to induce chromatin remodeling results in a chromatin conformation at the 5q locus that is not permissive for the expression of TH2 cytokines. Because GM-CSF
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production is rapidly upregulated after activation in both TH1 and TH2 cells, this would suggest that its gene lies outside the 5q core domain that requires remodeling during TH2 differentiation to permit expression of IL-4, IL-5, and IL-13. Although the regulation of IL-4 expression has been described as the main role for c-Maf in TH2 cells, recent studies have proposed IL-4 independent functions. Normal induction of CD25 (IL2Ra) in developing TH2 cells required c-Maf, because mice lacking c-Maf displayed delayed upregulation of CD25.26 Weigmann et al27 reported higher expression of c-Maf in T-bet expressing TH1 cells from the lamina propria of patients with Crohn disease and proposed a pathogenic role for c-Maf in memory T cells in colitis. In macrophages, c-Maf was found to be a potent activator of IL-10 and suppressor of IL-12 gene transcription and was proposed as a mediator of the immunosuppressive effects of IL-10.20 It is possible that c-Maf, in addition to its role in IL-4 expression, is required by both TH1 and TH2 cells for other, as yet unknown functions. On the basis of the evidence presented here, we propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. We demonstrate increased levels of endogenous GM-CSF mRNA and protein in response to overexpression of c-Maf and have localized binding of c-Maf to a region between -33 and -97 bp of the GM-CSF promoter. Furthermore, we find that, in contrast with the murine system, the expression pattern of c-Maf in differentiated human T cells is not restricted to TH2 cells and correlates with GM-CSF expression. We propose that c-Maf may have a broader role than previously described in the regulation of expression of inflammatory mediators. We thank Drs T. Kouzarides, T. Honjo, and J. Morgan for providing plasmids and Dr S. Hirst for assistance with statistical analysis.
REFERENCES 1. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human TH1/TH2 cell differentiation: direct evidence for coordinated expression of th2 cytokines. J Immunol 2002;169:2498-506. 2. Lee GR, Fields PE, Griffin TJ, Flavell RA. Regulation of the Th2 cytokine locus by a locus control region. Immunity 2003;19:145-53. 3. Takamoto M, Sugane K. Synergism of IL-3, IL-5, and GM-CSF on eosinophil differentiation and its application for an assay of murine IL-5 as an eosinophil differentiation factor. Immunol Lett 1995;45: 43-6. 4. Ebisawa M, Liu MC, Yamada T, Kato M, Lichtenstein LM, Bochner BS, et al. Eosinophil transendothelial migration induced by cytokines, II: potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J Immunol 1994;152:4590-6. 5. Burke LA, Hallsworth MP, Litchfield TM, Davidson R, Lee TH. Identification of the major activity derived from cultured human peripheral blood mononuclear cells, which enhances eosinophil viability, as granulocyte macrophage colony-stimulating factor (GM-CSF). J Allergy Clin Immunol 1991;88:226-35. 6. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176:1693-702.
7. Berger TG, Feuerstein B, Strasser E, Hirsch U, Schreiner D, Schuler G, et al. Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories. J Immunol Methods 2002;268: 131-40. 8. Blank V, Andrews NC. The Maf transcription factors: regulators of differentiation. Trends Biochem Sci 1997;22:437-41. 9. Kim JI, Ho IC, Grusby MJ, Glimcher LH. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 1999;10:745-51. 10. Ho IC, Hodge MR, Rooney JW, Glimcher LH. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 1996;85:973-83. 11. Ho IC, Lo D, Glimcher LH. c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms. J Exp Med 1998;188:1859-66. 12. Hausding M, Ho IC, Lehr HA, Weigmann B, Lux C, Schipp M, et al. A stage-specific functional role of the leucine zipper transcription factor c-Maf in lung Th2 cell differentiation. Eur J Immunol 2004;34: 3401-12. 13. Staynov DZ, Cousins DJ, Lee TH. A regulatory element in the promoter of the human granulocyte-macrophage colony-stimulating factor gene that has related sequences in other T-cell-expressed cytokine genes. Proc Natl Acad Sci U S A 1995;92:3606-10. 14. Codlin S, Soh C, Lee T, Lavender P. Characterization of a palindromic enhancer element in the promoters of IL4, IL5, and IL13 cytokine genes. J Allergy Clin Immunol 2003;111:826-32. 15. Richards DF, Fernandez M, Caulfield J, Hawrylowicz CM. Glucocorticoids drive human CD8(1) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production. Eur J Immunol 2000;30:2344-54. 16. Ramezani A, Hawley TS, Hawley RG. Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol Ther 2000;2:458-69. 17. Matsushima-Hibiya Y, Nishi S, Sakai M. Rat maf-related factors: the specificities of DNA binding and heterodimer formation. Biochem Biophys Res Commun 1998;245:412-8.
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18. Hegde SP, Kumar A, Kurschner C, Shapiro LH. c-Maf interacts with c-Myb to regulate transcription of an early myeloid gene during differentiation. Mol Cell Biol 1998;18:2729-37. 19. Sevinsky JR, Whalen AM, Ahn NG. Extracellular signal-regulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/ Kreisler. Mol Cell Biol 2004;24:4534-45. 20. Cao S, Liu J, Chesi M, Bergsagel PL, Ho IC, Donnelly RP, et al. Differential regulation of IL-12 and IL-10 gene expression in macrophages by the basic leucine zipper transcription factor c-Maf fibrosarcoma. J Immunol 2002;169:5715-25. 21. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 2002; 295:338-42. 22. Ho IC, Vorhees P, Marin N, Oakley BK, Tsai SF, Orkin SH, et al. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene. EMBO J 1991;10: 1187-92. 23. Henderson AJ, McDougall S, Leiden J, Calame KL. GATA elements are necessary for the activity and tissue specificity of the T-cell receptor betachain transcriptional enhancer. Mol Cell Biol 1994;14:4286-94. 24. Lee HJ, Takemoto N, Kurata H, Kamogawa Y, Miyatake S, O’Garra A, et al. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells. J Exp Med 2000; 192:105-15. 25. Takemoto N, Kamogawa Y, Jun Lee H, Kurata H, Arai KI, O’Garra A, et al. Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J Immunol 2000;165:6687-91. 26. Hwang ES, White IA, Ho IC. An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines. Proc Natl Acad Sci U S A 2002;99:13026-30. 27. Weigmann B, Nemetz A, Becker C, Schmidt J, Strand D, Lehr HA, et al. A critical regulatory role of leucin zipper transcription factor c-Maf in Th1-mediated experimental colitis. J Immunol 2004;173:3446-55.
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Mechanisms of asthma and allergic inflammation
Background: Inflammation is a key feature of asthma and allergic disease. The proinflammatory cytokines IL-4, IL-5, and IL-13 are clustered on chromosome 5q with GM-CSF in close proximity, and each of these cytokines has been implicated in the pathogenesis of inflammatory disease. Although the expression of IL-4, IL-5, and IL-13 is coordinately regulated, the TH2-associated transcription factor c-Maf is thought to be involved only in the regulation of IL-4, the cytokine thought to be the main driver of TH2 differentiation. Objective: We sought to determine whether c-Maf influenced the expression of proinflammatory cytokines other than IL-4 in the Jurkat human T-cell line. Methods: RT-PCR, ELISA, and promoter-driven CAT assays were used to determine the effect of c-Maf overexpression on cytokine genes. A biotinylated oligo pulldown assay was used to demonstrate recruitment of c-Maf to the GM-CSF promoter. Results: We found that in addition to induction of IL-4, c-Maf could upregulate GM-CSF expression at both mRNA and protein levels, and that c-Maf could strongly activate the promoters of GM-CSF and IL-4 but not IL-5. Recruitment of c-Maf to the -33 to -97 bp region of the GM-CSF promoter was demonstrated. Conclusion: We propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. Clinical implications: These data suggest that c-Maf may be a therapeutic target affecting both IL-4 and GM-CSF. (J Allergy Clin Immunol 2007;120:56-63.) Key words: Human, TH1/TH2 cells, cytokines, transcription factors, gene regulation
Inflammation is a key feature of asthma and allergy, and TH2 cells have been implicated in these diseases. The TH2associated cytokine genes IL-4, IL-5, and IL-13 are located on human chromosome 5q in proximity to the GM-CSF gene (Fig 1, A). The proximity of IL-4, IL-5, and IL-13 From the Medical Research Council and Asthma UK Center in Allergic Mechanisms of Asthma, King’s College London. Supported by the Medical Research Council (G9536930), Asthma UK 00/53, and the Guy’s and St Thomas’ Charitable Foundation Wellcome Trust (068642/Z/02/Z). Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest. Received for publication September 15, 2006; revised March 2, 2007; accepted for publication March 14, 2007. Available online May 17, 2007. Reprint requests: Paul Lavender, PhD, MRC and Asthma UK Center in Allergic Mechanisms of Asthma, Department of Asthma, Allergy and Respiratory Science, King’s College London, 5th Floor Thomas Guy House, Guy’s Hospital, London SE1 9RT, United Kingdom. E-mail: paul. [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2007.03.033
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Abbreviations used bZIP: Basic leucine zipper domain CAT: Chloramphenicol acetyl transferase CBA: Cytometric bead array PDBu: Phorbol dibutyrate PMA: Phorbol 12-myristate 13-acetate qRT-PCR: Quantitative RT-PCR UK: United Kingdom
and their importance in asthma have led to speculation about coordinate mechanisms of regulation. Coordinate expression of IL-4, IL-5, and IL-13 was demonstrated in in vitro differentiated human TH2 cells,1 and a locus control region for these genes was recently identified.2 Although GM-CSF is not coordinately expressed with the TH2-associated cytokine genes,1 it has been implicated in the pathogenesis of inflammatory diseases such as asthma. In addition to a role in myeloid cell function, GM-CSF has been shown to promote eosinophil migration, differentiation, and survival.3-5 GM-CSF is essential for the differentiation and maturation of dendritic cells in vitro, either alone or in combination with other factors including IL-4.6,7 The TH2-associated proto-oncogene c-Maf is a member of the Maf family of transcription factors, which homodimerize or heterodimerize through a basic leucine zipper (bZIP) domain to facilitate DNA binding and regulation of target gene expression.8 The Maf family shares significant homology with the AP-1 and CREB/ATF families and may interact with a range of other transcription factors. In contrast with the transcription factor GATA3, which is thought to drive TH2 differentiation, c-Maf has been suggested to initiate production of IL-4 only and have no effect on IL-5 or IL-13.9 Overexpression of c-Maf in murine TH1 clones and the murine M12 B cell lymphoma line transactivated an IL-4 promoter construct and in combination with NFATp induced low levels of endogenous IL-4 from M12 cells.10 Splenocytes from mice overexpressing c-Maf under the control of the CD4 promoter and differentiated under nonskewing conditions preferentially develop a TH2 phenotype and demonstrate attenuated expression of IFN-g.11 Conversely, purified naive CD41 T cells and splenocytes from a mouse line lacking c-Maf demonstrated impaired production of IL-4 and an inability to polarize to a TH2 phenotype.9 More recently, Hausding et al12 observed increased lung eosinophilia in mice overexpressing c-Maf and attributed this
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FIG 1. c-Maf activates GM-CSF expression. A, Schematic diagram of the human chromosome 5q cytokine locus. Conventional RT-PCR (B) and quantitative real-time RT-PCR (C) of the chromosome 5q cytokine locus. D, qRT-PCR of Amaxa transfected CD41 cells; average data from 5 experiments. qRT-PCR was performed for each gene as described in Methods.
to c-Maf–induced upregulation of IL-5 production by lung CD41 T cells at an early stage of TH2 differentiation. Differentiation of T cells to a polarized TH1 or TH2 phenotype has been well characterized in murine systems. The extent to which human T-cell differentiation reflects that of the mouse has not been clearly defined. To investigate the role of c-Maf in the regulation of human cytokine genes, we studied the response of IL-4, IL-5, IL-2, and GM-CSF to overexpression of c-Maf in a human T-cell line, and also its effect on IL-4 and GM-CSF in human CD41 T cells. We analyzed DNA binding by c-Maf and studied the distribution of c-Maf, GATA3, and T-bet protein in in vitro differentiated human T cells.
METHODS Cells and culture conditions The human Jurkat T-cell line was grown in RPMI 1640 medium (Invitrogen, Paisley, United Kingdom [UK]) supplemented with 10% FCS (Sigma, Poole, UK), L-glutamine (2 mmol/L), penicillin (100 U/ mL), and streptomycin (100 mg/mL; all Invitrogen) at 378C, 5% CO2, in humidified air. HEK-293 cells were grown in Dulbecco’s modified Eagle medium (Invitrogen) supplemented as described above.
In vitro differentiation and intracellular cytokine staining of human TH1 and TH2 cells CD41 naive T cells were isolated from peripheral blood and differentiated into either TH1 or TH2 cells in vitro for 28 days. Cells were assessed for intracellular cytokine staining either before or after
activation for 4 hours with 5 ng/mL PMA (Sigma) and 500 ng/mL ionomycin (CN Biosciences, Nottingham, UK) as described previously.1
Protein isolation and Western blotting Cytoplasmic and nuclear protein extracts were isolated from resting and activated TH1 and TH2 cells on day 28 of differentiation using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology, Cramlington, UK) according to the manufacturer’s instructions. Samples were separated on 10% SDS-PAGE gels, and Western blots were performed and incubated with rabbit anti– c-Maf (M-153), mouse anti–T-bet (4B10), or mouse anti-GATA3 antisera (HG3-31; all Santa Cruz Biotechnology, Santa Cruz, Calif). Goat anti-Lamin B (C-20; Santa Cruz Biotechnology) and mouse anti–glyceraldehyde-3-phosphate dehydrogenase (6C5; Abcam, Cambridge, UK) were used as loading controls. The blots were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies—goat antimouse IgG (sc-2005), donkey antigoat IgG (sc-2020), or goat antirabbit IgG (sc-2004; Santa Cruz Biotechnology) and visualized with ECL reagent (GE Healthcare, Giles, UK).
Plasmids The plasmids containing fragments of the human GM-CSF promoter linked to CAT reporter genes pGM-259, pGM-194, and pGM-152 were described previously.13 The IL-4 promoter construct pIL-4 was described previously.14 Cloning strategies for other plasmids along with oligonucleotides used are described in this article’s Appendix E1 in the Online Repository at www.jacionline.org.
Transfections Transfections and CAT assays for Jurkat cells were performed as previously described.13 Cells were activated with 100 ng/mL phorbol
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dibutyrate (PDBu) and 1 mg/mL ionomycin (CN Biosciences) 10 minutes posttransfection where indicated. Cells were incubated for 20 hours at 378C, 5% CO2 in humidified air and harvested by centrifugation, and cell lysates were assayed for CAT activity. Amaxa transfection of primary human CD41 T cells was performed according to the manufacturer’s instructions.
ELISA
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GM-CSF and IL-5 were measured in 72-hour or 24-hour supernatants from transfected, activated Jurkat cells using commercially available matched antibody pairs (BD Biosciences, Oxford, UK) according to the manufacturer’s instructions and as previously described.15
Cytometric bead array The cytometric bead array (CBA) was performed according to the manufacturer’s instructions on supernatants harvested at 24 hours from transfected, activated Jurkat cells using a human TH1/TH2 Cytokine Kit (BD Biosciences). Samples were run on a BD FACSCalibur, and the levels of IL-2, IL-4, and IL-5 were quantified using BD Cytometric Bead Array software.
RNA isolation and RT-PCR Total RNA was isolated from Jurkat cells harvested 20 hours posttransfection using the Qiagen RNeasy minikit (Qiagen, Crawley, UK) according to the manufacturer’s instructions. RNA was treated with RQ1 RNase free DNase (Promega, Southampton, UK) to remove residual genomic and plasmid DNA before being repurified using the RNeasy kit. RNA was reverse-transcribed with Revertaid Mouse Moloney Leukaemia Virus reverse-transcriptase enzyme (Fermentas, Vilnius, Lithuania) using 750 ng RNA in a 40-mL reaction with 0.4 mg random hexamers (GE Healthcare). RT-PCR was performed using the cDNA equivalent of 37.5 ng reversetranscribed RNA per reaction. The RT-PCR primers and conditions used are as previously described,1 or as in this article’s Appendix E2 in the Online Repository at www.jacionline.org. PCR products were analyzed by electrophoresis on 2.8% agarose gels run in glycine buffer (200 mmol/L glycine, 15 mmol/L NaOH, 2 mmol/L Na3EDTA).
Real-time qRT-PCR Real-time qRT-PCR was performed by using an ABI PRISM 7900 Sequence Detection System thermal cycler according to the manufacturer’s instructions (Applied Biosystems, Warrington, UK) with Real Time primer/probe sets purchased from Applied Biosystems: IL-4, Hs00174122_m1; GM-CSF, Hs00171266_m1; Kif3A, Hs00199901_m1; Rad50, Hs00194871_m1. SDS software was used to determine relative quantification of the target cDNA according to the 2-(DDct) method. The graphs shown (Fig 1, C and D) represent mean data from 3 experiments and show relative quantity for each gene using the pcDNA3 transfected sample as a calibrator.
Biotinylated oligo affinity pulldown To allow binding of protein to the oligonucleotides, 20 mg nuclear extract from c-Maf transfected 293 cells was incubated with 0.6 mmol/ L biotinylated oligonucleotides and 10 mg poly(dIdC)-poly(dIdC; GE Healthcare) in binding buffer (20 mmol/L TRIS pH 7.5; 150 mmol/L NaCl). Where competition was performed, the protein extract was preincubated with 5-fold molar excess of unbiotinylated oligonucleotides. Sense strand oligonucleotides were either 59 biotinylated, or unbiotinylated for competition studies. Antisense oligonucleotides were unbiotinylated. Oligonucleotide sequences are described in this article’s Appendix E3 in the Online Repository at www.jacionline.org.
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DNA-bound proteins were collected with streptavidin sepharose (GE Healthcare) for 1 hour, washed 4 times with PBS, and resuspended in sample loading buffer. Pulldown assays were performed with c-Maf transfected Jurkat nuclear extract as described with the exception that 1 mg instead of 10 mg poly(dIdC)-poly(dIdC) was used in the reactions. The oligonucleotides used as competitors in the Jurkat nuclear extract pulldown assays are described in the Plasmids section. The biotinylated oligonucleotide used was the 162/163 oligonucleotide. Western blots detected with anti–c-Maf antibody were performed as described above.
Statistical analysis Statistics were performed using the Student t test except where stated. Error bars are SEMs in all cases (*P < .05; **P < .01; ***P < .001).
RESULTS c-Maf upregulates GM-CSF and IL-4 mRNA To investigate its effect on T-cell cytokine expression, c-Maf was overexpressed in the Jurkat T-cell line, which does not produce measurable amounts of endogenous c-Maf. We examined the effect of overexpression of c-Maf on endogenous RNA of cytokine and noncytokine genes within the 5q locus using both conventional RT-PCR (Fig 1, B) and quantitative RT-PCR (qRT-PCR; Fig 1, C). Activation alone was not sufficient to induce IL-4 expression in Jurkat cells; however, overexpression of c-Maf in combination with activation initiated IL-4 expression (Fig 1, B, panel 2; compare lanes 4 and 2). Transcription of GM-CSF was initiated in response to activation of Jurkat cells (Fig 1, B, panel 1, lane 2) and was further increased in response to overexpression of c-Maf (Fig 1, B, panel 1; compare lanes 4 and 2). Overexpression of c-Maf without activation was not sufficient for expression of either GM-CSF or IL-4 (Fig 1, B, panels 1 and 2, lane 1) because CMV promoters are less active in hemopoietic cells, and stimulation is required for maximal expression.16 The levels of Rad50 mRNA and 18S rRNA were unchanged by activation or by overexpression of c-Maf (Fig 1, B, panels 3 and 5). Using qRT-PCR, a 3-fold increase in GM-CSF expression in c-Maf transfected Jurkat cells was demonstrated (Fig 1, C). Because only 40% of these cells were transfected as determined by fluorescence-activated cell sorting analysis (data not shown) after transient transfection of a GFP encoding vector, changes in mRNA levels observed will not fully reflect the effect of c-Maf at a single cell level. The difference in fold induction observed between IL-4 and GM-CSF is most likely a result of the existing background level of GM-CSF expression in activated Jurkat cells. Kif3A and Rad50 mRNA levels were unchanged by overexpression of c-Maf. IL-5 mRNA was undetectable in all samples (data not shown). Transient transfection of c-Maf into primary human CD41 cells showed a similar capacity to enhance both GM-CSF and IL-4 expression, causing an average 60% increase in GM-CSF expression and 120% increase in IL-4 mRNA (Fig 1, D). As with Jurkat cells, the transfection efficiency of CD41 cells was approximately 40%.
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Transactivation of the GM-CSF promoter by c-Maf c-Maf stimulates IL-4 expression through binding at the c-Maf response element (MARE) in the IL-4 proximal promoter.10 To investigate how c-Maf caused an increase in GM-CSF levels, we examined the effect of c-Maf on cytokine promoters driving a CAT reporter gene in transient transfection assays. Jurkat cells were transiently cotransfected with c-Maf and cytokine promoter reporter constructs and activated with PdBu and ionomycin. c-Maf specifically activated a 443-bp region of the human IL-4 promoter (pIL4) and a 194-bp region of the human GMCSF promoter (pGM-194) but had no effect on a 517-bp human IL-5 promoter construct (pIL-5) or the control empty expression vector pBLCAT3 (Fig 3, A). This suggests that c-Maf is acting at a site within the proximal 194 bp of the GM-CSF promoter. Comparison of the IL4 and GM-CSF promoter sequences failed to show any comparable MARE sites in the GM-CSF promoter (data not shown), suggesting that the mechanism of action of c-Maf on the GM-CSF promoter differs from that causing induction of IL-4 expression. Plasmids with sequential deletions of the GM-CSF promoter linked to a CAT reporter gene were then studied. These were cotransfected with c-Maf in Jurkat cells, and after overnight activation with PdBu and ionomycin, cells were harvested and CAT activity measured. CAT activity increased as the promoter fragment was shortened, with the highest level of CAT activity seen in the pGM-100 construct (Fig 3, B). This suggests that c-Maf is acting at a site contained within the proximal 100 bp of the GM-CSF promoter. c-Maf is recruited to the GM-CSF promoter The -100 bp region of the GM-CSF promoter does not contain an obvious MARE site; however, c-Maf is known to interact with several other protein families, so it could potentially heterodimerize with other transcription factors. To demonstrate recruitment of c-Maf to the GM-CSF promoter, we used a biotinylated oligonucleotide (oligo) affinity pulldown technique to capture bound c-Maf on a GM-CSF promoter oligo. The biotinylated oligo, termed 162/163, represented the sequence between -33 bp and -97 bp of the GM-CSF promoter and contains several
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c-Maf upregulates GM-CSF and IL-4 protein levels We examined cytokine levels in the supernatants from the transfected Jurkat cells described above by ELISA or CBA. Cells transfected with the c-Maf expression vector showed an average 3-fold increase in GM-CSF secretion (Fig 2, A), which mirrored the extent to which RNA was increased in the real-time qRT-PCR assay. IL-4 protein levels in the culture media were also increased in response to overexpression of c-Maf (Fig 2, B), whereas IL-2 levels were not significantly altered (Fig 2, C). IL-5 protein levels were undetectable with or without c-Maf overexpression when assayed by either ELISA or CBA (data not shown). Taken together, these data suggest that c-Maf can augment GM-CSF expression in Jurkat cells.
FIG 2. Increased GM-CSF and IL-4 production by c-Maf transfected Jurkat cells. Supernatants were taken from activated Jurkat cells transiently transfected with a plasmid containing c-Maf, the control plasmid pcDNA3, or no DNA. A, GM-CSF levels assayed by ELISA at 72 hours. B, IL-4 levels assayed by CBA at 24 hours. C, IL-2 levels assayed by CBA at 24 hours. Graphs represent mean data from 4, 6, and 6 separate experiments, respectively.
known binding sites for constitutive and activation responsive transcription factors, but lacks a consensus MARE (Fig 4, A). Nuclear extracts of HEK-293 cells overexpressing c-Maf were mixed with the biotinylated 162/ 163 oligo (Fig 4, B, lanes 1-4), a biotinylated IL-4 NFAT-MARE oligo (Fig 4, B, lanes 5-8), or a biotinylated IL-5 palindrome oligo (Fig 4, B, lane 9). The captured proteins were then run on a Western blot and tested for c-Maf immunoreactivity. Both the 162/163 oligo in lane 1 and the IL-4 NFAT-MARE oligo in lane 5 demonstrated pulldown of c-Maf. By contrast, an oligo representing a characterized palindromic element in the IL-5 promoter, used as a negative control (lane 9),13 did not interact with c-Maf protein. The interaction of c-Maf with the biotinylated 162/163 oligo could be competed with
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5-fold molar excess of an unbiotinylated 162/163 oligo (lane 2) or an unbiotinylated IL-4 NFAT-MARE oligo (lane 3) but not with the IL-5 palindrome oligo (lane 4). Similarly, for the biotinylated IL-4 NFAT-MARE sequence, there was self-competition with the IL-4 NFATMARE oligo (lane 7), and the 162/163 oligo (lane 6) competed for c-Maf to a greater extent than the IL-5 palindrome oligo (lane 8). This demonstrates recruitment of c-Maf to the GM-CSF promoter mapping to the region between -33 and -97 bp in HEK-293 cells overexpressing c-Maf. To define further the region of the GM-CSF promoter involved in c-Maf recruitment and to preclude the effect of cell-specific influences on c-Maf/DNA binding, a series of experiments were conducted using nuclear extracts from Jurkat cells overexpressing c-Maf. These experiments are described in this article’s Appendix E4 and results are shown in this article’s Fig E1 in the Online Repository at www.jacionline.org. These data suggest that c-Maf can be recruited to the GM-CSF promoter, but the lack of a consensus MARE site in the promoter and the lack of a tightly defined c-Maf binding site indicate a complex interaction mechanism. Interaction of c-Maf at the GM-CSF promoter may be as a result of tethering rather than direct DNA binding, and its recruitment may be stabilized by interaction with other DNA-binding proteins. These data are in keeping with previously reported modes of action of c-Maf.
Expression of c-Maf in human TH1 and TH2 cells In murine T-cell differentiation, c-Maf was selectively expressed in TH2 cells10 and would therefore not correlate with GM-CSF expression. If c-Maf plays a role in GMCSF expression, this would suggest a capacity for differential regulation of GM-CSF in murine TH1 and TH2 cells. To examine the role of c-Maf in GM-CSF expression in human primary cells, we used an in vitro differentiation assay to generate human TH1 and TH2 cells1 and to analyze the transcription factor and cytokine expression by these cells after 28 days in culture. In vitro differentiated TH1 and TH2 cells generated in this assay showed strong polarization toward either a TH1 or a TH2 phenotype as determined by multicolor intracellular cytokine staining for IL-5 and IFN-g (see this article’s Fig E2 in the Online Repository at www.jacionline.org). GM-CSF was expressed at high levels on activation in both TH1 and TH2 cells. Western blots were performed on nuclear and cytoplasmic extracts from both resting and activated TH1 and TH2 cells to assess transcription factor expression under these conditions (Fig 5, A). T-bet and GATA3 showed most abundant expression in TH1 and TH2 cells, respectively. c-Maf was expressed most strongly in resting TH2 cells (lane 7), and levels were substantially reduced after activation (lane 8). Surprisingly, the level of expression of c-Maf in activated TH2 cells was similar to that seen in resting and activated TH1 cells (lanes 3 and 4). Densitometry of western blots of c-Maf protein demonstrated a significant difference in the expression level of c-Maf between unactivated TH1 and TH2 cells and
FIG 3. Transactivation of the GM-CSF promoter by c-Maf. CAT activity was measured in extracts from activated Jurkat cells cotransfected with cytokine promoter constructs and either c-Maf or a control plasmid. Data are from 3 separate experiments and normalized to the sample cotransfected with c-Maf and pIL-4. *P < .05 and **P < .01 compared with the sample cotransfected with c-Maf and pBLCAT3. B, CAT activity was measured in extracts from activated Jurkat cells cotransfected with the GM-CSF promoter constructs and either a plasmid containing c-Maf or a control plasmid. Data are from 6 separate experiments and normalized to the sample cotransfected with c-Maf and pGM-100. **P < .01 compared with the sample cotransfected with c-Maf and pBLCAT3.
between unactivated and activated TH2 cells (Fig 5, B). The densitometry confirms the similarity in c-Maf protein levels between activated TH1 and activated TH2 cells. The protein expression profiles observed here strongly reflect the mRNA profiles previously documented for these transcription factors in human TH1 and TH2 cells.1
DISCUSSION In this study, we have shown that overexpression of c-Maf can increase expression of GM-CSF at both the mRNA and protein levels. We have used transient transfection assays to localize the effect of c-Maf to the GM-CSF proximal promoter and demonstrated recruitment of c-Maf to a region spanning -33 bp to -97 bp of the GMCSF proximal promoter. Furthermore, we have shown that both TH1 and TH2 cells express GM-CSF on activation
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FIG 4. c-Maf interacts with the GM-CSF proximal promoter. A, Sequence of the GM-CSF promoter region thought to be bound by c-Maf. Known binding sites are represented by black boxes. Oligos used as competitors in the affinity pulldown shown in Fig E1, A, and to construct the plasmids transfected in Fig E1 are represented by gray boxes. B, Biotinylated oligo affinity pulldown of the GM-CSF promoter in HEK-293 cells.
FIG 5. Analysis of 28-day in vitro differentiated human TH1 and TH2 cells. A, Western blot of nuclear (N) and cytoplasmic (C) extracts from TH1 and TH2 cells using the indicated primary antibodies and the relevant secondary antibodies. B, Densitometry of c-Maf protein expression. The graph represents mean data from 3 independent experiments normalized to the TH2 unstimulated sample. Data were analyzed by using a 2-way ANOVA test. NE, Nuclear extract.
and that c-Maf is found at similar levels in activated, in vitro differentiated human TH1 and TH2 cells. The Maf family of proteins can homodimerize or heterodimerize with other Maf proteins, other bZIP transcription factors, and a range of other non-bZIP factors.8
This property imparts on Maf proteins the ability to be recruited to a diverse range of DNA sequences and consequently be involved in regulation of genes that lack consensus Maf-response elements in their regulatory regions. Studies on the rat homologue of c-Maf (Maf-2)
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demonstrated altered specificity for DNA binding sites depending on its heterodimeric partner, with Maf-2 capable of interacting with c-Fos and to a lesser extent with Fos-B and Fra-2 but not with the Jun family of proteins.17 Interaction of c-Maf with c-Myb inhibited the cooperative activation of the CD13/APN gene by c-Myb and Ets-1 in human myeloid cell lines.18 Another Maf family member, MafB, synergistically transactivated the GPIIb promoter with GATA1 and Ets after cotransfection in K562 cells.19 No consensus MafB binding site was identified in the promoter, and the enhancement of transcriptional activity was presumed to be mediated by an interaction of MafB with GATA1 and Ets. c-Maf was found to inhibit IL-12 p40 gene expression in macrophages.20 Although a specific interaction domain could not be located in the promoter, overexpression of c-Maf affected the formation of several DNA-protein complexes as assessed by electrophoretic mobility shift assay. This suggested that c-Maf can impart an inhibitory activity without direct contact with DNA at the IL-12 proximal promoter and supports our suggestion that c-Maf may interact with multiple factors at more than 1 site in the GM-CSF proximal promoter to contribute to transcriptional activation. Polarization of CD41 T cells to a TH1 or TH2 lineage has been well characterized in mouse but less so in human T cells. In this study, we describe the protein expression patterns of the transcription factors T-bet, GATA3, and c-Maf in human TH1 and TH2 cells (Fig 5). We found that of the 3 factors, T-bet was the most lineage-restricted, with expression observed primarily in resting and activated TH1 cells, compatible with its role as a driving factor in TH1 differentiation and maintenance.21 Whether the low levels of T-bet observed in TH2 cells have a meaningful function is yet to be established, although it is unlikely to involve positive regulation of IFN-g because these cells are negative for IFN-g expression as determined by fluorescence-activated cell sorting. GATA3 showed strong expression in TH2 cells but also demonstrated low levels of expression in TH1 cells. This is unsurprising because in addition to its role in TH2 differentiation, GATA3 is also responsible for activation and tissue specific expression of the T-cell receptor a and b chains.22,23 Densitometric analysis of c-Maf Western blots demonstrated that the highest level of c-Maf protein was observed in resting TH2 cells, and that c-Maf was significantly downregulated in activated TH2 cells. The level of c-Maf expression in resting and activated TH1 cells was found to be similar to that seen in activated TH2 cells. The reason for this pattern of expression is currently unclear, but it was consistent with the pattern of c-Maf mRNA expression data that we have previously described.1 During T-cell differentiation, GATA3 is thought to induce chromatin remodeling at the 5q locus in TH2 cells and, in doing so, to increase accessibility of the IL-4 promoter to factors such as c-Maf, and therefore mediate its transcriptional regulation.24,25 The lack of sufficient GATA3 in TH1 cells to induce chromatin remodeling results in a chromatin conformation at the 5q locus that is not permissive for the expression of TH2 cytokines. Because GM-CSF
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production is rapidly upregulated after activation in both TH1 and TH2 cells, this would suggest that its gene lies outside the 5q core domain that requires remodeling during TH2 differentiation to permit expression of IL-4, IL-5, and IL-13. Although the regulation of IL-4 expression has been described as the main role for c-Maf in TH2 cells, recent studies have proposed IL-4 independent functions. Normal induction of CD25 (IL2Ra) in developing TH2 cells required c-Maf, because mice lacking c-Maf displayed delayed upregulation of CD25.26 Weigmann et al27 reported higher expression of c-Maf in T-bet expressing TH1 cells from the lamina propria of patients with Crohn disease and proposed a pathogenic role for c-Maf in memory T cells in colitis. In macrophages, c-Maf was found to be a potent activator of IL-10 and suppressor of IL-12 gene transcription and was proposed as a mediator of the immunosuppressive effects of IL-10.20 It is possible that c-Maf, in addition to its role in IL-4 expression, is required by both TH1 and TH2 cells for other, as yet unknown functions. On the basis of the evidence presented here, we propose a novel role for c-Maf in the transcriptional regulation of GM-CSF in human T cells. We demonstrate increased levels of endogenous GM-CSF mRNA and protein in response to overexpression of c-Maf and have localized binding of c-Maf to a region between -33 and -97 bp of the GM-CSF promoter. Furthermore, we find that, in contrast with the murine system, the expression pattern of c-Maf in differentiated human T cells is not restricted to TH2 cells and correlates with GM-CSF expression. We propose that c-Maf may have a broader role than previously described in the regulation of expression of inflammatory mediators. We thank Drs T. Kouzarides, T. Honjo, and J. Morgan for providing plasmids and Dr S. Hirst for assistance with statistical analysis.
REFERENCES 1. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human TH1/TH2 cell differentiation: direct evidence for coordinated expression of th2 cytokines. J Immunol 2002;169:2498-506. 2. Lee GR, Fields PE, Griffin TJ, Flavell RA. Regulation of the Th2 cytokine locus by a locus control region. Immunity 2003;19:145-53. 3. Takamoto M, Sugane K. Synergism of IL-3, IL-5, and GM-CSF on eosinophil differentiation and its application for an assay of murine IL-5 as an eosinophil differentiation factor. Immunol Lett 1995;45: 43-6. 4. Ebisawa M, Liu MC, Yamada T, Kato M, Lichtenstein LM, Bochner BS, et al. Eosinophil transendothelial migration induced by cytokines, II: potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J Immunol 1994;152:4590-6. 5. Burke LA, Hallsworth MP, Litchfield TM, Davidson R, Lee TH. Identification of the major activity derived from cultured human peripheral blood mononuclear cells, which enhances eosinophil viability, as granulocyte macrophage colony-stimulating factor (GM-CSF). J Allergy Clin Immunol 1991;88:226-35. 6. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176:1693-702.
7. Berger TG, Feuerstein B, Strasser E, Hirsch U, Schreiner D, Schuler G, et al. Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories. J Immunol Methods 2002;268: 131-40. 8. Blank V, Andrews NC. The Maf transcription factors: regulators of differentiation. Trends Biochem Sci 1997;22:437-41. 9. Kim JI, Ho IC, Grusby MJ, Glimcher LH. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 1999;10:745-51. 10. Ho IC, Hodge MR, Rooney JW, Glimcher LH. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 1996;85:973-83. 11. Ho IC, Lo D, Glimcher LH. c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms. J Exp Med 1998;188:1859-66. 12. Hausding M, Ho IC, Lehr HA, Weigmann B, Lux C, Schipp M, et al. A stage-specific functional role of the leucine zipper transcription factor c-Maf in lung Th2 cell differentiation. Eur J Immunol 2004;34: 3401-12. 13. Staynov DZ, Cousins DJ, Lee TH. A regulatory element in the promoter of the human granulocyte-macrophage colony-stimulating factor gene that has related sequences in other T-cell-expressed cytokine genes. Proc Natl Acad Sci U S A 1995;92:3606-10. 14. Codlin S, Soh C, Lee T, Lavender P. Characterization of a palindromic enhancer element in the promoters of IL4, IL5, and IL13 cytokine genes. J Allergy Clin Immunol 2003;111:826-32. 15. Richards DF, Fernandez M, Caulfield J, Hawrylowicz CM. Glucocorticoids drive human CD8(1) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production. Eur J Immunol 2000;30:2344-54. 16. Ramezani A, Hawley TS, Hawley RG. Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol Ther 2000;2:458-69. 17. Matsushima-Hibiya Y, Nishi S, Sakai M. Rat maf-related factors: the specificities of DNA binding and heterodimer formation. Biochem Biophys Res Commun 1998;245:412-8.
Gilmour et al 63
18. Hegde SP, Kumar A, Kurschner C, Shapiro LH. c-Maf interacts with c-Myb to regulate transcription of an early myeloid gene during differentiation. Mol Cell Biol 1998;18:2729-37. 19. Sevinsky JR, Whalen AM, Ahn NG. Extracellular signal-regulated kinase induces the megakaryocyte GPIIb/CD41 gene through MafB/ Kreisler. Mol Cell Biol 2004;24:4534-45. 20. Cao S, Liu J, Chesi M, Bergsagel PL, Ho IC, Donnelly RP, et al. Differential regulation of IL-12 and IL-10 gene expression in macrophages by the basic leucine zipper transcription factor c-Maf fibrosarcoma. J Immunol 2002;169:5715-25. 21. Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 2002; 295:338-42. 22. Ho IC, Vorhees P, Marin N, Oakley BK, Tsai SF, Orkin SH, et al. Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene. EMBO J 1991;10: 1187-92. 23. Henderson AJ, McDougall S, Leiden J, Calame KL. GATA elements are necessary for the activity and tissue specificity of the T-cell receptor betachain transcriptional enhancer. Mol Cell Biol 1994;14:4286-94. 24. Lee HJ, Takemoto N, Kurata H, Kamogawa Y, Miyatake S, O’Garra A, et al. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells. J Exp Med 2000; 192:105-15. 25. Takemoto N, Kamogawa Y, Jun Lee H, Kurata H, Arai KI, O’Garra A, et al. Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J Immunol 2000;165:6687-91. 26. Hwang ES, White IA, Ho IC. An IL-4-independent and CD25-mediated function of c-maf in promoting the production of Th2 cytokines. Proc Natl Acad Sci U S A 2002;99:13026-30. 27. Weigmann B, Nemetz A, Becker C, Schmidt J, Strand D, Lehr HA, et al. A critical regulatory role of leucin zipper transcription factor c-Maf in Th1-mediated experimental colitis. J Immunol 2004;173:3446-55.
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J ALLERGY CLIN IMMUNOL VOLUME 120, NUMBER 1