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The Journal of Immunology, 2004, 172: 7289-7296.
Copyright © 2004 by The American Association of Immunologists

c-Maf and JunB Mediation of Th2 Differentiation Induced by the Type 2 G Protein-Coupled Receptor (VPAC2) for Vasoactive Intestinal Peptide1

Julia Voice*, Samantha Donnelly{dagger}, Glenn Dorsam*, Gregory Dolganov{dagger}, Sudhir Paul{ddagger} and Edward J. Goetzl2,*

* Departments of Medicine and Microbiology/Immunology, {dagger} Division of Pulmonary Medicine, Cardiovascular Research Institute, University of California Medical Center, San Francisco, CA 94143; and {ddagger} Departments of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vasoactive intestinal peptide and its G protein-coupled receptors, VPAC1 and VPAC2, regulate critical aspects of innate and adaptive immunity. T cell VPAC2Rs mediate changes in cytokine generation, which potently increase the Th2/Th1 ratio and consequently shift the effector responses toward allergy and inflammation. To examine mechanisms of VPAC2 promotion of the Th2 phenotype, we analyzed controls of IL-4 transcription in CD4 T cells from T cell-targeted VPAC2 transgenic (Tg), VPAC2 knockout, and wild-type (WT) mice. c-maf and junB mRNA, protein, and activity were significantly up-regulated to a higher level in TCR-stimulated CD4 T cells from Tg mice compared with those from knockout and WT C57BL/6 mice. In contrast, GATA3, T-bet, and NFATc levels were identical in WT and Tg CD4 T cells. Vasoactive intestinal peptide binding to VPAC2 on CD4 T cells specifically induces an up-regulation of the Th2-type transcription factors c-Maf and JunB, which consequently enhances IL-4 and IL-5 production, leading to a Th2-type phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vasoactive intestinal peptide (VIP) 3 is a potent neuroendocrine mediator, which is generated and released by subsets of cholinergic and sensory nerves, including those in lymphoid organs, and by some T cells following immune activation (1). In T cells, VIP exerts its biological effect through two G protein-coupled receptors. Type 1 VIP receptors (VPAC1) are highly expressed constitutively on T cells, particularly Th cells, and type II VIP receptors (VPAC2) are absent or marginally expressed by unstimulated CD4 T cells (Th cells). Following immune stimulation of Th cells, VAPC1Rs are down-regulated, whereas VPAC2Rs are up-regulated, suggesting that VPAC2Rs are the major transducer of effects of VIP on activated T cells (2, 3). VIP affects proliferation, differentiation, and other immune functions of macrophages and T cells. In vitro, VIP modulates T cell cytokine production, with apparent preferential inhibition of cytokines typical of Th1 cells, such as IL-2, and enhancement of some from Th2 cells, such as IL-5. However, the results for the Th1 marker IFN-{gamma} and the Th2 marker IL-4 are sometimes conflicting (4, 5, 6, 7, 8). The ability to modulate the Th1/Th2 T cell cytokine ratio has profound immunological implications, because the development of immunopathologies may result from an imbalance in the Th1/Th2 cell ratio. Overproduction of Th1 cytokines has been implicated in autoimmune disease, and augmentation of Th2-type responses promotes the development and maintenance of allergic diseases.

To determine the role of VPAC2Rs in T cell function, a genetically based model was developed. A transgenic (Tg) C57BL/6 mouse line was established in which normally inducible VPAC2Rs are constitutively expressed at high levels in Th cells (9). The complementary model is represented by VPAC2R-null (knockout (KO)) C57BL/6 mice, generated by targeted insertion of a mutation in exon 1 of the VPAC2 gene (10).

Major differences were identified between these models and wild-type (WT) C57BL/6 mice in effector T cell phenotype, with striking deviations from normal CD4 T cell cytokine secretion profiles toward Th1 in KO mice and Th2 in Tg mice. Tg mice showed elevated blood IgE and eosinophil levels, dramatically increased immediate-type hypersensitivity, and decreased delayed-type hypersensitivity. In contrast, KO mice showed increased cutaneous delayed-type hypersensitivity and decreased immediate-type hypersensitivity. Subsequent studies demonstrated that these altered states of hypersensitivity are dependent on the CD4 T cell VPAC2-mediated deviations in cytokine profiles, and that the cytokine profile of Tg mice, which is biased toward a Th2 type, is dependent on T cell-derived VIP (11). The mechanisms by which VPAC2s are able to induce a bias toward the Th2 phenotype are unknown. We have shown that following stimulation, Tg mice have more IL-4-secreting CD4 T cells from splenic preparations than WT mice, but that the amount of IL-4 secreted per cell is not altered (11). Therefore, we explored the hypothesis that VIP/VPAC2 may be altering transcription factors and pathways involved in the initiation and propagation of Th2 polarization.

Several factors are known to regulate Th1/Th2 polarization selectively, of which the most powerful is the cytokine environment during primary T cell activation. IL-12 is a principal macrophage-derived determinant for Th1 cells and IL-4 for Th2 cells (12, 13, 14). Distinctive transcription factors, which act by changing cytokine profiles, are known to be crucial in determining the relative ratio of Th1/Th2 responses. These include NFAT, AP-1, and STAT proteins, and more selectively, GATA-3, c-Maf, and JunB which are preferentially expressed in Th2 cells, and T-bet, which is differentially up-regulated in Th1 cells (15, 16, 17, 18, 19, 20). GATA3, which is expressed at high levels in Th2 cells, has broad effects in CD4 T cell development, regulating expression of IL-4 indirectly, expression of IL-5 and IL-13 directly, and concurrently down-regulating the expression of Th1 cytokines (21, 22, 23, 24). In contrast to GATA3, the protooncogene, c-Maf, which has been shown to be critically involved in Th2 polarization, is more selective, and strongly transactivates the IL-4 promoter but not the IL-5 or IL-13 promoters. c-Maf, a member of the basic leucine zipper factor AP-1 family, cooperates with NFAT1, NFATc2, and NIP-45, and synergizes with JunB and IFN regulatory factor 4 to transactivate the IL-4 promoter (17, 25, 26, 27, 28). There is strong evidence to support requirements for c-Maf, JunB, and NFAT in promoting IL-4 production. JunB, a member of the AP-1 transcription factor family, acts synergistically with c-maf to strongly activate IL-4 expression, and has also been shown to be required for IL-5 expression in Th2 cells (17, 29).

The present results demonstrate a VIP/VPAC2-dependent modulation of some, but not all, Th2 transcription factors. c-Maf and JunB are specifically up-regulated in VPAC2 Tg CD4 T cells, at both the RNA and protein levels, but interestingly, neither T-bet nor GATA3 are modulated by constitutive VPAC2 expression. Other early factors associated with c-Maf and the expression of IL-4 were also examined, in particular the NFAT proteins. The NFATc/NFATp ratio has been shown to play a role in Th cell polarization, with a relative increase in NFATc correlating with an increase in IL-4 transcription, leading to a deviation of Th cells toward a Th2 phenotype (30, 31, 32). In this paper, we demonstrate that the VIP/VPAC2 axis does not alter the NFATc/NFATp ratio, indicating that the regulatory affects of VIP/VPAC2 on IL-4 transcription occurs specifically at the later c-Maf and JunB stages.


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

The VPAC2 Tg C57BL/6 mice were generated by injection of C57BL/6 oocytes with a minigene encoding LCK tyrosine kinase proximal promoter 5' to the human (h)VPAC2 before implantation. Founders expressing high levels of hVPAC2, quantified by real-time PCR, were bred to produce a stable line of mice with high expression of hVPAC2 in T cells, and especially high levels in CD4 T cells (9). VPAC2 KO mice were generated by gene-targeted insertion of a lacZ-neo cassette into the first coding exon of the VPAC2 gene. Stable KO mice were bred for characterization of their immune phenotype (10). WT C57BL/6 mice were purchased, and age- and sex-matched to the Tg in each experiment from Animal Technologies (Fremont, CA).

Preparation of CD4 T cells

Spleens were removed from groups of three to five VPAC2 Tg, WT, and KO mice at 8–12 wk of age. Splenic tissues were pushed through 70-µm pore nylon filters to disaggregate the cells in DMEM supplemented with 10% FBS, 1x penicillin G and streptomycin, and sodium pyruvate and glutamate (complete medium). Splenocytes were washed once in PBS containing 0.5% FBS and 2 mM EDTA (BD IMag buffer; BD Biosciences, Mountain View, CA), resuspended at 107 cells per 50 µl of IMag anti-mouse CD4 particle suspension, and incubated for 30 min at 4°C. The CD4+ cellular fraction of the splenocyte preparation was magnetically purified according to the manufacturers protocol (BD Biosciences). FACS analysis showed the purified cells to be >93% CD4 T cells.

Replicate aliquots of 106 cells/ml of the T cell-enriched suspensions in complete medium were preincubated with 1 µM IgG with VIP-cleaving activity (VIPase) or medium alone, and stimulated by incubation in 24-well plates containing 1 µg/well each of adherent anti-CD3 and anti-CD28 mouse mAb (BD PharMingen, San Diego, CA) at 37°C in 5% CO2 for 48, 72, and 96 h.

Quantitation of VPAC1 and VPAC2 expression following stimulation using real-time PCR was as follows. Levels of murine (m)VPAC1 and hVPAC2 RNA in CD4 T cells from both Tg and WT mice were quantitated following stimulation with plate-bound anti-CD3 and -CD28. After 1, 6, 24, 48, and 96 h of stimulation, high-quality DNase-treated RNA was obtained from the cells by the Qiagen (Valencia, CA) Qiashredder and RNeasy procedures as specified by the manufacturer. Reverse-transcriptase real-time PCR was performed using Applied Biosystems 7700 (Foster City, CA). Primers and probes were purchased from Integrated DNA Technologies (Coralville, IA). Probes were labeled with 5'-FAM/3'-TAMRA (Table I). Cycle threshold (CT) values for mVPAC1 and hVPAC2 were normalized to the housekeeping gene mHPRT.


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  		Table I. Primers and probes

 
Quantitation of cytokine genes using multiplex real-time PCR

High-quality RNA was purified from TCR-stimulated splenic CD4 T cells isolated from age- and sex-matched Tg and WT mice. The quality and quantity of the RNA was tested using Agilent Technologies (Palo Alto, CA) RNA Nano Chips. First-strand cDNA synthesis using random hexamers was conducted using the Invitrogen (Carlsbad, CA) SuperScript first-strand synthesis kit. Following reverse transcription, 4 µl of single-stranded cDNA (corresponding to 1 ng of total RNA) was used as a template in a multiplex PCR using 5 pmol each of the RTF and RTR primers listed in Table I. All PCR were conducted in a 50-µl volume using Advantage cDNA polymerase (Clontech, Palo Alto, CA) with the following cycling conditions: an initial incubation at 94°C for 15 min, followed by 18 cycles of 94°C for 20 s, 55°C for 20 s and 72°C for 20s, followed by a final incubation at 72°C for 10 min. Real-time PCR was as follows: All reactions were conducted in a 10-µl volume using 2x Universal Master Mix (Applied Biosystems) using ~2.4 pg of total RNA. All forward and reverse primers were individually optimized. All transcript quantifications were conducted in an ABI 7900 Sequence Detection System (Applied Biosystems). Real-time PCR data analysis was as follows: Baseline and CT values were selected according to the manufacturer’s protocols (User Bulletin No. 2; Applied Biosystems). Conversion of raw CT values to relative gene copy number was conducted as previously described (33).

Cytokine gene-specific primers were designed for the genes listed in Table I using Primer Express software (Applied Biosystems), using sequences available from National Center for Biotechnology Information. All primers and probes were purchased from Biosearch Technologies (Novato, CA). All probes were labeled with FAM and Black Hole Quencher at the 5'- and 3'-ends, respectively. Both sets of primers were nested, and amplicons were <250 bp.

Reverse-transcriptase real-time PCR quantitation of transcription factors and CD25 cDNA

For the transcription factors and CD25, reverse-transcriptase real-time PCR was performed using Applied Biosystems 7700. Primers and probes were purchased from Integrated DNA Technologies. Probes were labeled with 5'-FAM/3'-TAMRA.

Real-time PCR runs for transcription factors were optimized, and serial dilutions of mRNA were used in each run to ensure the CT values were in the linear range for each amplicon. Following the reverse-transcriptase reaction, the genes of interest were amplified over 40 cycles, primer annealing at 61°C for 15 s. Values for all genes were normalized to expression of the housekeeping gene HPRT.

Preparation of nuclear extracts and Western blot analysis

Nuclear and cytoplasmic extracts from 2 x 107 CD4 T cells, unstimulated and following TCR stimulation as described above, were prepared using NE-PER nuclear and cytoplasmic reagents (Pierce, Rockford, IL) according to the manufacturer’s protocol. Protein concentrations were determined using the BCA protein assay (Pierce). Twenty micrograms of nuclear extract protein in 6x reducing Laemmli buffer from each sample was loaded onto 7% SDS polyacrylamide minigel, electrophoresed at 60 V, followed by overnight transfer at 4°C and 20 V onto polyvinylidene difluoride membrane (0.2 mM). Membranes were blocked using 5% nonfat milk proteins in TBS- Tween 20 (0.5%), followed by incubation with rabbit anti-mouse c-Maf, -JunB, GATA3, actin B, and Oct1 (as a loading control) (Santa Cruz Biotechnology, Santa Cruz, CA), washed, and incubated with HRP-labeled anti-rabbit polyclonal Ab (Santa Cruz). Protein bands were visualized using standard chemiluminescent techniques, and the signal intensity quantitated using an Alpha Innotech (San Leandro, CA) Imaging system.

Transcription factor activation assay

TransAm AP-1 kits (Active Motif, Carlsbad, CA) were used to quantify levels of active JunB, JunD, and phosphorylated c-Jun from nuclear extracts prepared from WT and Tg CD4 T cells following 96-h TCR activation. Nuclear extracts were prepared using the Active Motif Nuclear Extract reagents. Five micrograms of the nuclear extracts were added to individual wells in a 96-well plate, and precoated with oligonucleotide containing a consensus sequence. Duplicate wells were then incubated with primary Ab specific for the active form of JunB, JunD or c-Jun. Wells were washed and incubated with an HRP-conjugated secondary Ab, followed by incubation with standard colorimetric developing solutions and quantification using an ELISA reader set to 450 nm (Dynex Technologies, Chantilly, VA).

Quantitation of fluid-phase IL-4 production from CD4 T cells

CD4 splenic T cells were isolated and purified from age- and sex-matched Tg, KO, and WT mice as described above. CD4 T cells at a concentration of 1.5 x 106 cells/ml were stimulated in 24-well plates by incubation on 1 µg/well of each plate-bound anti-CD3 and anti-CD28. Following incubation at 37°C for 72, 96, and 120 h, cell-free supernatants were harvested and analyzed for IL-4 levels by ELISA (Pierce).

EMSA

EMSAs for NFATs were performed using 3 µg of nuclear extract, incubated with 1 µg of poly(dI:dC) in 20 µl of 1x binding buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 10% glycerol, and 0.1% Nonidet P-40, with 40 fmol of biotin-labeled probe (Integrated DNA Technologies) for 20 min at room temperature. For detection of NFAT binding, a double-stranded oligonucleotide from the murine IL-4 promoter (–88 to –60) was used: 5'-CTGGTGTAATAAAATTTTCCAATGTAAAC-3'. For competition assays, 50-fold excess of unlabeled probe was added to the binding mix. The supershift Abs for NFATp and NFATc were from clone 4G6-G5 and 7A6 (Santa Cruz), respectively. Complexes were resolved on nondenaturing 5% polyacrylamide gels (37.5:1 cross-linking ratio) in 0.5x TBE for 2.5 h at 140 V. For supershift reactions, nuclear extracts were preincubated for 20 min on ice with the appropriate Ab before the addition of the labeled probe. Following gel resolution, DNA/protein complexes were electrophoretically transferred to nylon membrane at 280 mA for 1 h at 4°C; transferred DNA was cross-linked to the membrane using a UV light-cross-linker, and the biotin-labeled probe was detected and visualized using chemiluminescent techniques according to the manufacturer’s protocol (Pierce).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Predominance of Th2 cytokine genes in VPAC2 Tg CD4 T cells

The Th2-biased phenotype of Tg CD4 T cells was shown by their higher than normal production of IL-4 and IL-5, and lower than normal IFN-{gamma} in cells and supernatants after TCR stimulation (9, 11). Quantification of mRNAs encoding the cytokines IL-4, IL-5, and IFN-{gamma} now confirms our original findings of elevated Th2/Th1 level in VPAC2 Tg CD4 T cells and leads to investigations of responsible transcriptional mechanisms. Significantly higher levels of mRNA encoding IL-4 and IL-5, and a significantly lower level of mRNA encoding IFN-{gamma} were detected in Tg than WT CD4 T cells following 96 h of TCR stimulation (Fig. 1). That CD4 T cells are the predominant source of endogenous VIP for VPAC2-mediated increases in IL-4 and decreases in IFN-{gamma} secretion from TCR-stimulated Tg CD4 T cells was suggested initially by the inhibitory effects of an IgG Ab exhibiting catalytic activity toward VIP (34, 35). The ability of catalytic VIPase Ab during TCR stimulation to significantly inhibit the increase in Tg IL-4 message and, to a lesser extent, inhibit the decrease in Tg IFN-{gamma} message supported a transcriptional level of control. In contrast, the extremely high level of IL-5 message in TCR-stimulated Tg CD4 T cells compared with WT CD4 T cells was not significantly reduced by the addition of VIPase in Tg CD4 T cells. This suggests that the VPAC2-mediated increase in IL-5 observed in Tg T cells does not require either high concentrations or persistently elevated levels of VIP, but may result from a higher susceptibility of IL-5 production to lower levels of residual VIP or a persistence response to prior in vivo exposure of VPAC2 to VIP.



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  		FIGURE 1. Cytokine profiles of WT and Tg CD4 T cells. IL-4, IL-5, and IFN-{gamma} gene copy number in CD4 splenic T cells following 96-h TCR stimulation in the presence of inactive control VIPase IgG () or active catalytic VIPase ({blacksquare}). Gene copy number was determined by multiplex PCR, using 2.4 pg of starting total RNA. All data have been normalized to a panel of housekeeping genes, and represent the average of five independent experiments ± SD. *, p < 0.04.

 
Expression of VPAC1 and VPAC2 in CD4 T cells following stimulation

Although we have previously shown that the levels of hVPAC2 expressed in the Tg VPAC2 CD4 T cells far exceeds the expression of endogenous mVPAC1, it was important to establish which VIP receptor is predominantly expressed during persistent TCR-dependent stimulation. After TCR stimulation, the levels of VPAC1 dramatically drop at 24 h, and remain depressed. In contrast, the high levels of Tg hVPAC2 do not change following TCR activation of CD4 T cells, and remain high throughout the stimulation period (Fig. 2). Levels of endogenous mVPAC2 increase following 72-h TCR stimulation (data not shown), but only to a much lower maximum level than the Tg hVPAC2. These results clearly show that the effects of VIP and/or VIPase on the function of TCR-stimulated Tg CD4 T cells can be attributed to VPAC2.



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  		FIGURE 2. TCR-induced down-regulation of mVPAC1 but not hVPAC2. Tg splenic CD4 T cells purified by Ab-conjugated magnetic bead chromatography were seeded at 1 million cells per milliliter in 24-well plates and incubated with adherent anti-CD3 and -CD28. Over a 96-h time course, RNA was isolated and mVPAC1 (dashed line) and hVPAC2 (solid line) expression levels were determined by reverse-transcriptase real-time PCR, with CT values normalized to the housekeeping gene HPRT. Gene expression is presented as the percentage of expression in unstimulated cells (100%), and is representative of three independent experiments.

 
Enhanced expression of c-maf and junB

To ascertain whether constitutive VPAC2 expression in T cells induces a Th2 phenotype by altering the expression of transcription factors involved in CD4 T cell polarization, we used real-time PCR to quantitate the levels of c-maf, GATA3 and T-bet mRNA in resting and TCR-stimulated T cells over 120 h. We found no difference in the levels of expression of GATA3 or T-bet mRNA between WT and Tg CD4 T cells, either in naive T cells or in TCR-stimulated cells (data not shown). At zero time (constitutive levels), the expression levels of c-maf and junB were the same in WT and Tg CD4 T cells. However, there was a marked increase in mRNA levels of c-maf, starting after 72 h of TCR stimulation, and reaching a plateau following 96 h of TCR stimulation (Fig. 3A). Recent publications have shown that c-maf-related early up-regulation of junB leads to Th2 polarization (17). There is a significant increase in the level of mRNA encoding junB in Tg CD4 T cells following TCR stimulation compared with WT CD4 T cells following 72 and 96 h of TCR stimulation (Fig. 3B). To determine whether this up-regulation was mediated by the VIP/VPAC2 axis, we included the catalytic anti-VIP Ab or control inactive VIPase during 72 h of TCR stimulation in some samples. Elimination of endogenous VIP from the medium reduced the expression of c-maf and junB mRNA in Tg CD4 T cells back to the levels observed for WT CD4 T cells (Fig. 4). The active VIPase had no effect on the levels of expression of c-maf and junB in WT CD4 T cells. These mRNA data support our previous conclusion that the IL-4-driven Th2 phenotype of Tg mice depends on endogenous VIP (11).



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  		FIGURE 3. Greater up-regulation of c-maf and junB mRNA in Tg than WT CD4 T cells. Fold increase of c-maf (A) and junB (B) cDNA expression in Tg CD4 T cells () compared with WT CD4 T cells ({blacksquare}) following 72- and 96-h stimulation on plate-bound anti-CD3 and anti-CD28. Expression levels were determined by reverse-transcriptase real-time PCR, with CT values normalized to the housekeeping gene HPRT. Results represent the average of four independent experiments performed in duplicate ± SD. *, p < 0.05.

 


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  		FIGURE 4. Dependence on VIP for VPAC2-mediated up-regulation of c-maf and junB in Tg CD4 T cells. Fold increase of c-maf (A) and junB (B) cDNA expression in Tg CD4 T cells compared with WT CD4 T cells following 96-h TCR stimulation (for c-maf expression) and 72-h TCR stimulation (junB expression) in the presence of noncatalytic control IgG (), or active catalytic VIPase IgG ({blacksquare}). Levels of cDNA were determined by reverse-transcriptase real-time PCR, with CT values normalized to the housekeeping gene HPRT. Results are the average of three independent experiments ± SD. *, p < 0.02.

 
Increased levels of c-Maf and JunB but not GATA3 proteins in Tg CD4 T cells

Western blot analyses were performed on nuclear extracts taken from TCR-stimulated CD4 T cells to determine whether the increase in levels of mRNA encoding c-maf and junB correlate with an increase in nuclear protein expression in Tg CD4 T cells compared with WT. Twenty micrograms of protein per sample was loaded and run on SDS-PAGE gels, where loading efficiency was assessed by reprobing membranes with anti-{beta}-actin. Tg CD4 T cells clearly express higher levels of c-Maf and JunB protein compared with WT CD4 T cells following 72 h (not shown) and 96 h of TCR stimulation, whereas the levels of GATA3 protein were the same for Tg and WT CD4 T cells following 72 h (not shown) and 96 h of TCR stimulation (Fig. 5). Therefore, the VIP/VPAC2 axis-mediated specific up-regulation of junB and c-maf mRNA observed in stimulated Tg CD4 T cells, translates into an increase in their nuclear protein levels. The levels of c-Maf and JunB at earlier time points of 0, 24, and 48 h were too low to detect.



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  		FIGURE 5. Increased c-Maf and JunB, but not GATA3 protein expression in activated Tg compared with WT CD4 T cells. Western blot analysis of 20 ng of nuclear extract protein from WT and Tg CD4 T cells following 96-h stimulation on plate-bound anti-CD3 and anti-CD28. Western blots were probed with anti-c-Maf, -JunB, and -GATA3, followed by standard chemiluminescent techniques. Fold increase determined by densitometry readings: c-Maf protein, 5.57-fold higher in Tg than WT CD4 T cells; JunB protein, 1.8-fold higher in Tg than WT CD4 T cells.

 
Based on the data showing the dependence on endogenous VIP for the up-regulation of c-maf and junB mRNA, we anticipated that the increase in protein levels of c-Maf and JunB in the Tg CD4 T cells would also require T cell-derived VIP. Addition of catalytically active VIPase reduced the VIP/VPAC2-induced increase in both c-Maf and JunB protein in Tg CD4 T cells to a level comparable to WT for c-Maf, and to a level slightly higher than WT for JunB (Fig. 6). The fold increase of JunB expression for Tg over WT control CD4 T cells was calculated from densitometry readings using Alpha Innotech imaging of the JunB protein bands. The results were 2.2-, 1.6-, and 1.5-fold increases over WT control for Tg control, Tg plus VIPase, and WT plus VIPase CD4 T cells, respectively, indicating that the increase in JunB protein expression in Tg CD4 T cells is dependent on endogenous VIP.



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  		FIGURE 6. Dependence on VIP for VPAC2-mediated up-regulation of c-Maf and JunB protein in Tg CD4 T cells. Western blot analysis of 20 µg of nuclear extract protein from Tg and WT splenic CD4 T cells following 72-h TCR stimulation either in the presence of catalytically active VIPase Ab (VIPase +) or inactive control Ab (VIPase –). Fold increase of JunB expression over WT control was calculated from image capture and densitometry readings using Alpha Innotech imaging of the JunB protein bands, visualized by polyclonal anti-JunB followed by standard chemiluminescent techniques. The image is representative of three independent experiments.

 
Decreased levels of c-Maf and JunB protein in KO CD4 T cells following TCR stimulation

The ability of VIPase to prevent the increase in c-Maf and JunB expression in the Tg CD4 T cells (Fig. 6), combined with VIP receptor profiles following TCR stimulation (Fig. 2) implicate direct signaling from VPAC2. To definitively show that the increases in Tg T cell c-Maf and JunB are specifically due to the higher levels of VPAC2, the protein levels of c-Maf and JunB were examined in VPAC2 KO CD4 T cells following TCR stimulation, and compared with Tg and WT mice. At 96 h after TCR stimulation, KO CD4 T cells express significantly lower levels of c-Maf and JunB than WT CD4 T cells, which in turn express less c-Maf and JunB than Tg CD4 T cells (Fig. 7 and Table II). Equal nuclear extract loading was confirmed by probing the Western membranes for Oct1 and Actin, both of which are equivalent for all samples.



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  		FIGURE 7. Decreased levels of c-Maf and JunB protein in KO CD4 T cells following TCR stimulation. Western blot analysis of 20 ng of nuclear extract protein from WT and Tg CD4 T cells following 96-h stimulation on plate-bound anti-CD3 and -CD28. Western blots were probed with anti-c-Maf, -JunB, and Oct1 and Actin as loading controls, followed by standard chemiluminescent techniques. Image is representative of two independent experiments. Fold increase, normalized to Oct-1 housekeeping gene, was determined by densitometry readings, and is shown (±SD) in Table II.

 

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  		Table II. Dependence of relative increases in JunB and c-Maf or VPAC2

 
Time course of IL-4 production

Secreted IL-4 was measured in CD4 T cell supernatants following 72, 96, and 120 h of TCR stimulation to determine whether the increase in IL-4 production observed in Tg CD4 T cells following TCR stimulation temporally follows the increase in c-Maf and JunB message and protein levels in stimulated Tg CD4 T cells. Our previous studies (9, 10, 11) have shown that 24- and 48-h TCR stimulation induces a marginal increase in IL-4 secretion from Tg compared with WT CD4 T cells, but that 96 h is optimal. The present time course shows that increased IL-4 production in Tg CD4 T cells (due to increased number of IL-4-producing CD4 T cells (11)) occurs following 72-h TCR stimulation, with a dramatic increase following 96-h TCR stimulation. This time course mirrors the increased expression of the IL-4 transcription factors c-Maf and JunB (Figs. 3 and 5). IL-4 secreted from TCR-stimulated VPAC2 KO CD4 T cells, which have reduced levels of c-Maf and JunB upon TCR stimulation compared with WT and Tg CD4 T cells, was dramatically lower (Fig. 8), further confirming the association between VIP/VPAC2 and increased c-Maf and JunB expression with an increase in IL-4 secretion from a population of CD4 T cells. Although not excluding other factors that may also play a role in enhancing the development of IL-4 producing CD4 T cells in VPAC2 Tg mice, these data strongly suggest that VIP/VPAC2 up-regulates nuclear expression of c-Maf and JunB, which consequently leads to an increased proportion of IL-4-producing cells, i.e., a skewing of the CD4 T cells toward a Th2 phenotype.



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  		FIGURE 8. Time course of IL-4 production from Tg, WT, and KO CD4 splenocytes. IL-4 ELISAs were performed on aliquots of cell-free supernatants from magnetically purified KO (), WT (), and Tg ({blacksquare}) CD4 T cells, following in vitro TCR stimulation of 106 CD4 T cells/ml on plate-bound anti-CD3 and -CD28 for 72, 96, and 120 h. Results are shown as the average of three independent experiments performed in duplicate ± SD bars.

 
Levels of NFATc and NFATp in stimulated CD4 T cells of Tg and WT mice

An increase in the ratio of NFATc/NFATp has been shown to mediate a shift toward increased production of IL-4 (30). Therefore, to establish whether constitutive VPAC2 expression in CD4 T cells regulates transcriptional events possibly controlling expression of JunB and c-Maf, we examined the ratio of NFATp/NFATc in CD4 T cells following 48-h TCR stimulation. The ratio was determined by EMSA, using a region of the IL-4 promoter as the probe for NFAT proteins, followed by anti-NFATc or anti-NFATp supershift Abs to specifically retard the appropriate NFAT protein when run on a nondenaturing gel. Any increase in nuclear expression or localization of NFATc or decrease in NFATp (or vice versa) would be reflected in this ratio. The results shown in Fig. 9, indicate that NFAT protein in nuclear extracts from both WT and Tg CD4 T cells following 48-h TCR activation specifically bound the IL-4 promoter probe, illustrated by the ability of unlabelled probe to specifically compete for NFAT binding (Fig. 9A, lanes 2). Fig. 9B illustrates the supershift results from two separate nuclear extract preparations (i and ii). The intensity of the bands corresponding to the supershifted NFATc and NFATp proteins was quantitated using a phosphor imager (Alpha Innotech). The NFATp/NFATc ratios for WT and Tg nuclear extracts are as follows: experiment i, WT, 1:1.4, and Tg, 1:1.3; experiment ii, WT, 1:1.55, and Tg, 1:1.8. Comparison of these NFATp/NFATc ratios from Tg and WT nuclear extracts following 48-h TCR stimulation shows no significant difference, indicating that the VIP/VPAC2 interaction does not significantly induce either an increase in nuclear NFATc or a decrease in nuclear NFATp.



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  		FIGURE 9. NFATc/NFATp ratio. Nuclear protein fractions from WT and Tg CD4 T cells TCR stimulated for 48 h were subjected to EMSA using a biotinylated NFAT probe from the IL-4 promoter. The specificity of NFAT protein binding was determined competitively using unlabeled probe for both Tg and WT extracts (A). The ratio of NFATc/NFATp was determined with supershifting Abs specific for NFATc and NFATp (B). Two independent experiments (B, i and ii) are shown.

 
Increased levels of active nuclear JunB, but not JunD or c-Jun, in activated CD4 T cells from Tg compared with WT mice

The level of active nuclear JunB was determined for CD4 T cells following 96 h of TCR stimulation, to determine whether the significant increase in RNA and protein levels observed in the Tg CD4 T cells corresponds to an increase in functionally active JunB. Activation of the AP-1 proteins was determined using an ELISA-based transcription factor activation assay, in which transcription factors in the active conformation bind to plate-bound consensus DNA sequences, followed by visualization using HRP-conjugated Abs specific for the active forms of the AP-1 proteins. After 96 h of TCR stimulation, the levels of active nuclear JunB are much higher than those of JunD and c-Jun in both Tg and WT CD4 T cells (Fig. 10). The nuclear level of active JunB in stimulated Tg CD4 T cells is 1.8-fold higher than in WT CD4 T cells, which corresponds well to the observed increase in RNA and protein levels of JunB from Tg CD4 T cells compared with WT. The addition of VIPase has no effect on JunD or c-Jun levels in either Tg or WT CD4 T cells, but decreases the JunB in Tg CD4 T cells to a level similar to that observed for WT CD4 T cells, indicating a dependence on endogenous VIP for the VPAC2-mediated increase in JunB.



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  		FIGURE 10. Increased JunB activation in activated Tg compared with WT CD4 T cells. JunB ({blacksquare}), JunD (), and c-Jun () were quantified by TransAm ELISA using 5 µg of nuclear extract purified from WT and Tg CD4 T cells following 96-h stimulation on plate-bound anti-CD3 and -CD28, either in the presence of catalytically active VIPase Ab (+VIPase) or inactive control Ab. Results are the average of two independent experiments performed in duplicate ± SD. *, p < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major difference between effector CD4 T cell phenotypes as a function of the VIP/VPAC2 axis is a dramatic switch from the predominantly Th1 phenotype of the WT and VPAC2R-null mice to a Th2 phenotype in the VPAC2 Tg. We have demonstrated this switch to be contingent on the profile of T cell cytokines, in particular IL-4. In addition, we have shown this phenotypic difference to be heavily dependent on endogenous T cell-derived VIP (3, 9, 10, 11). Previous work has illustrated the ability of VIP and pituitary adenylate cyclase-activating polypeptide to modulate cytokine transcription by altering the expression levels of components of AP-1 complexes (36, 37, 38, 39); therefore, to determine at what level the VIP/VPAC2 axis is influencing the polarization of T cells, we examined transcription factors known to be involved in Th2 polarization. We specifically focused on transcription factors known to be unique to Th2 or Th1 development, such as GATA3, c-Maf, JunB, and T-bet. We have found that VIP/VPAC2 specifically up-regulated c-maf and junB at both the mRNA and protein levels in CD4 T cells following TCR stimulation, but did not affect the expression of GATA3 or T-bet. Examination of nuclear protein levels of STAT 6 also did not show any dependence on VIP/VPAC2 (data not shown).

In addition, elimination of endogenous VIP from the cell milieu during T cell activation dramatically inhibited this specific up-regulation. A marginal increase at the message level of CD25 was seen in Tg CD4 T cells following 96-h TCR stimulation compared with WT T cells (data not shown). However, although significant, this increase was extremely slight and does not occur during initial TCR-induced CD25 up-regulation, and is therefore most likely a minor consequence of VIP/VPAC2-induced Th2 polarization, mediated by other factors, such as c-Maf and JunB. The VPAC1 and VPAC2 receptor profiles, which show VPAC2 to be the prominent VIP receptor following TCR activation of Tg CD4 T cells, combined with the inhibitory effect of catalytic anti-VIP Ab on the up-regulation of c-Maf and JunB, and consequently IL-4 in Tg CD4 T cells, very strongly suggest that at least one major mechanism by which VIP/VPAC2 induces a shift toward a Th2 phenotype is by modulating the expression of IL-4 transcription factors. However, to definitively show that the VPAC2R is mediating these effects, we used VPAC2 KO CD4 T cells. Following TCR stimulation, KO CD4 T cells exhibited reduced levels of c-Maf and JunB, and low levels of IL-4 compared with WT and Tg CD4 T cells, confirming that the VIP/VPAC2 interaction is responsible for our observations.

A VIP-mediated increase in JunB and decrease in c-Jun in anti-CD3-stimulated splenic T cells from BALB/c mice have been previously shown, with consequent inhibition of IL-2 (38). In these early studies, VIP was added exogenously, and the VIP receptor responsible for mediating these changes was not addressed. Our data strongly suggest that it is the VPAC2R which is responsible for VIP-mediated up-regulation of both JunB and c-Maf, and that T cell-derived VIP is sufficient to induce deviation of CD4 T cells toward a Th2 phenotype when VPAC2 is constitutively expressed.

Some investigators have indicated that VIP can modulate the expression and/or nuclear localization of NFAT proteins, which are implicated in the expression of several cytokine genes and have been shown to have a critical role in the regulation of Th1 and Th2 cytokine gene transcription, whereas others argue against a direct role of VIP on nuclear NFAT (37, 39, 40). Recent observations have associated preferential nuclear localization of NFATc, or an increase in the nuclear NFATc/NFATp ratio with an increase in IL-4 production (30, 31). Therefore, we explored the possibility that the interaction of VIP with constitutively expressed VPAC2 could be inducing an increase in Th2 cytokines, specifically IL-4, by increasing the NFATc/NFATp ratio, which would also subsequently lead to an increase in c-Maf and JunB expression as the Th cells polarize toward a Th2 phenotype. Our results clearly demonstrate that the VIP/VPAC2 axis does not affect the nuclear ratio of NFAT proteins following TCR stimulation.

Taken together, our results indicate that the interaction of VIP with VPAC2 induces an increase in IL-4 secretion from CD4 T cells by increasing the number of IL-4-secreting CD4 T cells (i.e., induces an increase in Th2-type CD4 T cells) by specifically up-regulating functionally active c-Maf and JunB. Our previous publication has demonstrated that VIP/VPAC2 does not increase the rate of IL-4 gene transcription per cell. Indeed, the VIP/VPAC2 interaction is similar to that of a Th2-type cytokine with its respective receptor in the context of Th cell polarization. This is the first demonstration of a specific VIP/VPAC2-mediated modulation of the Th2-specific transcription factors, c-maf and junB, without the participation of GATA3, resulting in the deviation of TCR-stimulated CD4 T cells toward a Th2 phenotype. These data do not exclude the possibility that other VIP/VPAC2 mechanisms are also involved in Th cell polarization, such as changes in chromatin structure at the Th2 locus. In addition, constitutive exposure to VIP/VPAC2 signaling during T cell development in the thymus of Tg mice may confer a selective growth advantage to IL-4-secreting CD4 T cells. This could act in conjunction with the ability of VIP/VPAC2 to modulate IL-4 transcription factors in the more developed but still unpolarized CD4 splenic T cells, as presented in this study. Future studies will explore the effects of VIP/VPAC2 on thymic development.

The ability of VIP/VPAC2 to deviate an immune response toward a Th2 phenotype at the transcriptional level raises possible implications for VIP/VPAC2 abnormalities in the development and/or progression of allergy and autoimmune diseases. It also opens up possibilities for the development and use of VPAC2 agonists and antagonists in the treatment of autoimmune and allergic diseases, respectively.

The next phase is to examine the results of physiological Ag stimulation of Tg CD4 T cells compared with WT C57BL/6 CD4 T cells, to determine the potency of VIP/VPAC2-induced Th2 polarization following TCR stimulation with Ag. These studies will also enable the effect of VIP/VPAC2 on Ag dose-dependent Th cell polarization to be examined.


   Footnotes
 
1 This work was supported by a research grant from the American Lung Association of California (to J.V.) and Grants AI29912 (to E.J.G.) and AI3128 (to S.P.) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Edward J. Goetzl, Departments of Medicine and Microbiology/Immunology, University of California, UB8B, University of California Box 0711, 533 Parnassus at 4th, San Francisco, CA 94143-0711. E-mail address: egoetzl{at}itsa.ucsf.edu Back

3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; Tg, transgenic; KO, knockout; WT, wild type; m, murine; h, human; CT, cycle threshold; VPAC1, type I VIP receptor; VPAC2, type 2 VIP receptor. Back

Received for publication October 30, 2003. Accepted for publication April 6, 2004.


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