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International Immunology Advance Access originally published online on October 31, 2006
International Immunology 2006 18(12):1691-1700; doi:10.1093/intimm/dxl103
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Altered expression of vasoactive intestinal peptide receptors in T lymphocytes and aberrant Th1 immunity in multiple sclerosis

Wei Sun1,*, Jian Hong1,*, Ying C. Q. Zang1, Xin Liu1 and Jingwu Z. Zhang1,2

1 Department of Neurology, Baylor College of Medicine, Mail station NB302, One Baylor Plaza, Houston, TX 77030, USA
2 Joint Immunology Laboratory of Institute of Health Sciences and Shanghai Institute of Immunology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao-Tong University School of Medicine, Shanghai, China

Correspondence to: J. Z. Zhang; E-mail: jzang{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Vasoactive intestinal peptide (VIP) has a unique property of regulating Th1 and Th2 immunity of CD4+ T cells. In this study, we demonstrated, for the first time, that differential expression of VIP receptors and a compensatory mechanism directly affect the responsiveness of CD4+ T cells and their Th1 and Th2 properties to VIP. The expression of VIP receptor-1 (VPAC1) and VPAC2 in CD4+ T cells changed reciprocally in the context of the activation state. In activated CD4+ T cells of healthy individuals, markedly decreased VPAC1 expression was compensated for by increased expression of VPAC2 induced by T cell activation. In contrast, there was altered expression of VPAC2 in activated CD4+ T cells derived from multiple sclerosis (MS) patients, which rendered CD4+ T cells less responsive to VIP and skewed the system to a predominantly in a Th1 direction. Detailed characterization with agonist peptides of VIP showed that residues Met and Ser at positions 17 and 25 of VIP were critical to its regulatory properties through interaction with VAPC2. Furthermore, altered levels of VPAC2 expression in T cells of MS patients were not associated with single-nucleotide polymorphism in the encoding region of the VPAC2 gene but with gene regulation as characterized by a distinct DNA footprinting pattern in the promoter region of the VPAC2 gene in MS as compared with controls. This study has provided new evidence for an intrinsic mechanism associated with an aberrant, pro-inflammatory state of CD4+ T cells in MS.

Keywords: cytokines, multiple sclerosis, T cell activation, Th1 and Th2 immunity, vasoactive intestinal peptide


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Vasoactive intestinal peptide (VIP) is a 28-mer neuroendocrine peptide that is widely distributed in the central and peripheral nervous systems and interacts with tissues innervated by peptidergic neurons. General physiological effects of VIP include vasodilation (1), bronchodilation (2) and increases in gastric motility (3). VIP exerts its biological activities by binding to two closely related class II G-protein-coupled receptors VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2) (4, 5). Both VPAC1 and VPAC2 are membrane proteins of 457 and 438 amino acids in length, respectively (6, 7). Human VPAC1 receptor is encoded by VPAC1 gene located on chromosome 3p22 (8) while human VPAC2 receptor gene is localized on chromosomal site 7q36.3 (9). Although both receptors have distinct tissue distributions, they have similar affinities for VIP and are expressed on T lymphocytes (10). There is evidence that VPAC1 is constitutively expressed in resting T cells, especially in CD4 T cells while VPAC2 is expressed marginally or is absent in unstimulated T cells (11).

Research interest in VIP has recently extended to its immune regulatory properties and its role in maintaining Th1 and Th2 homeostasis in the immune system. Recent studies have demonstrated that VIP can be produced by T cells, especially Th2 cells, after antigen stimulation, and that it exerts immunological functions typically ascribed to Th2 cytokines in the immune system through its receptors expressed on T cells (12, 13). Macrophages and dendritic cells treated in vitro with VIP acquire the ability to induce Th2 immunity as characterized by the production of anti-inflammatory cytokines (IL-4 and IL-5). In addition, VIP treatment inhibits pro-inflammatory Th1 cytokines (IFN-{gamma} and IL-2) in antigen-primed CD4 T cells (14). There is also evidence that in vivo administration of VIP in mice results in a decreased number of Th1 cells and an increased number of Th2 cells (15). Delgado and colleagues (16) demonstrated recently that when given by injection, VIP has a potent therapeutic effect on the clinical course of collagen-induced arthritis, an animal model for rheumatoid arthritis in humans. In that study, VIP was found to significantly reduce inflammatory response by down-regulating the production of several pro-inflammatory cytokines in inflamed joints and synovial cells, including tumor necrosis factor-{alpha}, IL-18 and IL-12 and various chemokines (17).

The immune regulatory properties of VIP prompted us to investigate its role in multiple sclerosis (MS). In particular, we attempted to evaluate our proposed hypothesis that the pro-inflammatory Th1 trait commonly seen in T cells of MS patients might be associated with aberrant responsiveness of T cells to VIP. This might result directly from altered expression of VIP receptors in these T cells. In MS, there is ample evidence indicating that the disease is associated with hyperactivity and pro-inflammatory properties of, predominantly, CD4+ T cells (18). A markedly skewed Th1 immunity or a Th1 trait among T cells in the blood and cerebrospinal fluid represents one of the most significant immunopathologic features of MS, and is thought to contribute to the disease activity (19). The current treatment standard and emerging therapeutic strategies that are designed to regulate Th1 and Th2 immunity have shown treatment benefit in MS (20).

This study was undertaken to examine the differential expression of VPAC1 and VPAC2 and the compensatory mechanism involved in the Th1 and Th2 homeostasis of CD4+ T cells. We evaluated whether altered expression of the VIP receptors in T cells might render CD4+ T cells less responsive to endogenously produced VIP and exogenous VIP. A series of experiments were carried out to address whether aberrant responsiveness of CD4+ T cells to VIP was associated with the Th1 trait commonly seen in MS-derived T cells and to delineate the structural requirements, using a panel of VIP agonist peptides, for the regulatory function of VIP in the context of VPAC2. Genetic polymorphisms and gene regulation patterns within promoter regions of VPAC2 were analyzed in detail to provide explanations for altered expression of VPAC2 found in MS patients. The study has provided new evidence supporting the role of VIP and its altered receptor expression in the development of pro-inflammatory Th1 trait in CD4+ T cells of MS patients.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Preparation of PBMC and genomic DNA
A panel of blood specimens was obtained from 20 healthy individuals (9 males and 11 females aged between 20–44 years) and 20 MS patients (6 males and 14 females aged between 24–58 years) at Baylor MS Clinic for VIP receptor analyses and functional experiments. These specimens were pooled with a separate panel of 72 MS patients and 65 healthy individuals kindly provided by Yang Liu (Ohio State University, Cleveland, OH, USA) for the isolation of genomic DNA for single-nucleotide polymorphism (SNP) analysis. All specimens were obtained from MS patients who had not received treatment with immunomodulatory or immunosuppressive drugs, including IFN-ß and glatiramer acetate. The protocols were approved by the Institutional Review Boards of Baylor College of Medicine and Ohio State University.

Peptide synthesis
Wild-type VIP peptide (HSDAVFTDNYTRLRKQMAVKKYLNSILN) was purchased from Calbiochem (La Jolla, CA, USA). A small panel of VIP variant peptides used in this study and a control peptide of irrelevant sequence (FCASSLGRAGLTYEQYFGDG) were all synthesized by the Peptide Core Laboratory, Department of Molecular Pathology, University of Texas M. D. Anderson Cancer Center under a core grant CA-16672. The variant VIP peptides had the following amino acid sequences: VIP4-28, AVFTDNYTRLRKEMAVKKYLNSILN; VIP-M1, HSDAVFTDNYTRLRKQLAAK KYLNDIKKKRY; VIP-M2, HSDAVFTDNYTRLRKQMAAKKYLNSIKKKR and VIP-M3, HSDAVFTDNYTRLRKQMAAKKYLNSIKNKRY.

PBMC and CD4 T cell isolation
PBMCs were isolated from blood by Ficoll–Hypaque gradient density centrifugation (Amersham Biosciences, Uppsala, Sweden). CD4 T cells were separated using CD4 Negative Isolation Kit (Dynal Biotech, Oslo, Norway). Briefly, PBMCs in PBS/0.1% BSA were incubated with antibody mix for 10 min at 2–8°C to positively select all CD4-negative cells. After washing and removing the supernatant, depletion magnetic beads were added into the re-suspended cell pellet and incubated for 15 min at room temperature with gentle tilting and rotation. With a magnet, CD4 T cells were removed by pipetting the supernatant and the unwanted antibody-bound cells were discarded. Purity of enriched cells was >90% by flow cytometry.

PBMC and CD4 T cell activation and flow cytometric analysis of VIP receptors
PBMCs and fractionated CD4 T cells were cultured in complete RPMI 1640 medium (GIBCO RBL, Life Technologies, NY, USA) containing 10% fetal bovine serum and 1% penicillin–streptomycin–L-glutamine at a final concentration of 1 x 106 cells ml–1 in 96-well round-bottom tissue culture plates (Costar, Corning, NY, USA) in the presence or absence of 1 µg ml–1 preservative-free soluble anti-CD3 and anti-CD28 (Becton Dickinson, San Jose, CA, USA). Cultures were incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2 and harvested daily for total RNA extraction. Flow cytometric analysis of VIP receptors was performed using antibody to human VIP receptors (Sigma Chemical, St Louis, MO, USA) and conjugated antibodies to CD4 and CD25 (BD Bioscience, San Jose, CA, USA). The antibodies had no cross-reactivity between the two VIP receptors (Sigma, clone AS58 and clone AS69).

Quantification of endogenous VIP by ELISA
Culture supernatants were collected from CD4+ T cells stimulated with antibodies to CD3 and CD28. The concentrations of endogenous VIP were determined by an ELISA kit according to the manufacturer's instructions (Phoenix Pharmaceuticals, Belmont, CA, USA). Optical density was measured at 450 nm using an ELISA plate reader (Bio-Rad Laboratories, Hercules, CA, USA).

RNA isolation and first-strand cDNA synthesis
Total RNA was isolated from PBMCs and CD4 T cells using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. To obtain high-quality RNA, contaminating DNA was efficiently removed from RNA samples by digestion with DNase I (Qiagen) in the process of RNA isolation. cDNA was synthesized from RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories) in a 20-µl reaction.

Analysis of gene expressions by real-time PCR
Real-time PCR was performed in a MicroAmp Optical 96-well reaction plate (Applied Biosystems, Foster City, CA, USA) using 2 µl cDNA sample, 12.5 µl of 2x TaqMan Master Mix without AmpErase uracil N-glycosylase (Applied Biosystems), forward and reverse primer pairs (900 nM each), and 200 nM labeled probe for the target genes (including VPAC1, VPAC2, IFN-{gamma}, Stat1, Stat4, Stat6 and GATA-3). For genes of IL-5, IL-2R, FOXD2 and 18s rRNA, 1.25 µl of 20x Assay-on-Demand gene expression product (Applied Biosystems) was added into each reaction. The nucleotide sequences of real-time primers and probes are listed in Table 1. The reaction was performed in duplicate wells for 2 min at 50°C and for 10 min at 95°C as hot start activation, which was followed by 40 cycles of denaturation for 15 s at 95°C and an annealing/extension step of 1 min at 60°C. All reactions were conducted using an ABI 7000 Sequence Detection System (Applied Biosystems). The relative expression value of each target gene was calculated use the following formula: Formula ({Delta}CT = CT of target gene – CT of 18s rRNA). CT represents cycle threshold.


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  		Table 1 Nucleotide sequences of quantitative PCR primers and probes

 
Measurement of IFN-{gamma}-producing CD4 T cells by enzyme-linked immunospot
Fractionated CD4 T cells (3 x 105) were activated with soluble anti-CD3 and soluble anti-CD28 with or without 30 min of pre-incubation with 10–9 to 10–6 M wild-type VIP and VIP variant peptides. Human TCR peptide was used as a control. Plates were incubated at 37°C for 72 h in a humidified atmosphere of 5% CO2. CD4 T cells were removed from the wells, washed once with HBSS, re-suspended in 200 µl of complete RPMI medium and added to anti-IFN-{gamma} antibody-coated enzyme-linked immunospot (ELISPOT) plates, followed by an overnight incubation at 37°C. The ELISPOT plates were developed according to the manufacturer's instructions (BD PharMingen, San Diego, CA, USA). Spots were developed using 3-amino-9-ethylcarbazole (Pierce Pharmaceuticals, Rockford, IL, USA) solution for 10–20 min. The reaction was stopped by rinsing the plates with water until clear spots were visualized microscopically. The plates/membranes were then air-dried overnight before the plates were subjected to image analysis. The analysis was performed using a Series 1 ImmunoSpot Image Analyzer (Cellular Technology, Cleveland, OH, USA) specifically designed for ELISPOT assay. Digitized images were analyzed and results were expressed as the number of positive spots per total number of cells per well.

SNP genotyping of VPAC2
Two SNP sites, refSNP number rs885861 and rs2098349 of VPAC2 gene, were analyzed using TaqMan validated SNP Genotyping assays (Applied Biosystems). Briefly, PCR was run in a MicroAmp Optical 96-well reaction plate (Applied Biosystems) with 20 ng genomic DNA, 12.5 µl of 2x TaqMan Master Mix without AmpErase uracil N-glycosylase (Applied Biosystems) and 1.25 µl 20x Assay-on-Demand SNP genotyping reagent (assay IDs C_1245817_1 and C_16032096_10). The plate was thermal cycled for 2 min at 50°C and for 10 min at 95°C as hot start activation, which was followed by 40 cycles of denaturation for 15 s at 95°C and an annealing/extension step of 1 min at 60°C on the ABI 7000 Sequence Detection System (Applied Biosystems). The plate was post-read for allelic discrimination on the ABI 7000 and results were analyzed as described in User Bulletin #2 ABI Sequence Detection System (Applied Biosystems).

DNA footprinting
Purified CD4+ T cell preparations were pre-incubated in the presence and absence of VIP at a pre-determined concentration of 10–7 M for 30 min. Nuclear extracts were isolated using NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology). The VPAC2 promoter region (chromosome 7, positions 158,139,785 to 158,140,384) was first cloned from the genomic DNA of healthy individual PBMC using the TA cloning kit (Invitrogen, San Diego, CA, USA) and One Shot TOP10 Escherichia coli competent cells (Invitrogen) according to the manufacturer's protocol. The promoter gene was then obtained by PCR in a total volume of 50 µl, containing 100 ng of plasmid DNA, 2 pmol of each primer (Table 1), 250 µM diethylnitrophyenyl thiophosphate, 10 mM KCl, 20 mM Tris–HCl, 3.0 mM MgCl2 and 1 U Taq polymerase (Invitrogen). The PCR mixtures were then denatured at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 40 s, extension at 72°C for 40 s and final elongation at 72°C for 10 min. The PCR products were purified using the QIAquick PCR purification kit from Qiagen. DNase footprinting was performed using the Core Footprinting System from Promega (Madison, WI, USA). Briefly, purified DNA fragment was labeled with 32P (MP Biomedicals, Irvine, CA, USA) and digested with HinfI (New England Biolabs, Ipswich, MA, USA). The nuclear extract was incubated with the labeled DNA probe and then digested by incubating with RQ1 RNase-free DNase I. DNA was loaded onto a 6% denaturing urea/polyacrylamide gel and exposed on film at –80°C.

Statistical analysis
Relative expressions of the genes of interest and cell proliferation/activation in experimental groups were analyzed for their normality using the Shapiro–Wilk test. A difference of relative expression among groups was calculated using the Student's t-test for normally distributed variables and non-parametric Mann–Whitney test for non-normally distributed variables. A P-value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Differential expression of VPAC1 and VPAC2 in CD4 T cells derived from MS patients
The expression levels of both VPAC1 and VPAC2 were measured in purified CD4+ T cells derived from a group of MS patients and compared with those of healthy individuals. As shown in Fig. 1(A), mRNA expression of VPAC1 did not differ between the two groups and appeared to decrease significantly in activated CD4+ T cells of both MS patients and healthy individuals. A different pattern of mRNA expression was seen for VPAC2 in parallel experiments. VPAC2 mRNA was found at low expression in resting CD4+ T cells derived from both MS patients and controls and was markedly increased when CD4+ T cells were activated, which appeared to compensate for the decreased expression of VPAC1 in activated CD4+ T cells (Fig. 1B). However, the level of VPAC2 mRNA expression was markedly decreased in CD4+ T cells derived from MS patients as compared with that of controls (P < 0.05, Fig. 1B).


Figure 1
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  		Fig. 1 Differential expression of VPAC1 and VPAC2 genes in unstimulated and activated CD4+ T cells derived from MS patients and healthy controls. mRNA expression of VPAC1 (A) and VPAC2 (B) was analyzed by real-time PCR in purified CD4+ T cells that were either unstimulated (rCD4) or activated (aCD4) by antibodies to CD3 and CD28. Forty purified CD4+ T cell preparations derived from MS patients (n = 20) and healthy controls (n = 20) were included in the analysis. Results are shown as average mRNA expression level ± SEM. Asterisks represent statistically significant differences between the groups (P < 0.05). Dagger indicates significant difference between resting CD4+ T cells and activated CD4+ T cells.

 
Purified CD4+ T cells from both MS patients and controls were then subjected to activation and examined for the expression of both VIP receptors as a function of time. As illustrated in Fig. 2, the expression of VPAC1 significantly dropped over time to a level of 0.3 ± 0.1 in 72 h, which corresponded to a 16-fold decrease from baseline (Fig. 2A). In contrast, VPAC2 increased its expression in CD4+ T cells after activation. The VPAC2 expression was markedly altered in activated CD4+ T cells derived from MS patients, as characterized by the lack of increased expression in response to T cell activation (Fig. 2B). It was noticeable that from ~72 h onwards, there was minimal expression of both VIP receptors in activated CD4+ T cells of MS patients as opposed to that of control subjects. The discrepancies in the expression of the VIP receptors between the two groups were not associated with the activation state of CD4+ T cells examined as the expression of T cell activation markers, such as IL-2R, did not differ in the same T cell populations (Fig. 2C). The increased surface expression of the VIP receptors was confirmed by flow cytometry. A representative plot is shown in Fig. 3. The results indicate that VPAC2 exhibited an altered expression pattern in activated CD4+ T cells obtained from patients with MS.


Figure 2
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  		Fig. 2 The expression of VPAC1 (A) and VPAC2 (B) in CD4+ T cells after activation. mRNA expression of VPAC1 (A) and VPAC2 (B) was analyzed as a function of time in purified CD4+ T cell preparations derived from MS patients (n = 20) and healthy controls (n = 20). All T cell preparations were activated under the same conditions by antibodies to CD3 and CD28 and analyzed by real-time PCR. IL-2R (C) was examined in parallel in the same T cell preparations as a marker for T cell activation. Results are shown as average mRNA expression level ± SEM. Asterisks indicate significant differences in mRNA expression of activated CD4+ T cells between MS and normal subjects (NS) (P < 0.05).

 

Figure 3
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  		Fig. 3 The surface expression of VIP receptor in CD4+ T cells as determined by flow cytometry. Unstimulated or activated PBMCs from MS patients or controls (each n = 5) were examined for the surface expression of VIP receptor using flow cytometry. The plots shown are representative of five independent specimens analyzed.

 
Correlation of expression level of VIP receptors with Th1 and Th2 states of activated CD4+ T cells
We then addressed whether the altered expression level of VIP receptors on activated CD4+ T cells from MS patients had functional consequences and whether these correlated with responsiveness or with Th1 or Th2 state of the T cells. To this end, CD4+ T cells derived from both MS patients and controls were activated for 72 h and subjected to analysis for the representative Th1 and Th2 cytokines (i.e. IFN-{gamma} and IL-5) and transcription factors specifically associated with Th1 and Th2 responses (i.e. Stat1 for Th1 and Stat6 for Th2) since VIP is known to interact with its receptors to promote a Th2 deviation (21). We first showed that mRNA expression of VPAC2 was decreased in activated CD4+ T cells from MS patients by nearly 10-fold as compared with that of controls (0.04 ± 0.02 versus 0.39 ± 0.10) while the VPAC1 expression was at its lowest level in activated CD4+ T cells. As illustrated in Fig. 4, there was a significant Th1 deviation in activated CD4+ T cells derived from MS patients as compared with that of controls, as characterized by specific changes in mRNA expression of the selected cytokines and transcription factors. Analysis of the expression of VPAC2 and IL-10 by flow cytometry showed that VPAC2-positive T cells had a decreased production of IL-10 in MS as opposed to controls. It was determined that the concentrations of VIP endogenously produced by activated T cells did not differ between the MS-derived T cell culture and the control T cell culture (58.5 ± 15.2 pg ml–1 and 60.1 ± 11.3 pg ml–1). The results indicate that the altered Th1 and Th2 states of activated CD4+ T cells from MS patients were not associated with endogenously produced VIP but with the expression level of the VIP receptors that determined the sensitivity of CD4+ T cells to VIP towards a Th2 response. As shown in Fig. 5, there was a significant difference in mRNA expression of Stat6 but not Stat1 in CD4+ T cells treated with serial concentrations of exogenous VIP, ranging from 10–10 to 10–7 M, between the two groups (Fig. 5). Collectively, the findings suggest that the decreased expression of VIP receptors correlated with a marked Th1 deviation in activated CD4+ T cells derived from MS patients.


Figure 4
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  		Fig. 4 The expression of cytokines and transcription factors characteristic of Th1 and Th2 immunity in relation to the expression of VIP receptors. mRNA expression of selected cytokines and transcription factors characteristic of Th1 and Th2 immunity was analyzed in purified CD4+ T cell preparations obtained from MS patients (n = 20) and controls (n = 20). The same activated T cell preparations as described in legend to Fig. 2 were examined by real-time PCR (A). Results are shown as average mRNA expression level ± SEM. Purified CD4+ T cells from MS patients or controls were stimulated with anti-CD3/CD28 antibodies. The resulting cells were double stained with anti-IL-10 and anti-VPAC2 antibodies and analyzed by flow cytometry (B). Asterisks represent statistically significant differences between the groups (P < 0.05).

 

Figure 5
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  		Fig. 5 Effect of exogenously added VIP on Th1 and Th2 traits of activated CD4+ T cells. Purified CD4+ T cell preparations obtained from MS patients and controls were activated with antibodies to CD3 and CD28 in the presence or absence of exogenous VIP added at the indicated concentrations from 10-10 to 10-7 M. Cells were cultured for 1 day and collected for real-time PCR analysis for Stat1 and Stat6 as indicators for Th1 and Th2 traits. Results are shown as a ratio (exogenous VIP/control) of mRNA expression level ± SEM. The results are representative of separate experiments of five pairs of independent T cell preparations. Asterisks represent statistically significant differences between the groups (P < 0.05).

 
Structural requirements for the Th1 and Th2 traits of activated CD4+ T cells through VIP interaction with VPAC2
It was of interest to determine the structural requirements for VIP interaction with VPAC2 in relation to Th1 and Th2 traits of activated T cells. As shown in Fig. 6(A), activated CD4+ T cells obtained from MS patients and controls exhibited a different pattern of response to native VIP as measured by IFN-{gamma} ELISPOT, which was indicative of a Th1 shift in MS-derived T cells. VIP4–28 peptide is a potent agonist specific for VPAC1 and acts as an antagonist but not as an agonist for VPAC2. Our results showed that VIP4–28 peptide had a profound inhibitory effect on Th1 to Th2 deviation in control CD4+ T cells, as characterized by a marked decrease in IFN-{gamma} response by ELISPOT (Fig. 6B). The effect was absent when tested with MS-derived CD4+ T cells, suggesting that the inhibitory property of peptide VIP4–28 required sufficient expression of VIP receptors. Furthermore, a small panel of three VIP peptides with specific amino acid mutations was selected for their high binding affinity to VPAC2 (150- to 180-fold higher than that to VPAC1) (22). It was evident that the peptide VIP-M1 that had multiple amino acid substitutions exhibited an enhancing effect in reducing Th1 response in control CD4+ T cells and, to a lesser extent, in MS-derived T cells when given at higher concentrations (Fig. 6C). The two other agonist peptides, namely, VIP-M2 and VIP-M3, that differed from VIP-M1 in lacking amino acid substitutions at positions 17 and 25 induced a distinct reactivity pattern characterized by a markedly decreased Th1 response in CD4+ T cells of both groups (Fig. 6D and E). The findings suggest that both VIP agonist peptides rendered CD4+ T cells vulnerable to a Th1 to Th2 shift, even though VPAC2 was expressed at a minimal level in MS-derived CD4+ T cells. The observed effects of VIP and the agonist VIP peptides were determined to be specific as a control peptide of irrelevant sequence had no such effects on the same CD4+ T cells (Fig. 6F). The results were confirmed by measuring the production of IFN-{gamma} in supernatants of the same culture by ELISA (data not shown).


Figure 6
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  		Fig. 6 Effect of VIP and its altered peptide analogs on reactivity of purified CD4+ T cells. Purified CD4+ T cell preparations were activated with antibodies to CD3 and CD28 in the presence and absence of VIP (A) and altered peptide analog of VIP (B–E) used at the indicated concentrations. A peptide of irrelevant TCR CDR3 sequence was used as a control (F). IFN-{gamma}-secreting CD4+ T cells were measured by ELISPOT analysis. Results are presented as the number of IFN-{gamma}-producing T cells per million purified CD4+ T cells. The results are representative of separate experiments on five pairs of independent T cell preparations.

 
Relationship of altered expression of VPAC2 with genetic polymorphism
We further addressed whether the altered expression of VPAC2 in MS-derived CD4+ T cells was potentially associated with genetic polymorphisms using a large panel of MS specimens and control specimens (see Methods). Two validated SNP sites of the VPAC2 gene, rs885861 and rs2098349, were analyzed. Both SNP sites are within the coding region of the gene while SNP site rs885861 is identified in the 3' untranslated region with the highest minor allele frequency of 0.38 in the Caucasian population. SNP site rs2098349 represents a mis-sense mutation site with the lowest minor allele frequency of 0.07 in the Caucasian population. Genotyping of the selected SNP sites was performed using TaqMan validated SNP Genotyping assays. Table 2 shows the proportion of SNP genotypes and allele frequencies in both groups, indicating that the prevalence of genotype and allele frequencies at the two sites did not differ significantly between the MS group and the control group. Collectively, the findings do not support association of genetic polymorphisms of VPAC2 with its altered expression in MS.


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  		Table 2 SNP of VPAC2 gene and frequencies in MS and normal subject (NS) groups

 
Gene regulation pattern of the VPAC2 promoter region
We then attempted to address whether the regulation of gene expression within the promoter region of the VPAC2 gene might be associated with the altered expression of VPAC2 in MS. The VPAC2 promoter region is 600 bp in length and is located at chromosome 7, positions 158,139,785 to 158,140,384 (http://www.chr7.org, 23). Labeled promoter fragments of the VPAC2 gene were first allowed to bind to nuclear extracts from activated CD4+ T cells of both MS and healthy controls. DNase I was then added to generate specific cleavages of the probe for DNA footprinting. The results revealed that MS-derived T cell preparation had a specific DNA footprinting pattern characterized by intense bands at the positions of 66 and 110 bp (Fig. 7A, lane 3), which differed from that of controls (Fig. 7A, lane 2). This observation indicated significant differences in DNase digestion patterns that resulted from differential binding abilities of transcription factors to the promoter region. Sequence motif analysis of this region showed the involvement of six transcription factors, including GATA-3 and FOXD2 (Fig. 7B). It was of interest to note that GATA-3 and FOXD2 are known to control Th1 and Th2 properties and activation of CD4+ T cells (24, 25). We further analyzed mRNA expression levels of transcription factors GATA-3 and FOXD2 in a large panel of MS-derived and control CD4+ T cell preparations using real-time PCR. The data confirmed significantly decreased expression of both GATA-3 and FOXD2 in CD4+ T cells of MS patients as compared with controls (P < 0.05, Fig. 8). Taken together, the altered VPAC2 expression in MS-derived CD4+ T cells appeared associated with aberrant regulation of the binding of transcription factors, such as GATA-3 and FOXD2, to the promoter region of the VPAC2 gene.


Figure 7
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  		Fig. 7 DNA footprinting of the promoter region of the VPAC2 gene in purified CD4+ T cells. (A) Representative footprinting patterns of the VPAC2 promoter region from 66 to 110 bp in purified CD4+ T cells treated with VIP. 32P-labeled VPAC2 promoter fragments were used as a control (lane 1). Labeled fragments plus nuclear extract from control CD4+ T cell preparations (lane 2) and from MS-derived CD4+ T cells (lane 3). The results were representative of five independent pairs of CD4+ T cell preparations. (B) Schematic representation of putative nuclear protein binding to the regulatory element of the promoter region of the VPAC2 gene. The positions of the seven putative regulatory elements in this region are indicated. The shaded boxes (GATA-3 and FOXD2) are known transcription factors controlling the Th2 immunity of CD4+ T cells. The arrow indicates a possible transcriptional initiation site in exon 1 (29).

 

Figure 8
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  		Fig. 8 Expression of transcription factors GATA-3 and FOXD2 in CD4+ T cell preparations. Purified CD4+ T cell preparations obtained from MS patients and controls were pre-incubated in the presence and absence of VIP at a concentration range of 10–8 to 10–6 M and subsequently activated by antibodies to CD3 and CD28 for 72 h. The resulting T cells were examined for the expression of GATA-3 and FOXD2 by real-time PCR. Results are shown as relative mRNA expression ± SEM. The results are representative of separate experiments of five pairs of independent T cell preparations. Asterisks represent statistically significant differences between the groups (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Hyperactivity and the pro-inflammatory nature of CD4+ T cells remain as two of the most consistent and significant immunologic characteristics of MS, and are thought to directly contribute to the inflammatory processes involved in MS (19). However, despite intensive research in this area, the underlying mechanism remains unknown. In this study, we provide new evidence indicating that the expression level of VPAC2 in activated CD4+ T cells is critical to maintaining the homeostasis of Th1 and Th2 states in CD4+ T cells and that an altered expression level of VPAC2 is associated with the predominant Th1 trait found in activated CD4+ T cells of MS patients. The study has raised a number of important issues. We demonstrated, for the first time, that the expression of VPAC1 and VPAC2 in CD4+ T cells changes reciprocally, corresponding to the T cell activation state. The findings indicate that the reduced expression of VPAC1 in activated T cells is normally compensated for by the increased expression of VPAC2. This feature of CD4+ T cells is perhaps critical under normal physiological conditions to maintaining equilibrium of Th1 and Th2 homeostasis in the presence of endogenously produced VIP by activated CD4+ T cells and other cell types. However, in patients with MS, the altered expression of VPAC2 results in a loss of this compensatory mechanism for VIP receptors, which then leads to minimal expression of VIP receptors in activated CD4+ T cells. Our results show that altered expression of VPAC2 has direct functional consequences characterized by a shift toward a Th1 prominent pattern in CD4+-activated T cells derived from MS. These MS-derived T cells are less responsive to endogenously produced VIP and exogenously added VIP than are CD4+ T cells derived from healthy individuals, resulting in a skewed Th2 trait in activated CD4+ T cells. These findings are of importance and highly relevant to the understanding of the pathogenesis of MS as they have provided a possible explanation that potentially accounts for the pro-inflammatory nature of CD4+ T cells in MS.

In this study, we attempted to delineate the structural requirements for sensitivity/responsiveness of activated CD4+ T cells towards VIP. Our findings suggest that some of the known VIP agonist peptides are superior in shifting Th1 to Th2, even in MS-derived T cells in which VPAC2 expression is altered. When amino acid sequences of the selected agonist VIP peptides are compared, it becomes clear that Met at position 17 and Ser at position 25 are required to further reduce a Th1 response in CD4+-activated T cells that have minimal expression of VIP receptors. In this regard, as VIP has been shown to effectively treat autoimmune arthritis in an experimental animal model, these agonist VIP peptides that have a superior anti-inflammatory property may prove to have better therapeutic potential in reducing a Th1 response if used as an anti-inflammatory treatment. Potential therapeutic use of VIP in MS is also supported by the finding that poor susceptibility of CD4+ T cells to endogenously produced VIP, as a result of altered expression of VPAC2 in activated T cells, appears to be compensated for by the addition of exogenous VIP.

Furthermore, in this study, we attempted to determine whether the altered expression of VPAC2 was associated with genetic polymorphisms or with an aberrant gene regulation pattern in the promoter region of the VPAC2 gene in MS-derived CD4+ T cells. The findings described here failed to support genetic polymorphisms within the VPAC2-encoding regions. However, the data remain merely suggestive because of the limited number of samples analyzed. On the other hand, our results have pointed to an aberrant gene regulation pattern characterized by a distinct DNA footprinting pattern within the promoter region of VPAC2 in MS. It is likely that altered expression of VPAC2 is associated with the aberrant gene regulation of the VPAC2 gene upon activation of CD4+ T cells in MS. Little is known about the transcription mechanisms that regulate the expression of VPAC2 gene in T cells. We propose that transcription factors GATA-3 and FOXD2 may be involved in the induction of the VPAC2 gene in activated CD4+ T cells. VPAC1 is present constitutively on T cells while VPAC2 is expressed at a low level in resting CD4+ T cells and its expression rapidly increases after T cell activation. VIP activates adenylyl cyclase through interaction with VIP receptors and induces an accumulation of intracellular cyclic adenosine 3¢,5¢-monophosphate (cAMP). The interaction of VIP with its receptors initiates two signal transduction pathways, namely, the cAMP/protein kinase A (PKA)-dependent and the cAMP/PKA-independent pathways (26). The cAMP/PKA-dependent pathway inhibits IFN-{gamma}-induced Jak1/Jak2 and Stat1 phosphorylation while it induces GATA-3-dependent activation of IL-5 gene expression in Th2 cells (27). Elevation of cAMP could also promote phosphorylation of GATA-3 by p38 mitogen-activated protein kinase as part of the cAMP/PKA-independent pathway (28). Furthermore, PKA is activated by the elevation of intracellular cAMP and generates two active catalytic subunits from a regulatory dimer (R2). RI{alpha} or PKA type 1 is the dominating R isoform of the PKA in T cells that is recruited to the TCR–CD3 complex following T cell activation. FOXD2, a forkhead helix transcription factor, is associated with the protein level of the RI{alpha} subunit through induction of the RI{alpha}1b promoter, which in turn increases cAMP sensitivity and sets the threshold for cAMP-mediated negative modulation of T cell activation. This possibility is supported by our finding that the expression levels of both GATA-3 and FOXD2 in VIP-treated CD4+ T cells derived from MS patients are markedly decreased. In conclusion, decreased expression of VPAC2, as a result of altered gene regulation in the promoter region of VIP receptor, has functional consequences, which render CD4+ T cells more pro-inflammatory in MS.


   Abbreviations
 
cAMP, cyclic adenosine 3',5'-monophosphate
ELISPOT, enzyme-linked immunospot
MS, multiple sclerosis
PKA, protein kinase A
SNP, single-nucleotide polymorphism
VIP, vasoactive intestinal peptide

   Notes
 
* These authors contributed equally to this work. Back

Transmitting editor: C. Paige

Received 19 October 2005, accepted 13 September 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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M. Yadav, J. Rosenbaum, and E. J. Goetzl
Cutting Edge: Vasoactive Intestinal Peptide (VIP) Induces Differentiation of Th17 Cells with a Distinctive Cytokine Profile
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