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Cutting Edge: Is Vasoactive Intestinal Peptide a Type 2 Cytokine?

Mario Delgado^*, and Doina Ganea^*

^* Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid, Spain

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
A component of the chemical language shared by the immune and nervous system is the expression of neuropeptides by immune cells. Vasoactive intestinal peptide (VIP) was shown to be produced by T lymphocytes. Here we investigate whether T cell subsets differentially express VIP. Our studies indicate that, upon specific Ag stimulation, Th2 and T2 cells, but not Th1 and T1 cells derived from TCR transgenic (Tg) mice, express VIP mRNA and protein, and secrete VIP. Following immunization with the specific Ag, significant levels of VIP are present in the serum of syngeneic, non-Tg hosts that receive Th2, but not Th1 Tg cells. Th2 Tg cells recovered from the non-Tg hosts immunized with the specific Ag, but not with an irrelevant Ag, express intracellular VIP. Because VIP is produced by Ag-stimulated type 2 T cells, and differentially affects Th1 and Th2 cells, could VIP be viewed as a type 2 cytokine?

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The use of a common biochemical language by the immune and nervous system has been established recently. An important clue was the discovery that lymphocytes produce hormones and neuropeptides that act as endogenous immunomodulators. For example, immune cells produce substance P (1), corticotropin releasing factor (CRF) (2), -melanocyte stimulating factor (-MSH) (3), adrenocorticotropin hormone (4), -endorphins (5), somatostatin (6), calcitonin gene-related peptide (CGRP) (7), neuropeptide Y (NPY) (8), atrial natriuretic peptide (ANP) (9), and vasoactive intestinal peptide (VIP)³ (10, 11, 12, 13).

VIP, a 28-aa neuropeptide, affects both innate and acquired immunity (14, 15, 16, 17). Originally considered a negative regulator of several T cell and macrophage functions (16, 18), VIP appears to play a more complex role in immune homeostasis (19). Lately, this pleiotropic peptide was "rediscovered" in the immune system. Both the VIP-ergic innervation (15) and the immune cells (10, 11, 12, 13, 20, 21, 22, 23, 24) function as VIP sources. VIP was identified in thymocytes, peripheral macrophages, and lymphocytes (reviewed in Refs. 12, 14), with VIP_1–28 as the predominant secreted form (12). Recently, we reported that antigenic stimulation and inflammatory signals result in an increased VIP release (13). The aim of this study was to investigate the production and secretion of VIP by CD4⁺ Th1 and Th2 cells, and by CD8⁺ T1 and T2 cells. Our results show that antigenic stimulation preferentially induces VIP production by Th2 and T2 cells. As far as we know, this is the first report describing the differential production of a neuropeptide by specific T cell subsets.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

B10.A (I-E^k), AND TCR, and TCR-Cyt-5CC7-I/Rag2 transgenic (Tg) mice (25) were purchased from The Jackson Laboratory (Bar Harbor, ME), and Taconic Farms (Germantown, NY).

Reagents and Abs

Murine rIL-2, IL-4, IFN-, and mAbs to murine IL-4 (11B11), IFN- (XMG1.2), CD3 (145-2C11), PE-conjugated anti-V3 (RR4-7), and PerCP-conjugated CD4 mAbs were purchased from PharMingen (San Diego, CA); FITC-conjugated goat F(ab')₂ anti-mouse IgG, and OVA were purchased from Sigma (St. Louis, MO). Mouse anti-VIP mAb (Ab 55 raised against VIP) was provided by Center for Ulcer Research and Education (CURE)/Gastroenteric Biology Center, Ab/RIA Core. Pigeon cytochrome c fragment (PCCF) was synthesized and purified by Research Genetics (Huntsville, AL).

Cell preparations

Purified naive CD4⁺ T cells (>98% CD4⁺ by FACS analysis) were isolated by positive immunomagnetic selection (Miltenyi Biotec, Auburn, CA). B10.A T cell-depleted APCs were treated with 50 µg/ml mitomycin C (Sigma) for 30 min at 37°C.

Effector Th1 and Th2 cells from Cyt-5CC7-I/Rag2 and AND TCR mice were generated as described (26). Naive Tg CD4⁺ T cells (3 x 10⁵ cells/ml) were cultured with APC (10⁵ cells/ml) and PCCF (5 µM) in the presence of IL-2 (50 U/ml). IL-4 (200 U/ml) plus anti-IFN- Ab (10 µg/ml), or IFN- (1000 U/ml) plus anti-IL-4 Abs (10 µg/ml) were added to induce Th2 and Th1 polarization, respectively. Th1 and Th2 cell lines were established by regular cycles of stimulation with Ag (PCCF, 5 µg/ml) and APC (3 x 10⁵ cells/ml) of polarized Th1 and Th2 effectors (5 x 10⁵ cells/ml) for 3 days, followed by propagation in IL-2 (20 U/ml) for 5–8 days. The Th cell lines used for adoptive transfer experiments and cell cultures were at cycles 3–7 (1–2 months in culture). Th1 and Th2 cell lines were characterized through cytokine profiles. Typical results were as follows: IL-4, 43 ± 3 pg/ml for Th1 and 1234 ± 54 pg/ml for Th2; IFN-, 246 ± 23 ng/ml for Th1 and <8 ng/ml for Th2.

Allogeneic T1 and T2 CTL cell lines were generated as described (27). Purified B10.A CD8⁺ T cells (10⁶ cells/ml) were incubated for 5 days with BALB/c APCs (3 x 10⁶ cells/ml). To generate T1 cells, the cultures were treated with IL-2 (20 U/ml), IL-12 (20 U/ml), and anti-IL-4 mAb (2 µg/ml) for 5 days; for generation of T2, the cultures were treated with IL-2 (20 U/ml), IL-4 (40 U/ml), and anti-IFN- (80 µg/ml) for 5 days. The cultures were supplemented on day 3 with complete medium (RPMI 1640 with 10% FCS) containing IL-2 (20 U/ml) for T1, and IL-4 (40 U/ml) for T2, respectively. T1 and T2 cells were harvested on day 5 and characterized through cytokine profiles.

Cell cultures

TCR Tg Th1 or Th2 cells (5 x 10⁵ cells/ml) were incubated with B10.A APC (3 x 10⁵ cells/ml) and PCCF (5 µM, specific Ag), or OVA (5 µM, unrelated Ag). T1 and T2 effector cells (3 x 10⁵ cells/ml) were stimulated with anti-CD3 mAbs (5 µg/ml).

Adoptive transfer of Tg Th cell lines

On day -1, TCR Tg Th1 or Th2 cell lines (3 x 10⁶ cells) were inoculated i.v. into B10.A recipients. On day 0 and +2, 500 µg PCCF in balanced salt solution was injected i.p. On days 3 and 7, spleen T cells were isolated and examined by flow cytometry for the percentage of Tg T cells by staining with PE-conjugated anti-V3 and PerCP-conjugated CD4 mAbs, and for expression of VIP by staining with the anti-VIP mAb CURE-55, followed by FITC-conjugated goat F(ab')₂ anti-mouse IgG. On days 3, 5, and 7, VIP levels in serum were determined by ELISA.

FACS analysis

Cells (1 x 10⁶) were harvested in ice-cold RPMI 1640 complete medium, washed twice, incubated with PE-conjugated anti-V3 and PerCP-conjugated anti-CD4 mAbs (2.5 µg/ml) at 4°C for 1 h. After washing and fixing with 1% paraformaldehyde for 1 h at 4°C, the cells were incubated with mouse anti-VIP mAb (2 µg/ml) and stained with FITC-conjugated goat F(ab')₂ anti-mouse IgG (2.5 µg/ml) for 45 min at 4°C. The specificity of the anti-VIP mAb has been previously characterized by neutralization with excess VIP (10 mM for 12 h at 4°C) (13). Stained lymphocytes were analyzed in a FACSCalibur flow cytometer (Becton Dickinson, Palo Alto, CA). Fluorescence data were expressed as mean channel fluorescence and as a percentage of positive cells after subtraction of background isotype-matched values.

ELISA for VIP

VIP concentrations in culture supernatants and in serum of adoptively transferred mice were determined by using a specific competitive ELISA as previously described (13).

RNA extraction and Northern blot

Northern blot analysis was performed according to standard methods. Total RNA was extracted from 10⁷ Th1, Th2, T1, and T2 cells. The probes for murine VIP were generated by RT-PCR using the primers: 5'-CAGCAG TAGCATCTCGGAAGA-3' and 5'-CACAACACATTTTATTTGG-3'. Signal quantitation was performed in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).

Western blot

Cell lysates were prepared from 5 x 10⁶ cells, subjected to reducing SDS-PAGE (12.5%), followed by treatment with rabbit anti-mouse VIP Ab (Sigma) (1:1000) and peroxidase-conjugated goat anti-rabbit IgG (Sigma) (1:5000). The membranes were developed by chemiluminescence.

HPLC and RIA

VIP was extracted and purified from Th1 and Th2 cell suspensions and culture supernatants using Sep-Pak C18 cartridges (Waters, Milford, MA) and chromatographed by HPLC using a reverse-phase radial Novapak C18 column (Waters) as previously described (28). The VIP immunoreactivity in the HPLC fractions was determined by using RIA (Incstar, Stillwater, MN) (28). The sensitivity of the VIP RIA is 2 pmol/L (0.2 fmol/assay), with an intra-assay variation lower than 5% and an interassay variation lower than 8%. The cross-reactivity of the Ab with other neuropeptides was <0.1%.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Ag stimulation differentially induces VIP production in Th2, but not Th1, cells

Upon antigenic stimulation, CD4⁺ Th cells differentiate into two types of effector cells, with different cytokine profiles and functions. Th1 cells secrete IL-2, IFN-, and TNF-, critical for the generation of cellular immune responses. Th2 cells produce IL-4, IL-5, and IL-10, which play an important role in Ab production and down-regulate the cellular immune responses. We have previously demonstrated that VIP is produced by CD4⁺ T lymphocytes following stimulation through the TCR/CD3 complex (11, 12, 13). To investigate whether VIP is differentially expressed and produced in the two Th subsets, we generated Ag-specific Th1 and Th2 cell lines from TCR-Cyt-5CC7-I/Rag2 Tg mice. The Tg T cells recognize a fragment of the PCCF in the context of I-E^a/k and can be identified with anti-idiotypic Abs against V3 and V11 (25).

Two types of preproVIP mRNA can be generated, with immune cells expressing predominantly the 1-kb preproVIP mRNA (12). We designed primers that amplify 317 bp, encompassing the VIP coding region. Following Ag stimulation, Th2, but not Th1, cells express VIP mRNA (Fig. 1A). Similar results were observed at protein level. Western blots indicate that Th2, but not Th1, cells produce VIP following Ag stimulation in a time-dependent manner (Fig. 1B). To quantify intracellular and secreted VIP, we used FACS analysis and ELISA. After treatment with PCCF, Th2, but not Th1, cells contain intracellular VIP. Treatment with an unrelated Ag (OVA) did not induce VIP (Fig. 1C). The stimulated Th2 cells also secrete VIP in a time- and Ag dose-dependent manner (Fig. 1D). Similar to Th2, freshly isolated naive CD4⁺ Tg T cells secrete VIP (Fig. 1D). Similar results were obtained for Th1 and Th2 cell lines derived from AND TCR Tg mice. The four Th1 cell lines secrete on average 0.57 ± 0.05 ng VIP/ml, and the five Th2 cell lines secrete 5.89 ± 0.43 ng VIP/ml. In addition, Ag-stimulated D10.G4.1 cells (Th2) secrete 4.88 ± 0.43 ng VIP/ml.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. VIP production by Th2 cells. A, Tg Th1 and Th2 cells (5 x 105 cells/ml) were cultured with APC (3 x 105 cells/ml) and medium (unstimulated cells) or PCCF (5 µM). At different times VIP mRNA expression was assayed by Northern blot and normalized in respect to -actin expression. B, Th1 and Th2 cells (cultured as above) were analyzed for intracellular VIP protein by Western blots. C, Th1 and Th2 cells unstimulated (medium) or stimulated with PCCF or OVA were analyzed for intracellular VIP by FACS. D, VIP secretion was determined by ELISA in unstimulated (medium) or PCCF-stimulated freshly isolated naive CD4+ T, Th1, and Th2 cells. For the Western, Northern, and FACS analysis representative experiments are shown (three to six identical experiments were performed). The ELISA and densitometric data are the mean ± SD of three to five experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Characterization of Th2-derived VIP. Tg Th1 and Th2 cells were stimulated with PCCF or OVA as described in Fig. 1. The VIP content in cell extracts (12 h) and culture supernatants (24 h) was analyzed by HPLC-RIA as described in Materials and Methods. The arrows indicate the retention time for synthetic VIP. Results are representative of three identical experiments.

Naive CD8⁺ T cells cultured in vitro with Ag, APCs, and exogenous cytokines differentiate into T1 and T2 cells. We stimulated CD8⁺ T cells from B10.A mice with BALB/c APCs in the presence of IL-2, IL-12, and anti-IL-4 for T1 polarization, and with IL-2, IL-4, and anti-IFN- for T2 polarization. After 5 days, the H-2^k-anti-H-2^d CD8⁺ effectors were restimulated with anti-CD3 mAbs and assayed for their ability to produce and secrete VIP. Similar to the Th subsets, T2, but not T1, cells express VIP mRNA and protein (Fig. 3, A and B), and secrete VIP in a time-dependent manner (Fig. 3C, left panels). The amounts of secreted VIP increase with the degree of TCR-stimulation (Fig. 3C, right panels).

View larger version (38K): [in this window] [in a new window]   FIGURE 3. VIP production by CD8 T2 cells. A, B10.A T1 and T2 CTL cells (3 x 105 cells/ml) were cultured with medium (unstimulated cells) or anti-CD3 Abs (2.5 µg/ml). At different times VIP mRNA expression was assayed by Northern blot and normalized in respect to -actin expression. B, T1 and T2 cells were analyzed for intracellular VIP protein by Western blots. C, Freshly isolated CD8+ T, T1, and T2 cells cultured with medium (unstimulated cells) or anti-CD3 Abs (2.5 µg/ml in the left panels) for various times (24 h in the right panels) were analyzed for VIP secretion by ELISA. Western and Northern blots show a representative experiment of other four identical experiments. ELISA and densitometric data are the mean ± SD of three experiments performed in duplicate.

To investigate whether VIP production by type 2 T cells occurs in vivo, TCR Tg Th1 or Th2 cells were adoptively transferred into syngeneic, non-Tg hosts. The adoptive hosts were inoculated with PCCF or OVA. Several days later, serum VIP levels were determined by ELISA, and VIP expression in splenic CD4⁺ V3⁺ and V3^- T cells was determined by flow cytometry. Administration of PCCF resulted in increased serum VIP levels in hosts transferred with Th2, but not Th1, cells (Fig. 4A). The highest VIP levels were observed 3 days after Ag administration. Spleen cells were harvested from hosts receiving either Th1 or Th2 cells 3 days after Ag administration. We selected CD4⁺ V3⁺ T cells (adoptively transferred Tg cells) and V3^- (host T cells) and analyzed the content of intracellular VIP by flow cytometry. There was no increase in VIP in spleen cells obtained from hosts receiving Th1 cells, whether unimmunized (medium) or immunized (PCCF or OVA) (Fig. 4B, left panels). In contrast, V3⁺ cells from hosts that received Th2 cells and were immunized with the specific Ag (PCCF) expressed high levels of intracellular VIP (Fig. 4B, right panels) (92 ± 4% VIP⁺ cells with a mean fluorescence intensity of 62 ± 3). No such increase was observed in the V3^- population (22 ± 2% VIP⁺ cells with a mean fluorescence intensity of 12 ± 1). Also, V3⁺ cells from unimmunized hosts (medium) or from hosts injected with the unrelated Ag (OVA) did not exhibit an increase in VIP. Analysis of spleen cells 7 days after Ag stimulation showed nonsignificant numbers of V3⁺ cells, suggesting massive Ag-induced clonal deletion by apoptosis (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. In vivo VIP production by Th2 cells. Tg Th1 or Th2 cells (3 x 106/ml) were adoptively transferred into individual B10.A mice (three mice per group). Twenty-four hours after transfer, mice were injected i.p. with PCCF or OVA (500 µg). A, Serum VIP concentration was assayed by ELISA at 3, 5, and 7 days. The results represent the mean ± SD from three experiments performed in duplicate. B, Three days after initial Ag stimulation, spleen cells were isolated, counted, stained for CD4, Tg TCR (V3), and intracellular VIP, and analyzed by flow cytometry. Recovered Tg CD4+ T cells (V3+, solid lines) are compared with CD4+ V3- T cells (dashed lines) or freshly isolated CD4+ T cells (time 0). Data are representative of four separate experiments.

The difference in the ability of Th1/T1 and Th2/T2 cells to produce VIP is also reflected in their response to VIP. We have reported previously that VIP inhibits Th1, while stimulating Th2 differentiation in vivo (29), presumably through specific effects on B7.1/B7.2 expression (29) and through the inhibition of IL-12 production (30). In addition, we observed recently that VIP preferentially protects CD4⁺ Th2, but not Th1, cells from clonal deletion following antigenic stimulation (our unpublished data).

Because some cytokines act in an antagonistic manner, Th1 and Th2 cells can regulate each other’s development and function. For example, several Th2-derived cytokines inhibit Th1 differentiation. VIP appears to act in a similar manner. Because VIP is preferentially produced by type 2 T cells upon antigenic stimulation, and exerts a Th2-type function inhibiting cell-mediated immunity and favoring Th2 vs Th1 differentiation, could VIP be viewed as a type 2 cytokine?

	Acknowledgments

 
We thank Dr. John H. Walsh (University of California at Los Angeles Department of Medicine, Los Angeles, CA) for providing us with the CURE 55 mAb against VIP.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grant AI 41786 (to D.G.) and a Johnson & Johnson fellowship (to M.D.).

² Address correspondence and reprint requests to Dr. Doina Ganea, Department of Biological Sciences, Rutgers University, 101 Warren Street, Newark, NJ 07102.

³ Abbreviations used in this paper: VIP, vasoactive intestinal peptide; PCCF, pigeon cytochrome c fragment; Tg, transgenic.

Received for publication September 26, 2000. Accepted for publication January 2, 2001.

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Top Abstract Introduction Materials and Methods Results and Discussion References

 

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				E. J. Goetzl, J. K. Voice, S. Shen, G. Dorsam, Y. Kong, K. M. West, C. F. Morrison, and A. J. Harmar Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide PNAS, October 31, 2001; (2001) 241503798. [Abstract] [Full Text] [PDF]

				C. Martinez, C. Abad, M. Delgado, A. Arranz, M. G. Juarranz, N. Rodriguez-Henche, P. Brabet, J. Leceta, and R. P. Gomariz Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor PNAS, January 22, 2002; 99(2): 1053 - 1058. [Abstract] [Full Text] [PDF]

				E. J. Goetzl, J. K. Voice, S. Shen, G. Dorsam, Y. Kong, K. M. West, C. F. Morrison, and A. J. Harmar Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide PNAS, November 20, 2001; 98(24): 13854 - 13859. [Abstract] [Full Text] [PDF]

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