Vasoactive intestinal peptide downregulates the expression of IL-2 but not of IFNγ from stimulated murine T lymphocytes

Journal of Neuroimmunology, 47 (1993) 147-158

147

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00 JNI 02424

Vasoactive intestinal peptide downregulates the expression of IL-2 but not of IFNy from stimulated murine T lymphocytes Doina G a n e a and Lei Sun * Department of Biological Sciences, Rutgers University, Newark, NJ, USA (Received 22 January 1993) (Revision received 31 March 1993) (Accepted 31 March 1993)

Key words: Vasoactive intestinal peptide; Interleukin-2; Interferon gamma; Immunosuppression Summary

The neuropeptide vasoactive intestinal peptide (VIP) has been previously reported to inhibit T cell proliferation. Here we report on the effect of VIP on IL-2 and on IFN~/ production by murine T lymphocytes stimulated with mitogens (ConA), or activated through the antigen-specific T cell receptor. VIP inhibited IL-2 production by either unfractionated spleen cells, or by purified CD4 + T cells in a dose-dependent manner. The effect was specific, since structurally related peptides such as secretin and glucagon had little or no inhibitory effect. VIP induced a rapid increase in intracellular cAMP in CD4 ÷ T cells, suggesting that the inhibitory effect of VIP could be mediated through the induction of cAMP. Northern blots showed that VIP downregulated IL-2 mRNA, indicating the occurrence of a transcriptional regulatory event. In contrast with its effect on IL-2, VIP did not affect IFN3, production by either mitogen-stimulated normal T lymphocytes, or by the L12R4 murine T cell line which produces IFNy in response to PMA stimulation.

Introduction

Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide widely distributed in the central and peripheral nervous system, affects multiple cellular functions, including functions of the immune system (Said, 1984; O'Dorisio, 1987; Said and Mutt, 1988; Gozes and Brenneman, 1989; Huizinga and PintinQuezada, 1991). Several lines of evidence support the idea that neuropeptides such as VIP could act as a link between the nervous and the immune system in vivo. Primary and secondary lymphoid organs have been shown to possess a VIP-peptidergic innervation (Felten et al., 1985; Ottaway et al., 1987; Fink and Weihe, 1988; Gomariz et al., 1990; Nohr and Weihe, 1991). The presence of lymphoid cells which are VIP-imCorrespondence to: D. Ganea, Department of Biological Sciences, Rutgers University, 101 Warren Street, Newark, NJ 07102, USA. * In partial fulfilment of the requirements for the degree of Doctor in Philosophy in the Graduate College, Rutgers University, Newark, NJ 07102, USA.

munoreactive, and therefore potential VIP sources, has been also reported, primarily in thymic cortex and in the T-dependent areas of spleen and lymph nodes (Gomariz et al., 1992). The presence of VIP receptors on T and B lymphocytes, as well as on macrophage/ monocytes has been well documented through binding studies (Guerrero et al., 1981; Danek et al., 1983; Ottaway and Greenberg, 1983; Ottaway et al., 1983; Wiik et al., 1985; O'Dorisio et al., 1985, 1989; Wood and O'Dorisio, 1985; Calvo et al., 1986; Finch et al., 1989; Roberts et al., 1991; Segura et al., 1991). VIP has been also shown to modulate a variety of immune responses. VIP has been shown to decrease lymphocyte migration in and out of lymph nodes (Ottaway, 1984, 1985; Moore, 1984; Moore et al., 1987), to affect monocyte chemotaxis (Ruff et al., 1987; Bondesson et al., 1991), to mediate histamine release from mast cells (Johnson et al., 1973; Foreman and Piotrowski, 1984), and to modulate NK cell activity (Rola-Plesczynski et al., 1985). The activity of both T and B lymphocytes has also been shown to be affected by VIP. Stanitz et al. (1986) demonstrated a modulatory effect of VIP on Ig

148 production by murine B lymphocytes depending on the Ig isotype and on the organ source of the lymphocytes, and VIP has been reported to inhibit murine T cell proliferation in response to mitogens such as ConA or PHA (Ottaway and Greenberg, 1984; Ottaway, 1987; Boudard and Bastide, 1991). Soluble products of immune cells, i.e. immune cytokines or interleukins, have profound regulatory effects on the immune response, and the immunoregulatory effect of neuropeptides such as VIP could be mediated through their effect on cytokine expression. To this effect, VIP has been shown to stimulate IL-5 release from activated T cells in murine schistosomiasis (Mathew et al., 1992), and to inhibit IL-2 production in mitogen-stimulated murine T lymphocytes (Ottaway, 1987; Boudard and Bastide, 1991). Two major subsets of CD4 ÷ murine T cells have been described according to their lymphokine profile, i.e. the TH1 cells which upon mitogen- or antigen-stimulation secrete IL-2 and IFNy and the T H2 cells which are responsible for the production of IL-4, IL-5, and IL-6 (Mosmann et al., 1986). At the present time it is not known whether VIP can inhibit both IL-2 and IFNy production by mitogen- or antigen-stimulated cells. This study shows that VIP can indeed inhibit the production of IL-2 by murine splenocytes, or by purified CD4 ÷ T ceils, whereas it does not affect the production of IFNy by either murine splenocytes or by the L12R4 murine T cell line. This study also extends the previous observations regarding the inhibitory effect of VIP on IL-2 production and T cell proliferation by mitogen-stimulated cells to T lymphocytes stimulated by treatment with anti-CD3 monoclonal antibodies which mimic antigen stimulation through specific T cell receptors. Also, certain aspects of the molecular mechanisms involved in the downregulation of IL-2 production by VIP, such as involvement of cAMP and transcriptional versus posttranscriptional regulation, are examined.

Materials and Methods

Reagents Synthetic VIP, glucagon, secretin, and VIPI_I2, VIP10_28 were purchased from Peninsula Laboratories (Belmont, CA), Concanavalin A (ConA), phorbol 12myristate 13-acetate (PMA), and 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MT~) were purchased from Sigma (St. Louis, MO). All the peptides and ConA were dissolved in serum-free medium, and stored at - 20°C. PMA was dissolved in DMSO as a concentration of 1.0 mg ml-1, and stored at -20°C. Murine recombinant IL-2 (mrIL-2) and murine recombinant IFNy were generously provided by ScheringPlough Corp (Bloomfield, N J), and murine recombinant IL-4 (mrIL-4) was a generous gift from Genetics

Institute (Cambridge, MA). Anti-murine CD3-e monoclonal antibody (clone 145-2Cll) was purchased from Pharmingen (San Diego, CA). Anti-murine IL-2 ($4B6 mAb) and anti-murine IL-4 ( l l B l l mAb) were initially purchased from ATCC (Rockville, MD) and used as ascites at a 1/100 dilution. Anti-murine CD4 mAb (GK 1.5, ATCC) was used as a 1/10 dilution of culture supernatant. All cytokines and antibodies were stored at - 80°C.

Cell lines CTLL-2, an IL-2-dependent cell line, was purchased from the American Type Culture Collection (ATCC). $4B6 and l l B l l , two hybridomas that produce antimurine IL-2 and anti-murine IL-4, respectively, as well as GK 1.5, a hybridoma secreting anti-murine CD4 mAb, were also obtained from ATCC. L929, a murine fibroblast cell line, was also purchased from ATCC. All cell lines were maintained as recommended by ATCC. The L12R4 murine T lymphoma cell line (Sarzotti et al., 1983) was obtained from Dr. Howard Johnson (University of Florida, Gainsville, FL) and maintained in RPMI 1640 containing 10% FCS, and 5 × 10 -5 M 2-mercaptoethanol. Preparation of single cell suspensions and of purified CD4 + T lymphocytes The B a l b / c mice age 5-7 weeks (females, Charles River Laboratories, Wilmington, MA) were killed by cervical dislocation, the spleens were removed and single-cell suspensions were prepared in serum-free RPMI 1640 (Gibco, Grand Island, NY). After washing three times in serum-free RPMI 1640 medium, the cells were resuspended in RPMI 1640 containing 2% fetal calf serum (FCS, Hyclone Logan, UT) at a concentration of 2 × 106 cells ml-1. CD4 + T lymphocytes were separated in a two-step procedure: (a) CD4 ÷ T cells were first negatively selected through affinity immunocolumns (Biotex Labs Inc., Edmonton, Alberta) according to the manufacturer's instruction. 80-85% of the total recovered cells were determined to be CD4 ÷ cells by FACS analysis with anti-CD4-fluorescein coupled monoclonal antibodies (Becton-Dickinson, San Jose, CA); (b) the partially purified CD4 ÷ cell population was then depleted of residual adherent cells by adherence to plastic (2 × 106 cells m1-1 in RPMI 1640 containing 20% FCS) for 2 h at 37°. CD4 ÷ cells within the recovered non-adherent population were then positively selected with GK 1.5 mAbs, by resuspending the cells at a concentration of 5 × 106 cells m1-1 in RPMI 1640 containing 5% FCS and GK1.5 mAb (final dilution 1/100) at room temperature for 10 min followed by an additional 20 min incubation at 4°. Excess antibodies were removed by washing the cells three times in RPMI 1640 containing 5% FCS, and the cells were resuspended at a

149 concentration of 1 X 107 cells ml-1 and incubated with Dynabeads M-450 coupled to sheep anti-mouse IgG (Dynal, Great Neck, NY) (final concentration 10 /zg ml -~) for 2 h at 4°. The attached cells were then diluted to 2 x 106 cells ml-~ and detached from Dynabeads by incubation at 37° for approx. 4 h. After washing twice in RPMI 1640 containing 2% FCS the ceils were resuspended at a concentration of 1 x 106 cells ml-a in the same medium. FACS analysis indicated that > 95% of the selected cells were CD4 ÷.

CTLL-2 cells, as previously described (Tada et al., 1986). One IL-2 unit was defined as the amount which gave the half maximum OD value, and the number of IL-2 units in each supernatant was calculated by comparison with the standard curves obtained with traiL-2. The percent inhibition was calculated as follows: % Inhibition = 100 x [(number of IL-2 units for cells cultured with ConA) - (number of IL-2 units for cells cultured with ConA and peptides)]/(number of IL-2 units for cells cultured with ConA)

Cell culture and stimulation

IFNy bioassay. The IFN7 activity in 24-h super-

Cells were cultured in 24-well tissue culture plates at a concentration of 2 X 106 cells ml-1 for unfractionated spleen cells, 1 x 106 cells m1-1 for the CD4 + T ceils, and 5 x 105 cells ml -~ for the L12R4 cells in RPMI 1640 containing 2% FCS, 2 mM glutamine (Gibco), 100 U m1-1 penicillin, 10 mg m1-1 streptomycin (Gibco), and 5 x 10 -5 M 2-mercaptoethanol (Sigma, St. Louis, MO). The different concentrations of VIP or other peptides and stimuli (ConA, 1 /~g ml-~; anti-CD3 mAb 0.5 /zg ml-~; PMA 5 or 10 ng m1-1, final concentrations), were added first to the wells (10/xl/well), followed by 1 ml of cell suspension. Controls with medium alone, or with stimuli (ConA, anti-CD3, anti-CD3 plus PMA, PMA alone) in the absence of peptides were included. The culture plates were maintained at 37°C in a humidified incubator with 5% CO 2. Cell viability was determined by a Trypan blue exclusion assay. Supernatants collected at either 24 or 48 h were stored at 4°C and assayed for IL-2 or IFN7 activity.

natants was determined by an antiviral assay as previously described (Storch and Kirchner, 1985). Briefly, L929 cells (murine fibroblasts) were trypsinized and resuspended in DMEM medium (Gibco) supplemented with 5% FCS, 2 mM glutamine and antibiotics at a concentration of 5 x 105 cells m1-1. Serial dilutions of IFN7 containing samples were added to 96-well plates in a volume of 100/xl, followed by the addition of 50 /zl of resuspended L929 cells. The cells were incubated for 24 h in a humidified incubator (5% COz), followed by the addition of 50 ~1 of a previously titrated vesicular stomatitis virus suspension (VSV), and the plates were incubated for another 48 h. After removing the medium, the wells were washed three times with PBS, and stained with 100 /~1 of 0.05% Amidoblack (dissolved in 9% acetic acid containing 0.1 M sodium acetate) for 15-30 min. The stain was then removed and the cells were fixed with 150/xl of 10% formaldehyde (in 9% acetic acid containing 0.1 M sodium acetate) for 15 min. After washing in circulating distilled water for 10 min and drying overnight, the stain was dissolved in 100-150/xl of 50 mM NaOH, followed by the determination of OD590 values in an ELISA reader. The number of IFN7 units in the assayed samples were calculated by comparison to the standard curves using recombinant murine IFN7.

Cytokine determination IL-2 bioassay. The IL-2 activity in 24 or 48 h supernatants was determined by a modified colorimetric assay as previously reported (Mosmann, 1983; Tada et al., 1986). Briefly, the CTLL-2 cells were extensively washed in Phenol red-free RPMI 1640 medium supplemented with 5% FCS, 2 mM glutamine, 100 U m1-1 penicillin, 10 mg m1-1 streptomycin and 5 x 10 -5 M 2-mercaptoethanol, and resuspended in the same medium at a concentration of 1-2 X 105 cells m1-1. 50 /~1 of serial dilutions of IL-2-containing supernatants were added to each well in a 96-well tissue culture plate, followed by 50/xl of the CTLL-2 cell suspension. The cells were maintained at 37°C in a humidified incubator (5% CO 2) for 48 h, followed by the addition of 20 /~1 MT-I" (5 mg ml -~ in phosphate-buffered saline; PBS). The plates were incubated for another 5 h, followed by the addition of 100 /zl of a solution containing 10% SDS and 0.01 N HC1. After overnight incubation, the OD590 was determined using an ELISA reader (Cambridge Technology, Cambridge, MA). A590 represents a direct measure of the number of viable

ELISA assay for IFNy.

The amount of IFNy present in supernatants from stimulated splenocytes in the presence or absence of VIP was also determined by using a murine IFNy ELISA kit (Genzyme Co., Boston, MA). The kit was specific for mouse IFNy and the assay was performed according to the manufacturer's instructions.

Measurement of cyclic AMP CD4 + lymphocytes (1 X 106 cells m1-1) were resuspended in PBS containing 2% FCS and 50 uM of a cAMP phosphodiesterase inhibitor, 3-isobutyl-l-methyl-xanthine (IBMX, Sigma) in the presence of different concentrations of VIP. At different times the reaction was stopped by centrifugation, followed by the addition of 1 ml of 100% ethanol (Imboden et al., 1986). The ethanol phase was collected by centrifugation and the

150

pellets were extracted once more with ethanol. The ethanol was then dried under vacuum and the cAMP contents were determined by using a cAMP radioimmunoassay kit purchased from Amersham (Arlington Heights, IL). Samples were assayed in duplicate, and the amount of cAMP was determined by comparison with a standard curve obtained with predetermined concentrations of cAMP.

Northern blot The mRNA was analyzed by Northern blot according to standard methods. Briefly, 5 x 107 cells were stimulated with ConA or with anti-CD3 + PMA and harvested 6 and 20 h later, respectively. Total RNA was purified using the acid guanidinium thiocyanatephenol-chloroform extraction method (Chomczynski and Sacchi, 1987) and quantitated by spectrophotometric determination at 260 nm. 10-20 /zg of total RNA were size fractionated on 1.2% argarose-formaldehyde gels, transferred to S&S Nytran membranes (Schleicher and Schuell, Keene, NH), and baked at 80°C for 2

h under vacuum. The membranes were prehybridized for 10 h at 42°C, and hybridized with the 0.6-kb BamHI-BgllI fragment of the murine IL-2 cDNA probe pmut-1 (ATCC) for 12 h at 42°C. The probe was labeled by the random primer labeling method, according to the instructions of the manufacturer (Stratagene, La Jolla, CA). The prehybridization and hybridization buffers were purchased from 5' Prime-3' Prime Inc. (Boulder, CO). The membranes were washed twice at room temperature in 2 x SSC containing 0.1% SDS, once at 37°C, and once at 50°C in 1 x SSC containing 0.1% SDS (20-min washes). The membranes were then exposed to X-ray films (Kodak, Rochester, NY) for 36 h at -80° C in the presence of an intensifying screen.

Statistical analysis The statistical significance was determined by using one-way repeated measures, analysis of variance (ANOVA) followed by Turkey's multiple comparison at P < 0.05. All results were given as a two-tail P value.

0.6 0.2ug/ml of ConA 0.5 0.4 0.3 0.20.1 50 "1

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Con A ug/ml Fig. 1. ConA dose-response curve for the IL-2 production by murine splenocytes. Splenocytes (2 X 106 ml-1) were cultured with different amounts of ConA, in the presence or absence of 10 -7 M VIP. Supernatants were collected after 48 h and assayed for IL-2 activity by using the CTLL-2 bioassay (see Materials and Methods). Insert: titration curves for three ConA concentrations in the absence of VIP. The results from one representative experiment out of three are shown.

151 Results

VIP inhibits the IL-2 production by ConA-stimulated murine splenocytes To determine the effect of VIP on IL-2 production, we first established the dose-response curve for IL-2 production by murine splenocytes in response to ConA stimulation. A bell-shaped dose-response curve, with an optimal stimulation of approximately 45 U ml -I IL-2 for 1 /xg m1-1 of ConA, was obtained for the supernatants harvested after 48 h of culture (Fig. 1). Supernatants collected at 24 h showed similar results (data not shown). All the following experiments were done with supernatants collected after 48 h of culture. The optimal concentration of ConA (1 /zg m1-1) was selected to study the inhibitory effect of VIP on the IL-2 production. The splenocytes were cultured with ConA in the presence or absence of different concentrations of VIP (10 -7 M to 10-15 M) and supernatants harvested after 48 h of culture were assayed for IL-2 activity. VIP induced a significant decrease in IL-2 production by murine splenocytes (Fig. 2a). The inhibitory effect was dose-dependent, with inhibition values higher than 50% for 10-7-10 -9 M VIP. Inhibitions of approximately 25% were still recorded for very low VIP concentrations (10-13-10 -15 M) (Fig. 2a). Because VIP could act directly on the proliferation of the CTLL-2 cells used to measure the biological activity of IL-2, we determined the effect of VIP on the proliferative response of CTLL-2 cells to rlL-2 (Fig. 2b). The VIP concentrations which had an inhibitory effect in the murine splenocyte cultures (Fig. 2a) did not directly affect the proliferation of CTLL-2 cells in response to either high concentrations (10 U ml-1), or low concentrations (2.5 U m1-1) of rlL-2. This indicated that the inhibitory effect of VIP was indeed exerted at the level of IL-2 production by murine splenocytes. To determine whether VIP downregulated IL-2 production at different ConA concentrations, or whether it induced a shift in the bell-shaped dose-response curve, we incubated spleen cells with different concentrations of ConA (from 0.1 to 5 /zg m1-1) in the presence of a constant concentration of VIP (10 -7 M), and assayed the IL-2 concentrations in supernatants harvested 48 h later. VIP induced similar reductions for the entire ConA concentration range, and there was no apparent shift in the dose-response curve (Fig. 1). The inhibitory effect of VIP, especially in the high concentration range, could be due to a reduction in viability or cell number. Therefore, the viability and cell number in cultures containing ConA in the presence or absence of VIP (10-7-10 -9 M) were determined. There was no significant difference between the cell numbers and viabilities recorded in the presence or absence of VIP (1.6-1.7 × 106 cells m1-1, 92-94%

viability), which indicated that the inhibitory effect was a direct effect on IL-2 production and not the result of a reduction in cell growth. The specificity of the effect of VIP on IL-2 production was studied by comparing VIP with structurally related peptides, such as secretin and glucagon (Fig. 2a). Secretin exhibited an inhibitory effect on IL-2 production, but with an efficiency of at least two orders of magnitude lower than VIP. Approximately 50% inhibition was observed with 10-7 M secretin, and the inhibition dropped rapidly at lower secretin concentrations (Fig. 2a). No effect was observed with various concentrations of glucagon (10 -7 M to 10 -15 M) (Fig. 2a). The VIP fragments, VIPl_12 and VIP10_28 were also tested for their effects on IL-2 production. The results in Fig. 2c show no significant inhibition with either one of the VIP fragments, suggesting that the intact VIP molecule is required for its inhibitory activity. To further prove that the cytokine inhibited by VIP was IL-2, we first verified that the CTLL-2 cell line used in our experiments responded specifically to IL-2, as previously reported (Plaetinck et al., 1990; Rameshwar et al., 1992). When cultured with either rIL-2 or rIL-4, the CTLL-2 cells proliferated in response to rIL-2, but not rIL-4 (Fig. 3). In our next experiments, supernatants from ConA-stimulated splenocytes cultured in the absence or presence of 10 -8 M VIP were treated with neutralizing doses of the anti-IL-2 monoclonal antibody $4B6, or of the anti-IL-4 monoclonal antibody 11Bll. The $4B6 anti-IL-2 antibody blocked the proliferative activity of both types of supernatant (from 54.1 U m1-1 to 1 U m1-1 in the absence of VIP, and from 16.1 U m1-1 to 1.5 U m1-1 in the presence of VIP), whereas the 11Bll anti-IL-4 antibody had no effect (Table 1). These experiments definitely identified IL-2 as the cytokine whose production was inhibited by VIP.

Time-course for VIP inhibition of IL-2 production To determine the maximum time period after the onset of activation at which VIP could still inhibit IL-2 production, VIP was added at different times (0-36 h) after the initiation of stimulation with ConA. The addition of VIP up to 12 h after the initiation of ConA stimulation yielded similar degrees of inhibition (Table 2). The addition of VIP after 12 h of culture resulted in progressively lower degrees of inhibition (Table 2). We next determined the minimum time of exposure to VIP necessary for its inhibitory effect on IL-2 production. Ceils were cultured with ConA and VIP for different periods of time (from 5 to 180 min), washed extensively, and resuspended in medium containing ConA without VIP. The VIP had a surprisingly rapid effect, providing a strong inhibitory activity after only 5 min of incubation. The degree of inhibition observed

152

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VIP inhibits the IL-2 production by anti-CD3-stimulated murine splenocytes

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To determine whether VIP has an inhibitory effect on IL-2 production by antigen-stimulated T cells, we investigated its effect on murine splenocytes stimulated through the T cell receptor by treatment with anti-CD3 monoclonal antibodies, or with a combination of antiCD3 mAb and phorbol esters (PMA). These treatments have previously been reported to induce IL-2 production in murine and human T lymphocytes (Hara and Man Fu, 1985; Granelli-Piperno et al., 1986). Murine splenocytes were cultured in the presence of soluble anti-CD3 mAb (0.5/xg ml -]) or anti-CD3 mAb (0.5/zg m1-1) plus PMA (10 ng m1-1) in the absence or presence of VIP (10-7-10 -15 M), and supernatants harvested after 48 h of culture were assayed for IL-2 activity. In the absence of VIP, treatment with anti-CD3 mAb alone induced 75.2 IL-2 U m1-1, whereas the combined treatment with anti-CD3 mAb and PMA resulted in 1256.2 IL-2 U ml-1. The inhibition of IL-2 production by VIP was dose-dependent, with maximum inhibitory values (80% and 62.5% for anti-CD3 and anti-CD3 plus PMA activated splenocytes, respectively) for 10 -7 M VIP (Fig. 4). Significant inhibitory values (higher than 20%)were observed in the 10-7-10 -1° M concentration range (Fig. 4), whereas for ConAactivated splenocytes similar inhibitory values were observed at much lower VIP concentrations (10-13-10-15 M) (Fig. 2a). We concluded that although VIP inhibited IL-2 production by both mitogen- and antigen-induced splenocytes, the molecular mechanisms could be different.

Lack of effect of VIP on IFNT production by murine splenocytes A number of different stimuli, such as ConA or anti-CD3 with or without PMA, have been reported to induce both IL-2 and IFNy production in murine T 7

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Fig. 2. Effects of peptides on IL-2 production by ConA-stimulated murine splenocytes. Splenocytes (2 × 106 ml-1) were cultured with 1 /xg ml- i of ConA in the presence or absence of different concentrations of peptides. The 48-h supernatants were collected and assayed for IL-2 activity as described in Fig. 1. The results are presented as percent inhibition of controls without peptides (IL-2 U m l - l : unstimulated cells 2.4 _+1.1; ConA-stimulated cells 48.7_ 4.8). (a) Effect of VIP, secretin, and glucagon; P < 0.05 compared to ConA control; (b) effect of VIP on CTLL-2 cell proliferation in the presence of either high (10 U ml 1) or low (2.4 U m1-1) concentrations of rlL2; (c) effect of VIP, VIPx_12, and VIP10_28; P < 0.05 compared to ConA control. The results from one representative experiment out of four (2a), and out of three (2b and c) are shown. Asterisk indicates statistical significance.

TABLE 1 Identification of the CTLL-2 proliferative activity produced by ConA-stimulated splenocytes as IL-2 Sample

ConA ConA+ VIP

IL-2 activity (U m l - i ) No antibody

$4B6 mAb

11B11 mAb

54.1 + 4.2 16.1+2.4

1.0 + 0.8 1.5_+0.3

63.7 + 5.6 15.6_+3.1

Splenocytes (2×106 cells ml - I ) were stimulated with ConA (1 /zg m1-1) in the presence or absence of VIP (10 - s M). Supernatants collected after 48 h of culture were treated with 1/100 dilutions of $4B6 ascites (anti-murine IL-2 mAb), or 11Bll ascites (anti-murine IL-4 mAb) for 1 h at 37°, and then assayed for IL-2 activity in the CTLL-2 bioassay.

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lymphocytes (Farrar et al., 1982; Granelli-Piperno, 1986). Also, a certain type of murine CD4 + T cell clones, the TH1 , have been characterized through their TABLE 2 Time course for the VIP inhibitory activity on IL-2 production by ConA-stimulated splenocytes Time of VIP addition (h) a

% Inhibition

Time of VIP removal (min) b

% Inhibition

0 2 5 8 12 24 36

52.4 + 5.4 51.4+4.7 47.9 -+3.2 47.9 -+-4.8 47.2+3.2 20.6 + 5.0 18.7 + 2.1

5 15 30 60 180

46.0 -t-5.2 44.1 +4.8 44.0 + 5.5 40.0 + 3.2 45.8+4.5

No washing

56.5 + 5.0

" The splenocytes (2x 106 cells ml -~) were cultured with ConA (1 /zg ml-1). VIP (10 -8 M) was added at different times after the initiation of the cultures. Supernatants were collected after 48 h of culture and assayed for IL-2 activity. b The splenocytes (2× 106 cells m1-1) were cultured with ConA (1 /zg ml - I ) in the presence or absence of VIP (10 -8 M). The VIP was removed at different times by extensive washings (three times) with serum-free medium. Cells were then resuspended in medium containing 2% FCS and ConA (1 /zg ml-1), and cultured for another 48 h. Control cultures containing only ConA without VIP were washed and recultured exactly the same way as the experimental cultures. Supernatants collected after 48 h of culture were assayed for IL-2 activity, and the percent inhibition was calculated by comparing each experimental culture to its control.

8

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15

VIP concentration -Log[M] Fig. 4. Effect of VIP on IL-2 production by antigen-stimulated murine splenocytes. The splenocytes (2× 106 m1-1) were cultured with anti-CD3 mAb (0.5/zg ml-I), or anti-CD3 mAb (0.5/zg ml - I ) plus PMA (10 ng ml-]), in the presence or absence of different concentrations of VIP. Supernatants collected after 48 h were assayed for IL-2 activity as described in Fig. 1. The results were expressed as percent inhibtion by comparison with controls which consisted of activated cultures in the absence of VIP (IL-2 U ml-1 for unstimulated cells 2.1-t-0.9; cells stimulated with anti-CD3 rnAb 75.2+2.6; cells stimulated with anti-CD3 mAb and PMA 1256.2_+ 49.6. The results of one representative experiment out of three are shown, p < 0.05 compared to anti-CD3, or anti-CD3 + PMA, respectively. Asterisk indicates statistical significance.

specific lymphokine profile, i.e. production of IL-2 and IFNT, but not of IL-4, IL-5 or IL-6 (Mosmann et al., 1986). Therefore, we investigated the effect of VIP on ConA-induced IFNT production in murine splenocytes. Cells were cultured with ConA (1/zg m1-1) and different concentrations of VIP (from 10 -7 to 10 -15 M). Supernatants collected after 24 h of culture were assayed for IFNT activity by an antiviral biological assay, and by ELISA. In the absence of VIP, the supernatans from ConA-stimulated cells contained 70 IFNT U ml-1 (biological assay), and 15 ng m l - t IFNy protein (ELISA). VIP, over the entire concentration range used in these experiments, did not affect IFNT production (70 IFNT U ml-1 and 14.5 ng IFN T ml-1). Similar results were obtained with a murine T cell line L12R4 which produces IFNT upon stimulation with PMA (Sarzotti et al., 1983). The cells were stimulated with 5 or 10 ng ml-1 PMA with or without different concentrations of VIP (from 10 -7 to 10 -12 M), and supernatants collected after 24 h were assayed for IFNy in the biological assay. No inhibitory effect of VIP was observed (320 U ml-1 for 10 ng ml-1 PMA without VIP and 314 U ml -~ with VIP; 240 U m1-1 for 5 ng m1-1 PMA without VIP and 250 U ml-1 with VIP). These results indicate that the effect of VIP on

154 2-

200 ---l--

VIP ( 1 0 - 7 M )

150 E 100 (NI I ._1 I

50

=,

0

o I

Con A:

+

PMA: VIP (-LogM):

+

*

+

+

+

+

* 7

+ g

+ 11

+ 13

-

o

E

Fig. 5. Effects of VIP on IL-2 production by purified CD4 + T lymphocytes. Purified CD4 + cells (1 x l06 m1-1) were cocultured with ConA (1 /~g m1-1) and PMA (2 ng ml 1), in the absence or presence of various concentrations of VIP. Supernatants were collected after 48 h of culture and assayed for IL-2 activity as described in Fig. 1. Results are expressed as IL-2 U ml- 1. p < 0.05 compared to ConA + PMA control. Asterisk indicates statistical significance.

IL-2 p r o d u c t i o n is specific, a n d n o t d u e to a g e n e r a l s h u t - d o w n of l y m p h o k i n e p r o d u c t i o n by c e r t a i n T cell subpopulations.

Effect of PIP on purified CD4 + T cell subpopulations I n all the e x p e r i m e n t s r e p o r t e d above we u s e d u n f r a c t i o n a t e d s p l e e n cell cultures. T o investigate the cellular target for the V I P action, we p u r i f i e d C D 4 ÷ T cells by a c o m b i n a t i o n of negative a n d positive selections. T h e p u r i f i e d C D 4 ÷ cells ( > 95% C D 4 ÷) cult u r e d in the p r e s e n c e of C o n A (1 /zg m1-1) did n o t p r o d u c e IL-2 above cell c o n t r o l levels, as expected from m a c r o p h a g e - d e p l e t e d cultures. T h e r e f o r e , C D 4 ÷ T cells were c u l t u r e d in the p r e s e n c e of C o n A (1 /zg ml -~) plus P M A (2 ng m1-1) with or w i t h o u t different c o n c e n t r a t i o n s of V I P (from 10 - 7 to 10 -13 M), a n d

A

I L- 2

1

2

3

0

= 20

3'0

Time

(rain)

' 40

50

Fig. 6. Effect of VIP on the production of cAMP by purified CD4 + lymphocytes. Purified CD4 + T cells ( l x l 0 6 m1-1) were resuspended in PBS containing 2% FCS and 50/zM IBMX and incubated with different concentrations of VIP at room temperature. The reactions were terminated at different times and the contents of cAMP were measured by a radioimmunoassay as described in Materials and Methods. s u p e r n a t a n t s collected after 48 h of c u l t u r e were assayed for IL-2 activity. V I P i n h i b i t e d IL-2 p r o d u c t i o n in a d o s e - d e p e n d e n t m a n n e r , with m a x i m u m inhibitory values of 7 5 - 8 0 % for 1 0 - 7 - 1 0 -9 M V I P (Fig. 5). T h e s e results indicate that V I P can directly interact with C D 4 ÷ T cells a n d i n h i b i t IL-2 p r o d u c t i o n .

Effect of PIP on cAMP production in CD4 ÷ T cells V I P has b e e n previously r e p o r t e d to stimulate c A M P p r o d u c t i o n in a variety of cells (Calvo et al., 1986; Said,

4

,~- 1.0Kb B IL-2

28 S

r 10

0

1

2

3

4

5

6

o

1.0 Kb

Actin

Fig. 7. Effect of VIP on ConA- and anti-CD3-induced expression of ILo2. (a) 5 x 107 cells were cultured in medium alone (lane 1), 10 -8 M VIP (lane 2), 1/xg m1-1 ConA (lane 3) and ConA (1/zg m1-1) plus VIP (10 -s M) (lane 4). (b) 5 × 107 cells were cultured in medium alone (lane 1), 10 -7 M VIP (lane 2), 1/~g m1-1 anti-CD3 (lane 3), anti-CD3 plus VIP (10 -7 M) (lane 4), 1 p.g m1-1 anti-CD3 plus 10 ng ml -I PMA (lane 5), and anti-CD3, PMA and VIP (10 -7 M) (lane 6). Total RNA was isolated and analysed by Northern blotting (10/~g for lane 1 and 2, and 20/~g for the other lanes). The blots were probed with the 0.6-kb BamHI-BgllI fragment of a murine IL-2 cDNA probe as described in Materials and Methods. The autoradiograms were exposed to film for 36 h at - 80°C. Lower panel represents: (a) 28S ribosomal RNA visualized by ethidium bromide staining; (b) the blot reprobed with a chicken/3-actin probe.

155 1986; Robberecht et al., 1989; Wiik, 1989), and cAMPinducing agents such as PGE2, cholera toxin, or the cell-permeable analogue 8-bromo-cAMP have been reported to inhibit IL-2 production (Novak and Rothenberg, 1990; Anastassiou et al., 1992). To determine whether the inhibitory effect of VIP on IL-2 production observed in our experimental system could be mediated through an increase in intracellular cAMP, we investigated the effect of different concentrations of VIP on cAMP production in purified CD4 ÷ T cell cultures. CD4 ÷ T cells were cultured in the presence of IBMX, a phosphodiesterase inhibitor, and of three different VIP concentrations (10 -7, 10 -1°, and 10 -13 M) for different periods of time (from 0 to 40 min), and the amount of cAMP generated in the cells was determined as described in Materials and Methods. A significant increase in the amount of cAMP was observed for 10 -7 M VIP at 2 and 5 min after the addition of VIP (Fig. 6). 10 -1° M VIP still induced a small but significant increase at 5 rain, whereas 10-13 M did not cause any significant cAMP induction (Fig. 6). These results indicate that VIP (10-7-10 -1° M) can indeed induce cAMP production in CD4 ÷ T cells, and suggest that the inhibitory effect of these VIP concentrations on IL-2 production could be possibly mediated through the cAMP transduction pathway. VIP decreases the IL-2 mRNA level in the stimulated murine splenocytes VIP could exert an inhibitory effect on IL-2 production either at a transcriptional or at a posttranscriptional level. To address this question we cultured murine spleen cells with ConA, anti-CD3, or anti-CD3 plus PMA, in the presence or absence of 10-7-10 -8 M VIP. Total RNA prepared from 5 × 107 ceils was subjected to Northern blot analysis using a 0.6-kb mlL-2 cDNA probe. 28S ribosomal RNA or a chicken fl-actin probe was used as a control for equal loading. Cells cultured in medium alone (lane 1), or in the presence of VIP alone (lane 2) did not express IL-2 mRNA (Fig. 7a). Cells cultured in the presence of ConA expressed a high level of IL-2 mRNA (lane 3), which was significantly reduced in the presence of VIP (lane 4) (Fig. 7a). Similar results were obtained for cells activated with anti-CD3 or anti-CD3 plus PMA (Fig. 7b). These results indicate that treatment with VIP resulted in the transcriptional downregulation of IL-2 expression in ConA- or anti-CD3-stimulated splenocytes.

Discussion

Murine TH1 cells have been characterized by their lymphokine production profile, i.e. secretion of IL-2 and IFNy upon mitogen or antigen stimulation (Mosmann et al., 1986). VIP has been reported previously to

inhibit mitogen-induced murine T cell proliferation and IL-2 production (Ottaway, 1987; Boudard and Bastide, 1991). In this study we investigated the effect of VIP on IL-2 and IFNy production by mitogen or anti-CD3 stimulated murine T cells. VIP inhibited IL-2 production in a dose-response manner, but did not affect IFNy production, in both ConA- and anti-CD3stimulated T cells. A similar lack of effect was observed for IFNy production in the PMA-stimulated L12R4 murine T cell line. The inhibition of IL-2 required an intact VIP molecule, since the VIP fragments VIP1_12 and VIP10_28 lacked inhibitory activity. The specificity of VIP activity was confirmed by the fact that structurally related peptides, such as glucagon or secretin, either lacked or expressed a much reduced inhibitory activity. The effect of VIP was extremely rapid (5 min), and could still be observed for VIP additions up to 12 h after mitogen stimulation. CD4 ÷ T cells represent at least one of the cellular targets for VIP inhibition of IL-2 production. VIP exerted its inhibitory effect directly on purified CD4 ÷ T cells, and rapidly induced significant levels of intracellular cAMP in the CD4 ÷ T lymphocytes. At a molecular level, treatment of lymphocytes with VIP resulted in a transcriptional downregulation of IL-2 expression. The lack of VIP effect on IFNy production, in contrast to its effect on IL-2 production, was an unexpected finding. T n l cells produce both IL-2 and IFNy upon stimulation with either mitogens or antigens and, in general, inhibitory agents such as cyclosporin A or corticosteroids downregulate the production of both cytokines (Granelli-Piperno et al., 1986; Young and Hardy, 1990). Also, Johnson and Torres (1982) reported that agents which raise intracellular levels of cAMP, such as prostaglandins, inhibit IFNy production. As VIP in a certain concentration range (10 -710 -1° M) raises intracellular cAMP, we expected to find that VIP treatment induced a reduction in IFNy production. However, VIP at all concentrations tested (from 10 -7 to 10 -15 M) did not affect IFNy production by either ConA-stimulated normal T lymphocytes, or by the PMA-stimulated L12R4 T cell line, whereas the same VIP concentrations inhibited IL-2 production by either ConA- or anti-CD3-stimulated T cells. The differential effect of VIP on IL-2 versus IFNy production is shared by only one other naturally occurring factor, TGF/3, which selectively inhibits IFNy production, while having minimal effects on IL-2 production (Epsevic et al., 1987; Hardy et al., 1987). Although the molecular mechanism by which VIP inhibits IL-2 production without affecting IFNy production is not known, we could speculate that either an increase in intracellular cAMP does not modulate the expression of IFNy by itself, or that VIP, but not prostaglandins, induces a factor which counteracts the effect of increased intracellular cAMP.

156 The inhibitory effect of VIP on IL-2 production in ConA-stimulated spleen cell cultures was dose-dependent over a large concentration range with maximum inhibitory levels above 50% for 10-7-10 -1° M VIP. Since mitogen stimulation does not represent a typical physiological condition, we investigated whether VIP could affect IL-2 production in cells stimulated through the T cell receptor. Treatment with anti-CD3 mAbs, or with anti-CD3 plus PMA, has previously been shown to induce IL-2 production (Hara and Man Fu, 1985; Granelli-Piperno et al., 1986). VIP in the concentration range of 10-7-10 -1° M inhibited IL-2 production in both systems, with maximum inhibitory values of 80% and 62.5%, respectively. The active VIP concentration range for the inhibition of IL-2 production observed in our experiments is in agreement with the recently reported K d value for murine T lymphocytes (9 × 10 -9 M; Blum et al., 1992). VIP belongs to a family of structurally related peptides which include secretin and glucagon, two well characterized hormones (McDonald, 1991). Binding studies with different members of this gastrointestinal peptide family have been used to investigate the specificity of VIP receptors on lymphocytes, and the following pharmacological specificity has been determined: VIP > secretin > glucagon (Wenger et al., 1990). A similar order of potency was reported for the inhibition of ConA-induced T cell proliferation (Ottaway and Greenberg, 1984). Our results indicated a similar order of potency for the inhibition of IL-2 production. Distinctive fragments of VIPI_z8 have been isolated from in vitro cultures of lymphocytes and mast cells, suggesting the occurrence of posttranslational peptidolysis (Goetzl et al., 1989). Therefore, the possibility exists that various VIP fragments could play a physiological role in the lymphoid microenvironment. Some of these fragments have been shown to bind to VIP receptors on lymphocytes (Ottaway et al., 1983; Wenger et al., 1990), and therefore could act either as agonists or antagonists. We investigated the effect of the aminoand carboxyterminal VIP fragments VIPI_1e and VIP10_as on IL-2 production by ConA-stimulated lymphocytes and did not observe any effect over a concentration range of 10-7-10 -15 M. Experiments investigating the possible antagonistic effect of these fragments are now in progress. The inhibitory effect of VIP on ConA- or antiCD3-stimulated cells was initially determined in unfractionated spleen cell cultures. All types of immune cells, i.e. B and T lymphocytes, as well as m a c r o p h a g e / monocytes have been reported to possess VIP receptors (Guerrero et al., 1981; Ottaway et al., 1983; Danek et al., 1983; Ottaway and Greenberg, 1983; Wiik et al., 1985; O'Dorisio et al., 1985, 1989; Wood and O'Dorisio, 1985; Calvo et al., 1986; Finch et al., 1989; Roberts et al., 1991; Segura et al., 1991). Among T cell subpopula-

tions, CD4 + T cells have been shown to possess a higher number of high affinity VIP receptors than the CD8 + population (Ottaway, 1987). Based on the existence of VIP receptors on different types of immune cells, VIP could affect IL-2 production by acting directly on the CD4 ÷ IL-2 producing T cells, a n d / o r indirectly on a different cellular target. To investigate the cellular target(s) for the inhibition of IL-2 by VIP, we determined the effect of VIP on purified CD4 ÷ T cells stimulated with ConA and PMA. The purified CD4 ÷ T cells did not produce IL-2 in response to ConA alone, which was expected from a macrophagedepleted cell population, but produced significant levels of IL-2 when co-stimulated with ConA plus PMA. Our experiments showed that VIP inhibited IL-2 production in purified CD4 ÷ T cells, suggesting that VIP acted directly on the IL-2 producing CD4 ÷ T cells. The molecular mechanism by which VIP affects IL-2 production is not yet understood. Binding of VIP to its receptor has been shown to activate adenylate cyclase with concomitant cAMP accumulation in a variety of cell types, including human peripheral blood mononuclear cells, the MOLT-4b and SUP-T1 human lymphoblastoid cell lines, human B cells, and rat peritoneal macrophages (O'Dorisio et al., 1981, 1985, 1989; Beed et al., 1983; Robberecht et al., 1989; Segura et al., 1992). Boudard and Bastide (1991), however, reported no significant cAMP increase in murine T lymphocytes treated with 10-7-10 -11 M VIP, although significant inhibitions of both IL-2 production and T cell proliferation were observed within this concentration range. Due to this apparent contradiction, we decided to investigate whether an increase in cAMP upon VIP treatment could be detected in our experimental conditions. VIP in a concentration range of 10-7-10 -1° M induced significant cAMP levels in the purified CD4 ÷ T cells. An interesting observation was that the cAMP increase in the CD4 ÷ T cell cultures occurred very fast (within the first 5 min of incubation with VIP), which correlated with the observation that 5-min incubations with VIP were sufficient to induce a long-lasting inhibition of IL-2 production. The inhibitory role of cAMP on IL-2 production has been previously documented. Novak and Rothenberg (1990) showed that several activators of adenylate cyclase inhibit IL-2, but not IL-4 production, and further studies indicated that the inhibitory effect of cAMP involved both inhibition of nuclear IL-2 transcription and decreased stability of IL-2 m R N A (Anastassiou et al., 1992). Although our results support a possible involvement of cAMP in the inhibitory effect of VIP on IL-2 production, further experiments are necessary in order to determine such a direct correlation. Previous reports indicated that some cAMP-elevating agents such as prostaglandins, forskolin, and cholera toxin blocked IL-2 R N A induction in activated T cell

157

lines such as EL-4 and Jurkat, and in activated human peripheral blood T lymphocytes (Novak and Rothenberg, 1990; Anastassiou et al., 1992). Therefore, we expected that VIP would also downregulate the expression of the IL-2 gene in activated murine T lymphocytes, and indeed VIP treatment of ConA or antiCD3-activated murine splenocyte cultures resulted in a significantly reduced level of IL-2 mRNA. The reduced level of IL-2 mRNA could be due to a reduced transcriptional rate o r / a n d to a decrease in the stability of the IL-2 message. Both mechanisms have been reported to operate in the inhibitory effect of PGE 2 on IL-2 production by activated human T lymphocytes (Anastassiou et al., 1992). Experiments regarding the possible involvement of these two mechanisms in the downregulation of IL2 mRNA by VIP are now in progress in our laboratory.

Acknowledgements We thank Drs. Lee Sullivan and Satwant Narula from Schering-Plough Corp. for mrlL-2 and mrlFNy, the Genetics Institute for mrIL-4, and Drs. Howard Johnson and Marcella Sarzotti from University of Florida, Gainsville, FL, and VA Medical Center, Baltimore, MD, for the IFNy-producing L12R4 murine T lymphoma cell line. This work was supported in part by the 1991-1993 Johnson and Johnson Discovery Award.

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