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The Journal of Immunology, 2000, 165: 114-123.
Copyright © 2000 by The American Association of Immunologists

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Inhibit T Cell-Mediated Cytotoxicity by Inhibiting Fas Ligand Expression1

Mario Delgado*,{dagger} and Doina Ganea2,*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes, a constitutive part of the immune surveillance system, recognize and destroy foreign cells, or autologous cells expressing foreign or mutated self-proteins. CTL can kill target cells via two distinct lytic pathways (1, 2). In the calcium-dependent degranulation pathway, the secreted lytic protein perforin together with a family of CTL-specific granzymes are responsible for the lytic mechanism (3). In addition, most CTLs also induce target cell lysis via a calcium-independent pathway initiated by Fas ligand (FasL)3/Fas interactions (4, 5, 6, 7). CTL FasL expression and subsequent FasL-mediated cytotoxicity can be induced by a variety of stimuli, including stimulation with appropriate Ag-MHC complexes, TCR cross-linking with anti-CD3 Abs, and treatment with pharmacological agents that mimic TCR activation, such as phorbol esters and calcium ionophores (4, 8, 9). In contrast to the degranulation pathway that plays an essential role in the immunity against foreign or virally infected tissues, the FasL-mediated cytotoxicity appears to play an important role in immune regulation and tolerance induction (10, 11).

Vasoactive intestinal peptide (VIP) and the structurally related peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), two neuropeptides present in the immune microenvironment, modulate both innate and acquired immunity (reviewed in Refs. 12, 13, 14, 15, 16). Recently, VIP/PACAP were shown to also inhibit Ag-induced cell death in peripheral CD4+ T cells through the down-regulation of FasL expression (17).

In the present report we examine the effects of VIP and PACAP on the cytotoxic activity of both in vitro and in vivo generated CTLs and of T1/T2 CTL cell lines. Our results indicate that both neuropeptides inhibit CTL FasL expression and the subsequent lysis of Fas-expressing target cells.


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

Synthetic VIP and PACAP38 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1, VPAC2, and PAC1 agonists have been previously described (16, 17). mAbs to murine FasL (MFL3), IFN-{gamma}, IL-4, FITC-conjugated anti-mouse CD8 mAb, CD3{epsilon} mAb, and recombinant murine IL-2, IL-12, and IL-4 were purchased from PharMingen (San Diego, CA). PMA, ionomycin, PMSF, and protease inhibitors were obtained from Sigma (St. Louis, MO). Granzyme B-specific synthetic substrate Boc-Ala-Asp thiobenzyl ester was purchased from Enzyme Systems Products (Dublin, CA), and rapamycin was obtained from Research Biochemicals International (Natick, MA).

Mice, cell lines, and generation of CTL

Female 6- to 8-wk-old BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Target cells EL.4 (H-2b) and P815 (H-2d) were obtained from American Type Culture Collection (Manassas, VA), and L1210-Fas+, a Fas-transfected (DBA/2 leukemia cell, H-2d) variant, was provided by Dr. P. Golstein (Centre d’Immunologie, Marseille-Luminy, France) (4). BALB (H-2d) anti-B6 (H-2b) and B6 (H-2b) anti-BALB (H-2d) alloreactive CTL lines were generated in mixed lymphocyte cultures as previously described (18). BALB/c or B6 splenocytes (1–5 x 106) were cultured with 5 x 106 irradiated (3000 rad) B6 or BALB/c splenocytes, respectively, in the presence of 10 U/ml IL-2. Live cells harvested over Ficoll (density, 1.077) were cultured (1 x 105 cells/well) with periodic (7-day intervals) stimulation with irradiated B6 or BALB/c spleen cells and T cell growth factor (10% Con A-rat splenocyte supernatant). Effector cells for cytotoxicity assays and for mRNA and flow cytometry analysis were harvested on day 5 of the stimulation cycle. VIP and PACAP (10-8 M) were added simultaneously with CTL generation and with each periodical restimulation. In most experiments CTL were stimulated before the assay (see below).

T1 and T2 CTL cell lines were generated as previously described (19). B6 CD8+ cells (>97% CD8+) were purified by passage through nylon wool, followed by complement-mediated lysis of CD4+ and B cells. The purified B6 CD8+ T cells (1 x 106 cells/ml) were incubated for 5 days with T cell-depleted BALB/c splenocytes (3 x 106 cells/ml). To generate T1, the cells were cultured in the presence of IL-2 (20 U/ml), IL-12 (20 U/ml), and anti-IL-4 mAb (2 µg/ml) for 5 days; for T2, CD8+ T cells were cultured in the presence of IL-2 (20 U/ml), IL-4 (40 U/ml), and anti-IFN-{gamma} (80 µg/ml). The cultures were supplemented on day 3 with IL-2 (20 U/ml) and IL-4 (40 U/ml), respectively. T1 and T2 were harvested on day 5 and characterized through cytokine profiles (IFN-{gamma} and IL-5) following restimulation with T cell-depleted BALB/c splenocytes (3 x 106 cells/ml). T1 cells produce high amounts of IFN-{gamma} and no IL-5, whereas T2 cells secrete variable, but small, amounts of IFN-{gamma} and high levels of IL-5 (20).

Stimulation of CTL

CTL (5 x 104 cells/well) were activated in 96-well plates with immobilized anti-CD3 mAbs (2C11, 5 µg/ml) or with 10 ng/ml PMA and 3 µg/ml ionomycin in the presence or the absence of different concentrations of VIP or PACAP and cultured for 6 h at 37°C to allow FasL expression.

Preparation and culture of peritoneal exudate lymphocytes (PEL)

PEL were generated as previously described (21). Briefly, C57BL/6 and BALB/c mice were injected i.p. with allogeneic tumor cells L1210-Fas+ (H-2d) or EL.4 (H-2b; 25 x 106 cells/mouse) with or without VIP or PACAP (5 nmol/mouse). Eight to 10 days after the primary alloimmunization or 4–5 days after the secondary stimulation (which was administered 6–9 wk after priming) the mice were sacrificed, and the peritoneal exudate cells were harvested in PBS/0.5% FCS. The crude PEC were depleted of adherent cells on nylon wool. After 1 h, the nonadherent cells were eluted by rinsing the columns with cold PBS/0.5% FCS. The eluted cells (PEL) contained >95% T cells, 80–90% of which were CD8+, with about 50% of the PEL forming specific conjugates as described by Berke et al. (21).

Cytotoxicity assay

Cytotoxicity was assessed by 51Cr release (18). Activated CTL, PEL, and T1/T2 cells were added to 96-well V-bottom microtiter plate to obtain various E:T cell ratios. The target cells (P815, EL.4, L1210-wt, and L1210-Fas+ cells, 1 x 106/ml), were labeled for 2 h at 37°C with 200 µCi of sodium [51Cr]chromate (Amersham, Arlington Heights, IL), and were added at a concentration of 1 x 104 cells/well. The plates were centrifuged at 300 x g to promote conjugate formation and were incubated for 6 h at 37°C, followed by the removal of 100-µl supernatant aliquots for measurement in a gamma 8000 counter (Beckman, Fullerton, CA). The percent lysis was calculated as follows: % lysis = (E - S)/(M - S) x 100, where E is the release from experimental samples, S is the spontaneous release, and M is the maximum release upon lysis with 10% SDS. The spontaneous release was determined from target cells cultured in medium.

FACS analysis

CTL, PEL, or T1 cells (1 x 106 cells/ml) were harvested in ice-cold medium, washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS (wash buffer), and incubated in wash buffer containing 2.5 µg/ml mouse Igs for 15 min, followed by incubation at 4°C for 1 h with anti-FasL (MFL3) mAb (2.5 µg/ml). Isotype-matched mouse Abs were used as controls, and IgG block (Sigma) was used to block nonspecific binding. Cells were further stained with 2.5 µg/ml of FITC-conjugated goat F(ab')2 anti-hamster IgG (Sigma), for 30 min at 4°C. After extensive washing, the cells were fixed in 1% buffered paraformaldehyde. Stained lymphocytes, gated according to scatter characteristics, were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Fluorescence data were expressed as the mean channel fluorescence (MCF) and as the percentage of positive cells.

RNA extraction and Northern blot analysis

Northern blot analysis was performed according to standard methods. CTL and PEL were prepared and stimulated. At the various time points, 1 x 107 cells were harvested, and total RNA was extracted, electrophoresed on 1.2% agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher and Schuell, Keene, NJ), and cross-linked to the nylon membrane using UV light.

The probes for murine FasL and GAPDH were generated by RT-PCR as described previously (22), using the following primers: FasL, 5'-TCACCAACCAAAGCCTTAAAGTAT-3' and 5'-TCAACCTCTTCTCCTCCATTAGCA-3'; and GAPDH, 5'-TCCTGCACCACCAACTGCTTAGCC-3', and 5'-GTTCAGCTCTTGGATGACCTTGCC-3'. Oligonucleotides were end labeled with [{gamma}-32P]dATP (Amersham) with T4 polynucleotide kinase. The membranes were prehybridized for 16 h at 42°C, hybridized at 60°C for 16 h with the appropriate probes, and exposed to x-ray films (Eastman Kodak, Rochester, NY). Signal quantitation was performed in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).

RT-PCR for the detection of VPAC1, VPAC2, and PAC1 mRNA

Total RNA was isolated from CTL (1 x 107cells) using the Ultraspec RNA reagent (Biotecx, Houston, TX). Two micrograms of total RNA was reverse transcribed in the presence of 200 U of Moloney murine leukemia virus reverse transcriptase, 40 U of RNasin, 1 µg of random primers, 0.5 mM dNTPs, 3 µg of BSA, and Moloney murine leukemia virus reaction buffer (50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2) in a total volume of 30 µl at 37°C for 1 h. The cDNA was amplified with specific primers. Amplification with ß-actin primers was used as a control. The primers for VPAC1, VPAC2, and PAC1 receptors have been described previously (23). The expected sizes for the amplified fragments are: 450 bp for VPAC1, 572 bp for VPAC2, 317 bp for PAC1, and 660 bp for ß-actin. Five microliters of reverse transcribed cDNA was subjected to PCR in the presence of 0.5 U of pyrostase, 1 µM sense and antisense primers, 0.2 mM dNTPs, and polymerase buffer (50 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 20 mM (NH4)2SO4, and 50 µg/ml BSA). The PCR conditions were denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and primer extension at 72°C for 90 s for 35 cycles.

Analysis of functional FasL expression

The function of FasL expressed on anti-CD3 stimulated CTL was assessed by the ability of these cells to cause DNA fragmentation in Fas+ target cells as previously described (24). Briefly, BALB/c anti-H2b alloreactive CTL were activated with plate-bound anti-CD3 mAbs for 3 h to allow FasL expression in the presence or the absence of different concentrations of VIP or PACAP. The cells were washed twice and incubated for 8 h with [3H]TdR-labeled Fas+ L1210 target cells or Faslow L1210 as controls (2 x 104 cells/well). L1210 and L1210-Fas+ cells were labeled for 2 h with 5 µCi/ml [3H]TdR (79 Ci/mmol; Amersham) at 37°C in RPMI/5% FCS. [3H]TdR-labeled unfragmented high m.w. DNA was harvested on glass filters and counted in a liquid scintillation counter. DNA fragmentation was calculated as follows: % DNA fragmentation = 100 x (cpm control group - cpm experimental group/cpm control group) ± SD.

Detection of granzyme B activity and perforin protein expression

Cytosolic proteins were obtained from 5 x 106 CTL by treatment with 200 µl of ice-cold lysis buffer (1% Nonidet P-40, 5 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin) for 30 min. Cell lysates were cleared by centrifugation at 14,000 x g for 6 min, and the supernatants were stored at -20°C. The granzyme B activity was measured by a colorimetric enzyme assay as previously described (25) using the granzyme B-specific synthetic substrate Boc-Ala-Asp thiobenzyl ester. Absorbance of 0.01 at 405 nm was arbitrarily defined as 1 U of esterolytic activity. Detection of perforin in cytosolic proteins was determined by Western blotting as described by Makrigiannis and Hoskin (26).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VIP and PACAP inhibit FasL-mediated CTL cytotoxicity

Alloreactive CTL lyse target cells through both perforin/granzyme-dependent and FasL/Fas-dependent mechanisms. Because the perforin/granzyme pathway requires Ca2+-dependent degranulation, the only lytic mechanism operating in the presence of EGTA is the FasL/Fas-mediated lysis. We generated allogeneic BALB/c anti-H-2b or B6 anti-H-2d effector CTL in the presence or the absence of VIP or PACAP, activated them with immobilized anti-CD3 Abs in the presence or the absence of either neuropeptide, and determined the cytotoxicity against syngeneic and allogeneic targets. The anti-CD3-activated BALB/c anti-H-2b CTL lysed the allogeneic EL.4 cells (H-2b; Fig. 1GoA, upper panel), but failed to lyse the syngeneic L1210-wt (H-2d) or P815 cells (H-2d; Fig. 1GoA, lower panels). However, L1210 cells transfected with Fas cDNA (L1210-Fas+) were efficiently lysed (Fig. 1GoA, middle panels), in agreement with previous reports that lysis of syngeneic targets is entirely FasL/Fas mediated (18). VIP and PACAP had a poor inhibitory effect in the absence of EGTA, but almost completely inhibited the Ca2+-independent lysis of EL.4 cells (Fig. 1GoA, upper panels). The two neuropeptides dramatically inhibited the lysis of L1210-Fas+ cells (Fig. 1GoA, middle panels). Conversely, B6 anti-H-2d CTL effectively lysed the allogeneic L1210-wt, L1210-Fas+, and P815 targets (Fig. 1GoB, left panels), but not the syngeneic EL.4 targets (Fig. 1GoB, lower/right panel). In addition, whereas L1210-Fas+ cells were efficiently killed in the presence of EGTA, L1210-wt and P815 cells, which express very low levels of Fas, required Ca2+ for lysis (Fig. 1GoB). Again, VIP and PACAP showed little, if any, inhibitory effect on the cytotoxicity against L1210-wt and P815, although they decreased the lysis of L1210-Fas+ cells (Fig. 1GoB). In addition, both neuropeptides inhibited completely the Ca2+-independent cytotoxicity against L1210-Fas+ targets (Fig. 1GoB).



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  		FIGURE 1. VIP and PACAP inhibit the FasL/Fas-mediated cytotoxicity of alloreactive CTL. BALB/c anti-H2b (A) and B6 anti-H2d (B) alloreactive CTL (5 x 104 cells) generated in 5-day MLC as described in Materials and Methods in the absence (control) or the presence of VIP or PACAP (10-8 M) were activated by incubation in flat-bottom 96-well plates coated with anti-CD3 Ab (5 µg/ml) in the presence or the absence of VIP or PACAP (10-8 M). After 6-h activation 51Cr-labeled target cells (EL.4, L1210-wt, L1210-Fas+, or P815) were added at various E:T cell ratios, and the cultures were incubated in the presence or the absence of 4 mM EGTA/3 mM MgCl2 (+EGTA). After 12 h supernatants were harvested to measure 51Cr release. Cytotoxicity was expressed as the specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 18 and 29%. The results were calculated as the mean of triplicate samples,

 
VIP and PACAP inhibit the cytotoxic activity of BALB/c anti-H-2b CTLs stimulated with anti-CD3 Abs or with a combination of PMA and ionomycin against L1210-Fas+ targets (Fig. 2Go). The inhibition is dose-dependent, with maximum effects at 10 nM VIP/PACAP (Fig. 2Go).



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  		FIGURE 2. Dose-response effect for the inhibition of cytotoxicity by VIP and PACAP. BALB/c anti-H2b alloreactive CTL were generated in 5-day MLC as described in Materials and Methods in the absence (control) or the presence of different concentrations of VIP or PACAP. CTL (5 x 104 cells) were activated by incubation in flat-bottom 96-well plates coated with anti-CD3 Ab (5 µg/ml) or by treatment with 10 ng/ml PMA plus 3 µg/ml ionomycin in the presence or the absence of various concentrations of VIP or PACAP. After 6-h activation, 51Cr-labeled target cells (L1210-wt or L1210-Fas+) were added at a 10:1 E:T cell ratio, followed by incubation in the presence of 4 mM EGTA/3 mM MgCl2 (EGTA). After 12 h, supernatants were harvested to measure 51Cr release. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 11 and 23%. The results were calculated as the mean of triplicate samples.

 
These results suggest that VIP and PACAP inhibit the CTL Ca2+-independent, FasL/Fas-mediated lysis of syngeneic and allogeneic target cells, but do not affect the Ca2+-dependent, perforin/granzyme-mediated cytotoxicity.

The failure of VIP and PACAP to inhibit perforin/granzyme-mediated lysis correlates with their lack of effect on granzyme B activity or perforin expression. In contrast to rapamycin, a known inhibitor of perforin/granzyme-mediated cytotoxicity (26), VIP and PACAP did not affect granzyme B activity or perforin expression in anti-CD3-stimulated CTL (Fig. 3Go).



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  		FIGURE 3. VIP and PACAP do not affect granzyme B activity or perforin expression. BALB/c anti-H2b alloreactive CTL generated in 5-day MLC in the absence (control) or the presence of VIP or PACAP (10-8 M) were activated with anti-CD3-coated plates (5 µg/ml) in the presence or the absence of VIP or PACAP (10-8 M) or rapamycin (1 ng/ml). A, Following 48 h of culture, cytosolic lysates were obtained from equal numbers of CTL and added to a colorimetric reaction mixture containing synthetic granzyme B substrate as described in Materials and Methods. Data from a representative experiment (n = 3) are expressed as the mean units of estereolytic activity ± SD. B, Alternatively, after 48-h culture, perforin expression in cytosolic lysates was analyzed by Western blotting. Data are representative of two independent experiments.

 
Because FasL/Fas-mediated cytotoxicity leads to target cell apoptosis (27), we investigated whether VIP and PACAP inhibit DNA fragmentation in target cells. BALB/c anti-H-2b effector CTLs were generated and activated by CD3 cross-linking in the presence and the absence of VIP/PACAP. The effector cells were incubated with [3H]TdR-labeled L1210-Fas+ target cells in the presence of EGTA, and the percentage of DNA fragmentation was determined as a measure of apoptosis. TCR-stimulated CTL induced DNA fragmentation in L1210-Fas+ cells, but not in L1210-wt cells (Fig. 4GoA), and VIP/PACAP inhibited DNA fragmentation in a dose-dependent manner (Fig. 4GoB).



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  		FIGURE 4. VIP and PACAP inhibit CTL-induced apoptosis in Fas-bearing target cells. BALB/c anti-H2b alloreactive CTL (5 x 104 cells) generated in 5-day MLC in the absence (medium) or the presence of VIP or PACAP (10-8 M) were activated on plates coated with anti-CD3 mAbs (5 µg/ml) in the presence or the absence of different concentrations of VIP or PACAP (B) and 10-8 M (A). Cells were cultured for 3 h to allow FasL expression, harvested, washed twice, and incubated for an additional 8 h with [3H]TdR-labeled L1210 (wt) or L1210-Fas+ target cells at different E:T cell ratios in A and at a 10:1 E:T cell ratio in B. The percentage of DNA fragmentation was calculated as described in Materials and Methods. Each result is the mean ± SD of three independent experiments performed in duplicate.

 
VIP and PACAP affect the generation and/or activation of CTL

VIP and PACAP could affect the generation and/or stimulation of effector CTLs or/and the Fas signaling pathway in target cells. To distinguish between these possibilities, we measured the Ca2+-independent cytolysis of L1210-Fas+ by BALB/c anti-H-2b CTLs generated and activated in the absence or the presence of VIP or PACAP. Unstimulated effector CTL exhibit poor cytotoxicity, even toward L1210-Fas+ cells (Fig. 5GoA). However, if effector CTL are first activated with immobilized anti-CD3 Abs or with PMA/ionomycin, the cytotoxicity against L1210-Fas+ target cells is greatly increased (Fig. 5Go, B–F). Addition of VIP or PACAP during CTL generation (Fig. 5GoB) or during CTL activation (Fig. 5GoE) dramatically inhibited lysis, and addition of neuropeptides during both CTL generation and stimulation resulted in a higher inhibitory effect (Fig. 5Go, C and D). However, addition of VIP or PACAP during the effector phase (the cytotoxicity assay) failed to inhibit cytolytic activity (Fig. 5GoF). These results suggest that VIP and PACAP affect one or more events during the generation and stimulation of effector CTL, but not the signaling pathway of the Fas-mediated cytotoxic process.



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  		FIGURE 5. VIP and PACAP affect the generation and/or activation of CTL, but not the cytotoxic function. BALB/c anti-H2b alloreactive CTL were generated in 5-day MLC as described in Materials and Methods in the presence (A–D) or the absence (E and F) of VIP or PACAP (10-8 M). Cells (5 x 104 cells) were incubated for 3 h with medium alone (A) or were activated by incubation in flat-bottom 96-well plates with anti-CD3 Ab (5 µg/ml; B, C, E, and F) or by treatment with 10 ng/ml PMA plus 3 µg/ml ionomycin (D) in the absence (A, B, and F) or the presence (C–E) of 10-8 M VIP or PACAP. 51Cr-labeled target cells (L1210-wt or L1210-Fas+) were added at different E:T cell ratios to the activated CTL. After 12 h, supernatants were harvested to measure 51Cr release. All cultures contained EGTA during the 12-h cytotoxicity assay. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 14 and 22%. The results were calculated as the mean of triplicate samples.

 
VIP and PACAP inhibit CTL FasL expression

To determine whether the inhibition of CTL FasL/Fas-mediated cytotoxicity correlates with neuropeptide inhibition of FasL expression, BALB/c anti-H-2b and B6 anti-H-2d effector CTL generated and stimulated with or without VIP/PACAP were analyzed for FasL expression by flow cytometry and Northern blot analysis. Whereas unstimulated CTL express low levels of both protein and mRNA FasL, TCR stimulation through CD3 cross-linking results in a great increase in FasL expression (Fig. 6Go). Treatment with VIP or PACAP resulted in a significantly reduced expression of FasL expression at both protein and mRNA levels (Fig. 6Go). The inhibitory effect on FasL expression was dose dependent and specific, because neither VIP nor PACAP affected CD8 expression (Fig. 6Go). To address the question of whether VIP/PACAP affect already expressed FasL, we stimulated CTL with anti-CD3 and added VIP or PACAP at different times after stimulation. Additions at 0 and 30 min reduced both FasL expression and cytotoxicity, but additions at later times (1, 2, and 4 h) did not have any effect (Fig. 7Go).



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  		FIGURE 6. VIP and PACAP down-regulate FasL expression in CTL effectors. B6 anti-H2d (A) and BALB/c anti-H2b (B) were generated in 5-day MLC in the absence or the presence of 10-8 M VIP or PACAP. CTL (5 x 104 cells and 1 x 107 cells for flow cytometry and Northern blot analysis, respectively) were incubated with medium alone (unstimulated) or were activated with anti-CD3 (5 µg/ml) in the absence or the presence of VIP or PACAP (10-8 M) or with different neuropeptide concentrations (B, right panel). After 6-h incubation, expression of CD8 (percentage of CD8+ T cells) and FasL protein (fluorescence intensity in A; MCF in B) were analyzed by flow cytometry. Alternatively, after 3-h incubation, FasL mRNA expression was analyzed by Northern blot (results are expressed in arbitrary densitometric units normalized for the expression of GAPDH in each sample). The data presented are representative of three experiments.

 


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  		FIGURE 7. VIP and PACAP down-regulate de novo FasL expression, but not FasL already expressed following anti-CD3 stimulation. BALB/c anti-H2b alloreactive CTL were generated in 5-day MLC in the absence of VIP or PACAP. CTL (5 x 104 cells) were incubated with medium alone (unstimulated) or were activated with anti-CD3 (5 µg/ml), and VIP or PACAP (10-8 M) were added at different times after stimulation. A, After 6-h incubation, expression of FasL protein (MCF) was analyzed by flow cytometry. Each result is the mean ± SD of four independent experiments performed in duplicate. B, After 3-h incubation, 51Cr-labeled target cells (L1210-Fas+) were added at different E:T cell ratios to the activated CTL and incubated for an additional 12-h period. Supernatants were harvested to measure 51Cr release. All cultures contained EGTA during the 12-h cytotoxicity assay. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 11 and 18%. The results were calculated as the means of triplicate samples. Similar results were obtained for PACAP.

 
Involvement of VPAC1 and VPAC2 in the inhibitory effect of VIP and PACAP on FasL/Fas-mediated cytotoxicity and FasL expression in CTL

VIP and PACAP act through a family of receptors; VPAC1 and VPAC2 exhibit similar affinities for the two neuropeptides and activate primarily the adenylate cyclase system, whereas PAC1 exhibits a 300- to 1000-fold higher affinity for PACAP and activates both adenylate cyclase and phospholipase C (reviewed in Ref. 28). Although naive CD4+ and CD8+ T cells express both VPAC1 and VPAC2 following anti-CD3 stimulation (29, 30), the expression of VIP/PACAP receptors in effector CTL has not been studied. We investigated first the expression of VPAC1, VPAC2, and PAC1 mRNA by RT-PCR in allogeneic BALB/c anti-H-2b CTL. VPAC1- and VPAC2-specific, but not PAC1-specific, fragments were amplified (Fig. 8GoA). Next, we investigated the effects of VPAC1-, VPAC2-, and PAC1-specific agonists (31, 32, 33) on the induction of cytotoxic activity of BALB/c anti-H-2b effector CTL for L1210-Fas+ cells. Both VPAC1 and VPAC2 agonists, but not the PAC1 agonist, inhibited cytotoxic activity, with a potency similar to that of VIP/PACAP (Fig. 8GoB). A similar pattern was observed for the effect of VIP/PACAP agonists on FasL expression in CTL (Fig. 8GoC). These results suggest that the two neuropeptides exert their actions primarily through VPAC1 and VPAC2.



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  		FIGURE 8. VIP and PACAP inhibit Fas-mediated cytotoxicity and FasL expression in CTL through both VPAC1 and VPAC2. A, CTL express both VPAC1 and VPAC2. Total RNA extracted from BALB/c anti-H2b alloreactive CTLs (2 x 107 cells) was subjected to RT-PCR with specific primers for VPAC1, VPAC2, and PAC1 as described in Materials and Methods. Reactions without cDNA served as negative controls (not shown). One representative experiment of two is shown. B, VPAC1 and VPAC2 agonists inhibit Fas-mediated cytotoxicity. BALB/c anti-H2b alloreactive CTL (5 x 104 cells) generated in 5-day MLC as described in Materials and Methods in the absence (medium) or the presence of VPAC1, VPAC2, or PAC1 agonists (10-7 M) were activated by incubation in flat-bottom 96-well plates coated with anti-CD3 Ab (5 µg/ml) in the absence (medium) or the presence of 10-7 M VPAC1, VPAC2, or PAC1 agonists. After 3-h incubation, 51Cr-labeled target cells (L1210-wt or L1210-Fas+) were added at different E:T cell ratios in the absence or the presence of 4 mM EGTA/3 mM MgCl2 (+EGTA). After 12 h, supernatants were harvested to measure 51Cr release. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 16 and 21%. These results were calculated as the mean of triplicate samples. C, VPAC1 and VPAC2 inhibit FasL expression in CTL effectors. BALB/c anti-H2b alloreactive CTL (5 x 105 cells) generated in 5-day MLC in the absence (medium) or the presence of VPAC1, VPAC2, or PAC1 agonists (10-7 M) were activated for 6 h on anti-CD3-coated plates in the absence (medium) or the presence of 10-7 M VPAC1, VPAC2, or PAC1 agonists. Expression of FasL was analyzed by flow cytometry (MCF). Each result is the mean ± SD of three experiments performed in duplicate.

 
VIP and PACAP inhibit the FasL/Fas-mediated cytotoxicity of alloreactive T1 CD8+ T cell effectors

Polarized populations of cytokine-secreting CD8+ effector T cells (T1 and T2) can be generated in vitro (34, 35, 36). CD8+ T cells from B6 mice were stimulated with BALB/c APCs in the presence of IL-2, IL-12, and anti-IL-4 for T1 polarization or IL-2, IL-4, and anti-IFN-{gamma} for T2 polarization. After 5 days, the H-2b-anti-H-2d CD8+ effectors were restimulated and assayed for their ability to secrete representative type 1 (IFN-{gamma}) and type 2 (IL-5) cytokines. Both T1 and T2 populations were >92% CD8+, with T1 populations secreting large amounts of IFN-{gamma} and nondetectable amounts of IL-5, and T2 populations secreting substantial amounts of IL-5 and reduced levels of IFN-{gamma} (Fig. 9GoA). T1 and T2 effectors were assayed for their cytotoxic activity using L1210-wt and L1210-Fas+ targets in the presence or the absence of EGTA. T1 effectors killed both targets, but whereas cytotoxicity against L1210-wt was entirely Ca2+ dependent, substantial residual Ca2+-independent killing was observed for the L1210-Fas+ targets (Fig. 9GoB). As previously described (19), the T2 population displayed no significant Ca2+-independent cytotoxicity (Fig. 9GoB, right panels). Addition of VIP or PACAP during T1 generation resulted in a slight decrease in the lysis of L1210-Fas+ cells in the absence of EGTA and a complete inhibition of Ca2+-independent lysis (Fig. 8GoB). In contrast, the neuropeptides did not affect the lysis of L1210-wt cells (Fig. 9GoB). Also, VIP and PACAP failed to inhibit the cytotoxic activity of allogeneic T2 effectors (Fig. 9GoB). Therefore, VIP and PACAP inhibit only the FasL/Fas-mediated Ca2+-independent cytotoxic activity of alloreactive T1 effectors.



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  		FIGURE 9. VIP and PACAP inhibit the Fas-mediated cytotoxicity and down-regulate FasL expression on CD8+ T1 effectors. A, CD8 expression and cytokine profile for T1 and T2 effectors. T1 and T2 effectors were generated as described in Materials and Methods, stained with anti-CD8 Abs, and analyzed by flow cytometry. In addition, 4 x 105 T1 and T2 cells/ml were restimulated with 1 x 106 APC, and the 40-h supernatants were assayed for cytokines. Each data point represents the mean ± SD of three experiments performed in duplicate. B, VIP and PACAP inhibit the Fas-mediated cytotoxicity of T1 cells. CD8+ T1 (left panels) and T2 (right panels) effectors from C57BL/6 mice were generated in the absence (medium) or the presence of VIP or PACAP (10-8 M) and assayed for cytolytic activity in a 12-h assay with 51Cr-labeled target cells (L1210-wt or L1210-Fas+) in the presence or the absence of 4 mM EGTA/3 mM MgCl2 (+EGTA). Supernatants were harvested to measure 51Cr release. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 9 and 17%. The data are representative of three experiments. C, VIP and PACAP inhibit FasL expression in CD8+ T1 effectors. T1 cells were generated in the absence (medium) or the presence of VIP or PACAP (10-8 M). FasL expression was analyzed by flow cytometry. Each result is the mean ± SD of three separated experiments performed in duplicate.

 
To investigate whether the inhibitory effect of VIP/PACAP correlates with reduced FasL expression in T1 effectors, T1 cells were generated in the presence or the absence of VIP or PACAP, and FasL expression was assayed by flow cytometry. Both neuropeptides significantly decreased the expression of FasL protein in alloreactive T1 effectors (Fig. 9GoC).

VIP and PACAP inhibit the Fas-mediated cytotoxicity of alloreactive PEL

To investigate the in vivo effects of VIP and PACAP on FasL/Fas-mediated cytotoxicity, we used the murine model developed by Berke et al. (21, 37) for in vivo-generated peritoneal exudate cytotoxic CD8+ T cells (PEL). BALB/c and B6 mice were injected with EL-4 and L1210-Fas+ cells, with or without VIP or PACAP (5 nmol/mouse). Peritoneal exudate cells depleted of adherent cells, consisting of >95% T cells (80–90% CD8+), were tested for cytolytic activity against syngeneic and allogeneic targets. The BALB/c anti-EL.4 PEL exhibited potent, specific cytotoxicity toward EL.4 cells, with both Ca2+-dependent and -independent components. PEL obtained from mice injected with VIP or PACAP exhibited slightly reduced cytolytic activity toward EL-4 targets in the absence of EGTA, but showed significantly reduced Ca2+-independent lysis (Fig. 10GoA, upper panels). Although BALB/c anti-H-2b PEL did not kill syngeneic targets with low Fas expression (L1210-wt and P815; Fig. 10GoA, middle panels), they lysed L1210-Fas+ cells through a Ca2+-independent mechanism (Fig. 10GoA, lower panels). PEL obtained from mice injected with VIP or PACAP had a reduced lytic activity toward L1210-Fas+ targets (Fig. 10GoA, lower panels).



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  		FIGURE 10. In vivo inhibition of PEL cytotoxicity by VIP and PACAP. BALB/c and C57BL/6 mice were injected i.p. twice at a 4-wk interval with 25 x 106 EL.4 or L1210-Fas+, respectively, in the absence (medium) or the presence of VIP or PACAP (5 nmol). Four days after the second allostimulation, the BALB/c anti-EL.4 (A) and B6 anti-L1210-Fas+ (B) alloreactive PELs were harvested from the peritoneal cavity, mixed with the 51Cr-labeled targets (3 x 104 cells/well, EL.4, P815, L1210-wt, or L1210-Fas+), and incubated for 12 h at different PEL:target ratios with or without 4 mM EGTA/3 mM MgCl2 (+EGTA). After incubation, supernatants were harvested to measure 51Cr release. Cytotoxicity was expressed as specific 51Cr release after subtraction of spontaneous 51Cr release, which ranged between 14 and 18%. The data presented are representative of three experiments. C, VIP and PACAP inhibit FasL expression in PELs in vivo. BALB/c anti-EL.4 and B6 anti-L1210-Fas+ alloreactive PELs generated in the absence (medium) or the presence of VIP or PACAP (5 nmol/mouse) were analyzed for CD8 (% CD8+ cells) and FasL protein (fluorescence intensity) expression by flow cytometry and for FasL mRNA (arbitrary densitometric units) expression by Northern blot. The data presented are representative of three experiments.

 
On the other hand, B6 anti-H-2d PEL specifically lysed allogeneic L1210-wt, P815, and L1210-Fas+ targets, but not syngeneic EL.4 cells (Fig. 10GoB). Whereas in vivo administration of VIP and PACAP did not reduce PEL cytotoxicity against low Fas-expressing L1210-wt and P815 cells (Fig. 10GoB, middle and lower panels), it significantly inhibited lysis of L1210-Fas+ cells (Fig. 10GoB, upper panels). Therefore, similar to the in vitro results, the in vivo administration of VIP and PACAP inhibits the Ca2+-independent, FasL/Fas-mediated cytotoxicity of effector PEL.

Next we investigated whether the inhibitory effect of VIP and PACAP on PEL cytotoxicity correlates with a reduced FasL expression. BALB/c anti-EL.4 and B6 anti-L1210-Fas+ effector PEL generated in vivo in the presence or the absence of VIP and PACAP were analyzed for FasL expression by flow cytometry and Northern blots. The freshly isolated PEL, at peak lytic ability following immunization, express high levels of both FasL protein and mRNA without restimulation with cognate target cells. Although CD8 expression was not affected, FasL expression (both protein and mRNA) was significantly reduced following in vivo VIP/PACAP administration (Fig. 10GoC).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies suggested that the FasL/Fas-mediated pathway represents the basis for the Ca2+-independent CTL lysis reported previously (4, 5, 38, 39, 40). Once initiated, the FasL/Fas-mediated lysis is every bit as vigorous as lysis via the degranulation pathway, provided, of course, that the target cell is Fas+ (41). The FasL/Fas-mediated cytolytic activity is up-regulated in CTL effectors and clones through direct restimulation through the TCR complex (4, 41, 42, 43). Such stimulation results in an increase in CTL cytolytic activity and is accompanied by an increase in de novo FasL synthesis and surface expression. The requirement for new protein synthesis explains why the onset of the Ca2+-independent lysis is delayed with respect to Ca2+-dependent (degranulation) lysis in cloned CTL (44). In agreement with the reported FasL up-regulation following direct TCR restimulation, our study shows that anti-CD3-activated CTL lyse Fas+ targets, and that this ability is paralleled by an increase in FasL expression. In addition, the killing of Fas+ cells by activated CTL is blocked by the anti-Fas mAb Jo2 (results not shown). The expression of FasL in CTLs varies with activation and differentiation stage. The fact that naive and memory CTLs do not express FasL whereas cytotoxic effectors do (45) supports the idea that FasL expression occurs following specific CTL-target cell interaction and TCR triggering (2). This highly controlled FasL expression could explain the target cell specificity for FasL/Fas-mediated CTL cytotoxicity.

In the present study we demonstrate that whereas VIP and PACAP do not affect the Ca2+-dependent, perforin/granzyme-mediated CTL cytotoxicity, both neuropeptides significantly decrease the Ca2+-independent, FasL/Fas-mediated killing of Fas+ target cells. VIP/PACAP affect various CTL effectors, including in vitro-generated primary alloreactive CTL and T1 CD8+ effectors and in vivo-generated PEL effectors.

The inhibitory effect of both neuropeptides appears to be mediated by preventing FasL gene expression. Indeed, VIP and PACAP inhibit both the anti-CD3- and PMA/ionomycin-stimulated cytotoxic activity and FasL expression in primary alloreactive CTLs. The inhibition occurs if the neuropeptides are added during CTL generation and/or stimulation. In contrast, the addition of VIP/PACAP during the effector phase or 1 h after restimulation with anti-CD3 does not affect the cytotoxic activity. This is reminiscent of the lack of effect of VIP/PACAP on the apoptosis of Fas-bearing targets induced by soluble FasL (17) and supports the idea that VIP/PACAP interfere with FasL expression but not with Fas signaling. We reached similar conclusions from the effects of VIP/PACAP on cytolytic activity and FasL expression in the CD8+ cytotoxic subsets T1/T2. Although both subsets are cytotoxic and exhibit perforin/granzyme-mediated cytotoxicity, only the T1 subset uses the FasL/Fas-mediated pathway (19). VIP/PACAP inhibit the cytotoxic activity of T1, but not T2 cells, and this inhibition correlates with a decrease in T1 FasL expression.

Although the in vivo PEL effectors were reported to express functional FasL without apparent Ag stimulation and in the presence of transcriptional and translational inhibitors (42), our data indicate that administration of VIP/PACAP during the in vivo generation of PEL effectors results in the inhibition of both FasL expression and cytotoxicity against Fas+ target cells; in contrast, treatment of PEL with either neuropeptide after their isolation from the peritoneal cavity has no effect on FasL expression or cytotoxicity (results not shown). Taken together, these results suggest that VIP and PACAP act both in vivo and in vitro as modulators of the FasL/Fas-mediated cytotoxicity by inhibiting FasL expression in CTLs. This correlates with our previous finding that VIP/PACAP inhibit FasL gene expression in CD4+ T lymphocytes and T cell hybridomas (17).

Both CD4+ and CD8+ naive T cells stimulated in vitro with anti-CD3 mAbs were shown to express VPAC1 and VPAC2, but not PAC1, mRNA (29, 30). Here we report that CTL generated in response to alloreactive cells express both VPAC1 and VPAC2, but not PAC1, mRNA. The studies presented here are not quantitative. It remains to be established whether VPAC1/VPAC2 levels change during the inductive and effector phases of the cytotoxic response. Our agonist studies suggest that both VPAC1 and VPAC2 mediate the inhibitory effect on FasL-mediated cytotoxicity and expression in CTL. We reported similar results for the inhibition of FasL expression and subsequent clonal deletion of activated CD4+ T cells (17).

Recently, cytokines such as IL-2, RANTES, and IFN-{alpha} were shown to up-regulate FasL expression in T cells (46, 47, 48, 49). Although it is not known whether VIP/PACAP affect RANTES or IFN-{alpha}, the two neuropeptides inhibit IL-2 production (reviewed in Ref. 13). Therefore, the effect of VIP/PACAP on FasL expression might be mediated at least partially through a reduction in IL-2. However, this seems improbable, because we generated and stimulated the alloreactive CTLs in the presence of high levels of exogenous IL-2. However, the involvement of IL-2 in the regulation of FasL expression by VIP/PACAP in vivo cannot be excluded.

Because VIP and PACAP specifically inhibit FasL/Fas-mediated cytotoxicity without affecting the perforin/granzyme pathway, the physiological relevance of this immunoregulatory process depends on the function of the two cytotoxic pathways. During an allogeneic response, perforin/granzyme lysis represents the major mechanism for the destruction of specific allotargets (50). In contrast, FasL/Fas-mediated lysis is responsible for the observed bystander cytotoxic effect against Fas-bearing syngeneic cells (46). Although several studies describe CD4+ T effector cells as the major players in FasL/Fas-dependent lysis of syngeneic cells (8, 9, 50, 51), numerous reports, including the present study, demonstrate clearly that CD8+ primary alloreactive CTL, T1 cell lines, PEL effectors, CTL clones, and hybridomas express FasL/Fas-mediated cytotoxicity against allogeneic and syngeneic Fas+ targets (4, 5, 6, 19, 38, 41, 42, 43, 44, 52) This study demonstrates that VIP/PACAP inhibit CTL cytotoxicity against Fas+ syngeneic cells. In addition, preliminary experiments show that VIP/PACAP suppress the cytotoxicity against Fas+ bystanders by CD4+ T cells responding to specific Ag. Therefore, VIP and PACAP appear to be especially relevant for the control of FasL expression and the subsequent FasL/Fas-mediated cytotoxicity against bystander syngeneic cells.

Despite the important role of FasL/Fas-mediated cytotoxicity in immune homeostasis, unrestricted lysis of any Fas+ cell is potentially deleterious and may play a role in the pathology of some inflammatory and autoimmune diseases (reviewed in Ref. 53). Therefore, the existence of factors, such as VIP and PACAP, that control an excessive FasL/Fas-mediated bystander cytolysis by regulating FasL expression, may offer new avenues for an effective therapeutic intervention. On the other hand, it has been proposed that VIP and PACAP might favor the development and/or maintenance of a population of memory CD4+ T cells by inhibiting Ag-induced cell death through the down-regulation of FasL expression (17). Because memory CTL down-regulate FasL expression (45), the question arises of whether VIP/PACAP could participate in the establishment of the memory CD8+ T cell population. Further investigation will answer this question.

Previous studies defined VIP and PACAP as endogenous factors that play an important role in the control of immune homeostasis. The VIP/PACAP control of Ag-induced cell death and of cytotoxicity against syngeneic bystander targets through the down-regulation of FasL expression adds an additional dimension to their immunomodulatory properties.


   Acknowledgments
 
We thank Dr. Pierre Golstein (Centre d’Immunologie, Institut Nationale de la Santé et de la Recherche Médicale-Centre Nationale de la Recherche Scientifique, Marseilles, France) for the L1210 and L1210-Fas+ cells.


   Footnotes
 
1 This work was supported by Public Health Service Grant AI041786-02 (to D.G.), Busch Biomedical Award 98-00 (to D.G.), Grant PM98-0081 (to M.D.), and a postdoctoral fellowship from the Spanish Department of Education and Science (to M.D.). Back

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

3 Abbreviations used in this paper: FasL, Fas ligand; MCF, mean channel fluorescence; MLC, mixed lymphocyte culture; PACAP, pituitary adenylate cyclase-activating polypeptide; PAC1, PACAP-preferring receptor; PEL, peritoneal exudate lymphocytes; VIP, vasoactive intestinal peptide; VPAC1, type 1 VIP receptor; VPAC2, type 2 VIP receptor; wt, wild type. Back

Received for publication December 14, 1999. Accepted for publication April 17, 2000.


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