VIP and PACAP inhibit Fas ligand-mediated bystander lysis by CD4+ T cells

Journal of Neuroimmunology 112 (2001) 78–88www.elsevier.com/ locate / jneuroin

1VIP and PACAP inhibit Fas ligand-mediated bystander lysis by CD4 Tcellsa,b a ,*Mario Delgado , Doina Ganea

aDepartment of Biological Sciences, Rutgers University, Newark, NJ 07102, USAbDepartamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid 28040, Spain

Received 9 June 2000; received in revised form 18 September 2000; accepted 18 September 2000

Abstract

1FasL/Fas-mediated lysis represents the major cytotoxic mechanism for CD4 effectors, with important consequences for immune cell1homeostasis. Upon stimulation by specific antigen-presenting cells (APCs), CD4 effectors can lyse the cognate APCs (direct targets) and

neighboring innocent bystanders. Previously we showed that the neuropeptides VIP and PACAP prevent FasL expression andactivation-induced cell death in T cells. In this study we investigated the effects of VIP and PACAP on FasL expression and subsequent

1direct and bystander lysis by CD4 effectors generated in vivo. VIP/PACAP inhibit FasL expression in allogeneic effectors, and reduceFas-mediated cytotoxicity against specific allotargets and syngeneic bystanders. VIP/PACAP also inhibit FasL expression in antigen-

1specific CD4 effectors, and reduce their cytotoxic activity against both the stimulatory APC, and syngeneic or allogeneic bystanders.Since bystander lysis is involved in the pathogenesis of several autoimmune and inflammatory diseases, the identification of regulatoryfactors that limit this process is highly significant. 2001 Published by Elsevier Science B.V. All rights reserved.

Keywords: Neuropeptides; VIP; PACAP; CD41 cytotoxicity; Fas ligand; Bystander lysis

1. Introduction anti-CD3 Ab, and pharmacological agents that mimic TCRactivation, such as phorbol esters plus calcium ionophores

Two independent lytic pathways mediate T-cell cytotox- (Rouvier et al., 1993; Ju et al., 1994; Stalder et al., 1994).icity (Berke, 1995; Nagata and Golstein, 1995): a calcium- The perforin /granzyme pathway plays a major role in thedependent degranulation pathway, in which exocytosis of elimination of virally-infected cells or allogeneic grafts,perforin in combination with granzymes results in lysis of whereas Fas-mediated cytotoxicity appears to be the majortarget cells (Shiver et al., 1992), and a calcium-indepen- player in immune homeostasis and tolerance inductiondent pathway, which involves interactions between Fas (Vignaux and Golstein, 1994; Bellgrau et al., 1995).

1ligand (FasL) expressed on cytotoxic T cells (CTLs) and CD8 T cells using the perforin /granzyme pathway arethe Fas protein present on target cells (Rouvier et al., the classical effectors in response to viral antigens. How-

11993; Kagi et al., 1994; Walsh et al., 1994; Takahashi et ever, Th1 CD4 T cells display cytolytic activity throughal., 1994). Fas-mediated lysis is controlled mainly through the FasL/Fas pathway, lysing activated APCs (directthe regulation of FasL expression. FasL expression and targets), and neighboring bystanders (Fas-expressing non-subsequent Fas-mediated cytotoxicity are induced follow- APCs) (Erb et al., 1990; Lancki et al., 1991; Ju, 1991;ing treatment of T cells with a variety of stimuli, including Lowin et al., 1994; Kojima et al., 1994; Ju et al., 1994;

1appropriate Ag–MHC complexes, TCR cross-linking with Stalder et al., 1994). The APC activates the CD4 T cell ina MHC II-restricted manner, leading to the up-regulation

1of FasL, which then enables the CD4 T cell to lyseAbbreviations: APC, antigen presenting cell; CTL, cytotoxic T Fas-bearing targets found in the vicinity, in a MHC-

lymphocyte; FasL, Fas ligand; PACAP, pituitary adenylate cyclase nonrestricted and Ag-nonspecific manner. In vitro, bystan-1activating polypeptide; TCR, T cell receptor; VIP, vasoactive intestinal der lysis by CD4 T cells requires the simultaneous

peptide 1presence of the CD4 cytotoxic T cells, the cognate APC,*Corresponding author. Tel.: 11-973-353-1162; fax: 11-973-353-and the bystander (Wang et al., 1996; Smyth, 1997).1007.

E-mail address: [email protected] (D. Ganea). Although Fas-mediated cytotoxicity plays an important

0165-5728/01/$ – see front matter 2001 Published by Elsevier Science B.V. All rights reserved.PI I : S0165-5728( 00 )00414-8

M. Delgado, D. Ganea / Journal of Neuroimmunology 112 (2001) 78 –88 79

role in maintaining T cell homeostasis, the Fas-mediated mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessentiallysis of bystander targets has been implicated in the aminoacids, 2 mM glutamine, 50 mM 2-mercaptoethanol,pathogenesis of several autoimmune and inflammatory 100 U/ml penicillin, and 10 mg/ml streptomycin (com-diseases, especially in tissues with limited or restricted plete medium).MHC II expression (reviewed in De Maria and Testi, Spleen cells were harvested from Balb /c and C57Bl /61998). The mechanisms limiting the killing of innocent mice and cultured in complete medium with PMA (5bystanders are not understood, and the identification of ng/ml) and ionomycin (1 mg/ml) for 3 days. Spleen blastsfactors that regulate this process represents the focus of were washed three times before use as target cells in theconsiderable research. cytotoxicity assay.

Vasoactive intestinal peptide (VIP), and the structurally Purified macrophages were prepared following i.p.related peptide, the pituitary adenylate cyclase-activating injection of 2 ml of 3% thioglycollate broth (Difco,polypeptide (PACAP), are two neuropeptides released in Detroit, MI). After 4 days, the mice were killed, injectedthe immune microenvironment (Leceta et al., 1996; Bell- i.p. with 5 ml of cold DMEM medium, followed by theinger et al., 1996), affecting both natural and acquired harvesting of peritoneal fluid; the peritoneal exudate cellsimmunity, predominantly as anti-inflammatory agents were washed and macrophages were obtained after the(Ganea, 1996; Delgado et al., 1999a; Pozo et al., 2000). elimination of T and B cells through complement-mediatedRecently we reported that VIP and PACAP inhibit ac- lysis following treatment with anti-Thy-1 and anti-B220tivation-induced FasL expression and subsequent cell death mAbs. The purified macrophage preparations (.96% Mac-

1in T cells and T cell hybridomas (Delgado and Ganea, 1 by FACS analysis) were used as APC (specific targets)12000a), as well as the FasL-mediated cytotoxic activity of for Ag-primed CD4 CTLs in the cytotoxicity assay.

1alloreactive CD8 CTLs (Delgado and Ganea, 2000b). Inthis study, we investigate the effects of VIP and PACAP on 2.3. Generation of effector CTLsFasL/Fas-mediated cytotoxicity of in vivo primed effector

1CD4 T cells against direct and bystander targets. Our Effector CTL generated in response to allogeneic cellsstudies indicate that VIP/PACAP inhibit both the direct were prepared and purified as previously described with

1and bystander lysis of syngeneic targets by CD4 T cells, some modifications (Smyth, 1997). Briefly, C57BL/6 (H-b dthrough the down-regulation of FasL expression on the 2 ) and Balb /c (H-2 ) mice were injected three times at 2

cytotoxic effectors. week intervals into footpads with allogeneic cells: Balb /c1splenocytes, C57Bl /6 splenocytes, or L1210-Fas cells

d 7(H-2 ) (10 cells /mouse). Three days after the final2. Materials and methods immunization the mice were sacrificed, and popliteal

lymph node cells were harvested and incubated in 24-well62.1. Reagents plates (Corning Plastic, Corning, NJ, 4310 cells /ml), in

complete medium with immobilized anti-CD3 mAbsSynthetic VIP, PACAP38, and keyhole limpet (2C11, 5 mg/ml), in the presence or absence of VIP or

28hemocyanin (KLH) were purchased from Calbiochem (San PACAP (10 M) for 16 h at 378C to allow FasLDiego, CA). Monoclonal Abs to murine FasL (CD95L, expression. The lymph node effectors were washed, re-clone MFL3), Fas (CD95, clone Jo2), CD4, CD8 (clone suspended in complete medium, and diluted into V-shaped53-6.7), and CD3e (clone 2C11) were purchased from 96-well microtiter plates (Corning) for the cytotoxicityPharmingen (San Diego, CA). Ovalbumin (OVA), PMA assay.

1and ionomycin were purchased from Sigma Chemicals Co. To generate CD4 CTL in vitro, T cells were purified(St. Louis, MO). from Balb /c spleen by nylon wool filtration, followed by

1]complement-mediated lysis of CD8 T cells. The purified

12.2. Mice, cell lines and cell culture CD4 T cells (.97% by FACS analysis) were stimulatedwith immobilized anti-CD3 mAbs (5 mg/ml), in the

d 28Female 6- to 8-week-old BALB/c (H-2 ) and C57BL/6 presence or absence of VIP or PACAP (10 M) for 16 hb(H-2 ) mice were purchased from Jackson Laboratory (Bar at 378C to allow FasL expression. The cells were har-

Harbor, ME). vested, washed, resuspended in complete medium, anddThe Ia -positive B lymphoma A20.2J was obtained from diluted into 96-well V-bottom plates (Corning) for the

American Type Cell Culture (ATCC, Manassas, VA). cytotoxicity assay.1 d 1L1210-Fas is a L1210 (DBA/2 leukemia cell, H-2 ) To generate Ag-specific CD4 CTLs, Balb /c and

variant transfected with murine Fas (kindly provided by C57Bl /6 mice were immunized i.p. with 50 mg OVA orDr. P. Golstein, Centre d’Immunologie, Marseille-Luminy, KLH, respectively, in CFA as previously described (Erb etFrance) (Rouvier et al., 1993). All cells were grown in al., 1990; Wang et al., 1996). Ten days later, spleens wereRPMI 1640 medium supplemented with 10% heat-inacti- removed and T cells were purified by sequential passage ofvated fetal calf serum (FCS; GIBCO BRL), containing 10 a single cell suspension of splenocytes over two nylon

80 M. Delgado, D. Ganea / Journal of Neuroimmunology 112 (2001) 78 –88

1 1 1wool columns, followed by positive selection of CD4 T CD3 ), anti-CD4 mAb (2% CD4 ), anti-CD8 mAb (4%1cells using Dynabeads-bound anti-CD4 Abs according to CD8 ).

the manufacturer’s instructions (Dynal, Lake Success,1NY). Isolated CD4 T cells were used as Ag-primed 2.5. FACS analysis

effectors for direct or bystander cytolytic activity, by1 6incubating with allogeneic or syngeneic target cells, APC, Effectors and Ag-primed CD4 T cells (1310 cells /

and the corresponding Ag in the presence or absence of ml) prepared and stimulated under the conditions indicated28VIP or PACAP (10 M) as described below. above were harvested in ice-cold complete medium and

washed twice with PBS containing 0.1% sodium azide plus2% heat-inactivated FCS (wash buffer). Cells were incu-

2.4. Cytotoxicity assay bated in wash buffer containing 2.5 mg/ml normal mouseimmunoglobulin for 15 min, after which anti-FasL (MFL3)

51Cytotoxicity was assessed by Cr release assay as mAb was added at 2.5 mg/ml and cells were incubated atdescribed previously (Rogers et al., 1997; Thilenius et al., 48C for 1 h. Isotype-matched mouse Abs were used as1999). The target cells (Balb /c and C57Bl /6 spleen blasts, controls, and IgG block (Sigma) was used to block non-Balb /c and C57Bl /6 macrophages, L1210-wt and L1210- specific binding of the mAb to Fc receptors. Cells were

1 6Fas cells, 10 /ml), were labeled for 2 h at 378C with 200 then washed and further stained with 2.5 mg/ml of FITC-51

mCi of sodium [ Cr]chromate (Amersham, Arlington, IL), conjugated goat F(ab9) anti-hamster IgG (Sigma), for 302

washed three times with PBS/5% FCS, resuspended in min at 48C. After extensive washing, cells were fixed incomplete medium, and added to the wells of 96-well 1% buffered paraformaldehyde. Stained lymphocytes,V-bottom microtiter plates (Corning) at a concentration of gated according to scatter characteristics, were analyzed on

41310 cells /well. Activated lymph node effectors or Ag- a FACScan flow cytometer (Becton Dickinson). Fluores-1primed CD4 CTLs generated as described above were cence data were expressed as mean channel fluorescence

added to the microtiter plates in graded dilutions to obtain (MCF) and as percentage (%) of positive cells afterthe desired effector:target (E:T) ratios. In assays for direct subtraction of background isotype-matched values.

51targets, Cr-labeled macrophages from Balb /c mice were1incubated with OVA-primed Balb /c CD4 T cells, and 2.6. RNA extraction and Northern blot analysis

stimulated with 10 mg/ml OVA in the presence or absence28of VIP or PACAP (10 M). In assays for syngeneic Northern blot analysis was performed according to

51 1 1bystanders, Cr-labeled target cells (L1210-Fas cells) standard methods. Effectors and Ag-primed CD4 T cellswere incubated with Balb /c macrophages, OVA-primed were prepared and stimulated as described above. At the

1 7Balb /c CD4 T cells, and stimulated with OVA (10 various time points, 1310 cells were harvested and totalmg/ml) in the presence or absence of VIP or PACAP RNA was extracted by the acid guanidinium–phenol–

28 51(10 M). In assays for allogeneic bystanders, Cr-labeled chloroform method, electrophoresed on 1.2% agarose–1target cells (L1210-Fas cells) were incubated with formaldehyde gels, transferred to S&S Nytran membranes

1C57Bl /6 macrophages, KLH-primed C57Bl /6 CD4 T (Schleicher and Schuell, Keene, NJ), and cross-linked tocells, and stimulated with KLH (50 mg/ml) in the presence the nylon membrane using UV light. The probes for

28or absence of VIP or PACAP (10 M). The plates were murine FasL and GAPDH were generated by RT-PCR ascentrifuged at 300 g to promote conjugate formation and described previously (Zheng et al., 1998) using the follow-incubated 18 h at 378C, recentrifuged, and 100-ml superna- ing primers: FasL, 59-TCACCAACCAAAGCCTTAAAG-tant aliquots were removed for radioactivity measurements TAT-39 and 59-TCAACCTCTTCTCCTCCATTAGCA-39;in a Beckman Gamma 8000 counter (Beckman, Fullerton, and GAPDH, 59-TCCTGCACCACCAACTGCTTACC-39,CA). The percent lysis was determined by the following and 59-GTTCAGCTCTTGGATGACCTTGCC-39. Oligo-

32equation: % lysis5(E2S) /(M2S)3100, where E is the nucleotides were end-labeled with g P-dATP (Amersham,release from experimental samples, S is the spontaneous Arlington, IL) by using T4 polynucleotide kinase. Therelease, and M is the maximum release upon lysis with RNA-containing membranes were prehybridized for 16 h

5110% SDS. The spontaneous release of Cr was deter- at 428C, and then hybridized at 608C for 16 h with themined by incubating the target cells with medium alone. In appropriate probes. The membranes were then washedmost experiments, cytotoxicity assays were performed in twice in 23SSC containing 0.1% SDS at room tempera-the presence of 4 mM EGTA/3 mM MgCl (Sigma). In ture (20 min each time), once at 378C for 20 min, and once2

some experiments, lymph node effectors were depleted in 0.13SSC containing 0.1% SDS at 508C (20 min). Thewith anti-CD3, anti-CD4, or anti-CD8 mAb (10 mg/ml) prehybridization and hybridization buffers were purchasedfollowed by complement lysis (rabbit complement, Pel from 59 Prime-39 Prime (Boulder, CO). The membranesFreeze, Rogers, AR), before examining the cytotoxic were exposed to X-ray films (Kodak, Rochester, NY).activity. The remaining populations, assessed by flow Signal quantitation was performed in a PhosphorImager SIcytometry, were as follows: anti-CD3 mAb treated (3% (Molecular Dynamics, Sunnyvale, CA).


b3. Results generated allogeneic Balb /c anti-H-2 effector CTL invivo in response to C57Bl /6 spleen cells. The effector

3.1. VIP and PACAP inhibit the FasL /Fas-mediated CTL harvested from lymph nodes were activated in vitrocytotoxicity of allogeneic CTLs generated in vivo with immobilized anti-CD3 Abs in the presence or absence

of VIP or PACAP, and the cytotoxic activity against bothWe have previously shown that VIP and PACAP inhibit syngeneic and allogeneic targets was determined. Similar

FasL/Fas-mediated, but not the perforin /granzyme-me- to CTL hybridomas and primary CTLs (Young, 1996;diated cytotoxicity of in vitro generated CTLs against Suda et al., 1993; Rouvier et al., 1993; Glass et al., 1996),allogeneic targets (Delgado and Ganea, 2000b). Here, we the anti-CD3-activated CTLs expressed full lytic activity

21 7Fig. 1. VIP and PACAP inhibit the Ca -independent direct and bystander cytotoxicity of allogeneic CTLs. C57Bl /6 spleen cells (10 /50 ml) wereinjected three times at 2 week intervals into footpads of Balb /c mice (groups of 4). Three days after the final immunization, the Balb /c anti-B6 effectorcells were isolated from popliteal lymph nodes and activated by incubation with immobilized anti-CD3 mAbs in 96-well plates (Ab concentration: 5

28 51mg/ml), with or without VIP or PACAP (10 M) for 16 h. A. Activated effector cells were added at various effector:target ratios to Cr-labeled target

4cells (1310 cells /well). Target cells: allogeneic B6 spleen blasts (left and middle panels), and syngeneic Balb /c spleen blasts (right panel). The plates51were centrifuged and incubated with or without 4 mM EGTA/3 mM MgCl (1EGTA) for 12 h. Supernatants were harvested to determine Cr release.2

51Spontaneous Cr release ranged between 11 and 17%. The results were calculated as the mean of triplicate samples. B. Activated effector cells were added51 4at various effector:target ratios to a mix of Cr-labeled syngeneic Balb /c spleen blasts (bystander targets, 1310 cells /well) and unlabeled allogeneic B6

4 51spleen blasts (specific targets, 1310 cells /well). Supernatants were harvested 12 h later to determine Cr release. Cytotoxicity was expressed as specific51 51Cr release after subtraction of spontaneous Cr release, which ranges between 9 and 15%. The results were calculated as the mean of triplicate samples.

51 4C. Activated effector cells were added at various effector:target ratios to a mix of Cr-labeled allogeneic B6 spleen blasts (specific targets, 13104 51cells /well) and unlabeled syngeneic Balb /c spleen blasts (bystander targets, 1310 cells /well). Supernatants were harvested 12 h later to determine Cr

51release. Spontaneous Cr release ranged between 7 and 19%. The results were calculated as the mean of triplicate samples.


Fig. 2. VIP and PACAP inhibit the FasL-mediated bystander lysis of allogeneic CTLs. Allogeneic effectors were generated in vivo and activated in vitro as51 1 ddescribed in Fig. 1. Activated effector cells were added at various effector:target ratios to a mix of Cr-labeled syngeneic L1210-Fas cells (H-2 ) or

4 4L1210-wt cells (right panel) (1310 cells /well) and unlabeled allogeneic B6 spleen blasts (specific targets, 1310 cells /well). Supernatants were51 51harvested 12 h later to determine Cr release. Spontaneous Cr release ranged between 13 and 19%. The results were calculated as the mean of triplicate

samples.

Fig. 3. VIP and PACAP inhibit activation-induced FasL expression in the effector cells. Allogeneic effector cells were generated in vivo and activated invitro as described in Fig. 1. Effector cells cultured with medium alone (without anti-CD3, VIP, or PACAP) were used to measure the basal levels of FasLexpression (dotted lines). The expression surface FasL was analyzed by flow cytometry 12 h later (FI, fluorescence intensity; MCF, mean channelfluorescence). FasL mRNA expression was analyzed by Northern blot 4 h after activation (results are expressed in arbitrary densitometric units normalizedfor the expression of GAPDH in each sample). The data presented are representative of three experiments.


1Fig. 4. VIP and PACAP inhibit the FasL-mediated bystander cytotoxicity of allogeneic CD4 T cell effectors.A. The in vivo generated allogeneic effectorsacting on bystanders are CD41 T cells. Allogeneic effectors were generated in vivo and activated in vitro as described in Fig. 1 by incubation with PMA(5 ng/ml) and ionomycin (1 mg/ml) for 16 h. The activated effectors were divided into four aliquots, treated with various mAbs and C (closed circles, no

51Ab; open circles, anti-CD8; closed triangles, anti-CD4; open triangles, anti-CD3), and added at various effector:target ratios to a mix of Cr-labeled1 4 4syngeneic L1210-Fas cells (left panel) or Balb /c spleen blasts (right panel) (1310 cells /well) and unlabeled B6 spleen blasts (specific targets, 1310

51 51cells /well). Supernatants were harvested 12 h later to determine Cr release. Spontaneous Cr release ranged between 9 and 14%. The results were1calculated as the mean of triplicate samples. B. VIP and PACAP inhibit the Fas-mediated lysis of syngeneic targets by CD4 CTLs generated in vitro.

1Purified splenic CD4 T cells were incubated with immobilized anti-CD3 mAb in 96-well plates (Ab concentration: 5 mg/ml), with or without VIP or28 1PACAP (10 M) for 16 h as described in Materials and Methods. The activated CD4 T cells were added at various effector:target ratios to syngeneic

d 51 1 4(H-2 ) target cells, i.e. Cr-labeled syngeneic L1210-Fas cells (left panel), or A20.2J B cells (right panel) (1310 cells /well). The plates were51centrifuged and incubated in the presence of 4 mM EGTA/3 mM MgCl (1EGTA). Supernatants were harvested 12 h later to determine Cr release.2

51Spontaneous Cr release ranged between 10 and 21%. The results were calculated as the mean of triplicate samples.


against the allogeneic targets (Fig. 1A, left panel), and a targets (bystanders) in the presence of the specific al-significant reduction in the cytotoxic response was ob- lotargets, through the FasL/Fas-mediated pathway (Smyth,served upon treatment of EGTA (Fig. 1A, middle panel). 1997). To determine the effect of VIP and PACAP onIn contrast, CTL failed to lyse syngeneic Balb /c spleen bystander lysis, Balb /c anti-B6 effectors were activatedblasts (Fig. 1A, right panel). As shown before, VIP and with immobilized anti-CD3 in the presence or absence of

21PACAP had no effect on the Ca -dependent CTL-me- VIP or PACAP, and examined for lysis of cocultureddiated cytotoxic activity, but inhibited significantly the Balb /c spleen blasts (syngeneic) and specific allotargets

21Ca -independent lysis (Fig. 1A). (C57Bl /6 spleen blasts). The presence of bystanders didAllogeneic CTLs were reported to also lyse syngeneic not influence the cytotoxic activity against the allogeneic


targets (Fig. 1B, left and middle panels). Bystander lysis of CTL expressed low levels of both FasL protein andBalb /c spleen blasts was observed in response to the mRNA, TCR-stimulation by CD3 cross-linking resulted inallotargets, and was not affected by EGTA (Fig. 1B right a great increase in FasL expression (Fig. 3). Treatmentpanels). This indicates that the bystander lysis is mediated with VIP or PACAP significantly decreased TCR-inducedthrough the FasL/Fas pathway as previously described. FasL expression at both protein and mRNA levels (Fig. 3).VIP and PACAP significantly inhibited bystander lysis Therefore, inhibition of FasL/Fas-mediated lysis of(Fig. 1B right panels). bystander targets by VIP/PACAP correlates with the down-

To confirm these results, we examined the cytotoxicity regulation of FasL expression in the alloreactive CTL.1of Balb /c anti-B6 effectors against syngeneic L1210-Fas

bystander targets in cocultures with the specific allogeneic1 1targets. The Balb /c effectors lysed the L1210-Fas cells, 3.3. VIP and PACAP inhibit CD4 CTL FasL /Fas-

but not the L1210-wt controls, and this lysis was not dependent lysis of bystander targetsaffected by EGTA (Fig. 2). Again, VIP and PACAP

1inhibited the lysis of the syngeneic L1210-Fas bystanders Previous studies showed that FasL/Fas-mediated cyto-(Fig. 2). These results demonstrate that VIP/PACAP toxicity against bystander targets was mediated primarily

21 1inhibit the Ca -independent, FasL/Fas-mediated lysis of by effector CD4 T cells, particularly Th1 (Ju et al., 1994;bystander syngeneic targets in CTL responding to an Stalder et al., 1994; Hahn et al., 1995; Wang et al., 1996;allogeneic stimulus. Smyth, 1997). To determine the phenotype of the effectors

mediating bystander lysis, Balb /c anti-B6 effectors weredepleted of various T cell subsets by complement-mediated

3.2. VIP and PACAP inhibit activation-induced FasL lysis, and examined for lysis of syngeneic Balb /c blasts1expression in alloreactive CTLs generated in vivo and L1210-Fas cells in the presence of the specific

1 1allogeneic targets. Depletion of CD3 or CD4 T cellsSince FasL/Fas-mediated cytotoxicity is controlled significantly abrogated the FasL/Fas-mediated lysis of

1mostly through the regulation of FasL expression, and bystanders (Fig. 4A). In contrast, depletion of CD8 TVIP/PACAP were reported to inhibit activation-induced cells had a much lesser effect (Fig. 4A). This indicates thatFasL expression in T cells (Delgado and Ganea, 2000a), the FasL/Fas-mediated lysis of bystander targets is me-

1we determined whether VIP/PACAP downregulate FasL diated primarily by CD4 T cells.b 1expression in the activated effector CTL. Balb anti-H-2 To confirm that VIP and PACAP affect CD4 T cell

effectors were generated in vivo and stimulated with FasL/Fas-mediated cytotoxicity, we induced FasL expres-1immobilized anti-CD3 mAbs in the presence or absence of sion in vitro by incubating Balb /c spleen CD4 T cells

VIP or PACAP. The expression of FasL was determined at with immobilized anti-CD3 mAbs in the presence or21protein and mRNA level by flow cytometry and Northern absence of VIP or PACAP. The Ca -independent cytotox-

blot analysis, respectively. Whereas unstimulated effector icity was examined in the presence of EGTA against the

1Fig. 5. VIP and PACAP inhibit the FasL-dependent direct and bystander lysis by Ag-specific CD4 effectors generated in vivo. A.VIP and PACAP inhibit1FasL-mediated direct killing by Ag-specific CD4 effectors. Balb /c mice (groups of 4) were immunized i.p. with 50 mg OVA in CFA, and ten days later,

1 1 dsplenic CD4 T cells were isolated as described in Methods. Freshly isolated and purified OVA-primed CD4 T cells (H-2 ) were added at various4 51 28effector:target ratios to wells containing 1310 Cr-labeled syngeneic APC (Balb /c macrophages), 10 mg/ml OVA, and VIP or PACAP (10 M).

51Controls contained no Ag. After 16 h incubation in the presence of 4 mM EGTA/3 mM MgCl , supernatants were harvested to determine Cr release.251 51Spontaneous Cr release ranged between 11 and 16%. No significant Cr release was observed after incubation of cells with an irrelevant Ag (50 mg/ml

KLH). The results were calculated as the mean of triplicate samples. B. VIP and PACAP inhibit FasL-mediated syngeneic bystander killing by Ag-specific1 1 4CD4 effectors. Freshly isolated and purified OVA-primed CD4 T cells (see above) were added at various effector:target ratios to wells containing 1310

51 1 4 28Cr-labeled syngeneic non-APC targets (L1210-Fas cells), 1310 Balb /c macrophages, and 10 mg/ml OVA. VIP or PACAP (10 M) were added to the51 51cultures. After incubation for 16 h in the presence of EGTA, supernatants were harvested to determine Cr release. Spontaneous Cr release ranged

51between 16 and 24%. No significant Cr release was observed after incubation of cells with an irrelevant Ag (50 mg/ml KLH; closed rectangle). The1results were calculated as the mean of triplicate samples. C. VIP and PACAP inhibit FasL-mediated allogeneic bystander killing by Ag-specific CD4

1effectors. Groups of four C57Bl /6 mice were immunized i.p. with 50 mg KLH in CFA, and 10 days later, splenic CD4 T cells were isolated as described1 b 4in Methods. Freshly isolated and purified KLH-primed CD4 T cells (H-2 ) were added at various effector:target ratios to wells containing 1310

51 1 d 4 28Cr-labeled allogeneic target cells (L1210-Fas cells, H-2 ), 1310 syngeneic B6 macrophages, and 50 mg/ml KLH. VIP or PACAP (10 M) were51 51added to the cultures. After incubation for 16 h in the presence of EGTA, supernatants were harvested to determine Cr release. Spontaneous Cr release

51ranged between 13 and 24%. No significant Cr release was observed after incubation of cells with an irrelevant Ag (10 mg/ml OVA; closed rectangle).1The results were calculated as the mean of triplicate samples. D. VIP and PACAP inhibit FasL mRNA expression in CD4 effectors. Freshly isolated and

1 7 5purified OVA-primed Balb /c CD4 T cells (2310 ) were incubated for 8 h with 5310 syngeneic macrophages, and 10 mg/ml OVA, in the presence or28absence of VIP or PACAP (10 M). Cells incubated with medium alone without Ag (unstimulated) were used as controls. FasL mRNA expression was

determined by Northern blot analysis. The data presented are representative of three different experiments.


1 dsyngeneic targets L1210-Fas and A20.2J B cells (a H-2 4. Discussionlymphoma expressing high Fas levels). Both Fas-bearing

1targets were lysed by the activated CD4 T cells, and in FasL/Fas-mediated cytotoxicity plays a major role inboth cases VIP and PACAP inhibited the cytotoxic activity the homeostasis of the immune system. Apoptosis of

1(Fig. 4B). However, addition of VIP or PACAP 16 h after CD4 T cells during activation-induced cell death, whichanti-CD3 activation, at the time of the cytotoxic assay, did reduces the numbers of antigen-activated T cells followingnot affect the cytotoxicity against the two targets (Fig. 4C). an immune response, and the killing of activated antigen-

1This suggests that VIP and PACAP affect FasL expression, presenting cells by CD4 lytic effectors occur throughbut not events subsequent to FasL/Fas interactions. FasL/Fas interactions (Ju et al., 1994, 1995; Stalder et al.,

1994). In contrast, although Fas-mediated lysis operates in1the elimination of virally-infected targets by CD8 CTLs,

13.4. VIP and PACAP inhibit direct and bystander lysis the major lytic pathway for the CD8 effectors involves1by antigen-specific CD4 T cells perforin in conjunction with granzymes (Kagi et al., 1994;

Walsh et al., 1994; Lowin et al., 1995). Previous studiesIt has been previously described that antigen presenta- from our laboratory indicated that two structurally related

tion by macrophages and B cells stimulates antigen-spe- neuropeptides, VIP and PACAP, present in the lymphoid1cific CD4 T cells to express FasL and to mediate FasL/ microenvironment, prevent FasL induction on activated T

Fas-dependent lysis of the direct targets (the stimulating cells and inhibit the subsequent FasL/Fas-mediated lysis.APCs), and of bystander targets (Wang et al., 1996). To VIP and PACAP reduce activation-induced cell death ininvestigate the effects of VIP and PACAP on FasL/Fas- normal and hybridoma T cells (Delgado and Ganea,

21mediated direct and bystander lysis by antigen-specific 2000a), and inhibit the Ca -independent, Fas-mediated1 1 1CD4 T cells, we generated OVA-primed Balb /c CD4 T cytotoxicity of CD8 T cells and T1 effectors (Delgado

cells in vivo and incubated them in vitro in the presence of and Ganea, 2000b). In this study, we investigated the51EGTA with OVA and Cr-labeled Balb /c macrophages, effects of VIP and PACAP on FasL expression and

1 1plus or minus VIP or PACAP. The in vivo primed CD4 T cytolytic activity of CD4 lytic effectors. We used two1cells efficiently killed syngeneic macrophages (direct different systems for generating CD4 lytic effectors. In

1targets), and the treatment with VIP or PACAP signifi- the first system, CD4 effectors were generated in vivo by21cantly reduced this Ca -independent lysis (Fig. 5A). To allogeneic stimulation, followed by in vitro TCR stimula-

determine the effect of the neuropeptides on bystander tion. The lytic activity was determined against specific1lysis, OVA-primed Balb /c CD4 T cells were stimulated allogeneic targets and syngeneic, Fas-bearing bystanders.

1with OVA, syngeneic Balb /c macrophages (APCs), and In the second system, antigen-specific CD4 effectors51 1Cr-labeled syngeneic L1210-Fas cells, in the presence were generated in vivo, followed by in vitro incubationor absence of VIP and PACAP. The stimulated T cells with antigen, APCs, and syngeneic or allogeneic Fas-lysed the Fas-bearing bystanders, but not the FasL-insensi- bearing bystanders. The lytic activity was determined withtive L1210-wt cells. VIP and PACAP reduced the FasL/ APC (direct targets), and with bystander targets. In the first

21Fas-mediated cytotoxicity against bystanders (Fig. 5B). system, VIP and PACAP inhibited the Ca -independent,Finally, we determined the effects of VIP/PACAP on Fas-mediated lysis of both specific allotargets and

1antigen-specific CD4 T cell cytotoxicity against al- syngeneic bystanders. Similarly, in the second system, VIP1logeneic bystanders. KLH-primed C57Bl /6 CD4 T cells and PACAP inhibited the Fas-mediated lysis of both direct

were incubated in vitro with antigen (KLH), syngeneic (APCs) and bystander targets. In both cases, the inhibition51C57Bl /6 macrophages, and Cr-labeled allogeneic of the cytotoxic activity correlated with a reduction in

1 1L1210-Fas cells (which lack MHC-restricting element), FasL expression on the CD4 effectors.1in the presence or absence of VIP or PACAP. The activated FasL-mediated bystander lysis by CD4 effectors has

1CD4 T cells effectively lysed allogeneic Fas-bearing been previously described both in response to an al-bystanders, but failed to kill the FasL-insensitive L1210-wt logeneic stimulus and to specific antigen presentation by

1cells (Fig. 5C). In contrast, L1210-Fas cells were not APCs (Wang et al., 1996; Smyth, 1997). In both cases,1killed by unstimulated effector CD4 T cells, or by bystander lysis requires close contact between the effector

effectors stimulated with the irrelevant antigen OVA (Fig. T cell, the APC or the allotarget, and the bystander target.5C). Incubation with VIP and PACAP reduced the killing This requirement suggests that restimulation with the

1of allogeneic bystanders (Fig. 5C). The levels of bystander specific antigen is necessary to fully activate the CD41killing correlate with FasL expression in the effector CD4 effectors for bystander lysis (Smyth, 1997). In contrast to

1T cell, since antigen-stimulated CD4 T cells show a the direct targets (antigen-presenting APCs) which aresignificantly higher expression of FasL mRNA in com- lysed in a MHC-restricted, Ag-specific manner, the onlyparison with unstimulated cells, or with stimulated cells requirement for the lysis of bystanders appears to betreated with VIP or PACAP (Fig. 5D). surface Fas expression. Therefore, interactions between the


effector T cell and APC, not between T cells and bystan- Acknowledgements1ders, are critical for the ability of the CD4 effectors to

lyse bystander targets. Both the stimulatory signal, pro- We thank Dr. Pierre Golstein (Centre d’Immunologievided by TCR/Ag peptide /MHCII interactions, and the INSERM-CNRS, Marseille, France) for L1210 and L1210-

1costimulatory signal provided by CD28/B7 interaction Fas cells.appear to be required for bystander lysis, presumably This work was supported by grants PHS AI 041786-01through optimal upregulation of FasL expression (DG), and Busch Biomedical Award 98-00 (DG), by grant(Thilenius et al., 1999). We observed indeed upregulation PM98-0081 (MD), and by the postdoctoral fellowshipof FasL (protein and mRNA) in both allogeneic and from the Spanish Department of Education and Science

1antigen-specific CD4 effectors upon restimulation. (MD).The inhibitory effects of VIP and PACAP on direct and

1bystander lysis by CD4 effectors appear to be mediatedthrough down-regulation of FasL expression at both ReferencesmRNA and protein level. This is in agreement withprevious observations that both neuropeptides decrease Bellgrau, D., Gold, D., Selawry, H., Moore, J., Franzusoff, A., Duke,

R.C., 1995. A role for CD95 ligand in preventing graft rejection.activation-induced FasL expression in mature T cells and T1 Nature 377, 630–632.cell hybridomas and in CD8 CTL (Delgado and Ganea,

Bellinger, D.L., Lorton, D., Brouxhon, S., Felten, S., Felten, D.L., 1996.2000a,b). Downregulation of FasL could be due in part to The significance of vasoactive intestinal peptide (VIP) in immuno-the effect of VIP/PACAP on B7.1 expression. Previous modulation. Adv. Neuroimmunol. 6, 5–27.studies indicated that VIP and PACAP inhibit B7.1 /B7.2 Berke, G., 1995. The CTL kiss of death. Cell 81, 9–12.

De Maria, R., Testi, R., 1998. Fas-FasL interactions: a common patho-expression and the costimulatory function of LPS-stimu-genetic mechanism in organ-specific autoimmunity. Immunol. Todaylated macrophages (Delgado et al., 1999b), and the same19, 121–125.

phenomenon may occur in vivo during the generation of Delgado, M., Munoz-Elias, E.J., Martinez, C., Gomariz, R.P., Ganea, D.,1CD4 lytic effectors. 1999a. Vasoactive intestinal peptide (VIP) and pituitary adenylate

1The elimination of activated APCs by CD4 lytic cyclase-activating polypeptide (PACAP38) modulate cytokine andnitric oxide production in peritoneal macrophages and macrophageeffectors is thought to be one of the mechanisms forcell lines. Ann. NY Acad. Sci. 897, 401–414.immune homeostasis, by removing potentially harmful

Delgado, M., Sun, W., Leceta, J., Ganea, D., 1999b. VIP and PACAPcells after the completion of an immune response. How- differentially regulate the costimulatory activity of resting and acti-ever, bystander killing appears to represent a collateral vated macrophages through the modulation of B7.1 and B7.2 expres-damage in this process (Hahn et al., 1995). This is sion. J. Immunol. 163, 4213–4223.

Delgado, M., Ganea, D., 2000a. VIP and PACAP inhibit antigen-inducedparticularly relevant for tissues with limited MHC class IIapoptosis of mature T lymphocytes by inhibiting Fas ligand expres-expression such as the brain, where myelin basic protein-sion. J. Immunol. 165, 1200–1210.1specific CD4 cytotoxic cells contribute to the develop- Delgado, M., Ganea, D., 2000b. VIP and PACAP inhibit T-cell mediated

ment of experimental allergic encephalomyelitis (EAE), cytotoxicity by inhibiting FasL expression. J. Immunol. 165, 114–apparently through collateral damage to Ag-nonspecific 123.

Erb, P., Grogg, D., Troxler, M., Kennedy, M., Fluri, M., 1990. CD41 Tbystanders (Thilenius et al., 1999). Similar mechanismscell-mediated killing of MHC class II-positive antigen-presentingmay be responsible for damage in other autoimmunecells. Characterization of target cell recognition by in vivo or in vitro

diseases (insulin-dependent diabetes mellitus, Hashimoto’s activated CD41 killer T cells. J. Immunol. 144, 790–795.thyroiditis, autoimmune hepatitis, ulcerative colitis, aplas- Gagnon, S.J., Ennis, F.A., Rothman, A.L., 1999. Bystander target celltic anemia), or infectious diseases such as Dengue hemor- lysis and cytokine production by Dengue virus-specific human CD41

cytotoxic T lymphocyte clones. J. Virol. 73, 3623–3629.rhagic fever (De Maria and Testi, 1998; Gagnon et al.,Ganea, D., 1996. Regulatory effects of vasoactive intestinal peptide on1999). From this point of view, the ability to modulate the

cytokine production in central and peripheral lymphoid organs. Adv.Fas-FasL system in selected areas of the body may yield Neuroimmunol. 6, 61–74.new hopes for effective therapeutic intervention. Although Glass, A., Walsh, C.M., Lynch, D.H., Clark, W.R., 1996. Regulation of theFas is constitutively expressed in most cell types, FasL is Fas lytic pathway in cloned CTL. J. Immunol. 156, 3638–3644.

Hahn, S., Gehri, R., Erb, P., 1995. Mechanism and biological significancehighly upregulated in T lymphocytes upon activation.of CD4-mediated cytotoxicity. Immunol. Rev. 146, 57–79.Therefore, factors such as VIP and PACAP, which dow-

Ju, S.T., 1991. Distinct pathways of CD4 and CD8 cells induce rapidnregulate FasL expression, might be useful in the control target DNA fragmentation. J. Immunol. 146, 812–818.of Fas-mediated lysis of innocent bystanders. Based on the Ju, S., Ciu, H., Panaka, D.J., Ettinger, R., Marshak-Rothstein, A., 1994.fact that VIP and PACAP are normally present in the Participation of target Fas protein in the apoptosis pathway induced

by CD41 Th1 and CD81 cytotoxic cells. Proc. Natl. Acad. Sci. USAimmune microenvironment, and affect immune cells, in-91, 4185–4189.cluding T lymphocytes, through specific receptors, we

Ju, S.T., Panka, D.J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D.H.,propose that together with cytokines and hormones, these Stanger, B.A., Marshak-Rothstein, A., 1995. Fas (CD95) /FasL inter-two neuropeptides act as endogenous regulatory molecules actions required for programmed cell death after T-cell activation.that control immune homeostasis. Nature 373, 444–448.


Kagi, D., Vignaux, F., Ledermann, B., Burki, K., Depraetere, V., Nagata, Shiver, J.W., Su, L., Henkart, P.A., 1992. Cytotoxicity with target DNAS., Hengartner, H., Golstein, P., 1994. Fas and perforin pathways as breakdown by rat basophilic leukemia cells expressing both cytolysinmajor mechanisms of T cell-mediated cytotoxicity. Science 265, and granzyme A. Cell 71, 315–322.528–530. Smyth, M.J., 1997. Fas ligand-mediated bystander lysis of syngeneic cells

Kojima, H., Shinohara, N., Hanaoka, S., Someya-Shirota, Y., Takagaki, Y., in response to an allogeneic stimulus. J. Immunol. 158, 5765–5772.Ohno, H., Saito, T., Katayama, T., Yagita, H., Okumura, K., Shinkai, Stalder, T., Hahn, S., Erb, P., 1994. Fas antigen is the major targetY., Alt, F.W., Matsuzawa, A., Yonehara, S., Takayama, H., 1994. Two molecule for CD41 T cell-mediated cytotoxicity. J. Immunol. 152,distinct pathways of specific killing revealed by perforin mutant 1127–1133.cytotoxic T lymphocytes. Immunity 1, 357–364. Suda, T., Takahashi, T., Golstein, P., Nagata, S., 1993. Molecular cloning

Lancki, D.W., Hsieh, C.S., Fitch, F.W., 1991. Mechanism of lysis by an expression of the Fas ligand, a novel member of the tumor necrosiscytotoxic T lymphocyte clones: lytic activity and gene expression in factor family. Cell 75, 1169–1178.cloned antigen-specific CD41 and CD81 T lymphocytes. J. Im- Takahashi, T., Tanaka, M., Brannan, C.I., Jenkins, N.A., Copeland, N.G.,munol. 146, 3242–3249. Suda, T., Nagata, S., 1994. Generalized lymphoproliferative disease in

Leceta, J., Martinez, C., Delgado, M., Garrido, E., Gomariz, R.P., 1996. mice caused by a point mutation in the Fas ligand. Cell 76, 969–976.Expression of vasoactive intestinal peptide in lymphocytes: a possible Thilenius, A.R.B., Sabelko-Downes, K.A., Russell, J.H., 1999. The roleendogenous role in the regulation of the immune response. Adv. of the antigen-presenting cell in Fas-mediated direct and bystanderNeuroimmunol. 6, 29–36. killing: potential in vivo function of Fas in experimental allergic

Lowin, B., Hahne, M., Mattman, C., Tschopp, J., 1994. Cytolytic T cell encephalomyelitis. J. Immunol. 162, 643–650.cytotoxicity is mediated through perforin and Fas lytic pathways. Vignaux, F., Golstein, P., 1994. Fas-based lymphocyte-mediated cytotox-Nature 370, 650–652. icity against syngeneic activated lymphocytes: a regulatory pathway.

Lowin, B., Beerman, F., Schmidt, A., Tschopp, J., 1995. A null mutation Eur. J. Immunol. 24, 923–927.in the perforin gene impairs cytolytic T lymphocyte- and natural killer Walsh, C.M., Glass, A.A., Chiu,V., Clark, W.R., 1994. The role of the Fascell-mediated cytotoxicity. Proc. Natl. Acad. Sci. USA 91, 11571– lytic pathway in a perforin-less CTL hybridoma. J. Immunol. 153,11575. 2506–2514.

Nagata, S., Golstein, P., 1995. The Fas death factor. Science 267, Wang, R., Rogers, A.M., Ratliff, R.L., Russell, J.H., 1996. CD95-1449–1456. dependent bystander lysis caused by CD41 T helper 1 effectors. J.

Pozo, D., Delgado, M., Martinez, C., Leceta, J., Gomariz, R.P., Guerrero, Immunol. 157, 2961–2968.J.M., Calvo, J.R., 2000. Immunobiology of VIP. Immunol. Today 21, Young, J.D.E., 1996. Killing of target cells by lymphocytes: a mechanistic7–12. view. Physiol. Rev. 69, 250–314.

Rogers, A.M., Thilenius, A.R.B., Russell, J.H., 1997. Cyclosporine- Zheng, L., Trageser, C.L., Willerford, D.M., Lenardo, M.J., 1998. T cellinsensitive partial signaling and multiple roles of Ca21 in Fas growth cytokines cause the superinduction of molecules mediatinglingan-induced lysis. J. Immunol. 159, 3140–3149. antigen-induced T lymphocyte death. J. Immunol. 160, 763–769.

Rouvier, E., Luciani, F., Golstein, P., 1993. Fas involvement in Ca21-independent T cell-mediated cytotoxicity. J. Exp. Med. 177, 195–200.

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VIP and PACAP inhibit Fas ligand-mediated bystander lysis by CD4+ T cells