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The Journal of Immunology, 2006, 176: 97-110.
Copyright © 2006 by The American Association of Immunologists

Granzyme B, a New Player in Activation-Induced Cell Death, Is Down-Regulated by Vasoactive Intestinal Peptide in Th2 but Not Th1 Effectors1

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

* Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and {dagger} Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas, Granada, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Following antigenic stimulation and differentiation, Th1 and Th2 effector cells contribute differently to cellular and humoral immunity. Vasoactive intestinal peptide (VIP) induces Th2 responses by promoting Th2 differentiation and survival. In this study, we investigate the mechanisms for the protective effect of VIP against activation-induced cell death (AICD) of Th2 effectors. Surprisingly, microarray and protein data indicate that VIP prevents the up-regulation of granzyme B (GrB) in Th2 but not Th1 effectors. This is the first report of GrB expression in Th cells and of its involvement in activation-induced apoptosis. The enhanced responsiveness of Th2 cells to VIP is probably due to the higher expression of VIP receptors. The effect of VIP on Th2 survival and GrB expression is mediated through the VIP receptors 1 and 2 and cAMP signaling through exchange protein activated by cAMP and, to a lesser degree, protein kinase A. In addition to effects on GrB, VIP also down-regulates Fas ligand (FasL) and perforin (Pfr) expression. The extrinsic Fas/FasL pathway and the intrinsic GrB-dependent pathway act independently in inducing AICD. The mechanisms by which GrB induces cell death in Th1/Th2 effectors include both fratricide and suicide. Fratricide killing, prevalent in wild-type cells, is calcium and Pfr dependent, whereas the cell death of Pfr-deficient Th cells involves Fas and GrB but is calcium independent. This study identifies GrB as a new significant player in Th1/Th2 AICD and characterizes two mechanisms for the protective effect of VIP on Th2 survival, i.e., the down-regulation of GrB and FasL expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Following antigenic stimulation, CD4+ T cells differentiate into Th1 and Th2 effectors with different cytokine profiles and different physiological functions (1, 2). The differentiation into Th1/Th2 effectors is controlled by various factors, such as the nature of the APCs, the nature and amount of Ag, the genetic background of the host, and particularly the cytokine microenvironment (3, 4).

However, regardless of the initial Th1/Th2 balance, the final immune response depends to a large degree on the regulation of Th1 or Th2 survival under specific conditions. Differentiating Th1 and Th2 cells undergo several rounds of rapid divisions, resulting in a high number of Ag-specific effectors. This is followed at later time points by apoptosis, thus maintaining homeostasis and ensuring that only a defined number of specialized memory CD4+ T cells survive. Clonally expanded CD4+ T cells are eliminated primarily through activation-induced cell death (AICD),3 and the major mechanism involves signaling through the death receptor CD95 (Fas) (5, 6). Although both Th1 and Th2 effectors are ultimately eliminated, Th1 cells are more susceptible to AICD (7, 8, 9). Recent studies indicate that Th1 cells are more susceptible to death induced by either Fas ligand (FasL) or TRAIL, and that both Th1 and Th2 cells can kill Th1 but not Th2 targets (10, 11). The reason for the higher degree of resistance of the Th2 effectors is still under debate. In most cases, the resistance to AICD correlated with a lower expression of FasL (7, 11, 12), although this has not always been the case (8, 13). Some studies indicated that the Fas-associated protease Fas-associated phosphatase 1 (FAP-1), which blocks Fas-mediated signaling, was up-regulated in murine Th2 cells (9, 14), although this was not the case in human T cell clones (15). Recently, Pandiyan et al. (16) implicated CD152 (CTLA-4) as the determining factor in Th2 resistance. CD152 activates the PI3K, leading to the inactivation of the proapoptotic molecules Bad and the Forkhead transcription factor FKHRL1, which regulates FasL expression.

Endogenous factors such as progesterone, glucocorticoids, and neuropeptides, including vasoactive intestinal peptide (VIP) and pituitary adenylate-cyclase activating polypeptide (PACAP), have been reported to favor Th2 differentiation (17, 18). VIP/PACAP were shown to affect Th1/Th2 differentiation through effects on APCs and direct effects on the master transcriptional factors c-Maf and JunB (19, 20, 21, 22, 23). In addition, we reported previously that VIP/PACAP promote the specific survival of Th2 effectors in vivo (24). In this study, we investigated the mechanisms involved in the VIP-induced survival of Th2 effectors. Our experiments reveal a surprising new mechanism involved in Th1/Th2 AICD, i.e., the induction of enzymatically active granzyme B (GrB) upon CD3 restimulation. In wild-type (WT) Th1 and Th2 cells, apoptosis is mediated through both Fas signaling and GrB induction. In lpr (Fas-mutant) Th1 and Th2 effectors, CD3 restimulation results in apoptosis mediated solely by GrB, suggesting that GrB induction is independent of Fas signaling. VIP induces the specific survival of WT Th2 effectors by preventing the induction of GrB and the up-regulation of FasL expression in Th2 but not Th1 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

TCR-Cyt-5CC7-I/Rag1–/– transgenic (pigeon cytochrome c fragment (PCCF)-Tg, I-Ek) were purchased from Taconic Farms and bred in the animal facility at Rutgers-Newark. The B10.A (I-Ek), C57BL/6, Prf1tm1Sdz (perforin (Pfr)-deficient), and B6.MRL-Tnfrs6lpr (lpr) mice were purchased from The Jackson Laboratory. All mice used were males between 6 and 8 wk of age.

Reagents

Anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse FasL (MFL4 (11B11)), anti-mouse IL-12 (C17.8), and anti-Fas (Jo2) were purchased from BD Pharmingen. The rabbit anti-mouse GrB Ab was purchased from Lab Vision. FITC-conjugated goat anti-rabbit IgG F(ab')2 and PGE2 were purchased from Sigma-Aldrich. The granzyme inhibitor I, dibutyryl cAMP (dbcAMP), the protein kinase A (PKA) inhibitor H89, and VIP were purchased from Calbiochem. The adenylate cyclase (AC) inhibitor 2',5'-dideoxyadenosine, the exchange protein activated by cAMP (EPAC) activator 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP), the pancaspase inhibitor Z-VAD-FMK, the caspase-3 substrate Ac-IETD-pNA, the caspase-8 substrate Ac-DEVD-pNA, and the GrB substrate Ac-IEPD-pNA, were purchased from BIOMOL. The VIP receptor 1 (VPAC1) agonist [K15, R16, L27]-VIP1–7-GRF8–27 was a gift from Dr. P. Robberecht (Université Libre de Bruxelles, Brussels, Belgium), and the VPAC2 agonist Ro 25-1553 Ac-[Glu8, Lys12, Nle17, Ala19, Asp25, Leu26, Lys27,28, Gly29,30, Thr31]-VIP cyclo (21, 22, 23, 24, 25, 26, 27) was a gift from Drs. A. Welton and D. R. Bolin (both from Hoffmann-LaRoche, Nutley, NJ).

Cell isolation and culture

Effector CD4+ T cells were generated from the C57BL/6, perforin-deficient, lpr, and PCCF-Tg mice. Spleen CD4+ T cells were purified using anti-CD4 magnetic beads and the autoMACS system (Miltenyi Biotec) according to the manufacturer’s instructions. The purified T cells were >98% CD4+ by FACS analysis.

APCs were prepared by T depletion of B10.BR (I-Ek) spleen cells using anti-CD4 and anti-CD8 magnetic beads. Purified APCs were treated with 50 µg/ml mitomycin C (Sigma-Aldrich) for 20 min at 37°C.

To generate effector Th1 and Th2 cells, we used two different systems. In the first experimental system, we cultured CD4+ T cells (5 x 105 cells/ml; total volume, 1 ml) with APCs (3 x 105 cells/ml) pulsed with 5 µM PCCF. In the second experimental system, CD4+ T cells (1 x 106 cells/ml; total volume, 2 ml) were seeded in 12-well tissue culture plates containing immobilized anti-CD3 (3 µg/ml) and anti-CD28 (1 µg/ml) Abs. To generate Th1 effectors, the cells were cultured in the presence of IL-2 (10 ng/ml), IL-12 (10 ng/ml), and anti-IL-4 (1 µg/ml). To generate Th2 effectors, the cells were cultured with IL-2 (10 ng/ml), IL-4 (10 ng/ml), and anti-IL-12 (1 µg/ml). After 4–5 days, Th1 and Th2 effectors were harvested, and following removal of apoptotic cells with the Dead Cell Removal kit (Miltenyi Biotec), the CD4+ T cells were repurified and rested for 12–24 h in IL-2 (10 µg/ml). The Th1 and Th2 effectors were restimulated with immobilized anti-CD3 Abs (10 µg/ml) in the presence or absence of VIP, dbcAMP, PGE2, or inhibitors. Restimulation was performed in 48-well plates treated to maximize the efficiency of Ab binding (Sumitomo Chemical).

Th1 and Th2 cell lines were established by regular cycles of stimulation with PCCF (5 µg/ml) and APCs of polarized Th1 and Th2 effectors (5 x 105 cells/ml) for 3 days, followed by propagation in IL-2 (20 U/ml) for 5–8 days.

Splenic CD8+ T cells were positively selected with anti-CD8 magnetic beads and activated by plating on immobilized anti-CD3 and anti-CD28 Abs (see above).

Cytokine ELISA

The contents of IL-4 and IFN-{gamma} in the culture supernatants were determined by specific sandwich ELISAs. The Ab pairs used were as follows, listed by capture/biotinylated detection Abs (BD Pharmingen): IL-4, BVD4-1D11/BVD6-24G2; and IFN-{gamma}, R4-6A2/XMG1.2. The lower limits of detection for IL-4 and IFN-{gamma} were 0.1 and 0.2 ng/ml, respectively.

Macroarray

The expression of apoptotic genes in Th2 cells treated with or without VIP was compared using the Panorama mouse apoptosis gene array (Sigma-Genosys) according to the manufacturer’s protocol. A total of 273 genes was analyzed using the gene array. mRNA was extracted from Th2 cells restimulated with immobilized anti-CD3 Abs for 3 h. The mRNA was reverse transcribed using 33P-labeled dCTP, and the cDNA was used to hybridize the array. Hybridization was conducted overnight at 65°C in a hybridization chamber. The array was analyzed using a low-emission phosphorimager (Molecular Dynamics) and visualized in a Typhoon scanner (Amersham Biosciences). Fold induction or down-regulation was calculated using the software provided by the Typhoon manufacturer.

Real-time PCR

Total RNA was extracted from 5 x 106 purified CD4+ T cells using the Ultraspec RNA extraction reagent (Biotecx Laboratories) as recommended by the manufacturer. TaqMan real-time PCR was performed as described previously (28) for GrB and -actin. The primers for murine GrB were designed using the Primer Express software (Applied Biosystems). The primers and probe for GrB are as follows: (forward) 5'-CAAAGACTGGCTTCATGTCCATT-3', (reverse) 5'-GCAGAAGAGGTGTTCCATTGG3', and probe, 5'-FAM-ACAAGGACCAGCTCTGTCCTTGGCAGTAMRA-3'. The primers and probe for {beta}-actin are as follows: (forward) 5'-CGTGAAAAGATGACCCAGATCA-3', (reverse) 5'-CACAGCCTGGATGGCTACGT-3', and probe, 5'-FAM-TTTGAGACCTTCAACACCCCAGCCA-TAMRA-3'. TaqMan RT-PCR was conducted with the one-step master mix (Applied Biosystems). The real-time RT-PCR for VPAC1, VPAC2, EP-2, EP-4, Pfr, and Bcl-2 was performed by using the SYBR Green method. The following primers were used: VPAC1, (forward) 5'-CTCATCCCTCTGTTCGGAGTTC-3', (reverse) 5'-CGACGAGTTCGAAGACCATTTT-3'; VPAC2, (forward) 5'-GGACAGCAACTCGCCT CTCT-3', (reverse) 5'-AGAATGGGCATCCGAATGAC-3'; EP-2, (forward) 5'-TGCAAGAGTCGTCAGTGGCT-3', (reverse) 5'-AACAGTGCCAGTGCGATGAG-3'; EP-4, (forward) 5'-TTTCTTCGGTCTGTCGGGTC-3', (reverse) 5'-CGCTTGTCCACGTAGTGGCT-3'; and Pfr, (forward) 5'-CACGGCAGGGTGAAATTCTC-3', (reverse) 5'-CCATGCCAAGTGTCTGCCCC-3'. The primers for Bcl-2 are as follows: (forward) 5'-TGTGTGTGGAGAGCGTCAACA-3' and (reverse) 5'-GATGCCGGTTCAGGTACTCAGT-3'. Equal amounts of RNA were used for each reaction. mRNA from activated CD8+ T cells and naive CD4+ T cells were used to construct the standard curves.

Northern blot for FAP-1

Total RNA was isolated from Th1 and Th2 effectors (1 x 107 cells) using the Ultraspec RNA reagent (Biotecx Laboratories). Northern blot analysis was performed according to standard methods. The probe for murine FAP-1 was generated by RT-PCR as described previously (14).

FACS analysis

CD4+ T cells (1 x 106 cells/ml) were harvested in ice-cold RPMI 1640 medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS (wash buffer). The cells were then fixed and permeabilized with the Cytofix/Perm kit (BD Pharmingen), followed by incubation with PE-conjugated anti-CD4 or nonfluorescent anti-Fas, anti-FasL, or goat anti-mouse GrB (2.5 µg/ml final concentration) at 4°C for 1 h. Following incubation with anti-Fas and anti-FasL Abs, the cells were washed and further stained with 2.5 µg/ml FITC-conjugated goat F(ab')2 anti-hamster IgG. Following incubation with anti-GrB Ab, the cells were washed and further stained with 2.5 µg/ml FITC-conjugated rabbit F(ab')2 anti-goat IgG. Isotype-matched Ab and rabbit IgG were used as controls. The cells were analyzed on a FACScan flow cytometer (BD Biosciences). Samples in which isotype-matched Ab was used instead of specific Ab were used as negative controls to determine the proper region or window setting. Fluorescence data were expressed as geometric mean fluorescence and/or as percentage of positive cells after subtraction of background isotype-matched values. In some experiments, we stained activated Th1 effectors with the aliphatic fluorescent dye PKH26-GL (Sigma-Aldrich) according to the manufacturer’s directions.

Confocal laser microscopy

CD4+ and CD8+ T cells were fixed and permeabilized using the Cytofix/Perm kit, followed by incubation with rabbit anti-mouse GrB (2.5 µg/ml final concentration) at 4°C for 60 min. Cells were washed twice and incubated with FITC F(ab')2 goat anti-rabbit IgG (2.5 µg/ml final concentration) at 4°C for 1 h. Cells were washed three times and resuspended, mounted with antifade reagent (Molecular Probes), and visualized using epifluorescent microscopy. Activated CD8+ T cells (following exposure to anti-CD3 and anti-CD28 Abs) stained with both primary and secondary Abs were used as positive control, and activated CD8+ T cells stained only with the secondary Ab were used as negative control. Data are representative of at least 10 microscopic fields.

Measurement of apoptosis

Apoptosis was measured with the annexin V/PI apoptosis kit (BD Pharmingen) or by TUNEL staining using the In Situ Cell Death Detection kit (Roche) as per the manufacturer’s protocol. For the annexin V/PI measurements, the experimental groups were compared with the anti-CD3-restimulated T cell controls, which were considered as 100% apoptosis. For the TUNEL staining, the percentage of apoptosis represents the percentage of TUNEL+ cells.

Mitochondrial staining

Mitochondrial integrity can be detected by using the dye tetramethyl rhodamine (TMRE) (Cell Technology), which accumulates in the mitochondria depending on the properties of {Delta}{psi} (membrane potential). Intact mitochondria accumulate more dye and emit higher fluorescence. To detect mitochondrial integrity, the cells were incubated with 5 µl of TMRE dye for 60 min and analyzed by flow cytometry.

GrB enzyme assay

A total of 2 x 106 PBS-washed viable cells was resuspended in 50 µl of lysis buffer solution (150 mM NaCl, 20 mM Tris (pH 7.2), 1% (v/v) Triton X-100) for 10 min on ice. Supernatants were collected following a 10-min microfuge spin (10,000 x g). Five microliters of each lysate was used for cell lysates (5 µl) and were preincubated with the pancaspase inhibitor Z-VAD-FMK for 30 min before the addition of the GrB substrate. The paranitroanilide substrate, acetyl-Ile-Glu-Thr-Asp-paranitroanilide (Ac-IETD-pNA; BIOMOL), was used at 200 µM in reaction buffer containing 50 mM HEPES (pH 7.5), 10% (w/v) sucrose, 0.05% (w/v) CHAPS, and 5 mM DTT. GrB activity was determined by hydrolysis of the substrate at 37°C in 96-well flat-bottom tissue-culture plates (Nalge Nunc International) in a final volume of 100 µl. Released paranitroanilides were measured as absorbance at 405 nm on a fusion spectrophotometer (Packard Instrument). The enzymatic activity was quantified by using a standard curve with recombinant mouse GrB (BIOMOL) with dilutions from 2 to 200 U/ml and normalized to total protein content.

Statistics

The results are expressed as mean ± SD of at least three independent experiments. Where indicated, Student’s t test was used to compare control with experimental groups. Statistical significance was based on a value of p < 0.005.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
VIP protects Th2 cells from AICD

Previous reports indicated that VIP promotes Th2 responses in vivo through several mechanisms, including effects on Th2 survival. To investigate the molecular mechanisms by which VIP promotes the survival of Th2 cells, we focused on in vitro experiments using Th1 and Th2 effectors. Splenic CD4+ T cells from PCCF-specific TCR transgenic mice were stimulated with PCCF presented by MHC class II-compatible splenic APCs and polarized into Th1 and Th2 effectors as described in Materials and Methods. The polarization was verified by cytokine profile (IFN-{gamma} and IL-4) following restimulation (data not shown). Viable Th1 and Th2 effectors were first negatively selected with annexin V-coupled beads, followed by positive selection with anti-CD4-coupled magnetic beads, and subjected to AICD by plating on immobilized anti-CD3 Abs in the presence or absence of VIP. VIP protected Th2 but not Th1 effectors in a dose-dependent manner, with the maximum protective effect averaging 50% (Fig. 1, A and B). Similar results were obtained with Th1 and Th2 effectors generated following anti-CD3/anti-CD28 stimulation instead of antigenic peptide/APC (results not shown). A similar effect was observed for PCCF-specific Th1 and Th2 cell lines generated following repeated rounds of stimulations. In contrast to newly differentiated Th2 effectors that undergo apoptosis upon CD3 restimulation almost at the same level as Th1 effectors, Th2 cell lines are definitely more resistant, requiring several more days to reach the same apoptosis level as Th1 cells (Fig. 1C). However, regardless of this difference, VIP protects Th2 but not Th1 cell lines similar to its effect on newly differentiated effectors (Fig. 1C).



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  		FIGURE 1. VIP inhibits Th2 but not Th1 AICD. A and B, Th1 and Th2 effectors (A) and Th2 effectors (B) were preincubated with VIP (10–7 M for A and different concentrations for B) for 30 min, followed by restimulation with immobilized anti-CD3, and apoptosis was determined by annexin V/PI staining 24 h later. Annexin V+ and PI+ cells were considered apoptotic (A). Annexin V– and PI– were considered viable (B). **, Statistically significant (p < 0.005). C, Th1 and Th2 cell lines were generated as described in Materials and Methods. The Th1 and Th2 cells were restimulated with APC and PCCF in the presence or absence of IL-2 (20 U/ml) and/or VIP (10–7 M). At days 2, 4, and 7, the percentage of apoptotic cells was determined by using the TUNEL assay. The results are the mean ± SD of three independent experiments. NT, Nontreated cells.

 
Involvement of VPAC1/2 receptors in the VIP-induced survival of Th2 effectors

We showed previously that murine CD4+ T cells express VPAC1 and VPAC2 but not PAC1 receptors (29). To determine whether the differential effect of VIP on Th1 and Th2 survival is due to differences in the expression of VIP receptors, we analyzed VPAC1 and VPAC2 mRNA expression in Th1 and Th2 effectors by real-time RT-PCR. As previously reported, naive T cells express high levels of VPAC1 and low levels of VPAC2 mRNA. The expression of VPAC1 and VPAC2 in Th1 and Th2 effectors was normalized to the levels obtained for naive CD4 T cells. A significant increase was observed for both VPAC1 and VPAC2 mRNA in Th2 effectors, whereas the Th1 effectors express levels similar to that of naive T cells (Fig. 2A). To evaluate the role of VPAC1 and VPAC2 in the protective effect of VIP on Th2 survival, we used the VPAC1 agonist [K15, R16, L27]VIP1–7-GRF8–27 and the VPAC2 agonist Ro 25-1553 Ac-[Glu8, Lys12, Nle17, Ala19, Asp25, Leu26, Lys27,28, Gly29,30, Thr31]-VIP cyclo (21, 22, 23, 24, 25). Both agonists protected Th2 effectors from AICD (Fig. 2B), suggesting that both VPAC1 and VPAC2 mediate the protective effect of VIP.



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  		FIGURE 2. The role of VIP receptors and signaling pathways involved in the protective effect of VIP. A and C, Th1 and Th2 effectors were generated upon stimulation with anti-CD3 and anti-CD28 Abs as described in Materials and Methods. mRNA was extracted and subjected to real-time RT-PCR with primers and probes specific for VPAC1, VPAC2, EP-2, EP-4, and {beta}-actin. Fold change was calculated using the comparative Ct method. One representative experiment of three is shown. B, Th2 effectors were restimulated with immobilized anti-CD3 Abs in the presence or absence of VIP (10–7 M), VPAC1, or VPAC2 agonists (10–6 M). Apoptosis was determined by TUNEL 24 h later. The results are the mean ± SD of three independent experiments. D, Th1 and Th2 effectors were preincubated with various concentrations of VIP, PGE2, or dbcAMP, followed by restimulation with immobilized anti-CD3. Apoptosis was measured using the TUNEL staining. The data are represented as percentage of TUNEL+ cells. The results are the mean ± SD of three independent experiments. E, Th2 effectors were preincubated with either VIP (10–7 M) plus 2'-5'-dideoxyadenosine (10–4–10–6 M) (an AC inhibitor) or H89 (10–5–10–7 M) (a PKA inhibitor), or with the EPAC activator 8-CTP-2'-O-Me-cAMP (50–200 µM), followed by restimulation with immobilized anti-CD3 Abs. Apoptosis was determined by TUNEL 24 h later. The results are the mean ± SD of three independent experiments. NT, Nontreated cells.

 
Signaling through VPAC1 and VPAC2 results in AC activation and increases in intracellular cAMP. To evaluate the role of cAMP in the VIP-induced protection of Th2 cells, we compared the effects of VIP, PGE2, and dbcAMP. PGE2 has been reported to induce cAMP in CD4+ T cells through the PGE2 receptors (EP-2/EP-4) (30). Real-time RT-PCR experiments indicated that, similar to the VPAC1/2 receptors, the expression of EP-4 and particularly EP-2 is increased in Th2 cells (Fig. 2C). Similar to VIP, PGE2 and dbcAMP preferentially protect Th2 effectors (Fig. 2D).

To further substantiate the role of cAMP in the VIP-induced survival of Th2 effectors, we treated Th2 effectors with VIP in the presence or absence of different concentrations of an AC inhibitor before anti-CD3 restimulation. Higher concentrations of the AC inhibitor reversed the protective effect of VIP (Fig. 2E). The same AC inhibitor concentrations did not affect cell viability in the absence of anti-CD3 restimulation. Downstream cAMP targets include PKA and the recently described EPAC (31). We tested the involvement of PKA by treating Th2 effectors with VIP in the presence of various concentrations of H89, a specific PKA inhibitor, and we investigated the possible role of EPAC by treating Th2 effectors with the EPAC activator 8-CTP-2'-O-Me-cAMP before restimulation with anti-CD3. We observed a partial reversal of the protective effect of VIP only with high concentrations of H89, whereas the EPAC activator was highly efficient in protecting Th2 effectors against AICD (Fig. 2E). These experiments suggest the involvement of EPAC and, to a lesser degree, PKA in the VIP-induced cAMP signaling pathway.

VIP affects the expression of several apoptosis-related genes in Th2 effectors

To identify apoptosis-related genes whose expression is modified by VIP, we exposed Th2 cells to immobilized anti-CD3 Abs in the presence or absence of VIP for 3 h, followed by cDNA hybridization to an apoptosis gene macroarray. Among the 273 genes analyzed, VIP increased the expression of several well-known antiapoptotic factors such as FLIP, cIAP-1, XIAP, and Bcl-2 (Table I). However, we were surprised by the VIP-induced down-regulation of GrB expression. GrB is a well-known cytotoxic factor released by CTLs and NK cells involved in the killing of target cells. However, GrB has not been considered a relevant factor in the apoptosis of CD4 T cells following TCR restimulation.


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  		Table I. Changes in the expression of apoptosis-related genes in CD3-restimulated Th2 effectors treated with VIPa

 
Changes in GrB expression in Th1 and Th2 effectors undergoing AICD were confirmed by real-time RT-PCR. Th1 and Th2 effectors were restimulated with immobilized anti-CD3 Abs in the presence and absence of VIP. Restimulated Th1, regardless of the presence or absence of VIP, up-regulate GrB expression. Restimulated Th2 cells up-regulate GrB expression only in the absence of VIP (Fig. 3A). Up-regulation of GrB expression correlates with apoptosis in both Th1 and Th2 effectors, and the protective effect of VIP in Th2 cells correlates with the lack of GrB up-regulation.



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  		FIGURE 3. VIP prevents the induction of GrB mRNA expression in Th2 but not Th1 effectors. Th1 and Th2 effectors were preincubated with VIP (10–7 M) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 3 h. mRNA was extracted, and real-time RT-PCR was used to detect changes in GrB (A), Pfr (B), and Bcl-2 (C) mRNA expression. The data are expressed as fold increase in GrB, Pfr, and Bcl-2 mRNA, compared with nonstimulated controls (NT, nontreated). In each case, GrB, Pfr, and Bcl-2 expression was quantified relative to the {beta}-actin message in the same sample. One representative experiment of four is shown.

 
Because GrB activity has been associated in CTLs with Pfr, we determined Pfr expression in Th1/Th2 cells. Restimulation of both Th1 and particularly Th2 cells induces Pfr mRNA expression, although at about half of the levels of GrB induction (Fig. 3, A and B). In contrast with GrB expression, Pfr is down-regulated by VIP in both Th1 and Th2 cells (Fig. 3B).

Because the microarray data also indicated an increase in Bcl-2 upon VIP treatment, we performed real-time RT-PCR for Bcl-2 gene expression in Th2 effectors. Compared with GrB, Bcl-2 is expressed at much lower levels, and there is no significant change in Bcl-2 expression upon restimulation or VIP treatment (Fig. 3C).

Expression of GrB protein in Th1 and Th2 effectors

We restimulated Th1 and Th2 effectors with immobilized anti-CD3 Abs in the presence and absence of VIP for 6 h and analyzed intracellular GrB protein levels by FACS. The levels of GrB in cells restimulated with anti-CD3 in the absence of VIP increase dramatically, particularly in Th1 cells (Fig. 4, B and C). The presence of VIP during restimulation results in a much lower level of GrB in Th2 but not in Th1 effectors (Fig. 4, B and C). The specificity of the anti-GrB Ab was verified by preincubating the Ab with recombinant murine GrB, followed by FACS in restimulated Th1 effectors. Preincubation resulted in a reduction of staining to isotype controls (Fig. 4A). These results indicate that GrB protein levels correlate with apoptosis, i.e., high levels in Th1 and Th2 effectors undergoing AICD and low levels in VIP-treated Th2 cells, which are protected from apoptosis. Because both VPAC1 and VPAC2 mediate the VIP-protective effect in Th2 effectors, we measured the levels of intracellular GrB protein in Th2 cells treated with VPAC1 or VPAC2 agonists before anti-CD3 restimulation. Th2 cells treated with either VPAC1 or VPAC2 agonists expressed lower levels of GrB, compared with untreated controls exposed to anti-CD3 (Fig. 4D). Therefore, there is a good correlation between the effects of VIP and VPAC agonists on Th2 survival and GrB expression.



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  		FIGURE 4. VIP prevents GrB protein expression in Th2 but not Th1 effectors. A, The specificity of GrB Ab was confirmed by preincubating the Ab with recombinant murine GrB, followed by staining of restimulated Th1 effectors. B and D, Th1 and Th2 effectors were preincubated with VIP (10–7 M) (B) or VPAC1 and VPAC2 agonists (10–6 M) (D) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 6 h. Cells were fixed and permeabilized, followed by staining with rabbit anti-mouse GrB Ab. The secondary reagent was FITC-conjugated F(ab')2 goat anti-rabbit IgG. Normal goat IgG was used as control. The cells were analyzed by FACS. The data are presented as geometric mean fluorescence and as percentage of GrB+ cells. C, Quantification of the data presented in B. One representative experiment of three is shown.

 
Intracellular GrB protein was localized by confocal microscopy 6 h after restimulation with immobilized anti-CD3 Abs. Unstimulated Th1 and Th2 cells did not stain for GrB. Th1 cells restimulated in the presence or absence of VIP stain strongly for GrB; in contrast, only Th2 cells restimulated in the absence of VIP exhibit GrB staining (Fig. 5). The presence of GrB correlates with apoptosis, and the protective effect of VIP on Th2 cells correlates with the absence of GrB staining.



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  		FIGURE 5. Visualization of the GrB protein by confocal microscopy. Th1 and Th2 effectors were preincubated with VIP (10–7 M) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 6 h. The cells stained for GrB protein with the same Abs used in Fig. 4 and were subjected to epifluorescence microscopy. The cells were mounted with prolong antifade reagent to prevent photobleaching. Positive control, Activated CD8+ T cells treated with both primary and secondary Ab. Negative control, Activated CD8+ T cells treated only with the secondary Ab.

 
GrB activity in Th1 and Th2 cells

GrB is a serine protease activated by posttranslational processing. To determine whether the GrB protein detected in Th1/Th2 cells undergoing apoptosis is enzymatically active, we measured GrB enzyme activity with the synthetic substrate Ac-IEPD-pNA. Cell lysates prepared from Th1 and Th2 effectors restimulated for 24 h with immobilized anti-CD3 Abs in the presence or absence of VIP were preincubated with caspase inhibitors, followed by the addition of GrB substrate. Th1 cells restimulated in the absence or presence of VIP and Th2 cells restimulated without VIP exhibit high levels of GrB activity. In contrast, in VIP-treated Th2 cells, GrB activity does not exceed the levels observed in unstimulated cells (Fig. 6).



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  		FIGURE 6. VIP prevents GrB activity in Th2 but not Th1 effectors. Th1 and Th2 effectors were preincubated with VIP (10–7 M) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 24 h. Cell lysates were prepared from 1 x 106 cells, and GrB activity was determined as described in Materials and Methods using the synthetic colorimetric GrB substrate Ac-IEPD-pNA. The GrB activity was calculated based on a standard curve generated with recombinant murine GrB and normalized to the amounts of total protein in lysates. The results are the mean ± SD of three independent experiments.

 
Effects of VIP on mitochondrial potential in Th2 effectors

Because effects on mitochondrial membrane potential are important in the apoptotic pathways, we assessed the ability of VIP, PGE2, and dbcAMP to protect mitochondria in TCR-restimulated Th2 cells. We used the dye TMRE that fluoresces red upon accumulation in intact mitochondria. The fluorescence level decreased sharply in Th2 cells exposed to immobilized anti-CD3 Abs (Fig. 7). However, when the cells were restimulated in the presence of VIP, PGE2, or dbcAMP, fluorescence was ~50% higher (Fig. 7), indicating that a high number of the TCR-restimulated Th2 cells have intact mitochondria. These results correlate with the effects on apoptosis (Fig. 2D) and with the effects of VIP on GrB expression and activity (Figs. 3–6).



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  		FIGURE 7. Effects of Th2 restimulation on mitochondrial potential. Th2 effectors were pretreated for 30 min with VIP (10–7 M), dbcAMP (10–6 M), or PGE2 (10–6 M), followed by restimulation with immobilized anti-CD3 Abs for 6 h. The cells were harvested and incubated with the TMRE for 60 min at 37°C, followed by FACS analysis. One representative experiment of three is shown.

 
GrB is involved in AICD

Up to this point, the relationship between the effects of VIP on GrB expression in Th1 and Th2 effectors and AICD is only correlative. To demonstrate a direct cause-effect, we used the GrB inhibitor I (Z-AAD-CMK; GrB-Inh). To determine its specificity in our system, we tested the effects of the GrB inhibitor I on GrB activity in lysates from restimulated Th2 effectors. Th2 cell lysates prepared from cells restimulated for 24 h with anti-CD3 were treated with GrB-Inh (Z-AAD-CMK) or pancaspase inhibitor (Z-VAD-FMK), followed by the addition of GrB substrate (Ac-IEPD-pNA) or of a mix of caspase-3 and caspase-8 substrates (Ac-IETD-pNA and Ac-DEVD-pNA). In the absence of the inhibitors, the lysates exhibit high caspase and GrB activity (Fig. 8A). The pancaspase inhibitor inhibits the enzymatic activity for caspase-3 and caspase-8 substrates but not for the GrB substrate, whereas the GrB inhibitor inhibits the GrB but not caspase-3 and caspase-8 activity (Fig. 8A).



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  		FIGURE 8. GrB is involved in Th1 and Th2 effector AICD. A, Specificity of the GrB inhibitor I. Cell lysates were prepared from Th2 effectors restimulated with immobilized anti-CD3 Abs for 24 h as described in Materials and Methods. Equal amounts of lysate (5 µl; 1.5 µg protein/ml) were preincubated for 30 min with the pancaspase inhibitor Z-VAD-FMK (20 µM) or with the GrB inhibitor I (20 µM), followed by the addition of the GrB substrate (Ac-IEPD-pNA; 200 µM) or the caspase-3 plus caspase-8 substrate (Ac-IETD-pNA and Ac-DEVD-pNA, respectively; 200 µM) for 3 h at 37°C. The enzymatic activity was determined as the amount of pNA released per nanogram of protein. Lysis buffer instead of cell extracts was used in the No Lysate control. The results are represented as mean ± SD of three independent experiments. B, Th2 effectors were preincubated with the GrB inhibitor I (20 µM), the neutralizing anti-FasL Ab (10 µg/ml), or both for 30 min, followed by restimulation with immobilized anti-CD3 Abs. The concentrations of the GrB inhibitor and of the anti-FasL Ab were selected from previous dose-response measurements. Apoptosis was determined 24 h later by TUNEL assay. The results are the mean ± SD of three independent experiments. **, Statistically significant (p < 0.005). C, Th2 effectors were treated with etoposide (10 µM) in presence of the pancaspase inhibitor Z-VAD-FMK (10 µM), GrB inhibitor I (20 µM), or a combination of both inhibitors. Apoptosis was measured by TUNEL staining 24 h later. One representative experiments of three is presented. D, Th2 effectors were pretreated for 30 min with the pancaspase inhibitor Z-VAD-FMK (10 µM), the GrB inhibitor I (20 µM), or a combination of both inhibitors, followed by restimulation with immobilized anti-CD3 Abs for 6 h. The cells were harvested and incubated with the TMRE for 60 min at 37°C, followed by FACS analysis. One representative experiment of three is shown.

 
AICD in CD4+ effector T cells is considered to result primarily from suicide or fratricide performed through death-domain Fas receptors. To assess whether both Fas/FasL signaling and GrB participate in Th2 apoptosis, we pretreated Th2 effectors with an anti-FasL-neutralizing Ab, with the GrB inhibitor Z-AAD-CMK, or with both, followed by restimulation with immobilized anti-CD3 Abs. Both the anti-FasL Ab and the GrB inhibitor partially reduced apoptosis, whereas preincubation with both agents resulted in complete protection (Fig. 8B). Because there is always the possibility that high concentrations of inhibitors might act nonspecifically, and that Fas signaling results in caspase-8-dependent downstream caspase activation, we tested the GrB-Inh in T cell etoposide-induced death, a system where cell death is mediated through caspase-8- and caspase-2-dependent pathways (32, 33). The pancaspase inhibitor protected Th2 cells from etoposide-induced cell death, whereas the GrB-Inh had little if any effect (Fig. 8C). This indicates that the GrB-Inh, at the concentrations used in our experiments, does not directly affect caspase-8.

Fas signaling affects mitochondrial integrity through caspase-8-induced truncation of Bid, whereas GrB was reported to affect mitochondria through the direct cleavage of Bid (34, 35). To assess the role of Fas-activated caspases and of GrB on mitochondria in restimulated Th2 cells, we used the pancaspase inhibitor Z-VAD-FMK and the GrB inhibitor I (GrB-Inh). In the presence of the pancaspase inhibitor or the GrB-Inh, the fluorescence levels increased but remained below the levels of nontreated Th2 cells. Complete protection was observed in Th2 cells treated with both inhibitors (Fig. 8D), suggesting that mitochondria are affected in AICD by both caspase- and GrB-dependent pathways. These results confirm the involvement of GrB in CD4+ T cell apoptosis and suggest that the two pathways (the extrinsic Fas/FasL and an intrinsic GrB-dependent) might act independently following TCR restimulation.

Effects of VIP on Fas/FasL expression in Th1 and Th2 cells

In CD4+ T cells, AICD is considered to result primarily from signaling through Fas receptors. Several groups reported that, compared with Th1 cells, the Th2 effectors are more resistant to Fas/FasL-mediated AICD. The differential expression of Fas and FasL in Th1 vs Th2 cells has been proposed for the delayed apoptosis of Th2 cells in AICD. Therefore, we examined the effects of VIP on Fas/FasL expression in PCCF-specific Th1 and Th2 cell lines. Th1 and Th2 cells were restimulated with immobilized anti-CD3 Abs in the presence or absence of IL-2 and VIP, and surface Fas and FasL expression was determined by FACS. Both Th1 and Th2 cells cultured with IL-2 expressed lower levels of FasL, in agreement with their enhanced resistance to apoptosis (Figs. 1C and 9A). VIP did not affect FasL expression in Th1 cells, cultured with or without IL-2; in contrast, significant lower levels of FasL were detected in VIP-treated Th2 cells (Fig. 9A, upper panels). There was no effect of VIP on Fas or Bcl-2 protein levels in Th2 cells (Fig. 9A, lower panels).



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  		FIGURE 9. Effects of VIP on FasL, Fas, Bcl-2, and FAP-1 expression. A, Th1 and Th2 effectors were restimulated in the absence or presence of IL-2 and/or VIP (10–7 M). Expression of surface Fas, FasL, and intracellular Bcl-2 was determined by flow cytometry. Results are the mean ± SD of mean channel fluorescence (MCF) from three independent experiments. B, FAP-1 mRNA expression was analyzed by Northern blot. One representative experiment of three is shown.

 
In the extrinsic pathway, signaling through Fas can be prevented by antiapoptotic molecules such as FLIP and FAP-1. Our macroarrays indicate that VIP up-regulates FLIP in Th2 cells. Because FAP-1 was reported to be specific for Th2 effectors, we measured FAP-1 expression levels in cells treated with or without VIP. As expected, Th2 cells, but not Th1 cells, express FAP-1. However, VIP does not increase FAP-1 expression in Th2 cells (Fig. 9B). Taken together, these results suggest that VIP affects the extrinsic apoptotic pathway in Th2 cells by reducing FasL and increasing FLIP expression.

Both signaling through Fas and induction of GrB play a role in AICD

Our data indicate that VIP protects Th2 effectors from AICD by reducing Fas/FasL signaling (through down-regulation of FasL and up-regulation of FLIP) and by down-regulating GrB expression and suggest that the two pathways (the extrinsic Fas/FasL and an intrinsic GrB-dependent) might act independently following TCR restimulation.

To confirm that the GrB-dependent pathway can function independently of the Fas/FasL-mediated pathway, we assessed the role of GrB in AICD of Th1 and Th2 effectors generated from lpr (Fas-mutant) mice. As expected, the lpr effectors were not susceptible to the anti-Fas agonistic Ab Jo.2, whereas WT effectors underwent apoptosis when exposed to either the agonistic anti-Fas Ab Jo.2 or to restimulation through CD3 (Fig. 10A). In lpr Th1 and Th2 effectors, apoptosis induced by exposure to immobilized anti-CD3 Abs was prevented by the GrB inhibitor (Fig. 10B), indicating that restimulation through the TCR results in GrB-mediated cell death even in the absence of Fas signaling. Similar to WT effectors, exposure to VIP protected Th2 but not Th1 lpr effectors (Fig. 10B). The levels of apoptosis in the lpr Th1 and Th2 effectors correlated with the levels of GrB activity (Fig. 10C). These results indicate that the GrB-mediated apoptotic pathway functions independently of the FasL/Fas-mediated intrinsic pathway.



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  		FIGURE 10. GrB is solely responsible for activation-induced apoptosis in lpr (Fas-mutant) Th1 and Th2 effectors. A, Apoptosis in WT Th2 cells: WT Th2 effectors were preincubated with or without VIP (10–7 M) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 24 h, or treated with the agonistic anti-Fas Jo.2 Ab (20 µg/ml) for 24 h. Lpr Th2 effectors were treated with the agonistic anti-Fas Jo.2 Ab (20 µg/ml) for 24 h. Apoptosis was determined by annexin V/PI staining. B, Apoptosis in CD4+ T cells from lpr mice: lpr Th1 and Th2 effectors were preincubated with VIP (10–7 M) or GrB inhibitor I (20 µM) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 24 h. Apoptosis was determined by the TUNEL assay. C, GrB activity in lpr mice CD4+ T cells: lpr Th1 and Th2 effectors were preincubated with VIP (10–7 M) for 30 min, followed by restimulation with immobilized anti-CD3 Abs for 24 h. GrB enzymatic activity was measured in Th1 and Th2 cell lysates as described in Materials and Methods. The GrB activity was calculated based on the standard curve generated with recombinant murine GrB and normalized to the amounts of total protein in lysates. The results are the mean ± SD of three independent experiments.

 
GrB produced by TCR-restimulated Th1 effectors kills activated Fas-mutant CD4+ T cells

The cytotoxic mechanism for target cell killing by CTLs involves the directional release of GrB/Pfr granules from the activated CTLs, followed by endocytosis by target cells and intracellular release of GrB in a calcium-dependent, Pfr-mediated process. To determine whether GrB-dependent cell death of TCR-restimulated Th effector cells involves the transfer of GrB from one cell to another (fratricide), we generated Th1 effectors from WT (C57BL/6) and Fas-mutant lpr (mFas) mice. The mFas Th1 cells stimulated with anti-CD3 and anti-CD28 Abs were prestained with the fluorescent membrane dye PKH26. The WT Th1 cells were restimulated through contact with immobilized anti-CD3 Abs for 6 h and expressed GrB protein (results not shown). The two Th1 populations were cocultured for 24 h in the absence of anti-CD3 Abs, and apoptosis was determined through annexin staining. The mFas Th1 cells did not express GrB and could not initiate Fas signaling. In contrast, the WT-restimulated Th1 effectors expressed GrB and could initiate Fas signaling. WT Th1 cells undergoing apoptosis are PKH26–annexin+, whereas apoptotic mFas Th1 cells are PKH26+annexin+. In controls containing only mFas or WT Th1 cells, 3 and 48% of the cells are annexin+, respectively (Fig. 11A, upper panels). In contrast, in mixed cultures, 30% of the PKH26+ mFas cells are annexin+, and the GrB-Inh reduces the number of apoptotic mFas cells to 19% (Fig. 11A, lower panels). In addition, cell death also is reduced (18% apoptotic cells) in the presence of EGTA. Similar results were obtained in terms of annexin mean cell fluorescence (Fig. 11B). These results indicate that Th1 cells lacking functional Fas and not expressing GrB undergo apoptosis in a GrB-and calcium-dependent manner when exposed to restimulated Th1 cells that express GrB. This suggests that GrB is transferred from the restimulated WT Th1 effectors to activated (but not restimulated) mFas Th1 cells and induces cell death (fratricide).



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  		FIGURE 11. GrB is involved in fratricide killing of Th1 effectors. A and B, Th1 effectors were generated from WT (C57BL/6) and from lpr Fas-mutant (mFas) CD4+ splenic T cells. The mFas Th1 effectors were generated and activated for 3 days in the presence of IL-12, IL-2, anti-CD3, and anti-CD28 Abs. The WT Th1 cells were generated in a similar manner, followed by TCR restimulation with immobilized anti-CD3 Abs. mFas Th1 cells were fluorescently labeled with the plasma membrane intercalating dye PKH26. Equal numbers of mFas Th1 and WT Th1 cells (5 x 105 cells/ml) were cocultured for 24 h in the presence or absence of EGTA (5 mM) or GrB-inhibitor (20 µM). Following staining with Annexin VFITC, the cells were analyzed by FACS. A, The numbers in represent percentages of annexin+ cells in either the mFas (PKH26+) or the WT (PKH26–) population. B, Annexin fluorescence (mean channel fluorescence) for the gated mFas Th1 population (gating indicated by rectangles in A). One representative experiment of four is shown.

 
The restimulated WT Th1 effectors (PKH26–) undergo apoptosis, because they express both functional Fas and GrB. However, similar to mFas Th1 cells, the WT Th1 effectors in mixed cultures are partially protected from apoptosis by the GrB-Inh and EGTA (Fig. 11A, lower panels).

EGTA inhibits apoptosis in WT but not in perforin-deficient Th2 effectors

CTLs and NK cells exert their cytotoxic effect primarily through the directional release of GrB/Pfr-containing granules in the immediate vicinity of target cells. Granule exocytosis and Pfr polymerization are calcium-dependent processes. To determine whether calcium is required for Th1/Th2 AICD, we restimulated Th1 and Th2 effectors in the presence of EGTA. Apoptosis of both Th1 and Th2 effectors was significantly reduced by EGTA, indicating that AICD in Th1 and Th2 effectors occurs partially through a calcium-dependent mechanism (Fig. 12A).



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  		FIGURE 12. Role of calcium in Th1 and Th2 effector AICD. A, EGTA prevents AICD in both Th1 and Th2 cells. Th1 and Th2 effectors were restimulated with immobilized anti-CD3 in the presence of EGTA (5 mM), and apoptosis was determined by TUNEL staining 24 h later. The results are the mean ± SD of three independent experiments. B, Role of GrB and calcium in Pfr-deficient Th2 AICD. Th2 effectors generated from WT (C56BL/6) mice, and Pfr-deficient (Pfr–/–) mice were preincubated with blocking anti-FasL Abs (10 µg/ml), GrB inhibitor I (GrB-Inh; 20 µM), anti-Fas+Grb-Inh, VIP (10–7 M), or EGTA (5 mM) for 30 min, followed by restimulation with immobilized anti-CD3 Abs. Apoptosis was measured 24 h later by TUNEL. The mean fluorescence intensity of anti-CD3-only-treated cells was set as 100% for both WT and Pfr–/– Th2 cells and used as reference for apoptosis percentages. The results are the mean ± SD of four independent experiments.

 
Although Fas/FasL-induced apoptosis is calcium independent, EGTA could affect other apoptosis-related mechanisms. The delivery of GrB/Pfr to target cells by CTLs and NK requires calcium, and the release of GrB within the target cells requires Pfr. We observed increased Pfr expression in addition to GrB in restimulated Th1 and Th2 effectors (Fig. 3B), suggesting that the delivery of GrB/Pfr from one Th cell to another could result in apoptosis (fratricide). To assess the role of Pfr in AICD, we compared WT Th2 effectors with Th2 cells derived from Pfr-deficient (Pfr–/– mice). The expectation was that Th2 cells from Pfr–/– mice would undergo apoptosis following Fas/FasL signaling but not through GrB. Pfr–/– Th2 cells undergo less apoptosis than WT Th2 effectors (three times less increase in annexin fluorescence). Surprisingly, however, both a neutralizing anti-FasL Ab and the GrB-Inh partially prevented apoptosis in WT, and Pfr–/– restimulated Th2 effectors (Fig. 12B). Apoptosis was almost completely prevented in the presence of both inhibitors. VIP was equally protective for WT and Pfr–/– Th2 cells (Fig. 12B). These results suggest that, in Th effectors, AICD is partially mediated through GrB, even in the absence of Pfr. Because Pfr is required for the release of GrB from endolysosomes following transfer to target cells, GrB-mediated apoptosis in the absence of Pfr (in Pfr–/– Th2 cells) could result from GrB leakage from granules within the GrB-producing cell (suicide instead of fratricide). Because the suicide model does not involve calcium-dependent exocytosis and Pfr transfer, EGTA should not affect cell death. This is indeed the case, because EGTA inhibits AICD in WT Th2 effectors without affecting cell death in Pfr–/– Th2 cells (Fig. 12B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In response to antigenic stimulation, CD4+ T cells differentiate into Th1 and Th2 effectors with different cytokine profiles and immune functions. Differentiation into Th1 and Th2 effectors depends on Ag dose and site of entry, on the APC activation stage, and on the composition of the local milieu in terms of cytokines and immunomodulatory hormones, neuropeptides, and lipid mediators.

Following differentiation, Th1 and Th2 effectors proliferate rapidly, leading to high numbers of Ag-specific cells in a relatively short time. Homeostasis is re-established through apoptosis, primarily AICD that occurs upon TCR restimulation of previously activated T cells. Therefore, the Th1 and Th2 balance leading to the establishment of a particular type of immune response depends not only on factors that influence differentiation, but also on the regulation of Th1 vs Th2 survival. In general, Th1 effectors have been shown to be more susceptible to AICD than are Th2 cells, and the higher resistance of Th2 effectors has been attributed alternatively to lower levels of FasL expression, up-regulation of blockers of Fas signaling such as FAP-1 or c-FLIP, or higher levels of CTLA-4 expression (7, 9, 12, 14, 16). However, at the present time, the definite role of either one of these factors is still controversial.

We have reported previously that neuropeptides such as VIP and PACAP promote Th2-type responses, and that VIP/PACAP increase survival of Th2 effectors in vivo and in vitro (19, 24). In addition, studies with VPAC2-transgenic and VPAC2-knockout mice indicate an important role for endogenous VIP in the development of a prevalent Th2 immune bias (21, 23). In this study, we investigated the mechanisms by which VIP promotes Th2 survival and concluded that VIP prevents GrB expression and reduces FasL expression in Th2 but not Th1 effectors.

The involvement of GrB in the AICD of Th1 and Th2 effectors is surprising because GrB has been associated previously only with the killing of target cells by CD8+ CTL and NK cells (36, 37, 38, 39, 40). GrB expression also has been reported in activated CD4+ T cells, particularly adaptive regulatory T cells, with cytotoxic activity (41, 42). However, GrB message has been identified by microarray in cells apparently not involved in cytotoxicity. Particularly relevant to our study is the report by Hamalainen et al. (43) showing that Th1 cells polarized from human neonatal cord blood CD4+ T cells and subjected to several rounds of restimulation express high levels of GrB. Based on these observations and on our own studies, we would like to propose that, in addition to its well-established role in CTL- and NK-mediated cytotoxicity, the de novo expression of GrB in noncytotoxic cells represents a potent mechanism for apoptotic cell death.

The proposed role of GrB in AICD requires an enzymatically active molecule. In CTL, GrB gene expression is induced following Ag stimulation, and the GrB protein is shuttled as an inactive proprotease from the Golgi into lysosomal vesicles, where it is activated upon cleavage by cathepsin C. The enzymatically active GrB is stored in the cytotoxic granules until its release following conjugate formation with target cells (40, 44, 45). We observed early expression of the GrB gene in both CD3-restimulated Th1 and Th2 effectors and confirmed GrB expression at protein level by intracellular FACS staining and confocal microscopy. The immunoreactive GrB appears to be localized in discrete cytoplasmic patches, possibly granules, in both Th1 and Th2 effectors undergoing apoptosis. In addition, we confirmed that cell lysates from CD3-restimulated Th1 and Th2 cells contain enzymatically active GrB. These results suggest that GrB expression in CD3-restimulated Th1 and Th2 effectors contributes to AICD. This is indeed supported by the fact that the GrB inhibitor I, at concentrations that do not affect caspase-3 and caspase-8 activity, protects WT, lpr, and Pfr–/– Th1 and Th2 effectors from apoptosis.

In CTLs and NK cells, there is directional granule exocytosis and release of GrB, followed by GrB endocytosis by target cells. Although initial models envisioned GrB entering the target cells through pores created by polymerized Pfr, recent studies showed that GrB endocytosis can occur in the absence of Pfr. However, Pfr or other endolysomotropic agents, such as adenovirus or bacterial toxins, are required following GrB transfer for GrB release within the target cell and subsequent cell death (reviewed in Ref.46). The processes directly related to Pfr require calcium, and EGTA prevents CTL-induced, GrB/Pfr-mediated cell death. The fact that EGTA significantly reduces apoptosis in restimulated WT Th1 and Th2 effectors suggests that GrB/Pfr released by one cell induces cell death in a neighboring target (fratricide). Indeed, activated Fas-mutant Th1 cells that do not express GrB were killed when cultured with restimulated, GrB-expressing Th1 effectors, and the GrB inhibitor I and EGTA inhibited cell death.

However, the possibility also exists that TCR restimulation would promote local release of endogenous GrB and Th1 and Th2 cell death through suicide. Cell death due to cytoplasmic GrB leakage was reported for CTLs (reviewed in Ref.46). Because Pfr is considered essential for GrB release following transfer to target cells (47), the fact that AICD of Pfr-deficient Th1 effectors is still inhibited by the GrB inhibitor I suggests that, in the absence of Pfr, Th1 and Th2 effectors are killed through suicide rather than fratricide. In agreement with the role of calcium in Pfr-dependent mechanisms, EGTA does not prevent apoptosis in Pfr-deficient Th effectors.

In contrast to Th1 cells, Th2 effectors restimulated in the presence of VIP show significantly reduced levels of GrB message, intracellular protein, and GrB activity. These results suggest that VIP protects Th2 effectors from AICD at least partially by preventing GrB expression. The immunological effects of VIP are exerted through a family of receptors, i.e., VPAC1, VPAC2, and PAC1 (48). We reported previously that CD4+ T cells express VPAC1 and VPAC2 but not PAC1 (29, 49). Real-time RT-PCR data indicate that the Th2 effectors express higher levels of VPAC1 and VPAC2, compared with Th1 and naive CD4+ T cells. The higher density of VIP receptors on Th2 cells could be the reason for the prevalent protective effect of VIP on Th2 effectors. Both VPAC1 and VPAC2 mediate the protective effect of VIP and activate AC, resulting in increased cAMP levels (50). The role of cAMP in protecting Th2 cells from activation-induced apoptosis is supported by the fact that PGE2, another cAMP-inducing agent, and dbcAMP exerts similar effects. Similar to the VIP receptors, Th2 effectors express higher levels of EP-2/EP-4 receptors, the PGE2 receptors responsible for AC activation. The downstream signaling pathway involved in the protective effect of VIP on Th2 effectors appears to involve EPAC, and to a lesser degree, PKA activation. In addition to increased expression of VIP and PGE2 receptors, Th2 cells also might be more efficient in generating EPAC following AC activation.

The link between cAMP signaling and GrB gene expression remains to be established. Several transcription factors, including AP-1, CREB, CCAAT binding factor, and Ikaros, have been shown to bind and play a role in the activation of the murine GrB promoter (51). Effects of the VIP-induced cAMP signaling on these transcription factors or the possible induction of a transcription repressor remain to be determined.

We reported previously that VIP inhibits AICD in CD4+ T cells and that this correlates with a reduction in FasL expression (29). The effects of VIP on FasL expression are mediated through reductions in the expression or DNA binding activity of several transcription factors, i.e., c-myc, NF-{kappa}B, NF-AT, and Egr2/3 (52). The present study shows that VIP reduces FasL expression in Th2 but not Th1 effectors. This suggests the possibility that VIP protects Th2 cells from activation-induced apoptosis through two mechanisms, i.e., reduction in GrB and FasL expression. An important question is whether the two mechanisms are independent.

Our results indicate that, although the GrB inhibitor and an anti-FasL Ab partially reduced apoptosis in WT Th2 cells, together they completely prevented Th2 cell death. Finally, activation-induced apoptosis in lpr (Fas-mutant) Th1 and Th2 effectors is prevented by the GrB inhibitor, indicating that the GrB pathway functions independently of the Fas/FasL pathway. Similar to WT Th1/Th2 cells, VIP protected and reduced GrB activity only in lpr Th2 effectors. We conclude that GrB and the Fas/FasL pathways act independently in the activation-induced apoptosis of WT Th1 and Th2 effectors. The lack of linkage between the FasL/Fas and the GrB pathways is supported by the fact that Fas-mediated killing is normal in GrB-deficient animals (53).

Based on our results, we propose the following model for the VIP protection of Th2 cells. The clonally expanded Th1 and Th2 effectors are eliminated upon TCR restimulation through activation-induced apoptosis following expression of both FasL and GrB. Following differentiation, Th2 but not Th1 effectors up-regulate the expression of VIP receptors, becoming more responsive to VIP. VIP, released from the innervation in response to inflammatory signals such as NO (54, 55) or produced by Ag-specific Th2 cells (56), reduces both FasL and GrB expression in Th2 effectors and therefore promotes their survival. The VIP-induced survival of Th2 effectors is in agreement with its general anti-inflammatory function. By tilting the balance in favor of Th2 cells, through effects on both Th1 and Th2 differentiation and survival, VIP contributes to the reduction of the proinflammatory potential of the immune response. This is particularly relevant for sites with a high abundance of VIP sources, such as the gastrointestinal tract and the immune-privileged organs, where acute inflammatory processes are particularly harmful.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI47325 and AI52306 (to D.G.) and Johnson & Johnson Neuroimmunology Fellowships (to V.S.). Back

2 Address correspondence and reprint requests to Dr. Doina Ganea at the current address: Department of Physiology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. E-mail address: dganea{at}andromeda.rutgers.edu Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; FAP-1, Fas-associated phosphatase 1; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate-cyclase activating polypeptide; GrB, granzyme B; WT, wild type; PCCF, pigeon cytochrome c fragment; dbcAMP, dibutyryl cAMP; PKA, protein kinase A; AC, adenylate cyclase; EPAC, exchange protein activated by cAMP; VPAC1/2, VIP receptors 1 and 2; TMRE, tetramethyl rhodamine; Pfr, perforin. Back

Received for publication April 28, 2005. Accepted for publication October 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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